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CHARACTERIZATION OF FUNGAL PATHOGEN(S) CAUSING
WILT OF LENTIL AND THEIR MANAGEMENT
Faculty of Crop and Food Sciences
Arid Agriculture University Rawalpindi
CHARACTERIZATION OF FUNGAL PATHOGEN(S) CAUSING
WILT OF LENTIL AND THEIR MANAGEMENT
KHOLA RAFIQUE
03-arid-47
Department of Plant Pathology
Faculty of Crop and Food Sciences
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
Pakistan
2015
CHARACTERIZATION OF FUNGAL PATHOGEN(S) CAUSING
WILT OF LENTIL AND THEIR MANAGEMENT
Arid Agriculture University Rawalpindi
ii
CHARACTERIZATION OF FUNGAL PATHOGEN(S) CAUSING
WILT OF LENTIL AND THEIR MANAGEMENT
by
KHOLA RAFIQUE
(03-arid-47)
A thesis submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
in
Plant Pathology
Department of Plant Pathology
Faculty of Crop and Food Sciences
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
Pakistan
2015
iii
CERTIFICATION
I hereby undertake that this research is an original one and no part of this
thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible
for the consequences.
Student’s Name: Khola Rafique Signature: ____________
Registration No: 03-arid-47 Date: ____________
Certified that the contents and form of thesis entitled “Characterization of
Fungal Pathogen(s) Causing Wilt of Lentil and their Management” submitted
by Ms. Khola Rafique have been found satisfactory for the requirement of the
degree.
Supervisor: ______________________________ (Prof. Dr. Abdul Rauf)
Member: ______________________________ (Dr. Farah Naz)
Member: ______________________________
(Dr. Ghulam Shabbir)
Chairman: _________________________ Dean: __________________________ Director Advanced Studies: __________________________
iv
v
vi
CONTENTS
Page
List of Tables x
List of Figures xi
List of Abbreviations xvi
Acknowledgement xviii
Abstract xx
1. INTRODUCTON 1
2. REVIEW OF LITERATURE 8
2.1 THE IMPORTANCE OF LENTIL 8
2.2 LENTIL DISEASES 9
2.3 LENTIL WILT AND THE CAUSAL ORGANISMS 11
2.4 BIOLOGY AND MORPHOLOGICAL CHARACTERIZATION
OF FUSARIUM SPECIES ASSOCIATED WITH LENTIL
WILT
12
2.4.1 Fusarium oxysporum 13
2.4.2 Fusarium redolens 15
2.4.3 Fusarium nygamai 15
2.4.4 Fusarium commune 16
2.4.5 Fusarium equiseti 17
2.5 SURVIVAL AND DISEASE CYCLE OF FUSARIUM WILT OF
LENTIL
17
2.6 SYMPTOMS OF FUSARIUM WILT OF LENTIL 18
2.7 SILICA GEL PRESERVATION OF FUSARIUM ISOLATES 19
vii
2.8 MOLECULAR CHARACTERIZATION AND
PHYLOGENETIC ANALYSIS OF FUSARIUM ISOLATES
THROUGH DNA SEQUENCING
20
2.9 INCIDENCE, DISTRIBUTION AND YIELD LOSSES OF
LENTIL FUSARIUM WILT
24
2.10 PATHOGENICITY TEST 25
2.11 SCREENING FOR HOST RESISTANCE AGAINST LENTIL
WILT
26
2.12 BIOLOGICAL MANAGEMENT OF LENTIL WILT 28
2.13 CHEMICAL MANAGEMENT OF LENTIL WILT 30
3. MATERIALS AND METHODS 33
3.1 DISEASE SURVEY AND ASSESSMENT 33
3.1.1 Disease Survey 33
3.1.2 Disease Assessment and Sampling 35
3.2 ISOLATION AND IDENTIFICATION OF THE PATHOGENS 36
3.3 PRESERVATION OF FUSARIUM ISOLATES 37
3.4 MORPHOLOGICAL CHARACTERIZATION 38
3.5 PATHOGENICITY TEST 38
3.5.1 Disease Parameters 42
3.6 MOLECULAR CHARACTERIZATION 43
3.6.1 Mycelium Production and DNA Extraction 43
3.6.2 Polymerase Chain Reaction (PCR) Amplification of TEF-1α
Gene Region
45
3.6.3 PCR Product Analysis 47
viii
3.6.4 DNA Sequencing 47
3.6.5 Phylogenetic Analysis 48
3.7 MANAGEMENT OF FUSARIUM WILT 48
3.7.1 Management Through Host Plant Resistance 49
3.7.1.1 Disease parameters 49
3.7.2 Biological Management 50
3.7.2.1 Disease parameters 51
3.7.3 Chemical Management 51
3.7.3.1 In vitro evaluation of fungicides 51
3.7.3.2 In vivo evaluation of fungicides 53
3.7.3.2.1 Disease parameters 54
4. RESULTS AND DISCUSSION 55
4.1 DISEASE SURVEY AND ASSESSMENT 55
4.1.2 Disease Sampling 65
4.2 ISOLATION AND IDENTIFICATION OF THE PATHOGENS 67
4.3 PRESERVATION OF FUSARIUM ISOLATES 69
4.4 MORPHOLOGICAL CHARACTERIZATION 71
4.4.1 Colony Color 85
4.4.2 Growth Habit 85
4.4.3 Pigmentation 88
4.4.4 Days to Fill 9 cm Dish 88
4.4.5 Concentric Rings 90
4.4.6 Conidiophore and Phialide 90
4.4.7 Shape and Size of Micro-conidia 92
ix
4.4.8 Shape and Size of Macro-conidia 95
4.4.9 Shape of Apical and Basal Cells of Macro-conidia 104
4.4.10 Septation in Macro-conidia 106
4.4.11 Chlamydospores: Formation and Diameter 106
4.4.12 Interseptal Distance 108
4.5 PATHOGENICITY TEST 116
4.6 MOLECULAR CHARACTERIZATION 126
4.7 MANAGEMENT OF FUSARIUM WILT 150
4.7.1 Management Through Host Plant Resistance 150
4.7.2 Biological Management 155
4.7.3 Chemical Management 158
4.7.3.1 In vitro evaluation of fungicides 158
4.7.3.2 In vivo evaluation of fungicides 164
SUMMARY 171
CONCLUSION AND RECOMMENDATIONS 178
LITERATURE CITED 179
APPENDICES 205
x
List of Tables
Table No.
Page
3.1 Fungicides used for the management of lentil Fusarium wilt 52
4.1 Location-wise percent disease prevalence and incidence 60
4.2 Fusarium culture identification and morphological
characterization checklist
72
4.3 Mean/ standard deviation (S.D.) in four morphological
characteristics of Fusarium species
96
4.4 Virulence of morphologically identified and characterized
Fusarium species tested on lentil germplasm NARC-08-1 and
Masoor-93
117
4.5 Identification of Fusarium isolates based on DNA sequencing
of the translation elongation factor 1-α gene region
139
4.6 Involvement of major Fusarium species in lentil wilt and plant
mortality
144
4.7 Screening of lentil germplasm against Fusarium wilt 151
4.8 Effect of fungicides at different concentrations on mycelia
radial growth of Fusarium isolate
160
xi
List of Figures
Figure No.
Page
1.1 Lentil production zones 2
3.1 Map of Punjab Province showing major lentil districts
surveyed for the assessment of wilt prevalence, incidence and
collection of Fusarium isolates
34
3.2 Lentil seedlings in germinators (a) 8-days old, and (b) 15-days
old
40
3.3 Lentil roots dipping in fungal spore suspension (1 x
107conidia/ mL)
41
3.4 Inoculated lentil seedlings after transplantation to pots in
screen house
41
3.5 Schematic of part of translation elongation factor (TEF-1α)
gene region showing primer positions
46
4.1 Map of Pakistan and major lentil growing districts and sites of
Punjab province surveyed for the assessment of Fusarium wilt
prevalence, incidence and distribution
56
4.2 Wilted lentil fields: (a) Layyah-Fateh Pur, and (b) Bhakkar-
Garh Morr
57
4.3 District-wise disease incidence of lentil wilt at two plant
growth stages during 2011-12 and 2012-13 crop seasons.
58
4.4 Patches of wilted lentil plants in fields: (a) Chakwal-Piplee 66
xii
field, and (b) Bhakkar-Garh Morr field
4.5 Lentil plant samples: (Right) Wilted lentil plant, and (Left)
Healthy lentil plant (left)
68
4.6 Preserved Fusarium isolates on silica gel in glass vials stored
at 4±2oC (a), Gel crystals coated with fungal growth (b), and
Revived Fusarium culture using silica gel crystals after 5 days
of incubation at 25oC (c)
70
4.7 Isolates showing distinct White (a), Creamy white (b), and
Pinkish white (c) colony color
86
4.8 Petriplates showing variation in growth patterns of Fusarium
isolates: (a) Fluffy, (b) Compact, and (c) Flat
87
4.9 Petriplates showing presence and absence of pigmentation in
Fusarium isolates: (a) Isolate FWC1 without any colored
pigmentation, (b) Isolate FWC5 with dark violet pigmentation,
(c) Isolate FWB3 with violet pigmentation, (d) Isolate FWC3
with Pale brown pigmentation, (e) Isolate FWM1 with Light
brown pigmentation, and (f) Isolate FWB11 with Pink
pigmentation
89
4.10 Petriplates (9 cm) with mycelia at varied radial growth after 7
days of incubation at 25 °C
91
4.11 Isolates showing production of distinguished concentric rings
in 12 hours light/ darkness cycle: (a) With 1 concentric ring,
and (b) With 2 concentric rings
91
4.12 Phialide characteristics observed in Fusarium isolates under 93
xiii
light microscope at100X magnification: (a, b) Short and plump
monophialides, (c) Long monophialdes, and (d) Polyphialides,
and (a-d) Scale bar = 50 µm
4.13 Micro-conidia of Fusarium isolates under light microscope at
100X magnification: (a) Oval single-celled and two-celled
micro-conidia, (b) Obovoid micro-conidia, (c) Measurement of
length of a micro-conidium with a scale bar, (d) Measurement
of width of a micro-conidium with a scale bar, and (a-d) Scale
bar = 25 µm
94
4.14 Macro-conidia (a) and Macro-conidial shapes observed under
light microscope at 100X magnification (b-f): (b) Slightly
curved macro- conidia of isolate FW, (c) Straight-shape
macro-conidia of isolate FWC10, (d, e, f) Slender macro-
conidia of isolate, and (a-f) Scale bar = 25 µm
103
4.15 Macro-conidia with curved-shaped apical and foot-shaped
basal cells under light microscope at 100X magnification (a),
and Septation of macro-conidia (b-d): (b) Three-septate, (c)
Four-septate, (d) Five-septate, and (a-d) Scale bar = 25µm
105
4.16 Formation of Chlamydospores: (a) Singly, (b) Pairs, (c) Short
chains, (d) Clusters, (e) Rough-walled, (f) Smooth-walled, and
(a-d) Scale bar = 25 µm
107
4.17 Production of Chlamydospores: (a, b, c) Terminally, (d, e)
Intercalary, (f) Measurement of diameter of chlamydospore
with a scale bar at 100X magnification under light microscope,
109
xiv
and (a-f) Scale bar = 25 µm
4.18 Septate, hyaline and branched hyphae and mycelia (a and b),
measurement of Interseptal distance using a scale bar under
light microscope at 100X magnification (c), and Scale bar = 50
µm (a-c)
110
4.19 Pathogenicity testing of Fusarium isolates: (a) Screen house
pot experiment showing characteristic wilt symptoms on lentil
germplasm NARC-08-1 and Masoor-93, (b-e) Variation in wilt
incidence and severity on NARC-08-1: (b) Highly virulent
reaction with wilted and dead plants, (c) Moderately virulent
reaction, (d) Low virulent reaction, and (e) Avirulent reaction
with healthy plants
120
4.20 PCR amplification products (700bp) of genomic DNA of 67
(a-e) Fusarium isolates using TEF-1α primers
128
4.21 Phylogenetic tree based on Maximum likelihood analysis
generated from the translation elongation factor-1α gene
sequences of 67 Fusarium isolates from lentil along with the
sequences of GenBank accessions. F. beomiforme and F.
concolor were used to root the tree. Maximum likelihood
bootstrap values from 1000 maximum likelihood replications
are indicated on the branches
130
4.22 Phylogenetic tree based on Maximum likelihood analysis
generated from the translation elongation factor-1α gene
sequences of 13 most highly virulent Fusarium isolates from
135
xv
lentil along with the sequences of GenBank accessions. F.
beomiforme and F. concolor were used to root the tree.
Maximum likelihood bootstrap values from 1000 maximum
likelihood replications are indicated on the branches
4.23 Occurrence and frequency percentage of five species of
Fusarium associated with lentil wilt and plant mortality in
districts of Punjab
145
4.24 Influence of antagonists on percent disease severity index,
incidence and grain yield reduction. Different letters
indicated on bars represent significant differences in wilt
severity, incidence and yield reduction values (P<0.05)
156
4.25 Mycelial radial growth in MEA plates treated with fungicides
at 30 ppm concentration
161
4.26 Mycelial growth inhibition (%) at five concentrations.
Different letters indicated on bars represent significant
differences in inhibition values (P<0.05)
162
4.27 Percent disease severity index, incidence and grain yield
reduction. Different letters indicated on bars represent
significant differences in wilt severity, incidence and yield
reduction values (P<0.05)
165
xvi
List of Abbreviations
AARI Ayub Agriculture Research Institute
AFLP Amplified Fragment Length Polymorphism,
AZRI Arid Zone Research Institute
BLAST Basic Local Alignment Search Tool
BME β-mercaptoethanol
bp Base pair
BS Bootstrap
cm Centimeter
CSI Crop Sciences Institute
cv Cultivars
dia Diameter
ºC Degrees Celsius (Centigrade)
EDTA Ethylenediaminetetraacetic acid
et al. and others
gm Gram
HCl Hydrochloric acid
ICARDA International Centre for Agricultural Research in the Dry
Areas
ISSR Inter Simple Sequence Repeat
ITS Internal Transcribed Spacer
kg Kilogram
L Litre
xvii
min Minute
ML Maximum Likelihood
mL Milliliter
mm Millimeter
NARC National Agricultural Research Centre
NCBI National Center for Biotechnology Information
NIAB Nuclear Institute for Agriculture and Biology
PCR Polymerase Chain Reaction
pH Proportionate hydrogen ions
ppm Parts per million
% Percent
psi Pound-force per square inch
RAPD Random Amplified Polymorphic DNA
SDS Sodium dodecyl sulphate
sp. Species
SPSS Statistical package for the Social Sciences
SSR Simple Sequence Repeat
TEF Translation Elongation Factor
Tris Hydroxymethyl aminomethane
µg Microgram
µL Microliter
µm Micrometer
xviii
AKNOWLEDGEMENT
First and foremost, I praise “Allah”, the Benevolent and Clement, for
providing me this opportunity and granting me the capability to proceed
successfully. I offer my heartfelt thanks to “Holy Prophet Hazrat Muhammad”
(Peace Be Upon Him), a source of endless knowledge, blessing and mercy for
whole mankind, who enabled me to recognize my Creator and declared it to be an
obligatory duty of every Muslim to acquire knowledge.
It is quite delectable to avail this most propitious opportunity to aciculate
with utmost gratification, my profound and intense sense of indebtedness to my
research supervisor, Dr. Abdul Rauf , Professor, Department of Plant Pathology,
PMAS Arid Agriculture University Rawalpindi (PMAS-AAUR), for guiding me at
each step throughout my research period with patience, kindness and scholarly
knowledge whilst allowing me the room to work in my own way. He provided both
technical insight and a broad overview essential to this thesis. His truly scientist
intuitions exceptionally inspired and enriched my growth as a student, a researcher
and a scientist I want to be. I am indebted to him more than he knows.
It is a great pleasure and honor for me to pay tribute to my foreign advisor
Dr. Seogchan Kang, Professor, Department of Plant Pathology and Environmental
Microbiology (PPEM), Pennsylvania State University (PSU), USA for his endless
and kind support during my stay at PSU, USA. My cordial thanks are extended for
his warm encouragement, thoughtful guidance, insightful discussion and valuable
advice during the whole stay. I have been amazingly fortunate to have an advisor
like him. I am also thankful to Dr. David Geiser, Professor/ Director, Fusarium
Research Center, PPEM, PSU, USA for his expert advice and valuable suggestions
and for providing me the opportunity for attending his graduate course that helped
me understand my research area better and enriched my ideas.
I am unfathomably indebted to Dr. Farah Naz, Assistant Professor, PMAS-
AAUR and Dr. Ghulam Shabbir, Assistant Professor, PMAS-AAUR to be the
members of my supervisory committee. I am very much grateful to Dr. Farah Naz
for her kind and scholastic guidance, keen interest, consistent encouragement and
xix
well wishes during the course of my study. I value the indefatigable and skillful
way in which my thesis was shaped by her.
My appreciation is extended to my friend Sania Ahmed, Ph.D. Scholar,
PMAS-AAUR and also to all fellows and friends at PPEM, PSU, USA, specially
Dr. Jung Eum Kin , Post Doc Fellow and Ningxiao Li , Ph.D. Scholar. I greatly
value their friendship and deeply appreciate their belief in me. I will never forget
the time that we were together.
I would also like to acknowledge and appreciate the research funding
received from Pakistan Science Foundation, Islamabad for my Ph.D. study. I am
also grateful to Higher Education Commission, Islamabad for providing me the
opportunity to get International exposure and experience under their International
Research Support Initiative Program (IRSIP), which not only strengthened my
study but also enhanced my scientific approach. The laboratory and library
facilities of Pennsylvania State University, USA have been indispensably
advantageous as well.
Where would I be without my family? My family to whom this dissertation
is dedicated, has been a constant source of love, concern, support and strength all
these years. I would like to express my heart-felt gratitude to my family. My Father
Mr. Muhammad Rafique Ch. and Mother Mrs. Khadija Rafique deserve special
mention for their inseparable and unconditional love, support and prayers in all
aspects of my life. Words do not come out easily for me to mention the feelings of
obligations towards my parents who were always raising their hands for prayers for
the successful completion of this manuscript. I wish to thank my sister Sobia
Zulfiqar , brother-in-law Mr. Zulfiqar Ahmed , brother Khurram Rafique , sister-
in-law Mrs. Lubna Khurram , brother Haris Rafique, nieces Maryam, Aleeza
and nephews Zayyan and Aayan, whose constant encouragement, moral support
and love I have relied upon throughout my time in University. My career endeavor
would have been impossible without constant support, love and understanding of
my family members. May Allah bless them with long happy life (Ameen)!
Khola Rafique
xx
ABSTRACT
Vascular wilt of lentil caused by various ecologically and phylogenetically
diverse species of Fusarium is found in all the lentil growing areas of Pakistan and
the disease could be visualized at both seedling and adult stages of plant growth.
The disease is responsible for huge losses each year in Pakistan, yet, there is a
scarcity and lack of literature and information regarding its occurrence, incidence,
distribution, biology and management of wilt pathogens. Therefore, the study was
planned keeping in view the national interests to avoid future losses caused by
lentil wilt. The objectives of this study were to assess the wilt prevalence and
incidence in major lentil growing districts of Punjab, morpho-molecular and
pathogenic characterization of recovered wilt pathogens and the management
through host plant resistance, biological control agents and fungicides. A two year
field survey data (2011-12 and 2012-13) and laboratory isolations ascertained 213
isolates of Fusarium pathogen as associated wilt incidence identified in the fields.
Disease was found widespread with 100% prevalence in all the major lentil
growing districts of Punjab viz. Bhakkar, Layyah, Mianwali, Khushab, Sialkot,
Narowal, Chakwal, Attock, Gujrat and Jhelum. The mean wilt incidence was found
28% with maximum incidence recorded at adult plant stage (32.4%) than at
seedling (23.05%).
Morphological characterization showed significant variation among the
isolates and based on similar morphology, these were grouped into 67 type isolates
for subsequent study. The in vitro pathogenicity testing through root dip method
using line NARC-08-1 and cultivar Masoor-93 showed excellent production of wilt
symptoms for pathogenic characterization. High pathogenic variability was
xxi
revealed among the isolates. Based on disease reaction i.e. avirulent to highly
virulent observed on most susceptible line NARC-08-1, isolates showed 0 to 100%
disease incidence and severity index with significant (11.86 to 100%) reduction in
yield. The isolates were grouped into four categories viz. highly virulent (13
isolates, 19.40%), moderately virulent (41, 61.19%), low virulent (8, 11.94%) and
avirulent (5, 7.46%). The highly virulent isolates included FWC15, FWJ35,
FWJ49, FWG1, FWS11, FWS13, FWN2, FWL2, FWL6, FWL9, FWL12, FWB10
and FWK2.
Molecular characterization and DNA sequencing of isolates through PCR
amplification of translation elongation factor TEF-1α gene region using primers ef1
and ef2 confirmed the identity of the Fusarium isolates at species level. The
amplification produced a single DNA fragment of size 700bp in each of the
isolates. Phylogenetic analysis of 67 morphologically and pathogenically diverse
Fusarium isolates recovered from various lentil districts of the country revealed
that the isolates belonged to different clades under five distinct species. The
identified species included F. oxysporum, F. redolens, F. nygamai, F. commune
and F. equiseti. This data supported the morphological variation observed among
the isolates and divulged the association of these identified species in wilt disease
incidence as reported in the major lentil producing region of the country. The
findings revealed the highest prevalence of F. oxysporum (49.29%) in the region
followed by F. redolens (29.57%), F. equiseti (10.79%) and F. commune (7.98%),
while least prevalence was of F. nygamai (2.34%). The most virulent F. oxysporum
isolate FWL12 (GenBank accession number KP297995) was selected for the
management trials. Screening of the lentil germplasm revealed reduced wilt
infection in five cultivars viz. Markaz-09, Masoor-86, Masoor-2006, Punjab
xxii
Masoor-00518, Punjab Masoor-09 that showed 4.44 to 12.59% severity index, 20
to 46.67% incidence and 9.60 to 24.94% yield reduction. The biological
management revealed the best efficiency of T. harzianum in reducing wilt infection
on line NARC-08-1 and produced 8.9% disease severity index, 26.7% incidence
with 16.27% yield reduction. Chemical seed treatment identified systemic
fungicides as highly effective in disease management that resulted in improved
100% seed germination. Benomyl (6.7% incidence and 1.5% severity index) was
found superior than Thiophanate methyl (13.3%, 3%).
In conclusion, the results of this research study provide an overall current
status of wilt disease in the country and high lightened the areas under current high
risk of its spread. The findings also revealed the continuous reduction in the
acreage of this crop in the major lentil region. The revealed association of five
virulent and morpho-molecularly diverse species viz. F. oxysporum, F. redolens, F.
nygamai, F. commune and F. equiseti with the wilt disease is reported for the first
time in Pakistan. The screening suggests five cultivars viz. Markaz-09, Masoor-86,
Masoor-2006, Punjab Masoor-00518 and Punjab Masoor-09 as an important source
of resistance for lentil breeding against wilt. Moreover, T. harzianum proves an
efficient biological control agent, while seed treatment suggests Benomyl and
Thiophanate methyl as the most effective against the wilt pathogen.
1
Chapter 1
INTRODUCTION
Lentil (Lens culinaris Medikus) or masoor is a high value cool season pulse
crop. Split lentil (dhal) is important part of the diet as a source of protein in many
parts of the world, especially South Asian and Mediterranean regions, which have a
large vegetarian population. It serves as a second major source of dietary proteins
(25%) after soybeans in human and animal diet (Rahman et al., 2010). The crop
was first grown in South-West Asia about 7,000 B.C. (McVicar et al., 2006) and is
probably the oldest of grain legumes to be cultivated (Bahl et al., 1993). A variety
of lentil types exists with varying seed colors, which may be yellow, red-orange,
green, brown or black.
Lentils are commercially grown in more than fifty countries around the
world, however, major shares of global lentil production are attributed to only three
countries including Canada, India and Turkey. Collectively, these three countries
typically comprise about two-third of world lentil production. The recorded world
lentil production in 2012 was approximately 4.6 million metric tonnes, out of
which, the share of Canada was 37%, India 23% and Turkey 8% (Janzen et al.,
2014 and FAO, 2013). In Pakistan, lentil is the second highly grown winter season
legume crop next to chickpea in terms of quality and quantity (Ayub et al., 2001).
It is grown on an area of 30.8 thousand hectare annually, out of this, 24 thousand
hectare (77.41%) is planted in the Punjab province comprising of Sialkot, Narowal,
Gujrat, Rawalpindi, Jhelum, Chakwal and Thal districts where two-third of the area
is sown under rain-fed conditions (Figure 1.1). In Pakistan, about 9.7 thousand
tonnes production was recorded during 2012-13 (Saleem, 2013 and FAO, 2013),
1
2
Figure 1.1: Lentil Production Zones [Courtesy of Dr. Ahmed Bakhsh (CSI, NARC, Islamabad)].
3
which is much lower than main lentil producing countries, such as, Canada (1.5
million metric tonnes) (FAO, 2013).
Susceptibility to diseases is one of the main production constraints to the
lentil crop. The crop is vulnerable to a number of diseases, which adversely affects
seed yield and its quality. Among them, the most significant and serious soil-borne
threat is the occurrence of vascular wilt disease incited by several species of
Fusarium but the most devastating fungus is F. oxysporum Schlecht. emend.
Snyder & Hansen f. sp. lentis Vasudeva and Srinivasan (Khare, 1981) belongs to
the order Hypocreales of class Ascomycetes. While, it’s sexual state has not been
found on lentil (Taylor et al., 2007). It is the most significant disease of lentil
worldwide and is one of the devastating diseases of lentil in Asia (Erskine et al.,
2009).
Wilt symptoms become visible in the field in patches at both, the seedling
(seedling wilt) and at the reproductive (adult wilt) stage (Stoilova and Chavdarov,
2006). Seedling wilt can be distinguished by abrupt drooping followed by drying of
leaves and loss of seedlings. At the adult stage, symptoms appear from flowering to
the late pod filling stage and are characterized by drooping of the top leaflets of the
plant. Roots of such plants are mostly well developed with a minor reduction of
lateral roots and generally no discoloration of vascular structures is seen but roots
show reduced proliferation. Seeds of infected lentils are shrivelled.
The disease can cause total failure of the crop particularly in a hot spring
and dry, warm summer (Agarwal et al., 1993). The disease is favored by warm and
dry conditions (Bayaa and Erskine, 1998) with an optimal temperature of 22-25 °C.
4
The disease can cause huge lentil yield losses and under favorable environmental
conditions may result in entire failure of the crop and therefore, can be key limiting
factor for lentil cultivation in certain areas. According to rough estimates, 10-15%
losses occur due to lentil wilt each year in Pakistan. The wilt pathogens are seed- or
soil-borne and may live in the soil for many years devoid of a suitable host.
Chlamydospores (resting spores) are the most likely major fungal structures for
extended survival (Chaudhary and Amarjit, 2002).
A number of management strategies aiming at controlling Fusarium lentil
wilt are practiced, such as, cultural practices, biological control agents, chemicals
and other methods. Since, the pathogen is seed- or soil-borne and survives in soil
for extended period of time, management through cultural practices like crop
rotation is not much effective. Therefore, use of resistant cultivars, biological
control agents and chemical seed treatment are the most economical mean of
controlling Fusarium wilt of lentil (Akhtar et al., 2012 and Stoilova and
Chavdarov, 2006). Chemical seed treatment and biological control are considered
to be the most effective in eradicating the inoculum present in soil. Also, there is a
great need to replace the present low yielding and disease susceptible lentil
varieties with those producing higher yields and providing resistance against wilt
disease. The work on evaluation of lentil germplasm for disease resistance to
Fusarium has been done in different countries to test their germplasm resources
(Stoilova and Chavdarov, 2006).
In addition to germplasm screening, it is important to characterize the
pathogen. For this purpose, traditionally Fusarium species were characterized on
the basis of various morphological parameters such as presence or absence of
5
chlamydospores and size and shape of micro- and macro-conidia (Leslie et al.,
2007). Also, on the basis of vegetative compatibility groups (Puhalla, 1985) and
host specificity. Yet, such aspects are not constant and have certain limitations for
defining species and sub-generic groupings of Fusarium. Therefore, now-a-days,
the research is focused on molecular tools such as sequencing, SSR (simple
sequence repeat or microsatellites), RAPD (random amplified polymorphic DNA),
AFLP (amplified fragment length polymorphism) and RFLP (restriction fragment
length polymorphism) for identification and determination of evolutionary
relationships among Fusarium species (Visser, 2003 and Eujayl et al., 1998) and
for study of variability in pathogenic populations of Fusarium species (Belabid et
al., 2004). However, the formae speciales in F. oxysporum can only be determined
using the conventional time-consuming pathogenicity testing procedures to
different host plant species (Fravel et al., 2003).
Molecular techniques based on the polymerase chain reaction (PCR) have
been used as a tool in genetic mapping, molecular taxonomy, evolutionary studies
and diagnosis of several fungal species (McDonald, 1997). Sequence analysis of
certain informative regions of DNA is now also becoming interesting. In Fusarium
systematics, several molecular methods based on phylogenetic species concept
have been introduced and are now being employed for practical molecular
taxonomy of this genus (Geiser et al., 2004). The most commonly used sequences
based on DNA sequence analysis for distinguishing among the species of Fusarium
are portions of the genomic sequences encoding the translocation elongation factor
1-α (TEF) (Wulff et al., 2010), β-tubulin (tub2) (O’Donnell et al., 1998a),
calmodulin (O’Donnell et al., 2000), internally transcribed spacer regions in the
6
ribosomal repeat region (ITS1 and ITS2) (O’Donnell and Cigelnik, 1997) and the
intergenic spacer region (IGS) (Yli-Mattila and Gagkaeva, 2010).
Intron-rich protein coding genes have been shown more useful as compared
to ribosomal genes in fungal phylogenetics at species level because these genes
contain conserved exon regions, which can be aligned easily and intron sequences,
which provide more variable characters than the ITS gene region (Geiser et al.,
2004 and Geiser, 2003). Such protein coding genes include the translation
elongation factor 1-α (TEF) and the β-tubulin (tub2), which have been used
successfully in various fungal phylogenetic studies including the Fusarium species
allowing easy PCR and sequencing of these genes portions containing three or
more intron sequences. The TEF shows high levels of sequence polymorphism and
have been used to design species-specific markers as well as probes for the
identification, detection and quantification of pathogenic populations of Fusarium
(Arif et al., 2012; Nicolaisena et al., 2009 and Bogale et al., 2007). These tend to
evolve at a rate higher as compared to other markers that are used commonly in
fungi at the species and population level such as the ribosomal internal transcribed
spacer (ITS) regions of the nuclear ribosomal RNA gene repeat (O’Donnell et al.,
2000).
Fusarium wilt is a serious disease of lentil responsible for huge losses each
year in Pakistan, yet, there is a scarcity and lack of literature and information
regarding its occurrence, distribution, losses and pathogens involved.
Consequently, comprehensive studies pertaining to wilt incidence, distribution,
prevalent wilt pathogen species, their biology and disease management need to be
7
addressed. Therefore, the study was planned keeping in view the national interests
to avoid the future losses caused by lentil wilt.
This study was focused on the following objectives:
i. To investigate the incidence and distribution of Fusarium wilt in lentil
producing areas of Punjab.
ii. Morphological and molecular characterization of Fusarium isolates
recovered from wilted lentil plants.
iii. Management of disease through host plant resistance, biological control
agents and chemicals.
8
Chapter 2
REVIEW OF LITERATURE
2.1 THE IMPORTANCE OF LENTIL
Lentil (Lens culinaris Medikus), an oldest known protein-rich food legume
is also known as “poor man’s meat” (Bhatty, 1988). The Latin name of the species,
Lens culinaris, was first published by Medikus in 1787 (Hanelt, 2001). The crop is
drought resistant and can be grown in water logged and saline soils (Muehlbaur et
al., 2002). It belongs to the family Fabaceae and is native to sub-continent. Lentil
was first grown in southwest Asia about 7,000 B.C. (McVicar et al., 2006) and is
probably the oldest of grain legumes to be cultivated (Bahl et al., 1993). The
current cultivation of the crop has been reported from more than 50 countries
throughout the world on an area of 4.08 million hectares with the production of 4.6
million metric tonnes (FAO, 2013).
It is commonly known as masoor and is the second largest grown legume
crop after chickpea (Cicer arietinum L.) in Pakistan, both in quality and quantity
(Ayub et al., 2001). It is grown as a winter season Rabi crop on an area of 30.8
thousand hectares annually (FAO, 2010), out of this, 24 thousand hectare (77.41%)
is planted in Punjab province comprising of Sialkot, Narowal, Gujrat, Rawalpindi,
Jhelum, Chakwal and Thal districts. During 2012-13, 9.7 thousand tonnes
production was recorded (Saleem, 2013 and FAO, 2013).
Lentil serves as a second major source of dietary proteins after soybean in
human and animal diet (Rahman et al., 2010) with an average content of 25%,
though, they lack certain amino acids such as Methionine and Cystine (Janzen et
8
9
al., 2014 and Muehlbauer et al., 2002). According to Raymond (2006), they are
considered as one of the healthiest foods and one of the best vegetable sources of
iron. They are also rich in important vitamins, minerals, soluble and insoluble
dietary fiber (Ryan et al., 2007). As a legume, lentil crop restores the soil fertility
through fixation of atmospheric nitrogen resulting in increased grain yield from 23-
32% and straw yield up to 16% (Muscolo et al., 2014). Therefore, used as a
valuable green manure and forage crop. Their husks, dried leaves and stems are
also used as livestock feeds (Raza, 2003). On the other hand, lentil is usually
allocated as a low priority crop by the researchers in spite of all its important
dietary advantages and significant role in farming system and therefore, remains
the least researched and can be proclaimed the least understood of the cultivated
food legumes.
2.2 LENTIL DISEASES
Diseases are the important constraints in limiting lentil crop yields and
responsible for its yield instability. A wide range of plant pathogens are involved in
causing infectious diseases on lentil crop, among which, the diseases caused by
fungal pathogens are the most important. Such diseases reduce lentil production by
infecting the leaves, stems, roots and pods. These also result in lentil seed
discoloration that ultimately affects crop market value (Taylor et al., 2007). In
Pakistan, diseases including wilt, Ascochyta blight, rust, collar rot and root rot play
a significant role in greatly affecting the lentil crop production (Chaudhry et al.,
2008).
Among the fungal soil-borne diseases, Fusarium vascular wilt is the major
disease of lentil (Taylor et al., 2007). The disease has been reported to be caused
10
by several species of Fusarium but the most important fungal species is F.
oxysporum Schlecht. emend. Snyder & Hansen f. sp. lentis Vasudeva and
Srinivasan (Khare, 1981). Wilt is present significantly in areas where conditions
are dry and where foliar diseases are of minor importance. It has been reported in
every lentil producing continent except Australia (Tosi and Cappelli, 2001) and
causes great economic losses in South America, the Mediterranean basin and South
Asia (Bayaa et al., 1995). Chaudhary and Amarjit (2002) also documented that wilt
is considered as the key limiting factor for lentil cultivation in certain areas of
world as it leads to entire failure of crop under favorable environmental conditions
necessary for disease development. Among other diseases of lentil, collar rot
(Sclerotium rolfsii Sacc.) is prevalent globally under humid conditions (Chongo et
al., 2002). Also, root rot caused by fungal pathogens Aphanomyces euteiches C.
Drechsler, Phythium ultimum Trow and Rhioctonia solani Kuhn.
Foliar diseases are also a major threat to lentil production reducing its yield
in many parts of the world among which, ascochyta blight caused by Ascochyta
fabae Speg. f. sp. lentis, rust (Uromyces vicia-fabae), stemphylium blight
(Stemphylium botryosum), anthracnose (Colletotrichum truncatum), Sclerotinia
white mold (Sclerotinia sclerotiorum) and grey mould (Botrytis cinerea) are of
vital importance (Kumar, 2007 and Holzgang and Pearse, 2001). Of these,
ascochyta blight is regarded as the most important foliar disease of lentil causing
up to 40% yield losses in lentil producing countries such as Argentina, Australia,
Canada, Ethiopia, India, New Zealand, Pakistan and The Russian Federation
(Regan et al., 2006 and Ye et al., 2002). Whereas, stemphylium blight is a
significant problem in Bangladesh and Nepal and in recent years, its occurrence has
11
also been reported in North Dakota and Saskatchewan (Kumar, 2007 and Holzgang
and Pearse, 2001). Similarly, Chongo et al. (2002) reported anthracnose, botrytis
grey mould and sclerotinia white mold as devastating problems in North America.
Other foliar disease such as powdery mildew has been reported in USA and Canada
in the last few years by Attanayake et al. (2009) and Banniza et al. (2004).
2.3 LENTIL WILT AND THE CAUSAL ORGANISMS
Wilt is an economically important disease that is distributed universally,
especially in Asia (Erskine et al., 2009). The disease is favored by warm and dry
conditions (Bayaa and Erskine, 1998) with an optimal temperature of 22-25 °C.
The disease can cause total failure of the crop under favorable environmental
conditions particularly in hot spring and dry, warm summer (Agarwal et al., 1993)
and therefore, can be key limiting factor for lentil cultivation in certain areas
(Chaudhary and Amarjit, 2002).
The causal genus Fusarium, introduced for the first time by Link (1809) is
known for harboring a range of plant pathogenic fungal species (Zhang et al.,
2012). Lentil wilt, also known as vascular wilt is incited by several species of
Fusarium but the disease has often been attributed to the most devastating fungus
F. oxysporum Schlecht. emend. Snyder & Hansen f. sp. lentis Vasudeva and
Srinivasan (Khare, 1981) belonging to the family Nectriaceae (order Hypocreales).
Other species of Fusarium have also been reported to be associated with lentil wilt
and root rot diseases including F. acuminatum Ell. and Ev., F. avenaceum (Fr.)
Sacc., F. culmorum (W. G. Smith) Sacc., F. solani (Mart.) Appel and Wollenw.
Emend. Snyd. and Hans. (Burgess et al., 1988a), F. equiseti (Corda) Sacc. and F.
sporotrichioides Sherb. (Rauf and Banniza, 2007). Similarly, more species were
12
found to be associated with the wilt disease in lentil. Like F. oxysporum, isolates of
F. redolens Wollenw. [syn: F. oxysporum var. redolens (Wr.) Gordon] are
responsible for causing wilts, seedling damping-off and cortical rot (Booth, 1971).
In USA and Europe, F. redolens has been reported as the causal agent of vascular
wilt disease of lentil (Riccioni et al., 2008). Also, wilting-like symptoms on
chickpea produced by F. redolens were reported in Lebanon, Morocco, Pakistan
and Spain by Jimenez-Fernandez et al. (2011). Similarly, frequent isolation of F.
redolens from necrotic and discolored root and crown tissues of chickpea, pea,
lentil and durum wheat have also been reported by Taheri et al. (2011) in
Saskatchewan. Another species i.e. F. nygamai has also been found associated with
the rhizoplane of lentil plants in Egypt by Abdel-Hafez et al. (2012).
F. oxysporum is a specie complex comprised of different host plant specific
individuals known as formae specialis (f. sp.) (Beckman, 1987) and these are
further classified based on their virulence into cultivar specific sub-groups termed
as races (Corell, 1991). These pathogenic fungi cannot be distinguished
morphologically from each other and also from non-pathogens. However, its sexual
state (teleomorph) has not been found on lentil (Taylor et al., 2007). Formae
specialis (f. sp.) lentis has been reported on lentil (Hamdi and Hassanein, 1996)
and no known physiological races within F. oxysporum f. sp. lentis have been
reported (Belabid et al., 2004).
2.4 BIOLOGY AND MORPHOLOGICAL CHARACTERIZATION OF
FUSARIUM SPECIES ASSOCIATED WITH LENTIL WILT
The fungus Fusarium belongs to ascomycetes with unknown teleomorphic
(sexual) stage (Brayford, 1996). According to Leslie and Summerell (2006), it is
13
comprised of three teleomorphic genera viz. Gibberella, Haematonectria and
Albonectria. It is composed of distinct morphological characters that vary among
the species within the genus. Generally, Fusarium produces mycelium with three
kinds of asexual spores viz. micro-conidia, macro-conidia and chlamydospores.
Link (1809) who firstly introduced the genus Fusarium, diagnosed the presence of
the distinctive canoe- or banana-shaped conidia and suggested this as a primary
character for identifying the genus. Moreover, according to Toussoun and Nelson
(1976), Fusarium species are distinguished chiefly based on the shapes of the
macro-conidia. Their basal foot cells have a diagnostic hook or notch depending on
the species. Khare (1980) observed the hyaline, septate and branched mycelium of
Fusarium species in his study. Growth patterns of Fusarium also varied from fluffy
to appressed with colorless to pinkish culture on media. Similarly, Kontoyiannis et
al. (2000) showed that the colony color of Fusarium may vary from white, cream,
tan, salmon, cinnamon, yellow, red, violet, pink or purple and on the other hand, it
may be colorless, tan, red, dark purple or brown. Fusarium species can survive in
soil as mycelium and also in the form of spores known as chlamydospores (Agrios,
2005) which, help in extended survival under unfavorable conditions.
2.4.1 Fusarium oxysporum
F. oxysporum produces three kinds of spores (asexual) viz. micro-conidia,
macro-conidia and chlamydospores. The conidia are produced on monophialides
and in sporodochia and are scattered loosely over the mycelial surface. Macro-
conidia are multi-septate typically three or four septa, with a distinct foot cell and a
pointed apical cell (Agrios, 2005 and Nelson et al., 1983). Chlamydospores are
resting spores produced in or on older mycelium. They are composed of one or two
14
round cells with thick cell walls that provides cell defense against degradation and
antagonists. These spores also help in the fungal survival in the soil for longer
period of time even in the absence of the host.
Based on morphological characters, Nelson et al. (1983) demonstrated that
F. oxysporum can be differentiated from closely related species by the presence of
micro-conidia borne in false heads on short monophialides as compared to F.
moniliforme and F. solani. Similarly, chlamydospores are primarily produced
singly or in pairs, but occasionally in short chains or clumps. The macro-conidia
are slightly sickle-shaped and thin-walled having an attenuated apical cell and a
foot-shaped basal cell borne in abundant sporodochia. In a study, Murumkor and
Chavan (1985) reported that the fungal hyphae are septate and profusely branched.
Micro-conidia are produced on simple and short conidiophores. The macro-
conidial shape varied from straight to curved or oval to cylindrical and size
measured ranged from 2.5 to 3.5 μm in length and 5 to 11 μm in width. Later,
Gupta et al. (1986) and Saxena and Singh (1987) also reported septate hyphae i.e.
profusely branched. The colony color observed was white initially and later turned
light buff or deep brown. The growth was fluffy or submerged on potato dextrose
agar (PDA) at 25oC, which turned felted or wrinkled in old cultures. Pigmentation
of various colors such as yellow, brown and crimson was observed in cultures. On
solid medium, micro-conidia are borne on simple and short conidiophores which
arise laterally on the hypha. Thin-walled and 3 to 5 septate macro-conidia are borne
on the conidiophores. Both macro-conidial ends were pointed and size measured
ranged from 3.5-4.5 x 25–65 μm. Rough or smooth walled chlamydospores were
observed in old cultures, which were present intercalary or terminal and formed
15
singly, in chains or pairs. In another study, Gupta et al. (1987) reported similar
morphological observations i.e. thin walled, fused and 3 to 5 septate macro-conidia
with both end cells pointed and size ranged from 3.5 to 4.5 x 25 to 65 μm. Macro-
conidia were fewer in number as compared to micro-conidia in old culture. These
were rough or smooth walled, intercalary or terminal and may be formed singly, in
chains or pairs.
2.4.2 Fusarium redolens
The morphological identification of Fusarium species is very difficult as F.
redolens is very much similar to F. oxysporum (Leslie and Summerell, 2006) and
there is a controversy regarding its taxonomic status. In the past, F. redolens was
considered to be a variety of F. oxysporum (F. oxysporum var. redolens (Wr.)
Gordon) (Booth, 1971), whereas, later it was recognized as a synonym of F.
oxysporum by Nelson et al. (1983). However, present studies have shown that both
are different from each other (Baayen et al., 2001). Both species are mainly
differentiated morphologically on the basis of the sizes of their macro-conidia
(Gordon, 1952). F. redolens produces chlamydospores, short monophialides,
micro-conidia and stout macro-conidia (Shinmura, 2002). The disease symptoms
produced by F. redolens are similar to those produced by F. oxysporum, such as,
wilting symptoms, damping-off in seedlings and sometimes a cortical rot.
2.4.3 Fusarium nygamai
The key morphological identification criteria for F. nygamai Burgess and
Trimboli are the micro-conidia that are formed in short chains and false-heads and
also, the formation of chlamydospores in pairs, chains or clumps (Burgess et al.,
1989). According to Burgess and Trimboli (1986), the production of micro-conidia
16
in short chains and false head is a major distinguishing character between F.
nygamai and F. oxysporum. Regarding the resting spores i.e. chlamydospores,
Burgess and Trimboli (1986) observed these in both aerial and submerged hyphae
on carnation leaf agar (CLA) and soil agar. The macro-conidia produced by F.
nygamai are usually more slender as compared to those of F. oxysporum, however,
conidia of some F. nygamai isolates could be mistaken for those produced by F.
oxysporum isolates. The conidiogenous cells are characterized as monophialides
but polyphialiades have also been observed in few cultures of F. nygamai (Burgess
and Trimboli, 1986). However, later Burgess et al. (1989) proposed that
polyphialides should not be considered reliable character for identification of F.
nygamai because they are produced irregularly and are difficult to detect.
On PDA, the fungus forms floccose mycelium, which is white initially and
later in older cultures becomes dull violet to dark violet. In most of the isolates, a
central mass of spores of greyish orange or dark violet color is present and violet
grey to dark violet pigmentation has also been observed. The colony diameter
recorded after 3 days of incubation in dark ranged from 2.5 to 3.5 cm at 25ºC and
3.2 to 4.2 cm at 30ºC (Burgess and Liddell, 1983).
2.4.4 Fusarium commune
F. commune and F. oxysporum, both are morphologically similar in
characters like the production of conidia on short monophialides in false heads on
the aerial mycelium. The chlamydospores are formed singly or in pairs. The
distinctive features of F. commune are the formation of long and slender
monophialides as well as the occasional formation of polyphialides (Skovgaard et
al., 2003).
17
2.4.5 Fusarium equiseti
The species produces abundant mycelium, which is initially white in color
but later becomes brown age. It may form sporodochia in a central spore mass,
however, it may not be clear as they can be obscured by the mycelium. The color
of the spore mass varies from pale orange to dark brown and develops annular
zonations, when provided with a light-dark cycle. The colony produces
pigmentation at the point of contact with the agar that may be pale brown to dark
brown in color. Usually dark brown spots or flecks of pigment are produced in the
agar. Macro-conidia produced by this species are slender and have tapered,
elongate or whip-like apical cell, while foot-shaped basal cell (Leslie and
Summerell, 2006).
2.5 SURVIVAL AND DISEASE CYCLE OF FUSARIUM WILT OF
LENTIL
The fungus colonizes in vascular tissues of the plant and causes its wilting
(Rai et al., 2011). It is a filamentous genus that has a cosmopolitan distribution
occurring in many parts of the world (Saremi, 2003). It is seed or soil-borne and
may live in the soil for many years devoid of a suitable host (Burgess, 1981).
According to Singh et al. (2007), it can survive in the form of mycelium or
chlamydospores in seed, soil and also on infected crop residues, roots and stem
tissue buried in the soil for more than 6 years. Chlamydospores (resting spores) are
the major fungal structures for extended survival either in dormant form or
saprophytically (Chaudhary and Amarjit, 2002). The fungus transmits primarily
through plant debris and contaminated soil, where it causes infection through roots
and its transmission is also evident through seeds (Erskine et al., 1990).
18
Following infection of lentil roots, the pathogen enters the xylem vessels by
crossing the cortex. After entering, it spreads rapidly throughout the plant vascular
system thus becoming systemic within the host plant tissues and may possibly
results in direct seed infection or infestation. In soil contaminated with the fungus,
the root tips of healthy lentil plants are easily penetrated by the germ tube of fungal
spores or mycelium. This penetration of the fungus occurs either directly through
wounds or opportunistically at the point where the lateral roots are formed. The
mycelium then spreads intercellularly throughout the cortex and penetrates through
the pits into the xylem vessels. After entering into the vessels, the fungus remains
confined and the mycelium produces branches and micro-conidia. These conidia
then detach and move upward in the vascular system. At a point their upward
movement stops and they germinate and the resulting mycelium enters the adjacent
vessel through penetration of the wall. The lateral movement between the xylem
vessels occurs through the pits. Ultimately, the vessels become blocked and the
water contents of infected plants are strictly compromised. Finally, this results in
closing of the stomata, wilting and death of leaves, often followed by death of the
whole plant. Eventually, fungal invasion of all plant tissues occur where it
sporulates profusely to reach the plant surface. The spores dispersal may then occur
by wind and water or movement of soil or plant debris (Lindbeck, 2009 and Cho
and Muehlbauer, 2004).
2.6 SYMPTOMS OF FUSARIUM WILT OF LENTIL
Wilt disease occurs either in the early crop growth stage (seedling wilt) or
during reproductive growth (adult plant wilt) (Khare, 1981). Wilt symptoms
become visible in the field in patches. Seedling wilt can be distinguished by abrupt
19
drooping followed by drying of leaves and loss of seedlings. At the adult stage,
symptoms appear from flowering to the late pod filling stage and are characterized
by drooping of the top leaflets of the plant. Roots of such plants are mostly well
developed with a minor reduction of lateral roots and generally no discoloration of
vascular structures is seen but roots show reduced proliferation. Seeds of infected
lentils are shriveled (Bowers and Locke, 2000).
2.7 SILICA GEL PRESERVATION OF FUSARIUM ISOLATES
Permanent and long-term preservation is necessary for type specimens and
for strains with important characteristics (Greuter et al., 2000). Numerous methods
have been illustrated for long-term preservation of fungi (Ryan et al., 2000) such as
lyophilization (Fisher et al., 1982), liquid nitrogen (Booth, 1971), mineral oil
(Lima, 1991), sterile soil (Toussoun and Nelson, 1976) and silica gel (Windels et
al., 1988). Silica gel method developed by Perkins (1962) has been considered as a
convenient technique for preserving various genera of fungi including Fusarium
(Trollope, 1975), also bacteria (Sleesman and Leben, 1978 and Trollope, 1975) and
algae (Grivell and Jackson, 1969). Windels et al. (1988) through their five year
study suggested that 15 out of 17 Fusarium species well-survived on silica gel and
sterile soil. Later, in a study, Windels et al. (1993) proposed that silica gel method
should be preferred for storage of Fusarium because it is simple, much easier to
use, less-expensive, repeated isolations of cultures can be produced from a single
preserved tube and the chances of mutation are minimum as the cultures added to
silica do not colonize the substrate.
The method has also been considered as effective in retaining viability of
cultures, likewise, Windels et al. (1988) reported on the viability of Fusarium
20
species after storage for 10 years. Earlier, Perkins (1962) used this technique for
Neurospora species and proposed that sporulating fungi stored on anhydrous silica
gel crystals and protected by skim milk used, remain viable for 4-5 years. Smith
and Onions (1983) suggested that fungi have been stored successfully on silica gel
for up to 11 years. Later, Raper (1984) also proposed that spores and microcysts of
dictyostelids can be preserved for up to 11 years. It has been reported that micro-
organisms stored on silica gel retain their characteristics such as F. moniliforme
and F. proliferatum stored on silica for 5-6 months retained their pathogenicity and
caused corn stalk rot (Kommedahl et al., 1987). Similarly, silica gel preservation
method has also been found good for the phytopathogenic bacteria, which survived
and retained their pathogenicity (Sleesman and Leben, 1978). Recently, Sharma et
al. (2012) checked the survival, growth and pathogenicity of Fusarium isolates
using different preservation methods up to 36 month of storage and proposed that
best survival of isolates was obtained with filter paper which was followed by silica
gel, mineral oil, soil, water and slant, respectively. They also observed variations in
the growth and pathogenicity at different storage treatments, however, cultural and
morphological characters were the same.
2.8 MOLECULAR CHARACTERIZATION AND PHYLOGENETIC
ANALYSIS OF FUSARIUM ISOLATES THROUGH DNA
SEQUENCING
Classically, identification and characterization of plant pathogenic fungi
including the Fusarium species were based on morphological characters such as the
presence or absence of chlamydospores and size and shape of macro- and micro-
conidia (Leslie et al., 2007) along with the combination of diagnostic host
21
symptoms and fungal presence in the infected tissues. Such identification of the
pathogen based on morphological criteria or isolation from infected hosts is often
expensive, time consuming and necessitates great expertise in taxonomy in order to
differentiate among closely related species of Fusarium (Baayen et al., 2000). In
addition, Fusarium is both soil- and seed-borne and transmits through infected
seeds from one area to another, thus, for effective management of the disease, a
highly sensitive method for its early detection in an infection and in seeds was
needed. Therefore, the technological advancements in molecular biology help
avoided all the draw backs associated with the classical methods of pathogen
identification. It has opened several ways for the detection and enumeration of
fungal pathogens and information for the identification of unknown species from
their DNA sequences (Paplomatas, 2004).
According to Mule et al. (2005), a comparison at the DNA sequence levels
offers accurate classification of fungal species and is beginning to elucidate the
evolutionary and ecological relationships among diverse species. Such
phylogenetic techniques help identify new species, which is usually difficult and
often impossible by using conventional morphological characters (Aoki et al.,
2003). The DNA sequences are amplified employing polymerase chain reaction
(PCR), a molecular tool which has widespread application in the diagnosis and
detection of fungi (Louis et al., 2000). Abd-Elsalam et al. (2003) illustrated that
PCR does not involve use of viable organisms as used in fungal culture approaches
and may be executed using very small quantities of biological material. Likewise,
Waalwijk et al. (2004) and Williams et al. (2002) proposed that PCR provides
22
immense advantage over conventional methods and offers highly specific and
accurate detection and identification of the pathogen. DNA-based techniques
including AFLPs (amplified fragment length polymorphisms), RFLPs (restriction
fragment length polymorphisms), SSRs (simple sequence repeats), RAPD (random
amplified polymorphic DNA) and DNA sequence analysis have overcome all the
limitations related with the identification of sub-species taxa using morphology,
pathogenicity, vegetative compatibility and protein-based methods.
In Fusarium systematic, molecular methods have been introduced and are
now being employed for practical molecular taxonomy of this genus based on
phylogenetics species concept (Geiser et al., 2004). They play an important role in
Fusarium identification (Lee et al., 2000) by greatly increasing the accuracy in the
classification of unknown Fusarium isolates (Leslie et al., 2001 and Taylor et al.,
2000) and in understanding of genetic diversity among the members of this genus
(Bogale et al., 2006 and O’Donnell et al., 2000). In molecular phylogenetics or
DNA sequence analysis, the most commonly used sequences to distinguish species
of Fusarium are portions of the genomic sequences encoding the translocation
elongation factor 1-α (TEF) (Wulff et al., 2010), β-tubulin (tub2) (O’Donnell et al.,
1998a), calmodulin (O’Donnell et al., 2000), internally transcribed spacer regions
in the ribosomal repeat region (ITS1 and ITS2) (O’Donnell and Cigelnik, 1997)
and the intergenic spacer region (IGS) (Yli-Mattila and Gagkaeva, 2010).
Intron-rich protein coding genes have been shown more useful as compared
to ribosomal genes in fungal phylogenetics at species level because these genes
contain conserved exon regions, which can be aligned easily and also have intron
23
sequences, which provide more variable characters than the ITS region (Geiser et
al., 2004). Such protein coding genes include the translation elongation factor 1-α
(TEF) and the β-tubulin (tub2), which have been used successfully in various
fungal phylogenetic studies including the Fusarium species allowing easy PCR and
sequencing of these genes portions containing three or more intron sequences.
TEF gene was first employed as a marker in phylogenetics to infer species-
and generic-level relationships among Lepidoptera (Mitchell et al., 1997) and in
fungi, the TEF primers were first developed for the investigation of lineages within
the F. oxysporum complex (O’Donnell et al., 1998b). The TEF shows high levels
of sequence polymorphism and have been used to design species-specific markers
as well as probes for the identification, detection and quantification of pathogenic
populations of Fusarium (Arif et al., 2012; Nicolaisena et al., 2009 and Bogale et
al., 2007).
According to Geiser (2003), the markers of choice in fungal phylogenetics
at species-level are intron-rich portions of protein coding genes. These tend to
evolve at a rate higher as compared to other markers that are used commonly in
fungi at species and population level, such as, the ribosomal internal transcribed
spacer (ITS) regions of the nuclear ribosomal RNA gene repeat (O’Donnell et al.,
2000). Though, in F. oxysporum, the ITS gene region is not used for phylogenetic
analysis because some isolates possess non-orthologous copies of the ITS2 region,
which can lead to incorrect inferences (O’Donnell et al., 1998a). Conversely,
Geiser et al. (2004) suggested that TEF gene offers high phylogenetic efficacy, as it
is highly informative at the species level in Fusarium, non-orthologous copies of
24
the gene have not been detected in the genus and universal primers have been
designed that work across the phylogenetic breadth of the genus. TEF has also been
shown as the most commonly and successfully used marker in F. oxysporum by
O’Donnell et al. (2009), Geiser et al. (2004) and Baayen et al. (2000).
2.9 INCIDENCE, DISTRIBUTION AND YIELD LOSSES OF LENTIL
FUSARIUM WILT
Lentil wilt has a widespread distribution and has been reported to occur and
cause economic losses in as many as 26 countries of the world including the
regions of South Asia, Sub-Saharan Africa, West Asia and North Africa (WANA)
(Erskine et al., 1993). The first report of the disease was documented from
Hungary (Fleischmann, 1937). Afterwards, many other countries also reported the
disease such as India (Padwick, 1941), USA (Wilson and Brandsberg, 1965),
USSR (Kotava et al., 1965), Turkey (Bayya et al., 1998), Syria (Bayya et al.,
1986), Myanmar and Pakistan (Bahl et al., 1993), Nepal (Karki, 1993), Ethiopia
(Hulluka and Tadesse, 1994) and Egypt (El-Morsy et al., 1997).
The yield losses in lentil due to Fusarium wilt depend on various factors
including the stage of the crop at the time of infection (Khare et al., 1979),
environment and the crop variety. Wilt incidence can occur either at seedling stage
resulting in total failure of the crop or at adult plant stage (flowering and podding)
in which, the plants can produce some grain yield which could be shriveled. In
Pakistan, current data regarding losses caused by lentil wilt is not available, though,
according to an estimate provided by pulses program at Crop Science Institute,
NARC, Islamabad, Pakistan about 10-15% regular yield losses occur as a result of
this disease.
25
Wilt incidence calculated and correlated with yield loss estimates during
reproductive stage of lentil crop in Syria showed reduced seed yield per unit
change in wilt incidence of 0.846+0.118%. The disease response or incidence was
found positively correlated to inoculum density in susceptible lentils under
laboratory conditions whereas it was not correlated to inoculum density under field
conditions, thus, preventing the possibility of predicting disease incidence from
inoculum density (Erskine and Bayaa, 1996). Likewise, survey of 116 lentil
growing districts of India conducted by Chaudhary et al. (2010) for the calculation
of lentil wilt-root rot incidence at the crucial reproductive stage revealed a range of
0.7-9.3% mean plant mortality and an overall mean mortality of 6.3%. The main
pathogens associated with plant mortality were F. oxysporum f. sp. lentis (62.0%),
R. bataticola (25.2%) and S. rolfsii (9.8%) and the minor involvement of 1.8% was
that of F. solani, F. chlamydosporum, F. equiseti and R. solani.
In a similar study for the assessment of wilt disease damage, Bayaa et al.
(1986) surveyed 27 lentil fields in North-West Syria and found the proportion of
wilted plants in all fields varying from 2 to 70% with a mean of 12%. The isolation
of fungi from diseased samples showed a dominance of Fusarium species and
pathogenicity test reproduced the disease that showed vascular wilt symptoms
associated with the growth of F. oxysporum f. sp. lentis within infected tissues.
2.10 PATHOGENICITY TEST
Pathogenicity of 102 F. oxysporum f. sp. lentis isolates was checked by
Naimuddin and Chaudhary (2009) who isolated them from root samples collected
from Uttar Pradesh (India). Data was recorded on percent wilting induced in wilt
susceptible genotype of lentil "Vidokhar local" by these isolates. They observed
26
that out of 102 isolates 10 isolates were non pathogenic as they did not induce any
mortality in the wilt susceptible genotypes. Their study also revealed that there is
vast range of variability in pathogenic character of isolates of F. oxysporum f. sp.
lentis.
In an experimental study, Taheri et al. (2010) isolated 33 F. oxysporum
isolates from wilted lentil plants from different areas of Khorasan (Iran), to perform
pathogenicity tests on susceptible lines and found 27 pathogenic isolates. They also
inoculated these F. oxysporum isolates on different plants species to determine
their forma special, which was confirmed as F. oxysporum f. sp. lentis. Through
pathogenicity test of 32 isolates Belabid et al. (2004) obtained their results
indicating that the F. oxysporum f. sp. lentis isolates represent a single race but
differ in their virulence on the susceptible lines. Similarly, Belabid and Fortas
(2002) carried out pathogenicity tests on 32 Algerian F. oxysporum f. sp. lentis
isolates and found that the isolates represented a single race, but differed in their
virulence on susceptible lines.
2.11 SCREENING FOR HOST RESISTANCE AGAINST LENTIL WILT
Wilt pathogens remain in soil for many years (Chaudhary and Amarjit,
2002), consequently, simple control methods such as crop rotation is not greatly
effective. Therefore, use of resistant lentil varieties is an effective strategy against
this disease. Yet, their utilization is limited because of existence of location
specific pathogen races (Singh et al., 2006). A simple, rapid and reproducible
technique was developed by Bayaa and Erskine (1990) for screening lentil
germplasm at the seedling stage for resistance to F. oxysporum f. sp. lentis. They
planted one row of each of the test lines in metal trays filled with field soil with a
27
susceptible control at every 5th row and inoculated pathogen liquid culture to 14
days old plants. Eight weeks after sowing they noted final disease incidence that
out of 162 lines screened using this method, 29 showed no disease. The correlation
of results from repeated sowings of 25 lines was r = 0.86 (P<0.01). The reaction of
18 of the resistant lines was the same when tested in the field.
Evaluation of thirty lentil cultivars and accessions against F. oxysporum f.
sp. lentis in field and glasshouse by Pouralibaba and Alaii (2004) showed
differences in host response to the disease at different experimental conditions.
They found medium correlation between glasshouse responses and field results.
The cultivars ILL52, ILL795, ILL6806, ILL1878, FLIP 97-1L, ILL7531 and
ILL590 were found resistant in all conditions, while cultivars ILL7523 and
ILL6806 were moderately susceptible in glasshouse and resistant in the field.
Therefore, they recommended the release of all these lines. Whereas, ILL52 and
ILL7531 were recommended suitable for disease resistance sources in breeding
programs as were found highly resistant in all conditions. Later, Stoilova and
Chavdarov (2006) screened 32 lentil genotypes with diverse geographical origin
for reaction to wilt pathogen under greenhouse conditions and reported three
accessions (91-001, 91-028 and 98-001) susceptible with 45 and 50% of total
wilted plant.
A screening experiment for resistance sources by Chaudhry et al. (2008)
included 38 lentil lines and a local highly susceptible check line ILL-6031 in a wilt
sick plot. They rated disease incidence on a scale of 1-9 where 1= highly resistance
and 9= highly susceptible response and as a result found one line highly resistant,
seven lines resistant and ten lines as moderately resistant whereas rest of the lines
28
gave moderately susceptible to highly susceptible reaction. In a similar study,
Mohammadi et al. (2012) performed screening of germplasm using root dip
method under green house and also field tests in naturally infested plot and
identified three resistant lines (81S15, FLIP2007-42 L and FLIP2009-18 L) of
lentil against wilt.
2.12 BIOLOGICAL MANAGEMENT OF LENTIL WILT
Disease control using biocontrol agents is a potential substitute to chemical
fungicides (Parker et al., 1985) that offer advantages such as environment friendly,
cost effective and extended protection (Gohel et al., 2007). The genus Trichoderma
consists of most extensively used biocontrol agents among mycoparasites against
soil-borne, seed-borne as well as many other diseases (Etebarian, 2006). These are
filamentous saprophytic fungi that are most common in nature and found in soil
and plant litters in high population density (Samuels, 1996). They can be cultured
easily with the production of long life conidia in large quantities (Mohamed and
Haggag, 2006).
Among species within genus Trichoderma, T. harzianum is considered an
efficient biocontrol agent which has been successfully used for controlling
Fusarium wilt disease (Dubey et al., 2007). It is an active rhizosphere coloniser
(Tronsmo and Harman, 1992) that produces various substances which may be
involved in suppression of plant diseases or promotion of plant growth. Such
substances include antibiotics (trichodernin, trichodermol, gliotoxin, viridian)
(Kucuk and Kivanc, 2004), few enzymes involved in degrading cell wall
(chitinases, glucanases) that break down polysaccharides, chitins and glucanase
29
(Elad, 2000) and also biologically active heat-stable metabolites (ethyl acetate)
(Claydown et al., 1987). For effective control of Fusarium wilt, various other
antagonists in addition to Trichoderma species, such as, Burkholderia species,
Pseudomonas species, Bacillus species and non-pathogenic F. oxysporum have
been reported by Cao et al. (2011).
Recently, Kumar et al. (2013a) conducted field trials for the management of
vascular wilt of lentil using variety Pant L-639 and observed that seed treatment
with T. harizanum+P. fluorescence gave significant reduction in disease incidence
and maximum grain yield. Similarly, Dolatabadi et al. (2012) evaluated
effectiveness of four antagonistic fungi against Fusarium wilt of lentil in pot
experiment. These included T.viride, T. harzianum, Piriformospora indica and
Sebacina vermifera along with their combinations. The results revealed increased
plant height and reduced disease severity in pots treated with S. vermifera+T.
harzianum. For controlling Fusarium rot of lentil, Akrami et al. (2011) evaluated
three isolates of Trichoderma viz. T1 (T. harzianum), T2 (T. asperellum) and T3
(T. virens) alone and in combination. The green house experiment showed more
effectiveness of isolates T1 and T2 isolates and their combination as compared to
other treatments. Disease severity was found to be reduced ranging from 20 to
44%, while dry weight increased from 23 to 52%.
Various other studies have also reported disease control using Trichoderma
species, such as, Poddar et al. (2004) reported decreased chickpea wilt incidence
with isolate of T. harzianum. Correspondingly, Siddiqui and Singh (2004) found
maximum plant growth, increased transpiration and decreased wilt disease index
30
caused by F. oxysporum f. sp. ciceris through treatment with T. harzianum. Later,
Dubey et al. (2006) also reported reduced wilt disease incidence in chickpea using
Trichoderma species. Likewise, in a study, Ghahfarokhi and Goltapeh (2010)
reported Trichoderma species, P. indica and S. vermifera as the most effective
biocontrol agents against take-all diseases of wheat caused by Gaeumannomyces
graminis var. tritici .
2.13 CHEMICAL MANAGEMENT OF LENTIL WILT
The first fungicidal control of lentil wilt through dry seed treatment with
Captan (0.2%) and Thiram (0.2%) was reported by Kovacikova (1970).
Subsequently, Ahmed et al. (2002) determined the effect of different control
options including sowing dates, host plant resistance and fungicide seed treatment
on wilt disease parameters and lentil yields. The disease parameters were wilt
onset, duration, percent terminal wilt and areas under the disease progress curve.
They observed lentil genotype with greater effect on the onset and duration
of Fusarium wilt than planting date or fungicide seed treatment in two seasons.
Percent terminal wilt and areas under the disease progress curve were lowest
during November plantings for all genotypes. Straw yields were correspondingly
high for November plantings in both seasons. The correlation between percent
terminal wilt and area under the disease ± progress curve with yield parameters was
significantly negative. Since, November planting reduced Fusarium wilt and
increased straw and seed yields for the genotypes, therefore, they recommended
this practice to be adopted for lentil wilt management. Also, as fungicide seed
treatment showed no effect on disease onset, duration or on area under the disease
31
± progress curve, they suggested it not be a useful component in the integrated
management of the disease.
A report on minimum population (75-105 d) dynamics of F. oxysporum f.
sp. lentis with Carbendazim treatment was provided by Ahmed and Ahmed (2000).
Also, De et al. (2003) described the most effective seed treatment of F. oxysporum
f. sp. lentis with Carbendazim. Similar findings were also documented by Sinha
and Sinha (2004) and Karande et al. (2007) who found Carbendazim (0.1%) with
effective in vitro mycelial growth inhibition of F. oxysporum from cashew.
Likewise, Maheshwari et al. (2008) reported Carbendazim followed by Captan and
hexaconzole as most effective against F. oxysporum f .sp. lentis. Also, in a related
study, maximum inhibition of fungal growth was noted with Carbendazim (93.4)
which was followed by Captan (88.3%) (Kasyap et al., 2008). Later, Singh et al.
(2010) reported complete in vitro inhibition of growth with Carbendazim and
Carboxin, while Thiram and Captafol provided 87.5 and 83.1% growth inhibition.
In the same way, in vivo treatment with Carbendazim and Carboxin improved seed
germination, root and shoot length and vigor index. Separate foliar spray with both
fungicides also provided best results with reduced wilt incidence (37.5 to 5%). In
chickpea, Nikram et al. (2007) also showed the most effective seed treatment with
Thiram (0.15%) and Carbendazim (0.1%).
Other fungicides such as Benomyl and Bavistin at 500, 1000 and 1500 ppm
concentrations have also been used by Sharma et al. (2002) against F. oxysporum f.
sp. lini responsible for linseed wilt and they recorded no fungal growth. Seed
treatment with Bavistin (2 gm/ kg) was also used for lentil wilt fungus in field that
32
provided effective control and significant improvement in crop yield (De et al.,
2003). Later, similar 100% inhibition of F. oxysporum f. sp. pisi was found with
Bavistin (200 ppm) followed by Dithane M-45 (mancozeb) (Dabbas et al., 2008).
Recently, Hossain et al. (2013) reported absolute 100% inhibition of
mycelial growth of F. oxysporum f. sp. ciceris with fungicide Provax-200 at 100
ppm concentration. Furthermore, Garkoti et al. (2013) tested eight systemic and
non-systemic fungicides at four concentrations viz. 50, 100, 200, 400 µg/ L for
lentil wilt management in India. Through in vitro testing, they found complete
fungal growth inhibition with Benlate and Captaf at 400 µg/ L, whereas,
carbendazim with reduced fungal growth which was followed by thiram. Benlate
with maximum efficacy was tested in vivo under field conditions and resulted in
reduced wilt incidence (1%) with increased yield (608.7 kg/ ha) and maximum
1000-grain weight (15.1 gm) followed by captaf. In a recent integrated disease
management of Fusarium wilt of lentil, Kumar et al. (2013b) reported that seed
treatment with Bayleton+P. fluorescence (2+5 gm/ kg seed) produced significant
response to wilt incidence, 1000-grain weight (gm) and yield (kg/ ha). It was
followed by mancozeb+P. fluorescence (2.5+5 gm/ kg seed) and Captaf + T.
harzianum (2+5 gm/ kg seed). However, high wilt incidence and low yield was
observed with seed treatment using cow ghee and mustard oil.
33
Chapter 3
MATERIALS AND METHODS
The study comprised of the experiments viz. pathogen isolation,
identification, preservation, morphological characterization, pathogenicity testing
and the management trials was conducted at the Fungal Pathology Laboratory,
Department of Plant Pathology, PMAS-Arid Agriculture University Rawalpindi.
Molecular characterization through DNA sequencing and phylogenetic analysis
was performed at the Department of Plant Pathology and Environmental
Microbiology, Pennsylvania State University, USA. Following trials were
undertaken to achieve the objectives as defined at the end of Chapter 1.
3.1 DISEASE SURVEY AND ASSESSMENT
3.1.1 Disease Survey
The survey of farmer's field located in major lentil growing areas/ districts
of Punjab, Pakistan (Figure 3.1) viz. Chakwal (32°56'N; 72°53'E), Attock
(33°52'N; 72°20'E), Gujrat (32°40'N; 74°02'E), Jhelum (31°20'N; 72°10'E), Sialkot
(32°30'N; 74°31'E), Narowal (32°06'N; 74°52'E), Mianwali (32°38'N; 71°28'E),
Layyah (23°54'S; 21°55'E), Bhakkar (31°40'N; 71°05'E) and Khushab (32°20'N;
72°20'E) was conducted during 2011-12 and 2012-13 crop seasons for the
calculation of disease incidence, distribution and assembling of the diseased
specimen. The survey of fields were conducted twice, at two growth stages i.e.
seedling and reproductive (adult) of lentil in each crop season. Disease distribution
map of Punjab was developed by surveying wilt infected fields and documenting
disease incidence. Prior to disease survey, necessary information on lentil
33
34
Map of Punjab province showing major lentil districts surveyed for the assessment of wilt prevalence, incidence and collection of Fusarium isolates.
Figure 3.1:
35
plantation in various districts and years was discussed with Dr. Ahmed Bakhsh
(DG, Planning and Development, PARC) and Dr. Sheikh Muhammad Iqbal (PSO),
National Pulses Program, Crop Sciences Institute, NARC, Islamabad.
3.1.2 Disease Assessment and Sampling
A total of 41 lentil fields belonging to local farmers were assessed for wilt
disease during 2011-12 crop season, while 35 fields were visited during 2012-13.
Because of scarce and scattered lentil plantation, all the fields from each of the
districts were surveyed by travelling along the road side of the areas. For disease
assessment and sampling, 10 spots were randomly selected from each field. The
number of total plants and wilted or infected plants in 1 m2 were counted. These
observations were used to calculate the average wilt incidence in each field.
Disease prevalence and incidence were used to assess wilt distribution in
surveyed areas and calculated by using the following formula:
Disease Prevalence (%) = Number of Infected Fields x 100 Total Number of Fields Disease Incidence (%) = Number of Infected Plants x 100 Total Number of Plants
Wilted samples were collected by careful observations of typical wilt
disease symptoms (Bowers and Locke, 2000) excluding other lentil root-rot
diseased plants. The wilted lentil plants were examined for sudden dropping of
plants leaves, without premature shedding of brown leaves and with some
chlorosis. Internal brown discoloration of xylem vessels was also observed to avoid
root rot disease. The samples comprised of whole wilted plants were uprooted and
shaken to remove excess soil. Those were then placed in paper bags labeled with
36
required data including date, place, etc. and were brought to Fungal Pathology
laboratory, Department of Plant Pathology, PMAS-Arid Agriculture University
Rawalpindi (AAUR) and stored in a refrigerator at 5-6oC until used for the
isolation and further confirmation of associated Fusarium pathogens. Size of the
samples depended upon field size and on an average 25-30 samples per field were
collected with 92% samples were found positive for wilt.
3.2 ISOLATION AND IDENTIFICATION OF THE PATHO GENS
The isolation of the associated fungi was done from the collected wilted
lentil plants on Potato Dextrose Agar (PDA) (Starch = 20 gm, Dextrose = 20 gm,
Agar = 20 gm in 1 liter distilled water) medium (Johnson and Curl, 1972 and
Bayaa et al., 1994). The infected roots samples were cut into small pieces about 8-
10 mm, washed with tap water and then surface sterilized in 10 percent chlorox
solution for 1-2 minutes. The sterilized pieces were then washed in sterilized
distilled water twice and blotted on sterilized filter paper to remove excess
moisture contents. These sterilized pieces were then plated in petri plates (five
pieces per plate) containing PDA medium autoclaved at 121oC (15 psi) for 20
minutes. The needles, scissor and forceps used for cutting the pieces and
transferring them on the media were first sterilized in methylated spirit and then
flaming on spirit lamp. After plating, the plates were incubated at 25±2oC for 3-4
days.
After colonization of these pieces with different fungal pathogens, the
emerged fungi were isolated and purified, then identified and confirmed for the
associated Fusarium pathogen according to the identification keys of Fusarium
37
(Leslie and Summerell, 2006; Booth, 1977 and Toussoun and Nelson, 1976). The
prevalence of each fungal species in each location and their association with wilt
incidence were recorded. The identified 213 Fusarium isolates were then re-
cultured on Malt Extract Agar (MEA) medium (Malt = 20 gm, Agar = 20 gm,
Peptone = 2 gm in 1 liter distilled water) using single spore technique or hyphal-tip
method (Choi et al., 1999; Burgess et al., 1988b and Nelson et al., 1983) and
cultures of the Fusarium isolates were stored on MEA medium for short period of
time in refrigerator and later were preserved on silica gel in glass vials for long
term preservation and maintained for further studies.
3.3 PRESERVATION OF FUSARIUM ISOLATES
For silica gel preservation, procedure described by Leslie and Summerell
(2006) and Tuite (1969) was followed with minor modification. Screw capped
glass vials (30 mL) were filled with silica gel (one third or 60-75 percent of vial)
and then sterilized. These vials were instantly cooled in an ice bath before use. For
the preparation of conidial suspension, 2 mL of sterilized skim milk (25 gm in 500
mL distilled water) was added in a fresh culture of Fusarium isolate in a 9 cm
petriplate. Sterilized needle was used to tear the fungal growth to make a heavy
conidial suspension.
By using micropipette, spore suspension (1000 µL per vial) was sucked and
dispensed across silica gel in a glass vial. Vial was vortexed vigorously and placed
back into the ice bath instantly. This was done in order to cool the gel as addition
of any liquid material to the dried silica gel may kills the fungal spores. Loosely
capped vials were placed in incubator at 25±2oC for 10-14 days and after that their
38
caps were tightly closed and covered with parafilm M and stored at 4±2oC. To
check the viability of the isolates, few gel crystals were sprinkled on MEA
medium, the vials again recapped tightly and stored again at 4±2oC.
3.4 MORPHOLOGICAL CHARACTERIZATION
The collected 213 Fusarium isolates were characterized on the basis of their
morphological characteristics using the criteria of Leslie and Summerell (2006),
Skovgaard et al. (2003), Booth (1977) and Toussoun and Nelson (1976). Five mm
diameter mycelia plugs were taken from the periphery of the respective isolates by
using sterilized cork borer, placed in the center of 9 cm petri plates and incubated
at 25±2oC in dark. Temporary glass slide mounts of each Fusarium isolate were
made in lactophenol solution and assessed under light microscope (Nikon YS100)
at 100X magnification for observation of characters.
The morphological parameters including colony color, growth habit,
pigmentation, days to fill 9 cm dish, concentric rings, size of micro-conidia, shape
of micro-conidia, size of macro-conidia, shape of macro-conidia, phialide, shape of
apical and basal cells of macro-conidia, septation in macro-conidia, diameter and
formation of chlamydospores and interseptal distance, at random, were noted for
each isolate. Five readings were taken for each parameter and their mean was
calculated.
3.5 PATHOGENICITY TEST
For pathogenicity testing, the identified and morphologically characterized
213 isolates of Fusarium were grouped into 67 type isolates with similar
morphology and tested in a single batch for their virulence on lentil germplasm i.e.
39
line NARC-08-1 and cultivar (cv.) Masoor-93 collected from National Coordinated
Pulses Programme, Crop Sciences Institute, NARC, Islamabad under controlled
screen house conditions viz. 25-28 ºC day temperature, 20-22 ºC night temperature
and 60-70% moisture.
To carry out pathogenicity test following the method described by Taheri et
al. (2010), the fungal inoculum was prepared by growing in Erlenmeyer flasks (100
mL) containing 50 mL sterilized potato-dextrose broth. Each flask was inoculated
with a 5 mm diameter mycelial plug taken from pure culture. The inoculated flasks
were shaken in rotary shaker at 120 rpm for 3 days. The fungal spore suspensions
were adjusted to 1 x 107 conidia/ mL using haemocytometer. The lentil seeds were
surface sterilized using 0.5% sodium hypochlorite for 5 minutes, rinsed in sterile
water and germinated in germinators containing sterilized potting mixture
(sand/farmyard manure, 1:1) (Figure 3.2). After about 15 days, the lentil seedlings
were uprooted carefully from the germinators, dipped into the inoculum for about
10 minutes (Figure 3.3) and then sown in plastic pots (5 seedlings per pot)
containing sterilized potting mixture (sand/clay/farmyard manure, 1:1:1) (Figure
3.4). The pots with un-inoculated seedlings served as control. All the pots were
maintained in screen house and watered as required. The experiment was
conducted using completely randomized design (CRD) with 3 replications.
The potting mixture for pathogenicity test was sterilized following the
procedure of Naz et al. (2008) using formaldehyde (5%) prepared from 37%
commercial formulation (Merck, Germany). The mixture was mixed thoroughly
with prepared formaldehyde solution (100 mL/ kg of soil) and covered with
polythene sheets, the edges of which were air-sealed with ordinary soil. After two
a
b Figure 3.2: Lentil seedlings in germinators
40
: Lentil seedlings in germinators (a) 8-days old, and (b) 15
(b) 15-days old.
Figure 3.3: Lentil roots dipping in fungal
Figure 3.4: Inoculated lentil seedlings after transplantation to pots in screen house.
41
: Lentil roots dipping in fungal spore suspension (1 x 107
: Inoculated lentil seedlings after transplantation to pots in screen house.
7conidia/ mL).
: Inoculated lentil seedlings after transplantation to pots in screen house.
42
days, the polythene sheets were removed and the treated potting mixture was
exposed to air for 4-5 days and occasionally turned over to allow escape of fumes
of formaldehyde. The treated potting mixture was then used for partially filling
each pot.
3.5.1 Disease Parameters
After inoculation, data on perent disease parameters viz. percent disease
severity index, disease incidence and yield reduction was recorded. Disease
incidence was recorded based on number of plants wilted using the formula as
described earlier in disease survey and assessment. The disease severity was
recorded starting from the 5th day and continued up till maturity using a modified
scale of 0-9 (Bayaa et al., 1995), as 0 = no symptoms/ infection, 1-3 = yellowing of
the basal leaves only, 4-6 = yellowing of 50 percent of the foliage and 7-9 =
complete yellowing of the foliage with whole plant or part of the plant wilted and/
or dried. Based on disease symptoms and the rating scale, the virulence of isolates
was further characterized as avirulent (0 scale value; 0% infection), low virulent (1-
3 scale range; 1-25%), moderately virulent (4-6; 25-50%) and highly virulent (7-9;
50% or more). The severity index percentage was then calculated by using the
formula of Kranz (1988) as,
Disease severity index (%) = Ʃ (a x b) x 100 N.Z
Where,
Ʃ (a x b) = Sum of the symptomatic plant and their corresponding scale value.
N = Total number of plants per pot.
Z = Highest scale value.
43
Yield reduction was noted by harvesting the seeds and measuring the seed
weight per fifteen plants (five plants per three replications) and compared with the
control. After the final score, the fungus was re-isolated and confirmed for
associated fungus. The recorded data was analyzed statistically using software
program SPSS. The mean data of the readings was taken, analyzed statistically and
significance of results was expressed at 5% level.
3.6 MOLECULAR CHARACTERIZATION
The identified, morphologically and pathogenically characterized 67 type
isolates of Fusarium were molecularly characterized by sequencing and comparing
translation elongation factor (TEF-1α) gene region for the determination of genetic
diversity among the isolates, identification at the species level and phylogenetic
analysis.
3.6.1 Mycelium Production and DNA Extraction
The protocol of DNA extraction described by Cenis (1992) and Abd-
Elsalam et al. (2003) was followed with minor modifications. The fungal isolates
stored on silica gel crystals were revived on PDA medium for 7 days at 25 °C in
the dark (incubator). Both mycelium and conidia were collected from the plates by
scratching the fungal growth from the medium surface by using sterilized surgical
blade. The mycelial material was then transferred into a 2 mL screw capped
microcentrifuge tube with about 6 mm depth of glass beads (0.1 mm) for the
disruption of cell wall by bead beater. About 750 µL of lysis buffer (50 mM Tris
HCL pH7.2 or 8.0), 50 mM Ethylene Diamine Tetra Acetate (EDTA) pH7.5 or 8.0,
3% SDS, 1% BME/ mercaptoethanol, distilled water) was added to about 5 gm of
44
mycelia. Bead beating was done for 1 minute with highest speed. The tubes were
then vortexed and incubated at 65ºC for 30 minutes.
The tubes were cool down for a minute and 700 µL of phenol:chloroform
(1:1) was added. The tubes were again vortexed until liquid was mixed completely
(for about 15 seconds). Tubes were gently shaken by inverting for 2-3 minutes and
centrifuged for 10 minutes at 12000 rpm. After centrifugation, about 500 µL of
supernatant was transferred carefully to new 1.5 mL microcentrifuge tube. About
360 µL isopropanol with 60 µL of 3M sodium acetate (1/10 vol of transfer volume)
were added to precipitate DNA. The mixture was incubated at -20 ºC for 15
minutes. Centrifuged for 10 minutes at 12000 rpm at room temperature.
Supernatant was discarded and pellet was washed with 700 µL of 70% ethanol.
Centrifuged for 5 minutes at 12000 rpm at room temperature. Supernatant was
discarded and pellet was dried at room temperature for about 30 minutes in hood.
After drying, the pellet was dissolved completely in 300 µL 1x TE buffer
(10 mM Tris-HCl, 1 mM EDTA pH: 8.0 and distilled water) and incubated at 4ºC.
RNase (2 µL of 10 mg/ mL) was added to DNA and incubated at 37 ºC for 1 hour.
Phenol:chloroform (1:1) (400 µL) was added and vortexed (for about 15 seconds)
until liquid was mixed completely. Tubes were shaken by inverting for 2-3 minutes
and centrifuged for 10 minutes at 12000 rpm. About 250-300 µL of supernatant
was carefully transferred to new 1.5 mL microcentrifuge tube. Chilled 100%
ethanol (2X vol) 500-600 mL with 30 µL of 3M sodium acetate (1/10 vol of
transfer volume) were added to precipitate the DNA. Then, incubated the mixture
at -20 ºC for 15 minutes. Centrifuged for 10 minutes at 12000 rpm at room
45
temperature. Supernatant was discarded and pellet was washed with 750 µL of 70%
ethanol. Centrifuged for 5 minutes at 12000 rpm at room temperature. Supernatant
was again discarded and pellet was dried. After drying, the pellet was dissolved in
50-100 µL of 1X TE buffer (10 mM Tris, 1 mM EDTA and pH: 8.0).
After DNA extraction, to validate the successful DNA extraction and to
check the quality of the extracted DNA, agarose gel electrophoresis at 80-90V for
30-40 minutes was done using a 1 kb ladder (Biolabs). For electrophoresis, a 1.0%
(w/v) agarose gel was prepared in 1x TAE buffer (0.1 M Tris, 0.05M boric acid
and 0.001M EDTA) and added with 0.25 μg/ mL ethidium bromide (AppliChem).
Genomic DNA concentration and purity was measured using Nanodrop
spectrophotometer. The isolated DNA sample of each isolate dissolved in TE
buffer was placed at -20 °C for long term storage and use.
3.6.2 Polymerase Chain Reaction (PCR) Amplification of TEF-1α Gene
Region
The translation elongation factor (TEF-1α) gene region (Figure 3.5) was
amplified by PCR reaction using the primers ef1 (forward primer; 5'-
ATGGGTAAGGA(A/G)GACAAGAC-3') and ef2 (reverse primer; 5'-
GGA(G/A)GTACCAGT(G/C)ATCATGTT-3') (Geiser et al., 2004 and O’Donnell
et al., 1998b) with an annealing temperature of 53ºC (Geiser et al., 2004).
The protocol of Williams et al. (1990) was employed with minor
modification. 50 μL reaction mixture was used for PCR amplification containing 5
μL of 10X Taq Buffer, 0.2 mM of dNTPs mix (100 mM of each dNTPs), 1 μL of
each forward and reversed primer (25 ng each primer), 0.6 U (0.3 μL) of Taq
46
Figure 3.5: Schematic of part of translation elongation factor (TEF-1α) gene region showing primer positions.
47
polymerase (New England Biolabs, Ipswich, MA) with 25 ng of template DNA.
PCR conditions for the analysis were as follows; the PCR programme had one
initial denaturation step at 95 °C for 4 min, followed by 30 cycles of 95 °C for 1
min, annealing for 2 min at 53 °C and 72 °C for 1 min. The thermal cycles were
terminated by a final extension of 5 min at 72 °C.
3.6.3 PCR Product Analysis
The PCR amplified products were electrophoretically separated in a 1.0%
agarose gel in 1x TAE buffer at 80V for 30-40 minutes and stained with ethidium
bromide at 0.5 mg/ mL and visualized under Transilluminator. A 1kb ladder (New
England Biolabs, Ipswich, MA) was used as a molecular size standard, which was
remixed with 6X loading dye solution for direct loading on gel.
3.6.4 DNA Sequencing
The generated TEF PCR product employing the ef1 and ef2 primers was
used as a template for DNA sequencing after purification of the product using
ExoSap-IT (USB, Cleveland, USA) that removes non-target DNA, PCR primers
and spare dNTP’s. For purification using ExoSap-IT, the reagents included
Exonuclease-I (3 parts), Shrimp Alkaline Phosphatase (2 parts) and the 10X PCR
buffer (1 part) (0.5M KCl, 0.1M Tris HCl pH 8.3, 0.025M MgCl2). Two µL from
the final mix of these three reagents was added to 5 µL of PCR product. After
mixing, thermocycler was used following the program; 37ºC for 30 minutes, 80ºC
for 15 miutes and 4ºC to hold. After PCR, sequencing plate (96-well plate) was
loaded using 2 µL aliquots from the purified DNA along with 2 µL of both forward
and reverse primers. The sequencing was done from Genomics Core facility at
Pennsylvania State University, USA.
48
3.6.5 Phylogenetic Analysis
The obtained TEF sequences were edited from chromatograms using the
software program Sequencher v.4.1.4 (Gene Codes Corp.) and saved in FASTA
formatted files. All the TEF sequences of unknown isolates were compared with
sequences in the database using the FUSARIUM-ID server
(http://fusarium.cbio.psu.edu) (Geiser et al., 2004) and the BLAST server at US
National Center for Biotechnology Information (NCBI) GenBank database website
(http://www.ncbi.nlm.nih.gov) (Altschul et al., 1997) for species determination.
Representatives of all the species were included in the analysis and their sequences
were obtained from GenBank. TEF sequences of test isolates along with the
reference/ type sequences were aligned online at MAFFT website
(http://mafft.cbrc.jp/alignment/software/macportable.html). Maximum likelihood-
Bootstrap (ML-BS) analysis was performed on CIPRES Science Gateway website
using GARLI 2.01 on XSEDE. Bootstrap analysis performed using 1000
replications was used for the assessment of clade or branch support. F. beomiforme
and F. concolor served as outgroups in the phylogenetic analysis. The resulted
phylogenetic tree was edited and compiled using Adobe Illustrator program for
additional annotation of data. The sequences of all the isolates based on TEF
primers were deposited in NCBI GenBank database and accession numbers were
obtained.
3.7 MANAGEMENT OF FUSARIUM WILT
Three trials were undertaken for the management of lentil wilt disease. The
first trial comprised of the screening of lentil germplasm against selected most
highly virulent isolate of F. oxysporum (FWL12, GenBank accession number
49
KP297995). In the second trial, fungicides were evaluated for their efficacy against
the same isolate in vitro and in vivo. The third trial involved the evaluation of
biological control agents in vivo.
3.7.1 Management Through Host Plant Resistance
The lentil germplasm comprised of twenty three lines and cultivars was
screened in vitro against selected highly virulent isolate. The procedure for
screening and inoculum (1 x 107 conidia/ mL) preparation was similar to
pathogenicity test as described earlier (Taheri et al., 2010). For each lentil
germplasm line or cultivar, seeds were first grown in germinators and after 15 days,
seedlings were uprooted carefully, dipped into the inoculum for about 10 minutes
and then, sown in plastic pots (5 seedlings per pot) containing sterilized potting
mixture (sand/clay/farmyard manure, 1:1:1). Non-inoculated pots dipped in
sterilized distilled water served as control. The experiment formed CRD design
using 3 replications. Pots were maintained in the screen house and watered as
required.
3.7.1.1 Disease parameters
Disease scoring was started from the 5th day that continued up till maturity
and data on percent disease severity index, disease incidence and yield reduction
was calculated. Percent disease incidence was recorded based on number of plants
wilted using the formula as described earlier in disease survey and assessment. The
percent disease severity index was recorded using a modified 0-9 disease rating
scale (Bayaa et al., 1995). Infection types were characterized into four categories,
such as, plants having infection type 0 were considered immune (with 0%
50
infection), those with a score of 1-3 as resistant (1-25%), with 4-6 as moderately
susceptible (25-50%) and with 7-9 being susceptible (50% or more). The severity
index percentage was then calculated using the formula as described earlier in
pathogenicty test. Yield reduction was noted by harvesting the seeds and measuring
the seed weight per fifteen plants (five plants per three replications) and compared
with the control. The recorded data was analyzed statistically using software
program SPSS. The mean data of the readings was taken, analyzed statistically and
significance of results was expressed at 5% level.
3.7.2 Biological Management
The biological management was conducted using biocontrol agents viz.
Trichoderma harzianum and T. viridi were obtained from Crop Diseases Research
Institute (CDRI), NARC, Islamabad. The conidial suspensions of both agents were
prepared in 1 L sterilized distilled water using 5 mm mycelia disc from the margin
of actively growing colonies. For inoculation, 40 mL suspensions were mixed
separately using vortex mixture and adjusted to 5x108 conidia/ mL. The seeds of
the lentil line NARC-08-1 (proved susceptible through pathogenicity test) were
used for the experiment. Seeds (5 per pot) were sown in plastic pots containing
sterilized potting mixture (sand:soil:FYM, 1:1:1) prepared as described before.
After about 15 days, the seedlings were inoculated with 60 mL spore suspension
(1x107 conidia/ mL) of selected highly virulent isolate (FWL12: KP297995) and 40
mL suspensions of biocontrol agents by drenching. After inoculation, the pots were
watered as required and maintained in screen house. The inoculated pots with no
biocontrol agents and un-inoculated pots with sterilized distilled water served as
control. The experiment was conducted using CRD design with 3 replications.
51
3.7.2.1 Disease parameters
Biocontrol activity of both the microbes was measured by percent disease
severity index, disease incidence and yield reduction on treated plants. Disease
scoring was started upon appearance of wilt symptoms on uninoculated control
from 7th day and continued up till maturity. Disease incidence was recorded based
on number of plants wilted using the formula as described earlier. The disease
severity index was recorded based on modified 0-9 scale (Bayaa et al., 1995) and
calculated using the formula as described in pathogenicity test. Yield reduction was
noted by harvesting the seeds and measuring the seed weight per fifteen plants (5
plants per 3 replications) and compared with the control. The recorded data was
analyzed statistically using software program SPSS. The mean data of the readings
was taken, analyzed statistically and significance of results was expressed at 5%
level.
3.7.3 Chemical Management
The efficacy of four fungicides (Table 3.1) was evaluated in controlling the
lentil wilt.
3.7.3.1 In vitro evaluation of fungicides
In vitro evaluation of the fungicides was performed through poison food
technique (Nene and Thapilyal, 2000). Five concentrations viz. 10, 20, 30, 50 and
100 ppm of each test fungicide were used with each concentration replicated thrice
forming two factor factorial experiment.
A weighed quantity of each test fungicide was amended to the autoclaved
MEA medium to obtain required concentration. Twenty mL of the amended and
non-amended medium was poured in 9 cm petridishes. After solidification, MEA
52
Table 3.1: Fungicides used for the management of lentil Fusarium wilt.
Sr. No.
Common Name Trade Name Chemical Name Formulation Manufacturer Mode of Action
1 Captan Orthocide, Captane, Marpan, Vondcaptan
N- Trichloromethyl thio)-3a,4,7,7a tetrahydrophthalimide
50%WP ICI (Pvt) Ltd.
Non-systemic
2 Dithane M-45 Mancozeb 16% Mn, 2% Zn, 62% Ethylenebisdithio- Carbamate
80%WP Rohm & Hass Ltd.
Non-systemic
3 Benomyl Benlate, Sunlate Methyl-1-(butylcabamonyl)- 2-benzinidazol
50%WP R.B. Avari Entreprises Ltd.
Systemic
4 Thiophanate Methyl Topsin-M 1,2-di (3-ethoxycarboxyl) -2- thioureido Benzene
70%WP Pennwalt corp.
Systemic
53
media plates were inoculated with 5 mm mycelial disc taken from the edge of
freshly grown culture of selected highly virulent isolate using sterilized cork borer.
Non-amended MEA plates inoculated with the test isolate served as the control.
After inoculation, plates were incubated at 25±2 oC. After 7 days of incubation and
when control plates were filled completely with fungal growth, radial growth was
calculated. The efficacy of test fungicides was expressed as percent mycelial
growth inhibition over control and was calculated by applying the following
formula (Vincent, 1947).
Percent inhibition (I) = C – T x 100 C
Where,
C = Growth of test fungus in control (cm)
T = Growth of test fungus in treatment (cm)
The recorded data was analyzed statistically using software program SPSS.
The mean data of the readings was taken, analyzed statistically and significance of
results was expressed at 5% level.
3.7.3.2 In vivo evaluation of fungicides
For the management of lentil wilt through seed treatment, the tested
fungicides that showed best efficacy in vitro viz. Benomyl and Thiophanate methyl
were evaluated in vivo through seed treatment prior to sowing in plastic pots under
controlled screen house conditions. The seeds of the lentil line NARC-08-1 (proved
susceptible through pathogenicity test) were used and treated with each test
fungicide (2 gm/ kg seed). Seeds (5 per pot) were sown in plastic pots containing
sterilized potting mixture (sand:soil:FYM, 1:1:1) prepared as described before. The
54
potting mixture was inoculated with the 60 mL spore suspension (1x107 conidia/
mL) of selected highly virulent isolate (FWL12) 10 days before sowing the seeds.
After seed sowing, the pots were watered as required and maintained in screen
house (Figure 3.5). The inoculated pots with untreated seeds and un-inoculated pots
with untreated seeds and dipped in sterilized distilled water served as control. The
experiment was conducted using CRD design with 3 replications.
3.7.3.2.1 Disease parameters
Data on percent seed germination, percent disease severity index, disease
incidence and yield reduction was calculated. Disease scoring was started from the
5th day and continued up till maturity. Percent disease incidence was recorded
based on number of plants wilted using the formula as described earlier. The
disease severity was recorded using a modified 0-9 scale (Bayaa et al., 1995).
Yield reduction was noted by harvesting the seeds and measuring the seed weight
per fifteen plants (five plants per three replications) and compared with the control.
The recorded data was analyzed statistically using software program SPSS. The
mean data of the readings was taken, analyzed statistically and significance of
results was expressed at 5% level.
55
Chapter 4
RESULTS AND DISCUSSION
4.1 DISEASE SURVEY AND ASSESSMENT
Two year (2011-12 and 2012-13) systematic survey of lentil wilt disease
was conducted in major lentil growing districts of Punjab viz. Chakwal, Attock,
Sialkot, Narowal, Gujrat, Jhelum, Mianwali, Layyah, Bhakkar and Khushab
(Figure 4.1). Disease prevalence, incidence and distribution were calculated for
each district.
The disease prevalence was found 100% in all the visited districts during
both years. Lentil plants with wilt disease symptoms were found in all visited fields
but the incidence varied over fields during both study years (Figure 4.2a and b).
During the first year of study, average wilt incidence of 30% with mean plant
mortality of 25.1 and 34.3% was recorded at seedling stage and crucial
reproductive or adult stage, respectively. During the second year, 26% average wilt
incidence with 21 and 30.5% mean plant mortality was noted at seedling and adult
stage.
Significant variations in disease incidence were noticed during both years
and plant stages at different districts of Punjab as shown in Figure 4.3. During both
seasons, the maximum wilt incidence was however, observed in district Layyah
(85%, 80% with average mean of 82.5%) followed by Bhakkar (55%, 56%;
55.5%), while the lowest incidence was observed in districts Attock (10%, 5%;
7.5%) and Sialkot (10%, 5%; 7.5%). The rest of the districts showed severe to mild
incidence of disease.
55
Figure 4.1: Map of Pakistan and major Fusarium wilt prevalence, incidence and distribution
56
of Pakistan and major lentil growing districts and sites of Punjab province surveyed for thewilt prevalence, incidence and distribution.
surveyed for the assessment of
a
b Figure 4.2: Wilted lentil fields
57
entil fields: (a) Layyah-Fateh Pur, and (b) Bhakkar
(b) Bhakkar-Garh Morr.
58
Figure 4.3: District-wise disease incidence of lentil wilt at two plant growth stages during 2011-12 and 2012-13 crop seasons. Figures indicated above bars represent mean disease incidence (%).
59
During both survey, the number of lentil fields in each district varied and
was found reduced during the second year of the study. Locations of fields visited
are presented in Table 4.1. A total of 41 farmer’s field were visited in 2011-12 and
35 fields in 2012-13. Out of 41 fields visited in 2011-12, maximum fields were
found in Jhelum (8 fields), followed by district Gujrat (6), Bhakkar (4), Narowal
(4), Chakwal (4), Layyah (3), Mianwali (3), Khushab (3), Attock (3) and Sialkot
(3). Similarly, in 2012-13, fewer lentil fields were found and out of 35 lentil fields
visited, the maximum fields were found in Jhelum (8 fields), followed by district
Narowal (4), Chakwal (4), Layyah (3), Bhakkar (3), Khushab (3), Attock (3),
Sialkot (3), Mianwali (2) and Gujrat (2).
All the locations or fields surveyed showed 100% prevalence with
significant variation in percent disease incidence during both years ranging from
low to severe as shown in Table 4.1 and Appendix 1. Based on two year data, in
district Chakwal, the highest average mean wilt incidence was seen in Piplee (70%)
whereas the lowest incidence was observed in Bangali Gujar (8%), Dhudial (15%)
and Barani Agriculture Research Institute (BARI) (18%). In Attock, the lentil fields
at Tanazaya dam, Khaur and Jand, low incidence of 7%, 11% and 5%, respectively,
was observed. Within district Jhelum, significant variation in wilt incidence was
noted at different locations where mild incidence was found at Chanaal (35%)
followed by Khaiwal (30%) and Morha Skeiha (28%), while lowest was observed
at Panjaion (23%), Pindi Gujran (5%), Dhapai (13%), Dhuman Khanpur (15%) and
Dakhlee Karhan (13%). Similarly, relevant situation was found in six locations of
Gujrat, where Lambray (30%) field showed mild incidence, while Sombre (5%),
Naseera (5%), Shergarh (Dolat Nagar) (18%), Bhaddar (15%) and Jalalpur Jatan
(13%) showed lowest incidence. In Sialkot, two fields at Pasrur and one field at
60
Table 4.1: Location-wise percent disease prevalence and incidence.
No. District Area/ Location
Disease Prevalence* (%)
Disease Incidence* (%)
1
Chakwal
Bangali Gujar 100 8 2 Piplee 100 70 3 Dhudial 100 15 4 BARI 100 18 5
Attock Tanazaya Dam 100 7
6 Khaur 100 11 7 Jand 100 5 8
Jhelum
Pindi Gujran 100 5 9 Dhapai 100 13 10 Dhuman Khanpur 100 15 11 Panjaion 100 23 12 Khaiwal 100 30 13 Dakhlee Karhan 100 13 14 Morha Skeiha 100 28 15 Chanaal 100 35 16
Gujrat
Jalalpur Jatan 100 13 17 Shergarh (Dolat Nagar) 100 18 18 Sombre 100 5 19 Naseera 100 5 20 Bhaddar 100 15 21 Lambray 100 30 22
Sialkot Pasrur road, Field 1 100 8
23 Field 2 100 10 24 Chowinda 100 5 25
Narowal
Dongian 100 18 26 Behble 100 28 27 Zafarwal Road 100 10 28 Mureed Ke Road 100 45 29
Mianwali Chashma 100 13
30 Piplan 100 10 31 Harnoli 100 10 32
Layyah Fateh Pur 100 85
33 Chowk Azam 100 78 34 ARI, Karoor 100 85 35
Bhakkar
Mankera 100 60 36 Garh Morr 100 55 37 AZRI 100 52 38 Darya Khan 100 55 39
Khushab Nurpur 100 15
40 Adhikot 100 25 41 Hassan Pur Tiwana 100 28
*Data based on average mean of 2 years (Appendix 1).
61
Chowinda, low incidence of 8%, 10% and 5%, respectively, was noted. The sites of
Mureed Ke road (45%), Behble (28%), Dongian (18%) and Zafarwal Road (10%)
in Narowal district showed mild to low incidence. The four Thal districts showed
low to highest wilt incidence. Likewise, the Mianwali district fields at Chashma
(13%), Piplan (10%) and Harnoli (10%) showed lowest incidence. In district
Layyah, maximum incidence was witnessed at all three locations viz. Fateh Pur,
Chowk Azam and Agronomic Research Institute (ARI), Karoor showing 85%, 78%
and 85% wilt incidence, respectively. Similar situation was recorded in fields at
Bhakkar i.e. Mankera (60%), Garh Morr (55%), Arid Zone Research Institute
(AZRI) (52%) and Darya Khan (55%), which is also a significant figure. In
Khushab, fields at Nurpur (15%), Adhikot (25%) and Hassan Pur Tiwana (28%)
showed mild incidence.
The region was selected as around 77.41% of lentil is planted in Punjab
province and shares major part of country’s lentil production (FAO, 2010). This
region falls on the margin of the monsoon climate and the weather conditions
includes both the extremes i.e. hot and barren in south and much cooler in the
North. In general, the temperature is hot with distinct variations between summer
and winter seasons. Daily average temperature ranges from 30-10 °C and average
annual rainfall is low ranging from 180-580 mm. The soil is mostly fertile around
the river valleys, whereas, sparse deserts are also found near the border
with Rajasthan and the Sulaiman Range. District-wise significant variation in
disease incidence was noted, for which, various factors including the weather
conditions, soil type and the lentil varieties grown were also observed as these
might be all or one of the reason(s) of variation. Moreover, as the disease is
62
temperature-dependent, the lentil crop often has the opportunity of disease escape
and the inoculum level may also vary at different locations (Ahmad et al., 2010).
Ten major lentil producing districts of Punjab were surveyed, among which,
the Thal districts viz. Layyah and Bhakkar showed highest disease incidence. The
Thal districts are located in the neighborhood of Potohar region between Indus and
Jhelum rivers. The soil of this area is loamy and the weather conditions are much
warm and dry with low moisture contents in the soil than other areas (Abbas et al.,
2010). As, lentil wilt disease commonly occur in areas where high atmospheric
temperature prevails, therefore, all such conditions existing in these districts are
much suitable and conducive for the successful development of the disease as such
conditions favor the presence of inoculum in the soil. The plantation of lentil
varieties Masoor-2009, Masoor-2002 and Masoor-85 being more susceptible in this
area is also the reason of highest prevalence and incidence of wilt. On the other
hand, lowest incidence was observed in the fields of district Attock near Tanazaya
dam and also Sialkot with enough moisture contents in the soil, promoting
conditions that are unfavorable for the pathogen.
The rest of the districts showed severe to mild wilt incidence. Such as, mild
or moderate incidence in Thal districts Mianwali (11%) and Khushab (22.5%)
occurred due to less wilt damage observed on cultivar Masoor-2006 in Mianwali
and Masoor-85 in Khushab. The study area viz. districts Attock, Chakwal and
Jhelum fall under Potohar area and is bounded by river Jhelum on the east, Indus
river on the west, Kala Chitta Range and Margalla Hills on the North and by the
Salt range on the South. This is considered as semi-arid to sub-humid area which
receives average rainfall of 380-510 mm per annum. The land is poor to fertile
63
having sandy to silt loam and clay loam texture. During the season of lentil crop,
the maximum average temperature ranges from 19-35 °C, while minimum from 2-
18 °C. Irrigated agriculture is generally practiced to provide the crop with right
amount of water at right time (Haqqani et al., 2000). Commonly grown lentil
varieties were Masoor-2000, Masoor-2006, Masoor-2009 and Masoor-85, however,
local uncertified lentil seeds were also planted in some areas. The presence of low
to moderate (7.5-27.5%) wilt incidence in districts viz. Attock, Chakwal and
Jhelum might be due to the prevalence of less conducive environmental conditions
required for the wilt pathogen. In these areas, provision of irrigated water to lentils
also helps escaping the development of excessive dry conditions which
consequently reduces wilt pathogen attack.
The three districts viz. Sialkot, Gujrat and Narowal showed low to moderate
distribution of wilt i.e., 7.5, 17.5 and 25%, respectively. These districts consist of
plain and fertile land. The soils of these districts are silt loam, silty clay loam and
clay loams. The average rainfall ranges from 300 to 500 mm annually (Haqqani et
al., 2000). The Gujrat district is located in between river Jhelum and Chenab,
which creates conditions that prevents occurrence of extreme dry conditions in the
area and therefore, limits the development of wilt disease. As, the wilt pathogen
develops under low moisture conditions. Similar conditions are also prevalent in
district Sialkot, which is situated near the river Chenab at the foothills of Kashmir
bounded by district Narowal. Moreover, this reduced wilt damage might be due to
lentil varieties commonly grown in these areas viz. Masoor-2006, Masoor-86 and
local lentil seeds, which were assessed later as resistant in screening experiment
and did not allowed the pathogenicity to establish.
64
Considerable variation in mean wilt incidence during two growth stages
(32.4% at seedling and 23.05% at reproductive) of lentil may be attributed to high
temperature (24-27ºC) at reproductive stage during the months February and March
as compared the disease at seedling stage in the months of November, December
and January, where temperature remained low i.e., 5-20ºC (Haqqani et al., 2000).
In addition to disease assessment, the survey revealed continuous reduced
lentil plantation in the region. This was noticeable with the number of fields visited
during first survey year (41 fields) which reduced in the second year (35 fields).
The main reason behind this reduced plantation is the shiftment of land to other
crops such as wheat due to availability of irrigation water and the occurrence of
lentil diseases, thus, rating this valuable lentil as a low priority crop. Qasim et al.
(2013) also reported that lentil crop is being replaced by wheat and mustard crops
in Potohar lentil growing areas and therefore, these two crops are responsible for
reduced lentil acreage in this region. Moreover, local farmers identified additional
reasons, such as, lack of high yielding lentil varieties, decreased yield potential of
existing varieties, fluctuations in price and marketing problems. Similar socio-
economical production constraints of lentil and chick pea were reported by Kumar
and Bourai (2012).
Although lentil wilt is an important disease in Pakistan, no systematic
survey has been conducted in recent years for this disease. Such a detailed survey
work would provide useful information for prioritizing research on this disease.
Lentil wilt being an important disease problem globally, survey for lentil wilt
assessment had been conducted in various other countries. Such as, in India a study
65
was conducted for the calculation of lentil wilt-root rot incidence in 116 lentil
growing districts at crop reproductive stage by Chaudhary et al. (2010) that
revealed a range of 0.7-9.3% mean plant mortality and an overall mean mortality of
6.3%. The main pathogens responsible for plant mortality were F. oxysporum f. sp.
lentis (62.0%). Likewise, through the assessment of wilt disease damage in 27
lentil fields in Syria, Bayaa et al. (1986) found the proportion of wilted plants in all
fields that varied from 2 to 70% with a mean of 12% and the isolation of fungi
from diseased samples showed a dominance of Fusarium species. Similarly, Hamdi
and Hassanein (1996) conducted a systematic survey of lentil vascular wilt in
Egypt and observed F. oxysporum as the causal agent responsible for 0.5 to 10%
proportion of wilted plants. Later, Belabid et al. (2000) observed high incidence of
wilt in North-Western Algeria and found F. oxysporum as the major causal agent
along with F. moniliforme and F. equiseti as the minor pathogens.
4.1.1 Disease Sampling
The wilted lentil plant samples were collected on the basis of disease
symptoms which were pretty visible in the visited fields. Prominent patches of
wilted lentils were seen during the surveillence as shown in Figures 4.4a and b.
Seedling wilt was distinguished by drooping and drying of leaves with total loss of
seedlings in some cases. At the adult stage, symptoms were observed at flowering
stage to late pod filling stage. Drooping of top leaflets of plants was observed. In
some cases, upon cross section, roots of infected plants showed brown to black
discoloration of vascular structures. Such symptom observations were also noted
by Bowers and Locke (2000).
a
b Figure 4.4: Patches of wilted lentil (b) Bhakkar
66
Figure 4.4: Patches of wilted lentil plants in fields: (a) Chakwal-Piplee(b) Bhakkar-Garh Morr field.
Piplee field, and
67
4.2 ISOLATION AND IDENTIFICATION OF THE PATHOGENS
Collected lentil plant samples showing typical wilt symptoms (Figure 4.5)
were subjected to isolation of associated Fusarium pathogens in the laboratory. A
total of 213 Fusarium isolates were identified and purified from the diseased
specimens collected from different fields of visited districts. Details of these
isolates along with their collection sites are given in Appendix 1.
The isolations conducted for retrieval of wilt pathogens associated with
lentil plants mortality from all districts revealed the involvement of Fusarium
pathogens. These observations depict the species of Fusarium as the causal
organism and responsible for wilt disease damage witnessed in the fields at both
stages of the crop.
The frequency of Fusarium isolates varied from district to district.
Maximum number (70 isolates) of Fusarium isolates were recovered from the
samples collected from Jhelum district, whereas, minimum number (3 isolates) of
Fusarium isolates from Khushab district. Thirty one isolates were recovered from
wilted samples collected from district Chakwal. In contrast, in Attock district, five
Fusarium isolates have been recovered from diseased specimens. Samples from the
fields of district Gujrat revealed 26 isolates. In the district Sialkot, 13 isolates were
isolated from the diseased plant samples. Thirty one Fusarium isolates from district
Narowal were recovered. From district Mianwali, 4 isolates were isolated. The
Layyah district revealed 16 isolates, whereas, from Bhakkar fields, a total of 14
isolates were recovered. All these recovered isolates were stored, preserved and
maintained for further studies.
68
Figure 4.5: Lentil plant samples: (Right) Wilted lentil plant, and (Left) Healthy lentil plant (left).
69
4.3 PRESERVATION OF FUSARIUM ISOLATES
The preservation method of Fusarium isolates used in this research on silica
gel allows storage for 4-5 years with suitable results and reduced mutation (Figure
4.6a and b). The viability test for the recovery of stock cultures preserved with
silica conducted after 5 days after addition of conidia on gel crystals and also
before storage of isolates at 4±2oC in refrigerator provided almost 100% positive
results. The gel particles produced fungal colonies on media plates retaining similar
colony morphology and spore characteristics (Figure 4.6c).
The results of the study were similar to those reported by Sharma et al.
(2002) and Windels et al. (1988). The preservation method was effective in
producing positive cultures survival results and no effect on the pathogenicity of
the isolates. Similar results were also reported by Sharma et al. (2002), who
checked the survival, growth and pathogenicity of Fusarium isolates using different
preservation methods up to 36 month of storage and proposed the best survival of
isolates with filter paper, which was followed by silica gel, mineral oil, soil, water
and slant, respectively. Also, the storage of isolates at 4±2oC in refrigerator
increased the survival of isolates for longer period (Trollope, 1975). This
preservation method reported and proved to be inexpensive, rapid and very simple
to employ for various fungi as well as for other micro-organisms. Likewise,
Perkins (1962) used this method for the fungus Neurospora crassa by suspending
the spores or mycelia in skim milk and adsorbed to silica gel crystals. Silica gel
preservation method has also been found good for the phytopathogenic bacteria
(Sleesman and Leben, 1978). Likewise, Trollope (1975) used this technique for the
storage of both Gram (+) and Gram (-) bacteria.
Preserved Fusarium(a), Gel crystals coated with fungal growth (b), and culture using silica gel crystals after 5 days of incubation at
a
b c
Figure 4.6:
70
Fusarium isolates on silica gel in glass vials stored at (a), Gel crystals coated with fungal growth (b), and Revived culture using silica gel crystals after 5 days of incubation at
b c
isolates on silica gel in glass vials stored at 4±2oC Revived Fusarium
culture using silica gel crystals after 5 days of incubation at 25oC (c).
71
Preservation of fungal cultures on long-term basis is essential for further
detailed analysis and research. Though, the viability and stability of any living cell
must be ensured during the preservation phase. Silica gel preservation employed in
the study proved an easy and economical method that ensured the viability and
stability of Fusarium isolates. The cultures can be obtained using a single
preserved vial with reduced chances of contamination and mutation. Therefore, this
method is preferred and recommended for long-term storage of Fusarium species.
4.4 MORPHOLOGICAL CHARACTERIZATION
All the isolates were characterized and identified on the basis of
morphology according to Fusarium Laboratory Manuals (Leslie and Summerell,
2006; Booth, 1977 and Toussoun and Nelson, 1976). The 213 Fusarium isolates
identified in this study based on their morphological characters are similar to those
reported in these manuals. Tentatively, 208 isolates were characterized into three
species of Fusarium based on their close morphology viz. F. oxysporum, F.
equiseti and F. commune (according to Skovgaard et al., 2003), whereas, rest of the
5 isolates were designated as Fusarium species. The isolates used in this study,
their geographic origin along with their names given to each isolate are depicted in
Appendix 1.
The isolates were characterized morphologically and showed significant
variations among each other on the basis of parameters viz. colony color, growth
habit, pigmentation, days to fill 9 cm dish, concentric rings, size of micro-conidia,
shape of micro-conidia, size of macro-conidia, shape of macro-conidia, phialide,
shape of apical and basal cells of macro-conidia, septation in macro-conidia,
diameter and formation of chlamydospores and interseptal distance (Table 4.2).
72
Table 4.2: Fusarium culture identification and morphological characterization checklist (CC = Colony Color; GH = Growth Habit; P = Pigmentation; CR = Concentric ring, (+) present, (-) absent; D = Days to fill 9cm plate; S = Septation; Ph = Phialide, SM = Short Monophialide, LM = Long Monophialide, P = Polyphialide; Dia. = Diameter; IsD = Interseptal Distance). No. Isolate
ID. No. CC GH P CR D Macroconidia Microconidia Chlamydospores
IsD (µm)
Species
Size (µm)
Shape Apical S Size (µm)
Shape Ph Dia. (µm)
Formation
1 FWC1 Creamy White
Fluffy - - 9 19.2x2.5
Slightly Curved
Elongate 5 8.2x 2.1
Pyriform SM 12.4 Singly, Short Chains, Clusters
19.0 F. equiseti
2 FWC2 Creamy White
Fluffy Pale Brown
- 9 42.0x4.6
Slightly Curved
Elongate 5 9.6x 2.7
Pyriform SM 7.6 Singly, Short Chains, Clusters
26.4 F. equiseti
3 FWC3 Creamy White
Compact Pale Brown
- 9 28.6x3.8
Slightly Curved
Elongate 5 6.5x 3.6
Pyriform SM 9.8 Singly, Short Chains, Clusters
14.2 F. equiseti
4 FWC4 Creamy White
Fluffy - + 10 14.2x2.6
Slightly Curved
Elongate 5 6.4x 1.9
Pyriform SM 8.4 Singly, Short Chains, Clusters
16.6 F. equiseti
5 FWC5 White Fluffy Dark Violet
- 9 13.8x2.4
Straight Pointed 3-4 4.5x 2.0
2-celled Oval
SM 8.6 Singly, Pairs 11.0 F. oxysporum
6 FWC6 White Fluffy Dark Violet
- 9 17.8x3.0
Straight Pointed 3 5.8x 2.2
Oval SM 8.6 Singly, Pairs 23.6 F. oxysporum
7 FWC7 Creamy White
Compact - - 9 14.6x2.6
Slightly Curved
Elongate 5 5.6x 2.0
Pyriform SM 9.2 Singly, Short Chains
21.6 F. equiseti
8 FWC8 White Fluffy - - 9 19.8x3.8
Straight Pointed 3 6.9x 2.8
Oval SM 7.4 Singly, Pairs 16.6 F. oxysporum
9 FWC9 White Flat Light Brown
- 8 17.8x2.6
Straight Pointed 3-5 7.2x 2.5
Oval SM 9.8 Short Chains 12.8 F. oxysporum
10 FWC10 White Fluffy - - 8 22.4x3.3
Straight Pointed 3 8.8x 2.9
Oval SM 7.8 Singly, Pairs 10.2 F. oxysporum
11 FWC11 White Fluffy Violet - 9 24.6x4.0
Straight Pointed 3 5.4x 2.2
2-celled Oval
SM 11.4 Singly, Pairs 7.8 F. oxysporum
12 FWC12 White Flat - - 8 15.6x Straight Pointed 3-5 8.4x Oval SM 15.0 Singly, Short 16.0 F. oxysporum
Continued……
73
2.7 3.0 Chains 13 FWC13 White Flat - - 8 24.4x
3.0 Straight Pointed 3-5 7.4x
2.5 Oval SM 7.2 Singly, Pairs 9.4
F. oxysporum
14 FWC14 White Flat - - 8 25.6x3.6
Straight Pointed 3-5 7.8x 2.4
Oval SM 7.2 Singly, Pairs 11.6 F. oxysporum
15 FWC15 White Fluffy Dark Violet
- 8 26.2x3.2
Straight Pointed 3 6.8x 2.8
Oval SM 14.0 Singly, Pairs 19.4 F. oxysporum
16 FWC16 White Fluffy Dark Violet
- 8 25.0x3.7
Straight Pointed 3 7.8x 2.3
Oval SM 7.0 Singly, Pairs 18.4 F. oxysporum
17 FWC17 Creamy White
Fluffy Pale Brown
+ 9 20.6x3.0
Slightly Curved
Elongate 5 7.8x 2.8
Pyriform SM 9.4 Singly, Short Chains, Clusters
15.3 F. equiseti
18 FWC18 Creamy White
Compact - + 10 21.8x3.5
Slightly Curved
Elongate 5 7.6x 2.4
Pyriform SM 8.0 Singly, Short Chains, Clusters
13.6 F.equiseti
19 FWC19 Creamy White
Compact - + 10 25.8x3.1
Slightly Curved
Elongate 5 7.4x 2.4
Pyriform SM 7.6 Singly, Short Chains, Clusters
17.2 F. equiseti
20 FWC20 Creamy White
Compact - + 10 25.2x3.0
Slightly Curved
Elongate 5 7.2x 2.5
Pyriform SM 6.8 Singly, Short Chains, Clusters
12.4 F. equiseti
21 FWC21 White Fluffy - - 9 29.6x3.6
Straight Pointed 3 8.2x 2.9
Oval SM 12.0 Singly, Pairs 21.1 F. oxysporum
22 FWC22 White Fluffy - - 9 25.0x3.2
Straight Pointed 3 6.6x 2.5
Oval SM 12.6 Singly, Pairs 17.2 F. oxysporum
23 FWC23 White Flat Light Brown
- 9 26.4x3.2
Straight Pointed 3-5 8.9x 3.2
Oval SM 11.8 Short Chains 13.8 F. oxysporum
24 FWC24 White Flat Light Brown
- 9 27.8x3.0
Straight Pointed 3-5 8.2x 3.4
Oval SM 12.2 Short Chains 21.2 F. oxysporum
25 FWC25 White Flat - - 8 22.6x3.0
Straight Pointed 3-5 6.4x 2.6
Oval SM 11.6 Singly, Short Chains
18.2 F. oxysporum
26 FWC26 White Flat - - 9 24.6x3.6
Straight Pointed 3-5 7.5x 2.8
Oval SM 11.4 Singly, Short Chains
22.2 F. oxysporum
27 FWC27 White Flat - - 9 29.0x4.0
Straight Pointed 3-5 6.8x 2.6
Oval SM 11.4 Singly, Short Chains
21.8 F. oxysporum
28 FWC28 White Flat - - 8 23.6x3.3
Straight Pointed 3-5 7.8x 3.5
Oval SM 13.2 Singly, Short Chains
11.6 F. oxysporum
Continued……
74
29 FWC29 White Flat Light Brown
- 9 21.8x3.3
Straight Pointed 3-5 6.2x 2.6
Oval SM 12.4 Singly, Short Chains
16.2 F. oxysporum
30 FWC30 White Flat Light Brown
- 9 25.2x3.5
Straight Pointed 3-5 6.6x 2.8
Oval SM 11.0 Singly, Short Chains
17.4 F. oxysporum
31 FWC31 White Flat Light Brown
- 9 28.0x3.8
Straight Pointed 3-5 6.6x 2.75
Oval SM 10.2 Singly, Short Chains
13.8 F. oxysporum
32 FWA1 White Flat - - 9 19.4x2.8
Straight Pointed 3-5 7.5x 3.1
Oval SM 11.6 Singly, Short Chains
18.6 F. oxysporum
33 FWA2 White Flat - - 9 28.8x3.8
Straight Pointed 3-5 7.5x 3.3
Oval SM 9.2 Singly, Short Chains
17.4 F. oxysporum
34 FWA3 White Flat - - 9 28.2x4.2
Straight Pointed 3-5 6.4x 2.5
Oval SM 11.8 Singly, Short Chains
10.0 F. oxysporum
35 FWA4 White Flat Light Brown
- 9 19.8x3.1
Straight Pointed 3-5 6.55x 2.5
Oval SM 11.0 Singly, Short Chains
17.8 F. oxysporum
36 FWA5 White Flat - - 9 21.4x3.2
Straight Pointed 3-5 7.6x 3.0
Oval SM 12.0 Singly, Short Chains
14.8 F. oxysporum
37 FWJ1 White Fluffy - - 10 18.8x2.8
Straight Pointed 3 6.4x 2.2
2-celled Oval
SM 15.0 Singly, Pairs 18.6 F. oxysporum
38 FWJ2 PinkishWhite
Fluffy - - 9 18.0x2.6
Straight Pointed 3 7.1x 2.0
2-celled Oval
SM 10.8 Singly, Pairs 15.6 F. oxysporum
39 FWJ3 PinkishWhite
Fluffy Dark Violet
- 9 13.2x2.3
Straight Pointed 3 6.8x 2.1
2-celled Oval
SM 7.8 Singly, Pairs 10.6 F. oxysporum
40 FWJ4 White Fluffy Dark Violet
- 9 15.2x2.8
Straight Pointed 3 5.4x 2.1
Oval SM 10.4 Singly, Pairs 16.0 F. oxysporum
41 FWJ5 PinkishWhite
Fluffy - - 9 15.0x2.5
Straight Pointed 3 4.4x 2.5
Oval SM 9.0 Singly, Pairs 16.4 F. oxysporum
42 FWJ6 White Fluffy Violet - 9 17.4x2.9
Straight Pointed 3 5.0x 2.5
Oval SM 10.2 Singly, Pairs 14.8 F. oxysporum
43 FWJ7 White Fluffy - - 9 20.0x3.0
Straight Pointed 3 4.6x 2.6
Oval SM 10.8 Singly, Pairs 9.4 F. oxysporum
44 FWJ8 White Fluffy Violet - 8 12.2x2.5
Straight Pointed 3 5.1x 2.5
Oval-Globose
SM 9.6 Singly 17.6 F. oxysporum
45 FWJ9 White Fluffy Violet - 8 15.6x2.7
Straight Pointed 3 5.9x 2.65
Oval-Globose
SM 11.2 Singly 18.2 F. oxysporum
46 FWJ10 White Fluffy Dark Violet
- 8 18.4x2.6
Straight Pointed 3 5.8x 2.5
Oval-Globose
SM 11.2 Singly 13.6 F. oxysporum
47 FWJ11 Pinkish Fluffy - - 8 11.0x Straight Pointed 3-4 5.3x Oval SM 11.0 Singly 11.0 F. oxysporum
Continued……
75
White 2.4 2.05 48 FWJ12 Pinkish
White Fluffy - - 9 14.6x
2.2 Straight Pointed 3-4 6.6x
2.75 Oval SM 14.0 Singly 19.6 F. oxysporum
49 FWJ13 White Fluffy - - 8 17.4x2.5
Straight Pointed 3-4 6.6x 2.3
Oval SM 10.4 Singly 17.2 F. oxysporum
50 FWJ14 PinkishWhite
Fluffy Dark Violet
- 8 19.6x2.3
Straight Pointed 3 7.2x 2.0
Oval SM 11.4 Singly, Pairs 14.3 F. oxysporum
51 FWJ15 PinkishWhite
Fluffy Dark Violet
- 8 10.8x2.0
Straight Pointed 3 6.6x 2.5
Oval SM 12.0 Singly, Pairs 17.6 F. oxysporum
52 FWJ16 PinkishWhite
Fluffy - - 9 19.4x2.4
Straight Pointed 3 7.0x 2.1
Oval SM 9.4 Singly, Pairs 18.0 F. oxysporum
53 FWJ17 White Flat - - 9 15.8x2.3
Straight Pointed 3-5 6.2x 2.2
Oval SM 17.8 Singly, Short Chains
24.0 F. oxysporum
54 FWJ18 White Flat - - 8 17.0x2.3
Straight Pointed 3-5 6.6x 2.65
Oval SM 9.6 Singly, Short Chains
10.2 F. oxysporum
55 FWJ19 White Flat - - 8 16.6x2.3
Straight Pointed 3-5 7.2x 2.2
Oval SM 11.8 Short Chains 21.6 F. oxysporum
56 FWJ20 Creamy White
Compact - + 10 32.0x3.8
Slightly Curved
Elongate 5 6.4x 2.3
Pyriform SM 7.4 Singly, Short Chains
15.8 F. equiseti
57 FWJ21 Creamy White
Fluffy Pale Brown
+ 10 28.2x3.0
Slightly Curved
Elongate 5 6.0x 2.25
Pyriform SM 12.8 Singly, Short Chains
13.0 F. equiseti
58 FWJ22 Creamy White
Compact - + 10 30.8x3.6
Slightly Curved
Elongate 5 6.4x 2.7
Pyriform SM 7.8 Singly, Short Chains, Clusters
14.6 F. equiseti
59 FWJ23 Creamy White
Fluffy Pale Brown
- 8 27.6x2.8
Slightly Curved
Elongate 5 6.8x 2.85
Pyriform SM 9.2 Singly, Short Chains, Clusters
17.6 F. equiseti
60 FWJ24 Creamy White
Compact Pale Brown
- 10 20.0x2.7
Slightly Curved
Elongate 5 5.9x 2.65
Pyriform SM 7.4 Singly, Short Chains, Clusters
15.0 F. equiseti
61 FWJ25 Creamy White
Compact Pale Brown
+ 8 22.4x2.8
Slightly Curved
Elongate 5 6.8x 2.6
Pyriform SM 9.0 Singly, Short Chains
13.6 F. equiseti
62 FWJ26 Creamy White
Compact - - 10 23.8x2.9
Slightly Curved
Elongate 5 5.6x 2.5
Pyriform SM 9.8 Singly, Short Chains, Clusters
14.6 F. equiseti
63 FWJ27 Creamy White
Fluffy - - 10 21.0x2.9
Slightly Curved
Elongate 5 6.8x 2.75
Pyriform SM 8.0 Singly, Short Chains,
12.2 F. equiseti
Continued……
76
Clusters 64 FWJ28 White Flat - - 8 19.8x
2.8 Straight Pointed 3-5 8.0x
3.1 Oval SM 10.0 Singly, Short
Chains 14.0 F. oxysporum
65 FWJ29 White Flat - - 9 22.0x2.9
Straight Pointed 3-5 8.4x 3.15
Oval SM 11.8 Singly, Pairs 16.8 F. oxysporum
66 FWJ30 White Flat - - 9 18.2x2.7
Straight Pointed 3-5 6.2x 2.65
Oval SM 11.0 Singly, Pairs 17.0 F. oxysporum
67 FWJ31 White Flat - - 8 16.8x2.4
Straight Pointed 3-5 7.6x 3.0
Oval SM 10.4 Singly, Pairs 13.4 F. oxysporum
68 FWJ32 White Flat - - 8 20.8x2.6
Straight Pointed 3-5 6.4x 2.6
Oval SM 7.6 Singly, Pairs 13.0 F. oxysporum
69 FWJ33 White Flat - - 9 18.0x2.6
Straight Pointed 3-5 6.4x 2.7
Oval SM 10.0 Singly, Pairs 19.2 F. oxysporum
70 FWJ34 White Flat - - 8 17.0x2.6
Straight Pointed 3-5 7.2x 3.0
Oval SM 9.4 Singly, Pairs 10.2 F. oxysporum
71 FWJ35 White Fluffy Dark Violet
- 8 26.0x3.0
Straight Pointed 3 6.4x 2.5
Oval SM 7.2 Singly, Pairs 10.6 F. oxysporum
72 FWJ36 White Fluffy Violet - 8 12.6x2.5
Straight Pointed 3 5.4x 2.3
Oval SM 10.4 Singly, Pairs 12.8 F. oxysporum
73 FWJ37 White Fluffy Violet - 8 17.6x2.6
Straight Pointed 3 5.2x 2.2
Oval SM 11.2 Singly, Pairs 12.6 F. oxysporum
74 FWJ38 White Fluffy Violet - 8 18.3x2.5
Straight Pointed 3 4.6x 2.3
Oval SM 12.4 Singly, Pairs 12.2 F. oxysporum
75 FWJ39 White Fluffy Violet - 8 20.4x2.5
Straight Pointed 3 5.8x 2.1
Oval SM 11.0 Singly, Pairs 14.6 F. oxysporum
76 FWJ40 White Fluffy - - 8 21.8x2.7
Straight Pointed 3 5.4x 2.1
Oval SM 14.4 Singly, Pairs 15.4 F. oxysporum
77 FWJ41 White Fluffy - - 8 16.8x2.5
Straight Pointed 3 5.8x 2.4
Oval SM 13.8 Singly, Pairs 19.6 F. oxysporum
78 FWJ42 White Fluffy - - 8 19.1x2.5
Straight Pointed 3 5.2x 2.6
Oval SM 13.0 Singly, Pairs 17.2 F. oxysporum
79 FWJ43 White Fluffy - - 8 20.8x2.55
Straight Pointed 3 5.0x 2.0
Oval SM 10.4 Singly, Pairs 21.0 F. oxysporum
80 FWJ44 White Fluffy - - 8 19.2x2.5
Straight Pointed 3 6.2x 2.2
Oval SM 14.8 Singly, Pairs 18.6 F. oxysporum
81 FWJ45 White Fluffy - - 8 22.1x3.0
Straight Pointed 3 5.4x 2.5
Oval SM 9.8 Singly, Pairs 19.2 F. oxysporum
Continued……
77
82 FWJ46 White Fluffy Violet - 8 16.4x2.6
Straight Pointed 3 6.4x 2.7
Oval SM 10.4 Singly, Pairs 13.4 F. oxysporum
83 FWJ47 Creamy White
Fluffy Pale Brown
+ 10 16.9x2.5
Slightly Curved
Elongate 5 8.4x 2.6
Pyriform SM 9.2 Singly, Short Chains, Clusters
18.2 F. equiseti
84 FWJ48 White Compact Pale Brown
+ 8 18.6x2.75
Slightly Curved
Elongate 5 7.4x 2.8
Pyriform SM 12.2 Singly, Short Chains, Clusters
22.4 F. equiseti
85 FWJ49 White Fluffy - - 9 13.2x3.1
Slender Curved 3 8.5x 2.8
Oval-Comma
SM 9.6 Singly, Short Chains, Clusters
15.8 Fusarium sp.
86 FWJ50 Creamy White
Compact - + 9 22.0x2.9
Slightly Curved
Elongate 5 7.7x 2.8
Pyriform SM 12.4 Singly, Short Chains, Clusters
18.2 F. equiseti
87 FWJ51 White Compact - + 9 21.4x2.6
Slightly Curved
Elongate 5 6.6x 2.6
Pyriform SM 12.0 Singly, Short Chains, Clusters
18.0 F. equiseti
88 FWJ52 White Compact - + 9 23.2x2.9
Slightly Curved
Elongate 5 7.2x 2.85
Pyriform SM 7.0 Singly, Short Chains, Clusters
21.2 F. equiseti
89 FWJ53 Creamy White
Compact - + 9 21.4x2.8
Slightly Curved
Elongate 5 8.6x 3.0
Pyriform SM 10.0 Singly, Short Chains, Clusters
16.6 F. equiseti
90 FWJ54 White Fluffy - - 9 16.1x3.6
Straight Pointed 3 7.4x 3.2
Oval SM 13.0 Singly, Pairs, Short Chains
16.4 F. oxysporum
91 FWJ55 White Fluffy Violet - 9 17.6x3.0
Straight Pointed 3 6.6x 2.3
Oval SM 8.2 Singly, Pairs, Short Chains
19.2 F. oxysporum
92 FWJ56 White Fluffy Violet - 9 18.9x2.85
Straight Pointed 3 6.2x 2.6
Oval SM 12.8 Singly, Pairs, Short Chains
20.2 F. oxysporum
93 FWJ57 White Fluffy Violet - 9 17.2x2.9
Straight Pointed 3 7.2x 2.7
Oval SM 11.2 Singly, Pairs, Short Chains
14.6 F. oxysporum
94 FWJ58 White Fluffy Violet - 9 18.0x2.9
Straight Pointed 3 7.8x 3.1
Oval SM 7.8 Singly, Pairs, Short Chains
27.8 F. oxysporum
95 FWJ59 White Fluffy - - 9 17.6x2.7
Straight Pointed 3 7.0x 2.6
Oval SM 14.2 Singly, Pairs, Short Chains
18.0 F. oxysporum
96 FWJ60 White Fluffy - - 9 18.9x2.7
Straight Pointed 3 5.6x 2.4
Oval SM 10.0 Singly, Pairs, Short Chains
19.4 F. oxysporum
Continued……
78
97 FWJ61 White Fluffy - - 9 20.8x2.8
Straight Pointed 3 6.6x 2.4
Oval SM 9.4 Singly, Pairs, Short Chains
14.2 F. oxysporum
98 FWJ62 White Fluffy - - 8 15.2x2.6
Straight Pointed 3 6.7x 2.6
Oval SM 8.2 Singly, Pairs, Short Chains
20.2 F. oxysporum
99 FWJ63 White Fluffy - - 8 17.7x2.7
Straight Pointed 3 6.4x 2.4
Oval SM 14.0 Singly, Pairs, Short Chains
15.0 F. oxysporum
100 FWJ64 White Fluffy - - 8 16.5x2.5
Straight Pointed 3 5.7x 2.2
Oval SM 9.8 Singly, Pairs, Short Chains
16.2 F. oxysporum
101 FWJ65 White Fluffy - - 8 18.7x2.8
Straight Pointed 3 5.4x 2.3
Oval SM 13.8 Singly, Pairs, Short Chains
15.4 F. oxysporum
102 FWJ66 White Fluffy - - 8 20.4x2.85
Straight Pointed 3 6.8x 2.3
Oval SM 10.0 Singly, Pairs, Short Chains
18.2 F. oxysporum
103 FWJ67 White Fluffy - - 8 19.4x2.75
Straight Pointed 3 6.8x 2.6
Oval SM 13.6 Singly, Pairs, Short Chains
17.0 F. oxysporum
104 FWJ68 White Fluffy Violet - 8 22.4x2.9
Straight Pointed 3 6.4x 2.5
Oval SM 11.2 Singly, Pairs, Short Chains
14.4 F. oxysporum
105 FWJ69 White Fluffy Violet - 8 19.0x2.7
Straight Pointed 3 6.4x 2.3
Oval SM 8.6 Singly, Pairs, Short Chains
17.6 F. oxysporum
106 FWJ70 White Fluffy - - 8 16.9x2.5
Straight Pointed 3 6.2x 2.5
Oval SM 10.8 Singly, Pairs, Short Chains
22.6 F. oxysporum
107 FWG1 White Fluffy Dark Violet
- 10 16.2x2.8
Straight Pointed 3 7.4x 2.8
Oval SM 14.6 Singly 31.0 F. oxysporum
108 FWG2 White Fluffy Dark Violet
- 9 16.4x2.7
Straight Pointed 3 5.8x 2.4
Oval SM 12.6 Singly 29.8 F. oxysporum
109 FWG3 White Fluffy Violet - 9 20.3x3.1
Straight Pointed 3 6.0x 2.8
Oval SM 13.6 Singly 15.2 F. oxysporum
110 FWG4 PinkishWhite
Fluffy Dark Violet
- 9 17.0x2.7
Straight Pointed 3 6.0x 2.6
Oval SM 8.6 Singly 11.8 F. oxysporum
111 FWG5 PinkishWhite
Fluffy Dark Violet
- 9 20.0x2.9
Straight Pointed 3 6.0x 2.9
Oval SM 12.2 Singly 20.8 F. oxysporum
112 FWG6 White Fluffy Dark Violet
- 9 16.0x2.5
Straight Pointed 3 6.2x 2.5
Oval SM 15.6 Singly 21.2 F. oxysporum
113 FWG7 White Fluffy Violet - 9 17.8x2.65
Straight Pointed 3 5.8x 2.5
Oval SM 7.6 Singly 20.6 F. oxysporum
114 FWG8 White Fluffy Violet - 9 23.0x2.75
Straight Pointed 3 6.0x 2.4
Oval SM 9.8 Singly 16.6 F. oxysporum
115 FWG9 Pinkish Fluffy Dark - 9 14.4x Straight Pointed 3 5.8x Oval SM 13.2 Singly 16.2 F. oxysporum
Continued……
79
White Violet 2.5 3.0 116 FWG10 White Fluffy Dark
Violet - 9 20.4x
3.1 Straight Pointed 3 6.2x
2.8 Oval SM 10.4 Singly 24.0 F. oxysporum
117 FWG11 White Fluffy Dark Violet
- 10 18.4x2.85
Straight Pointed 3 6.8x 2.5
Oval SM 14.0 Singly 21.2 F. oxysporum
118 FWG12 White Fluffy Dark Violet
- 10 20.6x2.9
Straight Pointed 3 6.2x 2.7
Oval SM 9.0 Singly 24.0 F. oxysporum
119 FWG13 White Fluffy - - 8 19.4x3.8
Straight Pointed 3 5.6x 2.4
Oval SM 9.6 Singly, Pairs, Short Chains
12.2 F. oxysporum
120 FWG14 White Fluffy - - 8 18.6x3.4
Straight Pointed 3 5.8x 2.2
Oval SM 11.2 Singly, Pairs, Short Chains
16.0 F. oxysporum
121 FWG15 White Fluffy - - 8 19.6x3.5
Straight Pointed 3 6.0x 2.6
Oval SM 7.0 Singly, Pairs, Short Chains
14.4 F. oxysporum
122 FWG16 White Fluffy - - 9 20.0x3.4
Straight Pointed 3 5.0x 2.4
Oval SM 11.8 Singly, Pairs, Short Chains
12.2 F. oxysporum
123 FWG17 White Fluffy - - 9 18.8x3.7
Straight Pointed 3 5.4x 2.5
Oval SM 10.2 Singly, Pairs, Short Chains
16.0 F. oxysporum
124 FWG18 White Fluffy - - 8 19.2x3.0
Straight Pointed 3 5.6x 2.3
Oval SM 11.6 Singly, Pairs, Short Chains
15.0 F. oxysporum
125 FWG19 White Fluffy - - 8 19.0x3.4
Straight Pointed 3 6.8x 2.5
Oval SM 8.8 Singly, Pairs, Short Chains
11.6 F. oxysporum
126 FWG20 White Fluffy - - 9 19.0x3.3
Straight Pointed 3 5.6x 2.5
Oval SM 15.2 Singly, Pairs, Short Chains
13.6 F. oxysporum
127 FWG21 White Fluffy - - 8 17.4x3.1
Straight Pointed 3 6.4x 2.8
Oval SM 10.0 Singly, Pairs, Short Chains
18.6 F. oxysporum
128 FWG22 White Fluffy - - 7 20.0x3.2
Straight Pointed 3 6.2x 2.55
Oval SM 11.2 Singly, Pairs, Short Chains
13.4 F. oxysporum
129 FWG23 White Fluffy - - 8 17.6x3.0
Straight Pointed 3 5.4x 2.5
Oval SM 14.2 Singly, Pairs, Short Chains
19.2 F. oxysporum
130 FWG24 White Fluffy - - 9 19.4x3.2
Straight Pointed 3 5.6x 2.7
Oval SM 9.4 Singly, Pairs, Short Chains
18.8 F. oxysporum
131 FWG25 White Fluffy - - 9 17.6x2.8
Straight Pointed 3 6.0x 2.6
Oval SM 8.8 Singly, Pairs, Short Chains
14.6 F. oxysporum
132 FWG26 White Fluffy - - 8 19.4x3.1
Straight Pointed 3 5.4x 2.55
Oval SM 13.0 Singly, Pairs, Short Chains
15.0 F. oxysporum
133 FWS1 White Fluffy - - 9 20.6x2.9
Straight Pointed 3 8.0x 2.9
Oval SM 9.4 Singly, Pairs, Short Chains
12.2 F. oxysporum
Continued……
80
134 FWS2 White Fluffy - - 9 18.2x2.5
Straight Pointed 3 4.8x 2.5
Oval SM 12.0 Singly, Pairs, Short Chains
15.0 F. oxysporum
135 FWS3 White Fluffy - - 9 19.2x2.7
Straight Pointed 3 5.2x 2.5
Oval SM 8.4 Singly, Pairs, Short Chains
12.0 F. oxysporum
136 FWS4 White Fluffy - - 9 17.2x2.6
Straight Pointed 3 6.2x 2.6
Oval SM 8.6 Singly, Pairs, Short Chains
22.0 F. oxysporum
137 FWS5 White Fluffy - - 9 18.7x2.5
Straight Pointed 3 5.3x 2.5
Oval SM 14.0 Singly, Pairs, Short Chains
21.2 F. oxysporum
138 FWS6 White Fluffy - - 9 17.2x2.5
Straight Pointed 3 4.8x 2.4
Oval SM 7.6 Singly, Pairs, Short Chains
17.4 F. oxysporum
139 FWS7 White Fluffy - - 9 18.8x2.6
Straight Pointed 3 6.4x 2.7
Oval SM 9.8 Singly, Pairs, Short Chains
13.6 F. oxysporum
140 FWS8 White Fluffy - - 9 18.5x2.55
Straight Pointed 3 4.8x 2.45
Oval SM 8.6 Singly, Pairs, Short Chains
10.6 F. oxysporum
141 FWS9 White Fluffy - - 9 21.0x2.6
Straight Pointed 3 5.8x 2.6
Oval SM 9.2 Singly, Pairs, Short Chains
17.2 F. oxysporum
142 FWS10 White Fluffy - - 9 16.0x2.5
Straight Pointed 3 5.2x 2.5
Oval SM 13.0 Singly, Pairs, Short Chains
8.1 F. oxysporum
143 FWS11 White Fluffy Violet - 9 20.1x2.7
Straight Pointed 3 6.4x 2.6
Obovoid SM 7.6 Singly, Pairs 14.4 F. commune
144 FWS12 White Fluffy Violet - 9 13.8x2.5
Straight Pointed 3 5.6x 2.65
Obovoid SM 14.4 Singly, Short Chains
14.8 F. commune
145 FWS13 White Fluffy Violet - 9 16.4x2.6
Straight Pointed 3 5.8x 2.6
Obovoid SM 9.4 Singly, Short Chains
15.0 F. commune
146 FWN1 White Flat - - 9 16.0x4.1
Straight Pointed 3-5 5.4x 2.5
Oval SM 8.0 Singly, Short Chains
14.4 F. oxysporum
147 FWN2 White Flat Light Brown
- 8 19.4x2.8
Straight Pointed 3-5 5.6x 2.1
Oval SM 8.0 Singly, Short Chains
12.6 F. oxysporum
148 FWN3 White Flat Light Brown
- 8 16.6x2.6
Straight Pointed 3-5 5.4x 2.55
Oval SM 12.2 Singly, Short Chains
15.2 F. oxysporum
149 FWN4 White Flat - - 8 15.3x2.8
Straight Pointed 3-5 5.2x 2.2
Oval SM 8.4 Singly, Short Chains
9.6 F. oxysporum
150 FWN5 White Flat - - 8 19.0x2.7
Straight Pointed 3-5 5.8x 2.0
Oval SM 8.6 Singly, Short Chains
11.1 F. oxysporum
151 FWN6 White Flat - - 8 15.8x2.6
Straight Pointed 3-5 4.8x 2.2
Oval SM 8.2 Singly, Short Chains
11.6 F. oxysporum
152 FWN7 White Flat - - 9 11.9x Straight Pointed 3-5 5.6x Oval SM 10.8 Singly, Short 6.2 F. oxysporum
Continued……
81
2.5 2.25 Chains 153 FWN8 White Flat - - 8 15.2x
2.3 Straight Pointed 3-5 5.0x
2.75 Oval SM 9.4 Short Chains 9.8 F. oxysporum
154 FWN9 White Flat - - 9 11.9x2.5
Straight Pointed 3-5 4.95x 2.3
Oval SM 8.8 Singly, Short Chains
16.0 F. oxysporum
155 FWN10 White Flat - - 8 13.0x3.5
Straight Pointed 3-5 4.1x 1.8
Oval SM 8.6 Short Chains 14.8 F. oxysporum
156 FWN11 White Flat - - 9 17.5x2.6
Straight Pointed 3-5 6.4x 2.6
Oval SM 11.2 Short Chains 17.0 F. oxysporum
157 FWN12 White Flat - - 9 13.8x2.6
Straight Pointed 3-5 5.6x 2.4
Oval SM 10.8 Short Chains 9.2 F. oxysporum
158 FWN13 White Flat - - 7 15.1x2.6
Straight Pointed 3-5 5.8x 2.55
Oval SM 8.0 Singly, Short Chains
14.4 F. oxysporum
159 FWN14 White Flat Light Brown
- 9 12.1x2.5
Straight Pointed 3-5 5.0x 2.3
Oval SM 11.8 Singly, Short Chains
10.6 F. oxysporum
160 FWN15 White Flat Light Brown
- 7 16.8x2.7
Straight Pointed 3-5 5.8x 2.4
Oval SM 7.6 Singly, Short Chains
10.8 F. oxysporum
161 FWN16 White Flat Light Brown
- 8 18.8x2.75
Straight Pointed 3-5 3.5x 2.5
Oval SM 11.4 Singly, Short Chains
22.0 F. oxysporum
162 FWN17 White Flat - - 9 18.8x2.9
Straight Pointed 3-5 4.7x 2.5
Oval SM 11.4 Singly, Short Chains
26.4 F. oxysporum
163 FWN18 White Flat - - 9 19.0x2.6
Straight Pointed 3-5 5.6x 2.8
Oval SM 8.8 Singly, Short Chains
18.2 F. oxysporum
164 FWN19 White Flat - - 9 17.8x2.6
Straight Pointed 3-5 7.6x 3.0
Oval SM 12.4 Singly, Short Chains
17.2 F. oxysporum
165 FWN20 White Flat Light Brown
- 9 15.9x2.7
Straight Pointed 3-5 4.6x 2.4
Oval SM 8.2 Short Chains 18.4 F. oxysporum
166 FWN21 White Flat - - 9 13.0x2.5
Straight Pointed 3-5 4.6x 2.35
Oval SM 9.6 Short Chains 14.6 F. oxysporum
167 FWN22 White Flat Light Brown
- 9 20.2x2.7
Straight Pointed 3-5 6.2x 2.8
Oval SM 17.0 Singly, Short Chains
16.1 F. oxysporum
168 FWN23 White Flat - - 9 17.1x2.7
Straight Pointed 3-5 5.0x 2.5
Oval SM 13.6 Singly, Short Chains
14.6 F. oxysporum
169 FWN24 White Flat - - 9 18.4x2.5
Straight Pointed 3-5 6.3x 2.9
Oval SM 10.4 Short Chains 12.8 F. oxysporum
170 FWN25 White Flat - - 9 18.0x2.5
Straight Pointed 3-5 4.8x 2.6
Oval SM 9.4 Short Chains 17.6 F. oxysporum
Continued……
82
171 FWN26 White Flat - - 9 12.7x2.35
Straight Pointed 3-5 6.0x 2.8
Oval SM 7.8 Singly, Short Chains
14.0 F. oxysporum
172 FWN27 White Flat Light Brown
- 9 17.6x2.55
Straight Pointed 3-5 6.6x 3.0
Oval SM 9.2 Singly, Short Chains
22.8 F. oxysporum
173 FWN28 White Flat Light Brown
- 9 17.0x2.6
Straight Pointed 3-5 5.4x2.6
Oval SM 9.6 Singly, Short Chains
13.2 F. oxysporum
174 FWN29 White Flat - - 9 16.4x2.55
Straight Pointed 3-5 5.5x 2.7
Oval SM 9.6 Short Chains 17.6 F. oxysporum
175 FWN30 White Flat - - 9 19.8x2.6
Straight Pointed 3-5 5.0x 2.3
Oval SM 7.6 Short Chains 10.2 F. oxysporum
176 FWN31 White Flat - - 9 13.2x2.5
Straight Pointed 3-5 6.8x 2.85
Oval SM 12.4 Singly, Short Chains
19.8 F. oxysporum
177 FWM1 White Flat Light Brown
- 9 18.4x2.7
Straight Pointed 3-5 6.4x2.5
Oval SM 13.0 Short Chains 15.2 F. oxysporum
178 FWM2 White Flat Light Brown
- 9 16.2x2.7
Straight Pointed 3-5 6.4x2.5
Oval SM 13.0 Singly, Short Chains
14.6 F. oxysporum
179 FWM3 White Flat Light Brown
- 9 19.8x2.7
Straight
Pointed 3-5 5.7x2.5
Oval SM 10.8 Singly, Short Chains
12.4 F. oxysporum
180 FWM4 White Flat Light Brown
- 9 18.8x2.6
Straight
Pointed 3-5 6.2x2.6
Oval SM 12.4 Singly, Short Chains
11.8 F. oxysporum
181 FWL1 White Fluffy Violet - 8 13.0x2.4
Slender Curved 3 4.2x 1.95
Oval-Comma
SM 9.8 Singly, Short Chains, Clusters
15.8 Fusarium sp.
182 FWL2 White Fluffy Violet - 9 14.3x2.7
Slender Curved 3 4.8x 2.3
Oval-Comma
SM 8.4 Singly, Short Chains, Clusters
14.1 Fusarium sp.
183 FWL3 White Fluffy Violet - 9 15.0x3.0
Slender Curved 3 5.6x 2.7
Oval-Comma
SM 10.8 Singly, Short Chains, Clusters
13.6 Fusarium sp.
184 FWL4 White Fluffy Violet - 10 10.5x2.1
Slender Curved 3 4.0x 1.75
Oval-Comma
SM 7.8 Singly, Short Chains, Clusters
7.0 Fusarium sp.
185 FWL5 White Fluffy Dark Violet
- 11 10.2x2.2
Straight Pointed 3 4.0x 1.75
Oval SM 9.2 Singly, Short Chains
11.9 F. oxysporum
186 FWL6 White Fluffy Dark Violet
- 8 8.0x2.5
Straight Pointed 3 5.0x 2.4
Oval SM 7.0 Singly, Pairs, Short Chains
10.2 F. oxysporum
187 FWL7 Pinkish Fluffy - - 8 16.2x Straight Pointed 3 5.8x Oval SM 10.6 Singly, Pairs, 9.6 F. oxysporum
Continued……
83
White 3.0 2.1 Short Chains 188 FWL8 Pinkish
White Fluffy - - 11 10.0x
2.0 Straight Pointed 3 4.0x
1.75 Oval SM 8.8 Singly, Pairs,
Short Chains 15.4 F. oxysporum
189 FWL9 PinkishWhite
Fluffy - - 11 10.0x2.25
Straight Pointed 3 4.0x 1.75
Oval SM 8.8 Singly 11.6 F. oxysporum
190 FWL10 White Fluffy - - 11 19.0x3.0
Straight Pointed 3 6.0x 2.0
Oval SM 7.8 Singly 10.6 F. oxysporum
191 FWL11 White Fluffy Pink - 9 10.0x3.0
Straight Pointed 3-4 5.05x 2.9
Obovoid LM, P
10.4 Singly, Pairs 11.9 F. commune
192 FWL12 White Fluffy - - 8 13.0x2.4
Straight Pointed 3-4 4.2x 1.95
Oval SM 9.0 Singly 15.6 F. oxysporum
193 FWL13 White Fluffy Violet - 8 13.4x3.0
Straight Pointed 3-4 5.4x 2.8
Oval SM 10.2 Singly 10.0 F. oxysporum
194 FWL14 White Fluffy Violet - 8 14.0x2.9
Straight Pointed 3-4 5.6x 2.9
Oval SM 10.6 Singly 15.0 F. oxysporum
195 FWL15 White Fluffy Violet - 8 16.8x3.0
Straight Pointed 3-4 5.4x 2.7
Oval SM 11.4 Singly 16.2 F. oxysporum
196 FWL16 White Fluffy Violet - 8 17.0x3.0
Straight Pointed 3-4 6.0x 2.8
Oval SM 9.6 Singly 16.1 F. oxysporum
197 FWB1 White Fluffy Pink - 8 15.2x2.8
Straight Pointed 3 5.1x 1.9
Obovoid LM, P
11.6 Singly 11.2 F. commune
198 FWB2 White Fluffy Pink - 8 12.0x2.5
Straight Pointed 3 5.4x 2.5
Obovoid LM, P
7.8 Singly 9.4 F. commune
199 FWB3 White Fluffy Violet - 8 14.2x2.1
Straight Pointed 3 5.3x 1.9
Obovoid LM, P
10.0 Singly, Pairs 21.0 F. commune
200 FWB4 White Fluffy Violet - 8 10.5x2.0
Straight Pointed 3 5.0x 2.0
Obovoid LM, P
10.2 Singly, Pairs 5.6 F. commune
201 FWB5 White Fluffy - - 8 14.1x2.5
Straight Pointed 3 6.0x 2.2
Obovoid LM, P
10.6 Singly, Pairs 8.0 F. commune
202 FWB6 White Fluffy Pink - 8 12.0x2.5
Straight Pointed 3 5.0x 2.5
Obovoid LM, P
7.4 Singly, Pairs 13.2 F. commune
203 FWB7 White Fluffy Pink - 8 14.0x2.6
Straight Pointed 3 5.0x 1.9
Obovoid LM, P
9.8 Singly, Pairs 15.9 F. commune
204 FWB8 White Fluffy - - 9 10.0x2.6
Straight Pointed 3 4.7x 2.5
Obovoid LM, P
7.8 Singly, Pairs 7.0 F. commune
205 FWB9 White Fluffy - - 8 18.8x2.7
Straight Pointed 3 6.3x 3.1
Obovoid LM, P
11.4 Singly, Pairs 7.0 F. commune
Continued……
84
206 FWB10 White Fluffy Dark Violet
- 10 19.0x3.0
Straight Pointed 3 6.6x 2.9
Oval SM 8.8 Singly 14.2 F. oxysporum
207 FWB11 White Fluffy Pink - 8 14.6x2.6
Straight Pointed 3 6.1x 3.3
Obovoid LM, P
11.4 Singly, Pairs 6.6 F. commune
208 FWB12 White Fluffy Pink - 8 10.0x2.5
Straight Pointed 3 5.0x 2.0
Obovoid LM, P
11.0 Singly, Pairs 6.6 F. commune
209 FWB13 White Fluffy Pink - 8 10.4x2.5
Straight Pointed 3 5.0x 2.0
Obovoid LM, P
10.8 Singly, Pairs 6.0 F. commune
210 FWB14 White Fluffy Pink - 8 14.4x2.6
Straight Pointed 3 5.4x 2.1
Obovoid LM, P
15.0 Singly, Pairs 11.6 F. commune
211 FWK1 White Fluffy Dark Violet
- 8 18.8x2.6
Straight Pointed 3 5.8x 2.5
Oval SM 12.0 Singly, Pairs, Short Chains
16.2 F. oxysporum
212 FWK2 White Fluffy Dark Violet
- 8 19.7x2.55
Straight Pointed 3 6.0x 2.5
Oval SM 13.8 Singly, Pairs, Short Chains
18.6 F. oxysporum
213 FWK3 White Fluffy Dark Violet
- 8 15.8x2.6
Straight Pointed 3 6.4x 2.4
Oval SM 14.0 Singly, Pairs, Short Chains
12.6 F. oxysporum
85
4.4.1 Colony Color
The aerial mycelium above the surface of media appeared in different
colors. This variation in colony color helped in differentiating the isolates. The
colony color varied from white (179 isolates, 84.04%) to creamy white (20, 9.39%)
and pinkish white (14, 6.57%) among the isolates (Figure 4.7a, b and c). The
isolates viz. FWJ2, FWJ3, FWJ5, FWJ11, FWJ12, FWJ14-16, FWG4, FWG5,
FWG9 and FWL7-9 were found pinkish white in colony color. The isolates viz.
FWC1-4, FWC7, FWC17-20, FWJ20-27, FWJ47, FWJ50 and FWJ53 were creamy
white whereas rest of all the Fusarium isolates were white in their colony color
(Table 4.2). The observation on this parameter helped in identifying the cultures as
isolates of Fusarium. As, colony color is considered a secondary character in the
identification of a fungal species, therefore, it was difficult to identify the species
based on this character.
4.4.2 Growth Habit
Fusarium species are known to produce mycelia above the media surface
that have distinguished growth habit. The aerial mycelium of isolates under study
also exhibited different growth habits (Figure 4.8a, b and c). The isolates showed
distinct fluffy (Figure 4.8a), compact (Figure 4.8b) and flat (Figure 4.8c) mycelial
growth patterns. About fifteen isolates (7.04%) viz. FWC3, FWC7, FWC18-20,
FWJ20, FWJ22, FWJ24-26, FWJ48 and FWJ50-53 showed compact growth habit,
sixty three isolates (29.58%) including FWC9, FWC12-14, FWC23-31, FWA1-5,
FWJ17-19, FWJ28-34, FWN1-31 and FWM1-4 showed flat growth habit, while
remaining 135 isolates (63.38%) isolates exhibited fluffy growth pattern (Table
4.2).
Figure 4.7: Isolates showing distinct White (a), Creamy white (b) white (c) colony color.
86
a
b
c
Figure 4.7: Isolates showing distinct White (a), Creamy white (b),
white (c) colony color. , and Pinkish
Figure 4.8: Petriplates showing variation in growth patterns of (a) Fluffy, (b) Compact, and (c) Flat.
87
a
b
c
Figure 4.8: Petriplates showing variation in growth patterns of Fusarium(a) Fluffy, (b) Compact, and (c) Flat.
Fusarium isolates:
88
4.4.3 Pigmentation
Pigmentation is a secondary character in the identification of Fusarium
species. The isolates were checked for any pigmentation on MEA medium plates
after 15-20 days of incubation at 25oC. Most of the isolates (119 isolates; 55.87%)
were found without significant pigmentation (Figure 4.9a), while others showed
varied pigmentation (94; 44.13%) (Table 4.2). The presence of distinct dark violet
(Figure 4.9b), violet (Figure 4.9c), pale brown (Figure 4.9d), light brown (Figure
4.9e) and pink pigmentation (Figure 4.9f) was observed in isolates. Twenty five
isolates (11.74%) viz. FWC5, FWC6, FWC15, FWC16, FWJ3, FWJ4, FWJ10,
FWJ14, FWJ15, FWJ35, FWG1, FWG2, FWG4-6, FWG9-12, FWL5, FWL6,
FWB10 and FWK1-3 showed dark violet pigmentation. Thirty one isolates
(14.55%) viz. FWC11, FWJ6, FWJ8, FWJ9, FWJ36-39, FWJ46, FWJ55-58,
FWJ68, FWJ69, FWG3, FWG7, FWG8, FWS11-13, FWL1-4, FWL13-16, FWB3
and FWB4 showed violet-colored pigmentation. Nine isolates (4.23%) viz. FWC2,
FWC3, FWC17, FWJ21, FWJ23-25, FWJ47 and FWJ48 showed pale brown
pigmentation color, twenty isolates (8.92%) viz. FWC9, FWC23, FWC24, FWC29-
31, FWA4, FWN2, FWN3, FWN14-16, FWN20, FWN22, FWN27, FWN28 and
FWM1-4 showed light brown pigmentation and nine isolates (4.23%) i.e. FWL11,
FWB1, FWB2, FWB6, FWB7 and FWB11-14 showed distinct pink pigmentation.
The rest of the isolates were found without any visible pigmentation.
4.4.4 Days to Fill 9 cm Dish
The days required for each isolate to fill 9 cm petri plate on MEA medium
varied from 7-11 days (Table 4.2). Three isolates (1.40%) viz. FWG22, FWN13
and FWN15 were found to be fastest growing as compared to others and acquired
Petriplates showing presence and absence of pigmentation in Fusarium pigmentation, (b) Isolate FWC5 with dark violet pigmentation, (c) Isolate FWB3brown pigmentation, (e) Isolate FWM1 with Light brown pigmentation, and (f) Isolate FWB11 with Pink
a b
c d
e Figure 4.9:
89
Petriplates showing presence and absence of pigmentation in isolates: (a) Isolate FWC1 without any colored
pigmentation, (b) Isolate FWC5 with dark violet pigmentation, (c) Isolate FWB3 with violet pigmentation, (d) Isolate FWC3 with Pale brown pigmentation, (e) Isolate FWM1 with Light brown pigmentation, and (f) Isolate FWB11 with Pink pigmentation.
a b
c d
e f
Petriplates showing presence and absence of pigmentation in WC1 without any colored
pigmentation, (b) Isolate FWC5 with dark violet pigmentation, (c) with violet pigmentation, (d) Isolate FWC3 with Pale
brown pigmentation, (e) Isolate FWM1 with Light brown pigmentation.
90
minimum 7 days to fill the plate. On the other hand, four isolates (1.88%) viz.
FWL5, FWL8-10 were found to be the slow growing filling the plates in 11 days
maximum period. Among the rest of the isolates, 85 (39.91%) had taken a
minimum of 8 days to fill the plates, 104 (48.83%) filled the plates in 9 days, while
17 (7.98%) in 10 days. The data on this parameter helped in identifying the fast and
slow growing species of Fusarium. Figure 4.10 shows 9 cm petri plates filled with
fungal colonies at different growth rates observed after 7 days of incubation at 25
°C.
4.4.5 Concentric Rings
The Fusarium species also have the characteristic of producing ring-like
appearance (Figure 4.11) in the aerial mycelium above the medium surface called
concentric rings. All the isolates were observed for the presence of this unique
character. Though, fifteen isolates (7.04%) viz. FWC4, FWC17, FWC18, FWC19,
FWC20, FWJ20, FWJ21, FWJ22, FWJ25, FWJ47, FWJ48 and FWJ50-FWJ53
(Table 4.2) showed the presence of distinguished concentric rings whose number
varied from 1-2 in 12 hours light/ darkness cycle as shown in Figure 4.11a and b.
This unique formation of annular zonations or concentric rings is a character of
species of F. equiseti and therefore, these isolates were designated to this species.
4.4.6 Conidiophore and Phialide
Hyaline, septate and branched conidiophores, the common character of
Fusarium species, were observed in all the isolates. The conidiogenous cells have
openings through which conidia are produced and the number of these openings
may vary per cell. So, these may be monophialides with a single opening or poly-
Isolates showing production of distinguished concentric hours light/ darkness cycle: (a) With 1 concentric ring, and (b) With 2 concentric rings.
Figure 4.10: Petriplates (9 cm) with mycelia at varied radial growth after 7 days of incubation at 25
Figure 4.11:
91
Isolates showing production of distinguished concentric hours light/ darkness cycle: (a) With 1 concentric ring, and (b) With 2 concentric rings.
tes (9 cm) with mycelia at varied radial growth after 7 days ofincubation at 25 °C.
a
b
Isolates showing production of distinguished concentric rings in 12 hours light/ darkness cycle: (a) With 1 concentric ring, and (b) With
tes (9 cm) with mycelia at varied radial growth after 7 days of
92
phialides with more than one. The length of the conidiogenous cells may be short
or long and it is important to observe this character, so as to distinguish between
the species. In the study, most of the isolates (196; 92.01%) exhibited
monophialides, which were short in structure and plump in the aerial mycelium
(Figure 4.12a and b). It is a distinguishing feature that differentiates F. oxysporum
from F. solani exhibiting long and slender monophialides in the aerial mycelium.
This unique indication of short monophialides helped in the identification of F.
oxysporum isolates. Also, this character is present in F. equiseti, which was also
observed in this study. In the rest of the seventeen isolates (7.98%) viz. FWS11-13,
FWL11, FWB1-9 and FWB11-14, long monophialides along with polyphialides
were also seen (Figure 4.12c and d, Table 4.2). The presence of long
monophialides and polyphialides has been recorded in species of F. commune and
therefore, these isolates were referred to this species.
4.4.7 Shape and Size of Micro-conidia
Almost all the isolates produced micro-conidia after 7 days of incubation at
25±2oC in pure cultures, which were found in abundance in the aerial mycelium in
false heads on phialides and also in chains (Figure 4.13). The production in false
head provided an important diagnostic character for distinguishing isolates of F.
oxysporum from other species.
The shape of entire micro-conidial cell is also an important character and
this parameter further helped in differentiating the isolates into species. In this
study, micro-conidia of oval shape (Figure 4.13a) were observed mostly, however,
conidia of globose, pyriform, obovoid (Figure 4.13b) with a truncate base and
Phialide characteristics observed in microscope at 100X magnification: (a, b) Short and plump monophialides, (c) Long monophialdes, (d) Polyphialides, and (aScale bar = 50 µm.
a
c Figure 4.12:
93
Phialide characteristics observed in Fusarium isolates under light microscope at 100X magnification: (a, b) Short and plump monophialides, (c) Long monophialdes, (d) Polyphialides, and (aScale bar = 50 µm.
b
d
isolates under light microscope at 100X magnification: (a, b) Short and plump monophialides, (c) Long monophialdes, (d) Polyphialides, and (a-d)
Micro-conidia of magnification: (a) Oval singl(b) Obovoid microconidium with a scale bar, (d)conidium with a scale bar, and (a
a b
c Figure 4.13: .
94
conidia of Fusarium isolates under light microscope at 100X magnification: (a) Oval single-celled and two-celled micro(b) Obovoid micro-conidia, (c) Measurement of length of a microconidium with a scale bar, (d) Measurement of width of a microconidium with a scale bar, and (a-d) Scale bar = 25 µm.
a b
c d
under light microscope at 100X celled micro-conidia,
conidia, (c) Measurement of length of a micro-Measurement of width of a micro-d) Scale bar = 25 µm.
95
comma-shaped were also witnessed (Table 4.2). The micro-conidia produced by 23
isolates (10.79%) of F. equiseti viz. FWC1-4, FWC7, FWC17-20, FWJ20-27,
FWC47, FWC48 and FWC50-53 were found pyriform. Obovoid conidia were
observed in 17 isolates (7.98%) viz. FWS11-13, FWL11, FWB1-9 and FWB11-14
identified as F. commune, globose in 3 isolates of F. oxysporum viz. FWJ8-10
(1.40%) and comma-shaped in 5 isolates viz. FWJ49 and FWL1-4 (2.34%) of
Fusarium species. F. oxysporum are known to produce oval-shaped micro-conidia
and most of the isolates (173; 81.22%) in the study falling under this species were
found to produce single-celled oval conidia, out of which, 5 isolates (2.34%) viz.
FWC5, FWC11 and FWJ1-3 also produced 2-celled oval conidia.
The size of micro-conidia (length and width) was measured randomly 5
times using ocular micrometer under a light microscope at 100x magnification
(Figure 4.13c and d). Great variations were also observed in micro-conidia size.
The overall average mean size of micro-conidia measured was 6.12±0.46 in length
and 2.52±0.20 in width and ranged from 3.5±1.22 to 9.6±0.89 µm in length and
1.75±0 to 3.6±1.52 µm in width, respectively (Table 4.2, 4.3, Appendix 2).
4.4.8 Shape and Size of Macro-conidia
In this study, septate and thin-walled macro-conidia borne in sporodochia
produced were observed after 15-20 days of incubation at 25±2oC in pure cultures
(Figure 4.14a). The macro-conidia of different shapes (Figure 4.14b-f), such as,
slightly curved (Figure 4.14b), straight (Figure 4.14c) and slender (Figure 4.14d, e
and f) were observed. The macro-conidia produced by twenty three isolates viz.
FWC1-4, FWC7, FWC17-20, FWJ20-27, FWJ47, FWJ48 and FWJ50-53 were
96
Table 4.3: Mean/ standard deviation (S.D.) in four morphological characteristics of Fusarium species.
No. Isolate ID No.
Micro-conidia Macro-conidia Chlamydospores Interseptal Distance (µm)
Length (µm)
Width (µm)
Length (µm)
Width (µm)
Diameter (µm)
Mean/ S.D. Mean/ S.D. Mean/ S.D. Mean/ S.D. Mean/ S.D. Mean/ S.D. 1 FWC1 8.2±1.48 2.1±0.22 19.2±3.03 2.5±0 12.4±3.05 19±5.66 2 FWC2 9.6±0.89 2.7±0.445 42±7.348 4.6±0.42 7.6±1.67 26.4±6.73 3 FWC3 6.5±2.24 3.6±1.52 28.6±9.52 3.8±0.45 9.8±2.39 14.2±5.97 4 FWC4 6.4±0.553 1.9±0.22 14.2±1.30 2.6±0.42 8.4±1.67 16.6±4.83 5 FWC5 4.5±0.71 2±0 13.8±2.68 2.4±0.22 8.6±0.89 11±4.47 6 FWC6 5.8±0.84 2.2±0.45 17.8±2.28 3±0 8.6±0.89 23.6±5.59 7 FWC7 5.6±0.89 2±0 14.6±2.60 2.6±0.55 9.2±1.30 21.6±9.18 8 FWC8 6.9±2.07 2.8±0.27 19.8±2.68 3.8±1.30 7.4±1.67 16.6±6.88 9 FWC9 7.2±0.84 2.5±0.35 17.8±4.14 2.6±0.22 9.8±1.30 12.8±3.35 10 FWC10 8.8±1.30 2.9±0.22 22.4±5.17 3.3±0.45 7.8±1.09 10.2±3.49 11 FWC11 5.4±0.89 2.2±0.45 24.6±3.57 4±0.35 11.4±2.79 7.8±3.11 12 FWC12 8.4±1.14 3±0.612 15.6±5.94 2.7±0.84 15±5.09 16±3.39 13 FWC13 7.4±1.67 2.5±0.35 24.4±9.31 3±0.5 7.2±1.09 9.4±0.89 14 FWC14 7.8±1.48 2.4±0.42 25.6±10.45 3.6±0.65 7.2±1.64 11.6±2.97 15 FWC15 6.8±0.84 2.8±0.27 26.2±4.02 3.2±0.45 14±4 19.4±4.09 16 FWC16 7.8±1.48 2.3±0.27 25±8.77 3.7±0.83 7±1.41 18.4±7.30 17 FWC17 7.8±1.48 2.8±0.27 20.6±9.58 3±0.61 9.4±3.51 15.3±9.27 18 FWC18 7.6±1.52 2.4±0.42 21.8±12.13 3.5±0.5 8±1.22 13.6±10.31 19 FWC19 7.4±0.89 2.4±0.42 25.8±9.44 3.1±0.65 7.6±1.14 17.2±2.39 20 FWC20 7.2±0.84 2.5±0.35 25.2±7.56 3±0.71 6.8±0.84 12.4±2.19 21 FWC21 8.2±1.48 2.9±0.22 29.6±5.18 3.6±0.55 12±4.74 21.1±2.75 22 FWC22 6.6±0.89 2.5±0 25±7.681 3.2±0.67 12.6±3.37 17.2±5.17 23 FWC23 8.9±0.74 3.2±0.27 26.4±2.19 3.2±0.45 11.8±1.79 13.8±5.02 24 FWC24 8.2±1.30 3.4±0.65 27.8±2.59 3±0 12.2±1.09 21.2±8.07 25 FWC25 6.4±1.52 2.6±0.22 22.6±3.21 3±0 11.6±2.88 18.2±7.95 26 FWC26 7.5±2.35 3±0.79 24.6±3.85 3.6±0.55 11.4±1.14 22.2±5.40 27 FWC27 6.8±1.92 2.6±0.65 29±4.89 4±0 11.4±2.97 21.8±7.89 28 FWC28 7.8±1.48 3.5±0.35 23.6±5.18 3.3±0.67 13.2±4.66 11.6±4.72
Continued……
97
29 FWC29 6.2±1.30 2.6±0.42 21.8±6.09 3.3±0.67 12.4±4.28 16.2±7.49 30 FWC30 6.6±1.52 2.8±0.45 25.2±7.29 3.5±0.71 11±3.16 17.4±6.73 31 FWC31 6.6±1.82 2.75±0.56 28±4.06 3.8±0.45 10.2±2.86 13.8±4.15 32 FWA1 7.5±2.35 3.1±0.74 19.4±6.07 2.8±0.45 11.6±1.14 18.6±2.97 33 FWA2 7.5±0.87 3.3±0.57 28.8±4.55 3.8±0.45 9.2±1.92 17.4±4.34 34 FWA3 6.4±1.14 2.5±0 28.2±5.76 4.2±0.57 11.8±1.79 10±1.41 35 FWA4 6.6±1.12 2.5±0.35 19.8±4.92 3.1±0.22 11±5.19 17.8±3.49 36 FWA5 7.6±1.52 3±0.71 21.4±6.31 3.2±0.76 12±3.08 14.8±4.15 37 FWJ1 6.4±1.14 2.2±0.27 18.8±2.28 2.8±0.27 15±5.09 18.6±6.23 38 FWJ2 7.1±2.07 2±0 18±3.16 2.6±0.42 10.8±3.03 15.6±6.23 39 FWJ3 6.8±0.84 2.1±0.22 13.2±3.56 2.3±0.27 7.8±1.48 10.6±3.36 40 FWJ4 5.4±0.55 2.1±0.22 15.2±2.28 2.8±0.27 10.4±1.52 16±2.24 41 FWJ5 4.4±0.55 2.5±0 15±4.79 2.5±0.35 9±0.71 16.4±5.73 42 FWJ6 5±0.71 2.5±0 17.4±2.79 2.9±0.22 10.2±1.30 14.8±5.02 43 FWJ7 4.6±0.55 2.6±0.22 20±1.41 3±0 10.8±1.30 9.4±2.61 44 FWJ8 5.1±0.22 2.5±0.87 12.2±1.89 2.5±0 9.6±2.61 17.6±3.85 45 FWJ9 5.9±0.74 2.65±0.22 15.6±7.64 2.7±0.45 11.2±2.59 18.2±6.49 46 FWJ10 5.8±0.87 2.5±0.35 18±3.46 2.6±0.41 11.2±5.06 13.6±3.51 47 FWJ11 5.3±0.45 2.05±0.59 11±1.41 2.4±0.41 11±1 11±6.48 48 FWJ12 6.6±0.89 2.75±0.25 14.6±4.34 2.2±0.27 14±3.16 19.6±3.29 49 FWJ13 6.6±0.89 2.3±0.27 17.4±7.33 2.5±0.35 10.4±1.67 17.2±9.65 50 FWJ14 7.2±0.84 2±0 19.6±5.13 2.3±0.27 11.4±1.67 14.3±4.12 51 FWJ15 6.6±1.34 2.5±0 10.8±2.28 2±0 12±4.74 17.6±8.08 52 FWJ16 7±0.71 2.1±0.22 19.4±7.13 2.4±0.42 9.4±1.34 18±10.07 53 FWJ17 6.2±1.09 2.2±0.27 15.8±3.49 2.3±0.27 17.8±4.15 24±8.12 54 FWJ18 6.6±0.89 2.65±0.22 17±7.14 2.3±0.27 9.6±1.67 10.2±1.79 55 FWJ19 7.2±1.09 2.2±0.27 16.6±3.71 2.3±0.27 11.8±1.48 21.6±13.94 56 FWJ20 6.4±1.14 2.3±0.27 32±13.71 3.8±0.91 7.4±1.34 15.8±7.16 57 FWJ21 6±1.36 2.25±0.35 28.2±2.49 3±0 12.8±3.3 13±1.41 58 FWJ22 6.4±1.14 2.7±0.27 30.8±11.29 3.6±1.08 7.8±1.64 14.6±4.77 59 FWJ23 6.8±0.84 2.85±0.33 27.6±3.58 2.8±0.27 9.2±1.48 17.6±5.86 60 FWJ24 5.9±0.74 2.65±0.22 20±6.36 2.7±0.27 7.4±1.52 15±9.64 61 FWJ25 6.8±0.84 2.6±0.22 22.4±6.58 2.8±0.27 9±1 13.6±4.77 62 FWJ26 5.6±0.55 2.5±0 23.8±12.01 2.9±0.65 9.8±3.03 14.6±4.77
Continued……
98
63 FWJ27 6.8±0.84 2.75±0.25 21±9.57 2.9±0.65 8±0.71 12.2±4.71 64 FWJ28 8±1.22 3.1±0.22 19.8±10.21 2.8±0.45 10±2 14±4.47 65 FWJ29 8.4±1.14 3.15±0.34 22±3.74 2.9±0.22 11.8±3.77 16.8±7.29 66 FWJ30 6.2±0.45 2.65±0.2207 18.2±6.34 2.7±0.27 11±2.12 17±2.65 67 FWJ31 7.6±0.89 3±0 16.8±3.89 2.4±0.22 10.4±0.89 13.4±4.56 68 FWJ32 6.4±1.52 2.6±0.22 20.8±6.14 2.6±0.22 7.6±1.82 13±7.38 69 FWJ33 6.4±1.52 2.7±0.27 18±6.28 2.6±0.22 10±2.83 19.2±3.03 70 FWJ34 7.2±0.84 3±0 17±5 2.6±0.22 9.4±2.61 10.2±1.09 71 FWJ35 6.4±1.14 2.5±0.5 26±8.37 3±0.35 7.2±1.30 10.6±0.89 72 FWJ36 5.4±1.14 2.3±0.45 12.6±3.29 2.5±0 10.4±2.61 12.8±4.55 73 FWJ37 5.2±0.84 2.2±0.45 17.6±3.21 2.6±0.22 11.2±3.83 12.6±3.78 74 FWJ38 4.6±1.34 2.3±0.45 18.3±1.79 2.5±0 12.4±3.85 12.2±4.71 75 FWJ39 5.8±0.84 2.1±0.22 20.4±4.84 2.5±0 11±2.65 14.6±4.77 76 FWJ40 5.4±0.55 2.1±0.22 21.8±6.09 2.7±0.45 14.4±4.39 15.4±5.13 77 FWJ41 5.8±0.45 2.4±0.42 16.8±5.93 2.5±0.18 13.8±4.15 19.6±7.92 78 FWJ42 5.2±0.84 2.6±0.55 19.1±4.85 2.5±0 13±3.61 17.2±6.22 79 FWJ43 5±0.70 2±0 20.8±3.03 2.55±0.11 10.4±1.52 21±4.85 80 FWJ44 6.2±1.09 2.2±0.27 19.2±3.96 2.5±0 14.8±5.76 18.6±8.05 81 FWJ45 5.4±1.52 2.5±0.5 22.1±6.47 3±0.35 9.8±2.49 19.2±5.26 82 FWJ46 6.4±1.14 2.7±0.45 16.4±4.34 2.6±0.42 10.4±2.70 13.4±3.13 83 FWJ47 8.4±1.47 2.6±0.22 16.9±2.25 2.5±0 9.2±2.17 18.2±9.91 84 FWJ48 7.4±1.14 2.8±0.45 18.6±5.94 2.75±0.25 12.2±4.82 22.4±5.73 85 FWJ49 8.5±1.41 2.8±0.27 13.2±2.48 3.1±0.74 9.6±1.14 15.8±7.36 86 FWJ50 7.7±1.09 2.8±0.27 22±3.16 2.9±0.22 12.4±3.51 18.2±2.86 87 FWJ51 6.6±0.89 2.6±0.22 21.4±2.79 2.6±0.22 12±5.29 18±8.03 88 FWJ52 7.2±1.92 2.85±0.22 23.2±3.89 2.9±0.22 7±1.41 21.2±5.81 89 FWJ53 8.6±1.34 3±0 21.4±4.88 2.8±0.27 10±1.41 16.6±9.94 90 FWJ54 7.4±1.67 3.2±0.45 16.1±3.05 3.6±0.55 13±6.32 16.4±4.39 91 FWJ55 6.6±0.89 2.3±0.45 17.6±2.70 3±0.35 8.2±2.05 19.2±10.06 92 FWJ56 6.2±1.30 2.6±0.42 18.9±3.94 2.8±0.22 12.8±6.72 20.2±9.86 93 FWJ57 7.2±1.92 2.7±0.57 17.2±4.87 2.9±0.22 11.2±1.79 14.6±8.05 94 FWJ58 7.8±1.79 2.9±0.65 18±1.41 2.9±0.22 7.8±3.70 27.8±8.14 95 FWJ59 7±2.12 2.6±0.55 17.6±4.93 2.7±0.27 14.2±3.77 18±8.37 96 FWJ60 5.6±0.89 2.4±0.42 18.9±2.61 2.7±0.27 10±2.45 19.4±6.99
Continued……
99
97 FWJ61 6.6±2.30 2.4±0.65 20.8±2.68 2.8±0.27 9.4±2.61 14.2±6.02 98 FWJ62 6.7±1.48 2.6±0.55 15.2±3.42 2.6±0.22 8.2±3.11 20.2±9.58 99 FWJ63 6.4±1.95 2.4±0.55 17.7±5.78 2.7±0.27 14±4 15±7 100 FWJ64 5.7±2.22 2.2±0.27 16.5±1.41 2.5±0 9.8±2.49 16.2±4.60 101 FWJ65 5.4±1.82 2.3±0.45 18.7±4.99 2.8±0.27 13.8±4.38 15.4±7.60 102 FWJ66 6.8±1.09 2.3±0.45 20.4±6.84 2.85±0.42 10±2.55 18.2±2.86 103 FWJ67 6.8±1.30 2.6±0.42 19.4±6.19 2.75±0.43 13.6±3.78 17±4 104 FWJ68 6.4±1.52 2.5±0.35 22.4±6.23 2.9±0.41 11.2±2.39 14.4±6.22 105 FWJ69 6.4±1.52 2.3±0.45 19±7.48 2.7±0.45 8.6±2.19 17.6±4.56 106 FWJ70 6.2±1.30 2.5±0.5 16.9±3.65 2.5±0 10.8±2.95 22.6±4.34 107 FWG1 7.4±2.30 2.8±0.45 16.2±3.03 2.8±0.27 14.6±5.73 31±10.29 108 FWG2 5.8±0.84 2.4±0.42 16.4±2.61 2.7±0.27 12.6±3.13 29.8±13.68 109 FWG3 6±1.87 2.8±0.45 20.3±2.77 3.1±0.22 13.6±4.34 15.2±4.09 110 FWG4 6±1.22 2.6±0.42 17±2.83 2.7±0.27 8.6±0.89 11.8±2.49 111 FWG5 6±1.22 2.9±0.22 20±6.32 2.9±0.42 12.2±1.79 20.8±2.28 112 FWG6 6.2±1.30 2.5±0.5 16±2.55 2.5±0 15.6±3.85 21.2±5.17 113 FWG7 5.8±0.84 2.5±0.35 17.8±5.59 2.65±0.22 7.6±1.52 20.6±7.99 114 FWG8 6±1.22 2.4±0.42 23±5.39 2.75±0.25 9.8±2.28 16.6±4.67 115 FWG9 5.8±1.30 3±0 14.4±2.61 2.5±0 13.2±6.09 16.2±6.06 116 FWG10 6.2±1.30 2.8±0.27 20.4±5.18 3.1±0.55 10.4±4.56 24±8.69 117 FWG11 6.8±2.17 2.5±0.5 18.4±7.27 2.85±0.42 14±5.48 21.2±6.46 118 FWG12 6.2±1.09 2.7±0.45 20.6±5.98 2.9±0.42 9±1.73 24±7.38 119 FWG13 5.6±2.07 2.4±0.42 19.4±3.13 3.8±0.27 9.6±3.21 12.2±2.28 120 FWG14 5.8±1.48 2.2±0.27 18.6±2.41 3.4±0.42 11.2±2.78 16±5.05 121 FWG15 6±1.41 2.6±0.22 19.6±6.02 3.5±0.61 7±1.41 14.4±4.34 122 FWG16 5±1 2.4±0.22 20±4.47 3.4±0.42 11.8±6.02 12.2±3.63 123 FWG17 5.4±0.545 2.5±0 18.8±4.76 3.7±0.67 10.2±2.68 16±4.69 124 FWG18 5.6±1.52 2.3±0.27 19.2±3.35 3±0.35 11.6±2.70 15±7 125 FWG19 6.8±1.64 2.5±0.35 19±4.36 3.4±0.65 8.8±1.79 11.6±3.85 126 FWG20 5.6±1.14 2.5±0.36 19.08±5.55 3.3±0.57 15.2±5.02 13.6±4.33 127 FWG21 6.4±1.14 2.8±0.27 17.4±6.31 3.1±0.82 10±1.41 18.6±3.29 128 FWG22 6.2±1.30 2.55±0.37 20±6.32 3.2±0.57 11.2±1.79 13.4±5.64 129 FWG23 5.4±0.55 2.5±0 17.6±3.91 3±0.61 14.2±5.12 19.2±1.30 130 FWG24 5.6±1.14 2.7±0.27 19.4±4.88 3.2±0.57 9.4±0.89 18.8±4.60
Continued……
100
131 FWG25 6±1.22 2.6±0.42 17.6±3.58 2.8±0.45 8.8±3.56 14.6±6.07 132 FWG26 5.4±1.52 2.55±0.37 19.4±6.69 3.1±0.82 13±4.12 15±5.43 133 FWS1 8±1.58 2.9±0.22 20.6±5.08 2.9±0.22 9.4±1.14 12.2±4.38 134 FWS2 4.8±0.91 2.5±0 18.2±4.82 2.5±0 12±0.71 15±6.59 135 FWS3 5.2±0.45 2.5±0 19.2±8.44 2.7±0.27 8.4±1.67 12±4.64 136 FWS4 6.2±1.304 2.6±0.42 15.2±6.30 2.6±0.22 8.6±0.89 22±8 137 FWS5 5.3±0.672 2.5±0 18.7±1.20 2.5±0 14±5.70 21.2±5.40 138 FWS6 4.8±0.91 2.4±0.22 17.2±3.27 2.5±0 7.6±1.82 17.4±12.24 139 FWS7 6.4±0.55 2.7±0.27 18.8±5.22 2.6±0.22 9.8±3.19 13.6±2.61 140 FWS8 4.8±0.84 2.45±0.27 18.5±3.04 2.55±0.11 8.6±0.89 10.6±3.29 141 FWS9 6±1.73 2.6±0.22 21±3.46 2.6±0.22 9.2±1.30 17.2±4.87 142 FWS10 5.2±0.84 2.5±0 16±4.85 2.5±0 13±4.47 8.1±1.95 143 FWS11 6.4±2.30 2.6±0.42 20.1±5.32 2.7±0.27 7.6±1.52 14.4±5.37 144 FWS12 5.6±0.89 2.65±0.22 13.8±4.82 2.5±0 14.4±5.37 14.8±5.72 145 FWS13 5.8±1.30 2.6±0.22 16.4±4.34 2.6±0.22 9.4±1.67 15±6.63 146 FWN1 5.4±0.55 2.5±0 16±4.06 3.7±1.20 8±1.41 14.4±4.77 147 FWN2 5.6±0.89 2.1±0.22 19.4±2.97 2.8±0.27 8±1 12.6±5.18 148 FWN3 5.4±0.55 2.55±0.11 16.6±3.05 2.6±0.22 12.2±4.49 15.2±5.36 149 FWN4 5.2±0.84 2.2±0.27 15.3±6.18 2.8±0.27 8.4±2.19 9.6±6.23 150 FWN5 5.8±1.09 2±0 19±2.65 2.7±0.45 8.6±1.14 11.1±7.42 151 FWN6 4.8±0.84 2.2±0.27 15.8±6.87 2.6±0.22 8.2±2.86 11.6±5.18 152 FWN7 5.6±0.89 2.25±0.43 11.9±2.07 2.5±0 10.8±2.17 6.2±2.17 153 FWN8 5±0 2.75±0.25 15.2±0.76 2.3±0.27 9.4±0.89 9.8±3.96 154 FWN9 4.95±0.97 2.3±0.27 11.9±3.44 2.5±0 8.8±1.79 16±6.12 155 FWN10 4.1±1.24 1.8±0.65 13±5.42 3.5±2.24 8.6±1.14 14.8±3.27 156 FWN11 6.4±1.14 2.6±0.22 17.5±4.03 2.6±0.22 11.2±1.79 17±6.40 157 FWN12 5.6±1.52 2.4±0.42 13.8±6.34 2.6±0.22 10.8±1.30 9.2±3.03 158 FWN13 5.8±0.84 2.55±0.11 15.1±6.54 2.6±0.22 8±1.41 14.4±3.78 159 FWN14 5±0.71 2.3±0.27 12.1±1.43 2.5±0 11.8±6.26 10.6±3.58 160 FWN15 5.8±0.84 2.4±0.42 16.8±6.42 2.7±0.27 7.6±0.89 10.8±2.59 161 FWN16 3.5±1.22 2.5±0.71 18.8±7.56 2.75±0.43 11.4±0.89 22±6.20 162 FWN17 4.7±0.97 2.5±0.35 18.8±5.02 2.9±0.22 11.4±5.03 26.4±6.73 163 FWN18 5.6±1.52 2.8±0.27 19±7.21 2.6±0.22 8.8±2.28 18.2±10.49 164 FWN19 7.6±0.55 3±0 17.8±6.26 2.6±0.22 12.4±3.29 17.2±4.76
Continued……
101
165 FWN20 4.6±0.65 2.4±0.22 15.9±5.75 2.7±0.27 8.2±3.89 18.4±9.24 166 FWN21 4.6±0.89 2.35±0.34 13±1.73 2.5±0 9.6±0.55 14.6±10.85 167 FWN22 6.2±1.30 2.8±0.45 20.2±8.96 2.7±0.27 17±4.47 16.1±1.52 168 FWN23 5±1.22 2.5±0.5 17.1±4.22 2.7±0.27 13.6±3.29 14.6±8.32 169 FWN24 6.3±0.97 2.9±0.22 18.4±3.21 2.5±0 10.4±3.05 12.8±2.39 170 FWN25 4.8±0.84 2.6±0.55 18±6.44 2.7±0.45 9.4±1.34 17.6±4.34 171 FWN26 6±1.41 2.8±0.27 12.7±3.15 2.45±0.11 7.8±1.79 14±5.79 172 FWN27 6.6±1.14 3±0 17.6±6.95 2.55±0.27 9.2±2.17 22.8±8.32 173 FWN28 5.4±0.55 2.6±0.22 17±6.16 2.6±0.22 9.6±6.02 13.2±4.44 174 FWN29 5.5±1.12 2.6±0.42 16.4±2.97 2.55±0.11 9.6±0.89 17.6±5.55 175 FWN30 5±1 2.3±0.27 19.8±7.09 2.6±0.22 7.6±0.89 10.2±2.77 176 FWN31 6.8±0.84 2.85±0.22 13.2±3.63 2.5±0 12.4±4.77 19.8±9.86 177 FWM1 6.4±1.14 2.5±0.5 18.4±3.05 2.7±0.27 13±2 15.2±5.40 178 FWM2 6.4±1.95 2.5±0.5 16.2±5.59 2.7±0.27 13±2.34 14.6±6.07 179 FWM3 5.7±1.39 2.5±0.35 19.8±5.97 2.7±0.45 10.8±2.59 12.4±2.88 180 FWM4 6.2±1.09 2.6±0.22 18.8±4.82 2.6±0.22 12.4±4.34 11.8±2.86 181 FWL1 4.2±0.84 1.95±0.45 13±2.98 2.4±0.42 9.8±0.84 15.8±4.15 182 FWL2 4.8±0.84 2.3±0.27 14.3±4.74 2.7±0.45 8.4±1.14 14.1±3.51 183 FWL3 5.6±0.89 2.7±0.45 15±3.32 3±0 10.8±1.09 13.6±5.94 184 FWL4 4±0.94 1.75±0.68 10.5±2.74 2.1±0.22 7.8±1.30 7±2.83 185 FWL5 4±0.94 1.75±0.5 10.2±1.44 2.2±0.27 9.2±0.45 11.9±5.44 186 FWL6 5±0 2.4±0.22 8±1.17 2.5±0.35 7±1 10.2±6.87 187 FWL7 5.8±1.09 2.1±0.22 16.2±3.63 3±0 10.6±3.13 9.6±3.51 188 FWL8 4±0 1.75±0 10±1.06 2±0.35 8.8±0.84 15.4±9.53 189 FWL9 4±0.94 1.75±0.5 10±1.66 2.2±0.31 8.8±0.84 11.6±6.73 190 FWL10 6±1.87 2±0 19±5.39 3±0.35 7.8±1.48 10.6±6.99 191 FWL11 5.05±0.72 2.9±0.14 10±1.66 3±0.5 10.4±1.54 11.9±0.22 192 FWL12 4.2±0.84 1.95±0.45 13±2.98 2.4±0.42 9±0.71 15.6±8.56 193 FWL13 5.4±0.55 2.8±0.27 13.4±1.95 3±0 10.2±1.30 10±3.54 194 FWL14 5.6±0.89 2.9±0.22 14±1.87 2.9±0.22 10.6±1.67 15±5.19 195 FWL15 5.4±0.55 2.7±0.27 16.8±3.63 3±0 11.4±0.89 16.2±5.67 196 FWL16 6±1 2.8±0.27 17±2 3±0.35 9.6±0.89 16.1±6.43 197 FWB1 5.1±1.14 1.9±0.22 15.2±2.17 2.8±0.45 11.6±1.52 11.2±6.46 198 FWB2 5.4±0.89 2.5±0.35 12±2.09 2.5±0 7.8±1.48 9.4±3.97
Continued……
102
199 FWB3 5.3±2.05 1.9±0.42 14.2±3.19 2.1±0.22 10±1.22 21±4.47 200 FWB4 5±0.79 2±0.5 10.5±2.74 2±0 10.2±1.09 5.6±2.70 201 FWB5 6±1.22 2.2±0.27 14.1±1.75 2.5±0 10.6±0.89 8±1.41 202 FWB6 5±0.88 2.5±0 12±2.09 2.5±0 7.4±1.67 13.2±7.15 203 FWB7 5±1.06 1.9±0.22 14±1.87 2.6±0.42 9.8±1.30 15.9±9.17 204 FWB8 4.7±0.97 2.5±0.35 10±1.41 2.6±0.22 7.8±1.09 7±2.45 205 FWB9 6.3±1.30 3.1±0.22 18.8±8.79 2.7±0.27 11.4±0.89 7±2.83 206 FWB10 6.6±0.89 2.9±0.42 19±2.65 3±0 8.8±1.09 14.2±7.53 207 FWB11 6.1±0.89 3.3±0.45 14.6±5.18 2.6±0.22 11.4±1.67 6.6±1.82 208 FWB12 5±1.27 2±0.53 10±1.41 2.5±0 11±1 6.6±1.95 209 FWB13 5±0.35 2±0.77 10.4±1.98 2.5±0 10.8±1.30 6±2.45 210 FWB14 5.4±0.89 2.1±0.22 14.4±2.51 2.6±0.42 15±5.09 11.6±5.50 211 FWK1 5.8±1.30 2.5±0 18.8±4.55 2.6±0.22 12±5.48 16.2±6.09 212 FWK2 6±1 2.5±0.5 19.7±1.30 2.55±0.11 13.8±3.03 18.6±2.71 213 FWK3 6.4±0.89 2.4±0.42 15.8±3.63 2.6±0.22 14±5.15 12.6±3.97
Macro-conidia (a) and Macromicroscope at conidia of isolate FW,FWC10, (d, e, f) Slender macro= 25 µm.
a
b c
d e Figure 4.14:
103
conidia (a) and Macro-conidial shapes observed under light microscope at 100X magnification (b-f): (b) Slightly curved macroconidia of isolate FW, (c) Straight-shape macro-conidia of isolateFWC10, (d, e, f) Slender macro-conidia of isolate, and (a
b c
d e f
conidial shapes observed under light (b) Slightly curved macro-
conidia of isolate conidia of isolate, and (a-f) Scale bar
104
found to be slightly curved (8.92%), slender shaped (2.81%) in five isolates viz.
FWJ49 and FWL1-4, whereas, straight (86.85%) in rest of all the isolates (Table
4.2). The slightly curved shaped macro-conidia were observed in isolates of F.
equiseti, straight conidia were observed in both F. oxysporum and F. commune
isolates, whereas, slender conidia were noted in isolates identified as Fusarium
species.
The size of macro-conidia was also measured randomly 5 times using
ocular micrometer under a light microscope at 100X magnification. Significant
variability was also observed in macro-conidia size. The overall average mean size
of macro-conidia measured was 18.39±2.35 in length and 2.82±0.27 in width,
while ranged from 8±1.17 to 42±7.35 µm in average mean length and 2±0 to
4.6±0.42µm in average mean width, respectively (Table 4.2, 4.3, Appendix 3).
4.4.9 Shape of Apical and Basal Cells of Macro-conidia
The macro-conidial ends are very important in morphological identification
of Fusarium species as shown in Figure 4.15. In this study, the apical cells of
macro-conidia in five Fusarium species isolates (2.34%) viz. FWJ49 and FWL1-4
had a distinct bend and characterized with curved apical cells (Figure 4.15a).
Whereas, in twenty three isolates (10.79%) viz. FWC1-4, FWC7, FWC17-20,
FWJ20-27, FWJ47, FWJ48, FWJ50-53 characterized as F. equiseti, the apical cells
were elongated with no distinct bend (Table 4.2). However, in rest of the 185
isolates (86.85%) of F. oxysporum and F. commune, the apical cell was found to be
pointed and tapered. Similarly, in all of the isolates (100%), the cell at the base of
the macro-conidia had a distinct notch and therefore, was characterized as foot
shaped basal cell (Figure 4.15a).
Macro-conidia with curvedunder light microscope at 100X magnification (a), and Septation of macro-conidia (bseptate, and (a
a
b c d Figure 4.15:
105
conidia with curved-shaped apical and foot-shaped basal cells under light microscope at 100X magnification (a), and Septation of
conidia (b-d): (b) Three-septate, (c) Four-septate, (d) Fiveseptate, and (a-d) Scale bar = 25µm.
b c d
shaped basal cells under light microscope at 100X magnification (a), and Septation of
septate, (d) Five-
106
4.4.10 Septation in Macro-conidia
The macro-conidia have diagnostic septation, whose number varies in
different species. The number of septa was counted for each isolate per spore that
showed variation in number (Figure 4.15b, c and d). The macro-conidial septation
ranged from 3 to 5. Macro-conidia with 3 septa were produced by 117 isolates
(54.93%) (Figure 4.15b), while 5 septa were observed in 23 isolates (10.80%) that
showed the characters of F. equiseti (Figure 4.15d). Ten isolates (4.69%) produced
3 to 4 conidial septation range (Figure 4.15c), whereas, 63 isolates (29.58%)
produced septation ranged from 3 to 5 as shown in Figure 4.15d (Table 4.2).
4.4.11 Chlamydospores: Formation and Diameter
Chlamydospores are thick-walled swollen storage cells produced aerially or
in sporodochial macro-conidia (Figure 4.16). The presence and absence of
chlamydospores is also an important criterion for distinguishing Fusarium species.
In this study, the data for chlamydospores was recorded using old cultures after 25-
30 days of incubation, which showed the presence and formation of
chlamydospores singly (Figure 4.16a), in pairs (Figure 4.16b), short chains (Figure
4.16c) and clusters (Figure 4.16d). All the isolates produced chlamydospores,
where 198 isolates (92.96%) showed production of spores singly, 99 isolates
(46.48%) in pairs, 24 isolates (11.27%) in clusters and 132 isolates (61.97%) in
short chains. Table 4.2 shows the formation of chlamydospores either produced
singly, in pairs, short chains or clusters. The presence of spores singly, in short
chains and clusters further assisted in the identification of F. equiseti isolates.
Likewise, indication of spores as singly, in pairs and short chains supported the
identification of F oxysporum isolates. The chlamydospores were found to be
Formation of Chlamydospores: (a) Singly, (b) Pairs, (c) Short chains, (d) Clusters, (e) Rough= 25 µm.
a b
c d
e f Figure 4.16:
107
Formation of Chlamydospores: (a) Singly, (b) Pairs, (c) Short chains, (d) Clusters, (e) Rough-walled, (f) Smooth-walled, and (a
a b
c d
e f
Formation of Chlamydospores: (a) Singly, (b) Pairs, (c) Short chains, walled, and (a-d) Scale bar
108
rough (Figure 4.16e) or smooth-walled (Figure 4.16f) and produced either at
terminal (Figure 4.17a, b and c) or intercalary (Figure 4.17d and e).
The diameter of chlamydospores was recorded in old cultures after 25-30
days of incubation at 25±2oC and was measured randomly five times for each
isolate using ocular micrometer under light microscope at 100X magnification
(Figure 4.17f). A high degree of variation was observed in the diameter of
chlamydospores. The overall diameter of chlamydospores (average mean) recorded
was 10.50±1.50 µm and ranged from 6.8±0.84 to 17.8±4.15 µm. The isolate
FWC20 showed the shortest diameter, while the isolate FWJ17 showed the longest
diameter of the chlamydospores (Table 4.2, 4.3, Appendix 4).
4.4.12 Interseptal Distance
The hyphae and mycelia (Figure 4.18a and b) of all the isolates were
hyaline, septate and branched. The distance between two septa was observed and
measured. Significant variation was found in interseptal distance (Figure 4.18c)
that ranged from 5.6±2.70 to 31±10.29 µm (mean) with overall average mean
distance of 15.49±2.48 µm (Table 4.2, 4.3, Appendix 5).
The findings of this study obtained from morphological characterization
section was supported by the concept given by various scientists including Leslie
and Summerell (2006), Skovgaard et al. (2003), Nelson et al. (1983), Booth (1977)
and Toussoun and Nelson (1976). The morphological study revealed difference
among the isolates, which showed that the isolates belonged to different species
within the genus Fusarium. Based on morphological characterization and
identification, 213 isolates were tentatively characterized into three species based
Production of Chlamydospores: (a, b, c) Terminally,(f) Measurement o100X magnification under light microscope, and (aµm.
a b
c d
e
Figure 4.17: .
109
Production of Chlamydospores: (a, b, c) Terminally, (d, e) Intercalary, (f) Measurement of diameter of chlamydospore with a scale bar at 100X magnification under light microscope, and (a-f) Scale bar = 25
a b
c d
e f
(d, e) Intercalary, f diameter of chlamydospore with a scale bar at
f) Scale bar = 25
Septate, hyaline and branched hyphae and mycelia (a and b), measurement of Interseptal distance using a scale barmicroscope at 100X magnification (c), and Scale bar
Figure 4.18:
110
Septate, hyaline and branched hyphae and mycelia (a and b), measurement of Interseptal distance using a scale barmicroscope at 100X magnification (c), and Scale bar = 50 µm (a
a
b
c
Septate, hyaline and branched hyphae and mycelia (a and b), measurement of Interseptal distance using a scale bar under light
= 50 µm (a-c).
111
on their close morphology viz. F. oxysporum (168 isolates), F. commune (17
isolates), F. equiseti (23 isolates), while rest of the 5 isolates were unidentified at
species level and characterized as Fusarium species. Different microscopic
characters important for distinguishing Fusarium species were recorded and used
to identify and distinguish the isolates. The characteristics of conidiophores
including hyaline, profusely branched septate and branched hyphae observed in this
study have also been noted by Leslie and Summerell (2006), Saxena and Singh
(1987), Gupta et al. (1986), Nelson et al. (1983) and Khare (1980) who described
similar characteristics of conidiophores.
Leslie and Summerell (2006) and Nelson et al. (1983) proposed that F.
oxysporum is a distinct species with distinguished morphology within the genus
Fusarium, which was also found in the study. Among the important characters used
for the characterization and identification of F. oxysporum isolates, the unique
indication of distinct short and plump monophialides helped in the identification of
the isolates as F. oxysporum. It is a distinguishing feature that differentiates F.
oxysporum from F. solani with long and slender monophialides in the aerial
mycelium has been supported by concept given by Seifert (2001). Similarly, the
presence of long monophialides along with polyphialides in species of F. commune
reported by Skovgaard et al. (2003) was observed in seventeen isolates and
therefore, those were characterized as F. commune. The isolates characterized as F.
equiseti were found with short monophialides. In case of unidentified Fusarium
species (molecularly identified F. nygamai), the conidiogenous cells were
characterized as monophialides. However, in a study (Burgess and Trimboli, 1986),
polyphialiades have also been observed in few cultures of F. nygamai (Burgess and
112
Trimboli, 1986). However, later Burgess et al. (1989) proposed that polyphialides
should not be considered reliable character for identification of this species because
they are produced irregularly and are difficult to detect.
Macro-conidia are the most important character for the identification of
species of Fusarium which are differentiated mainly on the basis of the shapes of
the macro-conidia they produce (Nelson et al., 1983). In the present study, the
shapes recorded were straight, slightly curved and slender. The isolates with
straight macro-conidia were characterized as F. oxysporum and F. commune as
proposed by Leslie and Summerell (2006). The isolates with slightly curved-shaped
conidia were tentatively indicated as F. equiseti. Such curvature in macro-conidia
was reported by Leslie and Summerell (2006). The rest of the five isolates with
slender conidia were unidentified and named Fusarium species. Moreover,
considerable variability shown in macro-conidial size in the study has also been
observed by Mandhare et al. (2011) and Booth (1977).
The macro-conidial ends are considered very important in morphological
identification of Fusarium species and similar observations have been shown in
studies, such as, according to Nelson et al. (1983) and Toussoun and Nelson
(1976), the macro-conidia are comprised of apical cell with a diagnostic hook like
shape or notch depending on the species. As well, Leslie and Summerell (2006)
proposed that F. oxysporum has tapered and curved or occasionally a slightly
hooked apical cells, while the basal cells are foot-shaped to pointed. Such
observation of presence of pointed and tapered apical cells in isolates of F.
oxysporum was recorded in this study. Similarly, elongate apical cells with no
distinct bend identified in twenty three isolates and characterized as F. equiseti
have been reported by Leslie and Summerell (2006).
113
In addition, Leslie and Summerell (2006) reported that the number of
septation in macro-conidia is also an important criterion for identification because
the number of septation in macro-conidia varies depending upon the species. Based
on their report, the septation of macro-conidia were observed in this study to
distinguish among the species. Septate and thin-walled macro-conidia observed
have also been shown by Nelson et al. (1983). The diagnostic septation of macro-
conidia showed variation in number and ranged from 3 to 5 that helped in the
identification and characterization of the isolates into species. Similar results have
been documented by Leslie and Summerell (2006) and Nelson et al. (1983), such
as, isolates of F. oxysporum exhibited 3 to 5-septate macro-conidia, F. equiseti
isolates consisted of 5 septations in the conidia and F. commune showed 3
septations per spore.
Moreover, the concept that the presence of micro-conidia is an important
character for the identification of species because all Fusarium species do not
produce them has been demonstrated by Leslie and Summerell (2006). Following
this, presence of micro-conidia was observed and found that all the isolates
produced conidia. Similarly, the production of micro-conidia in false heads on
short monophialides and not in chains provided an important diagnostic character
for distinguishing F. oxysporum from other species has been confirmed by Nelson
et al. (1983) and Burgess et al. (1989). The size (length and width) of micro-
conidia measured showed a considerable variation among the isolates. In the same
way, the studies of Mandhare et al. (2011) and Booth (1977) also showed similar
variations in conidial size of F. oxysporum isolates from chickpea. The
morphological unidentified isolates of Fusarium species, which were later
114
identified molecularly as F. nygamai in this study, were found to exhibit the key
characters of F. nygamai species viz. formation of micro-conidia in short chains
and false-heads as reported by Burgess et al. (1989). Earlier, the production of
micro-conidia in short chains and false head was also proved a major
distinguishing character between F. nygamai and F. oxysporum by Burgess and
Trimboli (1986).
Chlamydospores produced aerially or in sporodochial macro-conidia were
observed to be present in different forms and their presence or absence is also an
important criterion for distinguishing Fusarium species. Likewise, these are
primarily produced singly or in pairs, but occasionally in short chains or clumps
(Nelson et al., 1983). Similar findings were observed in this study and
chlamydospores were seen as singly, in pairs, short chains as well as in clusters.
Most of the isolates regarded as F. oxysporum and F. commune species produced
spores singly and in pairs. Leslie and Summerell (2006) reported the same
chlamydospore characters of both species. Similarly, chlamydospores produced
singly, in short chains and clusters observed in the isolates identified as F. equiseti
has also been shown by Leslie and Summerell (2006) A high degree of variation
was observed in the diameter of chlamydospores (range 6.8-17.8 µm), which was
also recorded in isolates of F. oxysporum from chickpea in studies conducted by
Mandhare et al. (2011) and Booth (1977).
Other characters including pigmentation, growth habit and growth rate at
which the isolates fill the petri plates are generally considered as secondary
characters in the identification of Fusarium species and primarily employed for
115
identification and differentiating between slowly and rapidly growing species
(Summerell et al., 2003 and Leslie and Summerell, 2006). Based on these three
parameters (pigmentation, growth habit and rate), distinct variation was observed
among the isolates. The isolates produced distinct dark violet, violet, pale brown,
light brown and pink pigmentation, while most of the isolates were also observed
without any visible pigmentation (Table 4.2). The production of violet to dark
violet pigmentation observed in F. oxysporum isolates has also been reported by
Leslie and Summerell (2006). Therefore, those isolates that produced violet and
dark violet pigmentation were characterized as F. oxysporum. Likewise, presence
of pale brown pigmentation as illustrated by Leslie and Summerell (2006) helped
in the identification of F. equiseti isolates. Growth patterns of Fusarium isolates
varied from fluffy, flat or compact with white to pinkish colony color on media.
Similar results were observed by Kontoyiannis et al. (2000) who found that the
colony color of Fusarium may vary from white, cream, tan, salmon, cinnamon,
yellow, red, violet, pink or purple and on the other hand, it may be colorless, tan,
red, dark purple or brown. The data on growth rate clearly distinguished the slow
and fast growing isolates as shown in Table 4.2.
Significant variations were observed in different morphological characters
discussed above among the Fusarium isolates recovered from Punjab province.
Based on morphology, identified Fusarium isolates were tentatively characterized
into F. oxysporum, F. commune, F. equiseti and Fusarium sp. The variability in
morphology between the isolates might be as a result of genetic differences among
them, since all the isolates used in this study were cultured and maintained under
116
same laboratory conditions. Therefore, further molecular and pathogenic study was
important to distinguish and confirm the species of the Fusarium isolates.
4.5 PATHOGENICITY TEST
The identified and morphologically characterized 213 Fusarium isolates
were grouped on the basis of similar morphology and 67 type isolates were
subjected to pathogenicity test on available lentil germplasm viz. cultivar (cv.)
Masoor-93 and line NARC-08-1 under controlled screen house conditions for the
analysis of their virulence. Disease parameters including percent disease severity
index, disease incidence and yield reduction were recorded. The results of the
experiment showed high pathogenic variability among the isolates on the basis of
wilt disease severity index, incidence and yield (Table 4.4, Appendix 6, 7 and 8).
The isolates were characterized pathogenically on the basis of typical wilt
disease symptoms (Figure 4.19a, b, c, d and e). Characteristic wilt disease
symptoms such as drooping of lentil plants and yellowing of lower lentil plants
leaves were recorded 15-18 days after inoculation, while prominent discoloration
of wilted lentil roots was also noted internally. In the later stage, some plants dried
and died (Figure 4.19b). The plants in control treatment were found healthy
without any disease symptoms. The incubation period i.e. number of days between
inoculation and death of the seedlings was recorded varied from 20 (3rd week) to 25
(4th week) days in case of line NARC-08-1, while 30 (5th week) to 40 days (6th
week) in Masoor-93 after inoculation under artificial inoculation conditions in
screen house and proved to be pathogenic. Variable disease reactions among the
characterized isolates was observed based on disease parameters viz. disease
incidence measured by counting the number of wilted plants and severity index
using 0-9 disease rating scale.
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Table 4.4: Virulence of morphologically identified and characterized Fusarium species tested on lentil germplasm NARC-08-1 and Masoor-93.
No. Isolate ID
Species District of Origin
NARC-08-1 Masoor-93 Disease
Severity* (%)
Disease Incidence
(%)
Yield Reduction
(%)
DR Disease Severity*
(%)
Disease Incidence
(%)
Yield Reduction
(%)
DR
1 FWC1 F. equiseti Chakwal 0 m 0 b 11.86 t A 0 o 0 g 27.53 klmnop A 2 FWC2 F. equiseti Chakwal 0 m 0 b 17.48 st A 0 o 0 g 19.05 qrstuvw A 3 FWC4 F. equiseti Chakwal 0 m 0 b 30.37 op A 0 o 0 g 28.16 klmn A 4 FWC5 F. oxysporum Chakwal 53.3 gh 100 a 51.71 defghi M 3.7 mn 26.67 cd 26.81 lmnopq L 5 FWC6 F. oxysporum Chakwal 54.81fg 100 a 47.69 efghijklm M 11.11 l 100 a 26.31 lmnopqr L 6 FWC7 F. equiseti Chakwal 0 m 0 b 16.45 st A 0 o 0 g 19.21 qrstuvw A 7 FWC8 F. oxysporum Chakwal 55.55 efg 100 a 47.26 efghijklm M 0 o 0 g 6.47 xy A 8 FWC9 F. redolens Chakwal 22.22 k 100 a 17.98 rst L 0 o 0 g 18.62 rstuvw A 9 FWC10 F. oxysporum Chakwal 0 m 0 b 19.33 qrst A 0 o 0 g 21.62 nopqrstuvw A 10 FWC11 F. oxysporum Chakwal 60 d 100 a 42.62 jklmn M 1.48 no 13.33 ef 34.67 hijk L 11 FWC12 F. redolens Chakwal 64.44 c 100 a 43.27 jklmn M 55.55 de 100 a 42.52 cdefg M 12 FWC15 F. oxysporum Chakwal 88.14 b 100 a 100 a H 65.92 ab 100 a 47.12 abcde M 13 FWC17 F. equiseti Chakwal 52.59 gh 100 a 46.08 fghijklm M 22.22 j 13.33 ef 37.97 fghi L 14 FWC21 F. oxysporum Chakwal 45.18 i 100 a 37.66 no M 0 o 0 g 19.88 pqrstuvw A 15 FWC22 F. oxysporum Chakwal 54.07 fgh 100 a 49.90 defghij M 57.03 d 86.7 b 39.63 efgh M 16 FWC23 F. redolens Chakwal 57.03 def 100 a 48.20 efghijklm M 2.22 no 20 de 34.69 hijk L 17 FWA1 F. redolens Attock 55.55 efg 100 a 45.18 hijklm M 0 o 0 g 19.36 qrstuvw A 18 FWJ2 F. oxysporum Jhelum 60 d 100 a 48.63 efghijkl M 16.29 k 100 a 36.68 ghij L 19 FWJ4 F. oxysporum Jhelum 63.7 c 100 a 52.95 defg M 65.92 ab 100 a 37.97 fghi M 20 FWJ8 F. oxysporum Jhelum 64.44 c 100 a 49.12 defghijk M 45.92 gh 100 a 40.56 defgh M 21 FWJ11 F. oxysporum Jhelum 66.66c 100 a 45.21 hijklm M 0 o 0 g 22.86 mnopqrstuv A 22 FWJ14 F. oxysporum Jhelum 45.18 i 100 a 41.61 lmn M 48.14 fg 100 a 47.00 abcde M 23 FWJ15 F. oxysporum Jhelum 45.92 i 100 a 49.09 defghijkl M 0 o 0 g 23.64 lmnopqrstu A
Continued……
118
24 FWJ16 F. oxysporum Jhelum 58.51 de 100 a 49.67 defghij M 0 o 0 g 27.70 klmno A 25 FWJ17 F. redolens Jhelum 30.36 j 100 a 20.79 qrs L 0 o 0 g 21.23 nopqrstuvw A 26 FWJ18 F. redolens Jhelum 47.4 i 100 a 44.29 ijk lmn M 3.7 mn 20 de 25.75 lmnopqrs L 27 FWJ19 F. redolens Jhelum 54.07 fgh 100 a 45.75 ghijk lm M 11.85 l 26.67 cd 27.41 klmnop L 28 FWJ20 F. equiseti Jhelum 66.66 c 100 a 48.33 efghijkl M 48.89 f 100 a 49.62 abc M 29 FWJ26 F. equiseti Jhelum 45.92 i 100 a 40.83 mn M 12.59 l 20 de 28.22 klmn L 30 FWJ28 F. redolens Jhelum 55.55 efg 100 a 54.59 de M 0 o 0 g 20.59 nopqrstuvw A 31 FWJ35 F. oxysporum Jhelum 100 a 100 a 100 a H 49.63 f 100 a 46.51 abcde M 32 FWJ47 F. equiseti Jhelum 21.48 k 100 a 21.04 qrs L 0 o 0 g 23.43 lmnopqrstuv A 33 FWJ48 F. equiseti Jhelum 20.74 k 100 a 32.30 op L 5.92 m 20 de 28.32 klmn L 34 FWJ49 F. nygamai Jhelum 89.62 b 100 a 100 a H 54.07 e 100 a 44.05 bcdefg M 35 FWJ50 F. equiseti Jhelum 11.11 l 100 a 19.61 qrs L 0 o 0 g 16.58 tuvw A 36 FWJ53 F. equiseti Jhelum 22.96 k 100 a 25.14 pqr L 0 o 0 g 20.56 nopqrstuvw A 37 FWJ62 F. equiseti Jhelum 21.48 k 100 a 25.96 pq L 0 o 0 g 20.73 nopqrstuvw A 38 FWG1 F. oxysporum Gujrat 89.62 b 100 a 100 a H 57.77 cd 100 a 46.87 abcde M 39 FWG13 F. oxysporum Gujrat 46.66 i 100 a 49.07 defghijkl M 12.59 l 33.33 c 30.15 jklm L 40 FWS1 F. oxysporum Sialkot 44.44 i 100 a 48.23 efghijklm M 2.22 no 20 de 28.11 klmn L 41 FWS3 F. oxysporum Sialkot 66.66 c 100 a 66.32 b M 2.22 no 20 de 25.66 lmnopqrs L 42 FWS5 F. oxysporum Sialkot 53.33 gh 100 a 52.93 defg M 48.89 f 86.7 b 45.09 bcdef M 43 FWS7 F. oxysporum Sialkot 66.66 c 100 a 42.65 jklmn M 44.44 h 100 a 45.29 bcdef M 44 FWS9 F. oxysporum Sialkot 59.26 d 100 a 52.24 defgh M 0 o 0 g 17.13 tuvw A 45 FWS11 F. commune Sialkot 100 a 100 a 100 a H 65.92 ab 100 a 51.72 ab M 46 FWS13 F. commune Sialkot 100 a 100 a 100 a H 29.63 i 100 a 48.98 abc M 47 FWN2 F. redolens Narowal 100 a 100 a 100 a H 55.55 de 100 a 48.05 abcd M 48 FWN4 F. redolens Narowal 54.07 fgh 100 a 51.34 defghi M 2.22 no 26.67 cd 29.77 jklm L 49 FWN5 F. redolens Narowal 64.44 c 100 a 56.52 cd M 0 o 0 g 20.08 opqrstuvw A 50 FWN6 F. redolens Narowal 54.81fg 100 a 41.76 klmn M 0.74 o 6.67 fg 29.71 jklm L 51 FWM1 F. redolens Mianwali 22.96 k 100 a 18.86 qrst L 0 o 0 g 23.89 lmnopqrst A 52 FWL1 F. nygamai Layyah 66.66 c 100 a 52.48 defgh M 60 c 100 a 46.42 abcde M 53 FWL2 F. nygamai Layyah 90.37 b 100 a 100 a H 66.66 a 100 a 42.57 cdefg M 54 FWL4 F. nygamai Layyah 54.81 fg 100 a 51.35 defghi M 44.44 h 100 a 43.12 cdefg M
Continued……
119
55 FWL5 F. oxysporum Layyah 65.18 c 100 a 67.74 b M 0 o 0 g 16.47 tuvw A 56 FWL6 F. oxysporum Layyah 100 a 100 a 100 a H 44.44 h 100 a 43.12 cdefg M 57 FWL7 F. oxysporum Layyah 55.55 efg 100 a 47.46 efghijklm M 0 o 0 g 23.52 lmnopqrstu A 58 FWL9 F. oxysporum Layyah 100 a 100 a 100 a H 63.7 b 100 a 46.54 abcde M 59 FWL11 F. commune Layyah 44.44 i 100 a 48.64 efghijkl M 0 o 0 g 15.65 vw A 60 FWL12 F. oxysporum Layyah 100 a 100 a 100 a H 66.66 a 100 53.68 a M 61 FWB3 F. commune Bhakkar 46.66 i 100 a 49.78 defghij M 0 o 0 g 18.03 stuvw A 62 FWB4 F. commune Bhakkar 51.11h 100 a 52.87 defg M 0 o 0 g 13.86 wx A 63 FWB9 F. commune Bhakkar 46.66 i 100 a 63.49 bc M 0 o 0 g 15.93 uvw A 64 FWB10 F. oxysporum Bhakkar 100 a 100 a 100 a H 54.07 e 100 a 44.71 bcdef M 65 FWB11 F. commune Bhakkar 44.44 i 100 a 51.74 defghi M 55.55 de 100 a 45.31 bcdef M 66 FWK1 F. oxysporum Khushab 60 d 100 a 53.24 def M 1.48 no 20 de 30.69 ijkl L 67 FWK2 F. oxysporum Khushab 89.62 b 100 a 100 a H 44.44 h 100 a 46.77 abcde M 68 Control - 0 m 0 b 0 u A 0 o 0 g 0 y A LSD Value at α = 0.05 3.08 0 7.49 - 2.72 7.83 7.81 -
At α=0.05 level of significance means sharing same letters are non-significant. *Disease severity index percentage on the basis of 0-9 scale. Data based on mean of three replications. DR = Disease Reaction; H= Highly, M= Moderately, L= Low Virulent and A= Avirulent.
Pathogenicity testing of experiment showing characteristic wilt symptoms on lentil germplasm NARCincidence and severity on NARCwith wilted and dead plants, (c) Moderately viruvirulent reaction, and (e) Avirulent reaction with healthy plants.
a
b c
d Figure 4.19:
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Pathogenicity testing of Fusarium isolates: (a) Screen house pot experiment showing characteristic wilt symptoms on lentil germplasm NARC-08-1 and Masoor-93, (b-e) Variation in wilt incidence and severity on NARC-08-1: (b) Highly virulent reaction with wilted and dead plants, (c) Moderately virulent reaction, (d) Low virulent reaction, and (e) Avirulent reaction with healthy plants.
b c
e
es: (a) Screen house pot experiment showing characteristic wilt symptoms on lentil
e) Variation in wilt 1: (b) Highly virulent reaction
lent reaction, (d) Low virulent reaction, and (e) Avirulent reaction with healthy plants.
121
Considerable difference was observed in virulence of the isolates
particularly on line NARC-08-1, when compared with control, where no incidence
of wilt was observed (Table 4.4). On lentil line NARC-08-1, disease incidence and
severity index ranged from 0 to 100% with the reduction in yield observed after
harvest ranged from 11.86 to 100 %, whereas, the control was observed with 0%
wilt infection and yield reduction. This data showed that disease had great negative
effect on yield. On the other germplasm cv. Masoor-93, disease incidence and
severity index ranged from 0 to 100% and 0 to 66.66%, respectively, with the
reduction in harvested yield ranged from 6.47 to 53.68%. The control produced 0%
infection and yield reduction.
Overall, on the basis of 0-9 disease rating scale and wide disease reaction,
the isolates were characterized into the following 4 groups as shown in Table 4.4.
a) Highly virulent (7-9 scale range),
b) Moderately virulent (4-6),
c) Low virulent (1-3), and
d) Avirulent (0)
The disease rating showed significant variability among the Fusarium
isolates response on lentil germplasm tested with significant difference in percent
plant mortality. Avirulent to highly virulent reaction was recorded on NARC-08-1
and isolates were characterized using all 0 to 7-9 scale categories (Figure 4.19b, c,
d and e). While, cv. Masoor-93 produced avirulent to moderately virulent disease
reactions against the tested isolates and characterized using three scale categories
i.e. 0, 1-3 and 4-6.
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The highest wilt incidence and severity on line NARC-08-1 was caused by
13 isolates (19.40%) viz. FWC15, FWJ35, FWJ49, FWG1, FWS11, FWS13,
FWN2, FWL2, FWL6, FWL9, FWL12, FWB10 and FWK2. Concurrently, the
third parameter i.e. yield further strengthened their aggressiveness where the
reduction was 100% as all the seedlings died earlier (Table 4.4). This showed that
these isolates prevalent in our region are highly pathogenic and could be
responsible for low lentil yield. The forty one (61.19%) isolates including FWC5,
FWC6, FWC8, FWC11, FWC12, FWC17, FWC21, FWC22, FWC23, FWA1,
FWJ2, FWJ4, FWJ8, FWJ11, FWJ14, FWJ15, FWJ16, FWJ18, FWJ19, FWJ20,
FWJ26, FWJ28, FWG13, FWS1, FWS3, FWS5, FWS7, FWS9, FWN4, FWN5,
FWN6, FWL1, FWL4, FWL5, FWL7, FWL11, FWB3, FWB4, FWB9, FWB11
and FWK1 showed intermediate (moderately virulent) response and scored
between 4-6 on the rating scale. The reduction in yield recorded in plants infected
by these isolates ranged from 37.66 to 67.74%, which was significantly different
statistically compared with the control. This reduced yield showed that the wilted
plants produced grains which were not only inferior in quantity but also in quality
means the plants can produce some grain yield which could be shriveled.
Therefore, can be assumed that yield was highly affected by these isolates.
The eight low virulent isolates (11.94%) that scored 1-3 included FWC9,
FWJ17, FWJ47, FWJ48, FWJ50, FWJ53, FWJ62 and FWM1. These isolates
infected the plants at low level and therefore, allowed the plants to resist and had
minor negative effect on grain yield (17.98 to 32.30% reduction). However, few of
the plants produced only shriveled seeds similar to moderately infected plants. The
five isolates (7.46%) that showed no infection at all and characterized avirulent
123
included FWC1, FWC2, FWC4, FWC7 and FWC10. These showed 0% severity
and incidence but showed minute reduction in yield compared with the control i.e.
11.86 to 30.37%. This showed that yield is somewhat reduced in few uninfected
plants.
On the other hand, different pathogenicity response was obtained using
same isolates on cv. Masoor-93 as shown in Table 4.4. The isolates produced
avirulent to moderately virulent disease reactions. None of the isolates were found
to produce highly virulent response. Out of the total 67 isolates characterized
pathogenically, twenty four isolates (35.82%) were found moderately virulent on 4-
6 scale value and these included FWC12, FWC15, FWC22, FWJ4, FWJ8, FWJ14,
FWJ20, FWJ35, FWJ49, FWG1, FWS5, FWS7, FWS11, FWS13, FWN2, FWL1,
FWL2, FWL4, FWL6, FWL9, FWL12, FWB10, FWB11 and FWK2. These
isolates showed 29.63 to 66.66% severity index and 86.7 to 100% incidence. It was
observed that most of these isolates were found highly virulent on line NARC-08-1
with total loss of yield. The result of harvested yield confirmed that these
moderately virulent isolates significantly (p ≤ 0.05) reduced the yield of Masoor-93
i.e. 37.97 to 53.68% as compared to the control (0%).
Out of the rest, sixteen isolates (23.88%) gave low virulent reaction and
scored 1-3 on rating scale. These included FWC5, FWC6, FWC11, FWC17,
FWC23, FWJ2, FWJ18, FWJ19, FWJ26, FWJ48, FWG13, FWS1, FWS3, FWN4,
FWN6 and FWK1. These isolates showed 0.74 to 22.22% severity index and 6.67
to 100% incidence while yield reduction observed from plants infected with these
isolates ranged from 25.66 to 37.97% with the production of shrivelled seeds in
some of the infected plants. The rest of the twenty seven isolates (40.30%) viz.
124
FWC1, FWC2, FWC4, FWC7, FWC8, FWC9, FWC10, FWC21, FWA1, FWJ11,
FWJ15, FWJ16, FWJ17, FWJ28, FWJ47, FWJ50, FWJ53, FWJ62, FWS9, FWN5,
FWM1, FWL5, FWL7, FWL11, FWB3, FWB4 and FWB9 were found totally
avirulent on cultivar falling 0 on disease rating scale. Similar to NARC-08-1,
minute yield reduction was also observed that ranged from 6.47 to 28.16%
compared with the control. After scoring for wilt, the re-isolation of pathogenic
Fusarium isolates from wilted roots confirmed the disease caused by them.
The wilt pathogen causes huge damage to lentils under favorable hot and
dry conditions and therefore, produced effective disease symptoms in the
experiment when provided with controlled conditions (temperature and appropriate
humidity), conducive for disease development (Bayaa and Erskine, 1998). The
method followed for the inoculation of the isolates i.e. root dip method was found
very much effective and reproducible in producing clear wilt symptoms on the
plants. Similar results with this method were also achieved by Taheri et al. (2010).
Common wilt disease symptoms (Bowers and Locke, 2000) were used for the
pathogenic characterization of the Fusarium isolates. The isolates of F. oxysporum
belonging to a single race differed in their virulence on tested germplasm in this
study as observed by Belabid et al. (2004) and Belabid and Fortas (2002). These
isolates showed a wide variety of pathogenic variability as reported in a study
conducted by Naimuddin and Chaudhary (2009). Out of 31 tested F. oxysporum
isolates, 30 (96.77%) were found pathogenic. Close results were also achieved by
Taheri et al. (2010) who tested 33 isolates and found 27 (81.82%) pathogenic
isolates. Varied yield reduction was observed in the study due to lentil wilt on two
125
different lentil germplasm. This result was supported by the concept given by
Khare et al. (1979) who proposed that yield losses depend on the crop variety.
The results of the experiment showed significant difference among the
response of the isolates tested on two different germplasm. The variation recorded
towards the disease reaction of the two different germplasm suggested that in the
availability of same environmental conditions and amount of pathogen inoculum,
the genetic makeup of the plants also plays role in resistance reaction of the plants
towards the inoculated pathogens (Mohammadi et al., 2012). Employment of
modified 0-9 disease rating scale as described by Bayaa et al. (1995) helped in the
characterization of the isolates into categories viz. avirulent, low virulent,
moderately virulent and highly virulent. This helped in the identification of the
highly virulent isolates prevailing in the country.
The Fusarium isolates from lentil growing areas of Punjab districts have not
been previously studied in detail and this is the first ever morpho-molecular and
pathogenic characterization of Fusarium isolates prevalent in the lentil growing
areas of country. This experiment, therefore, revealed their pathogenicity at various
levels which was very much essential to identify the prevailing pathogenic species
of Fusarium responsible for yield losses in the country. The most highly virulent as
well as moderately and low virulent isolates identified during the study would be
helpful in managing the disease by screening the available lentil germplasm (lines,
cultivars or varieties) used in the country for cultivation. Also the information
could also be used by the plant breeders for the development of new resistant
sources against the specific pathogenic strains of the fungus.
126
4.6 MOLECULAR CHARACTERIZATION
Morphologically and pathogenically characterized 67 type isolates of
Fusarium were further subjected to molecular characterization by sequencing and
comparing translation elongation factor (TEF-1α) gene region for the determination
of genetic diversity among the isolates, species identification and phylogenetic
analysis.
The TEF PCR reaction employing sequence specific primers ef1 and ef2
amplified a single DNA fragment of size 700bp in each isolate (Figure 4.20a, b, c,
d and e). The gel electrophoresis produced clear single band of amplified product
in each of the isolate which was used for sequencing. Significant genetic diversity
was observed on the basis of sequenced data. This data when used in phylogenetic
analysis greatly helped in revealing the species identity and determining the
evolutionary relationship between the isolates and the representative species. The
results revealed that isolates from different district of origin had varying levels of
phylogenetic diversity.
The phylogenetic analysis of TEF gene region resulted in sufficient
resolution and resolved the isolates into different clades and differentiated into five
species including F. oxysporum, F. nygamai, F. redolens, F. commune and F.
equiseti. Figure 4.21 shows the maximum likelihood analysis based on TEF
primers among 67 Fusarium isolates along with the reference sequences. The
phylogenetic analysis using all the isolates also produced sufficient resolution and
isolates were clearly distinguished into separate clades. The analyzed TEF gene
region showed enough variation among the sequences of isolates thereby resulted
in clustering of the tree that helped in differentiating the isolates into distinct
a
b
c
127
PCR amplification products (700bp) of genomic DNA of 67 (aFusarium
d
Figure 4.20:
128
PCR amplification products (700bp) of genomic DNA of 67 (aFusarium isolates using TEF-1α primers.
e
PCR amplification products (700bp) of genomic DNA of 67 (a-e)
129
Continued……
130
Phylogenetic tree based on Maximum likelihood analysis generated from the translation elongation factor-1α gene sequences of 67 Fusarium isolates from lentil along with the sequences of Genbank accessions. F. beomiforme and F. concolor were used to root the tree. Maximum likelihood bootstrap values from 1000 maximum likelihood replications are indicated on the branches.
Figure 4.21:
131
species prevalent in different geographical locations and exhibited variable
virulence response. The tree was divided into six major clades belonging to F.
oxysporum, F. commune, F. nygamai, F. fujikuroi, F. redolens and F. equiseti and
rooted with sequences of F. concolor and F. beomiforme. Maximum number of
isolates was falling under the clade of F. oxysporum followed by F. redolens, F.
equiseti, F. commune and minimum under F. nygamai clade.
The earliest diverging lineage moderately supported with 68% maximum
likelihood bootstrap value (ML-BS) with the two basal branches forming the rooted
outgroups phylogeny comprised of F. equiseti clade. This clade consisted of
strongly to weekly supported internodes (99 to 31% ML-BS) and contained twelve
isolates recovered from districts Chakwal and Jhelum viz. FWC2, FWJ26, FWC7,
FWJ62, FWJ53, FWJ47, FWC4, FWJ48, FWJ50, FWC17, FWC1 and FWJ20
grouped with F. equiseti strains viz. NRRL32175, NRRL26417, NRRL20423,
NRRL20697, NRRL13402, NRRL43680, NRRL34037, NRRL45997 and 286-09.
These isolates showed avirulent to moderately virulent disease response in
pathogenicity testing.
The next clade to diverge was represented by species of F. redolens, which
was resolved with strong support (87% ML-BS) with other clades. The clade
showed strong support (99% ML-BS) of the isolates with the strain NRRL22901.
The internodes within the clade showed strong to moderate support (91 to 24% ML-
BS). Thirteen isolates belonging to five districts (Chakwal, Attock, Jhelum,
Narowal and Mianwali) viz. FWC9, FWA1, FWN4, FWJ18, FWN5, FWJ28,
FWN6, FWM1, FWN2, FWC12, FWC23, FWJ17 and FWJ19 were differentiated
as species of F. redolens under this clade with the strains NRRL54967, FF-00411,
132
NRRL25123, NRRL52814 and NRRL52619. The isolates produced wide wilt
infection range i.e. avirulent to highly virulent.
The lineage next to this clade was F. fujikuroi complex in which none of the
tested isolates were falling. This clade was resolved as sister group with the next F.
nygamai clade with strong branch support (77% ML-BS). The F. nygamai clade
was weekly supported (38% ML-BS) with NRRL22045. However, 98% strong
ML-BS nodal support was observed within the clade. The isolates from district
Layyah viz. FWL4, FWL2 and FWJ49 were falling with two F. nygamai strains
NRRL52708 and NRRL13488 with moderate internodal support (BS=66%). The
isolate FWL1 grouped separately with F. nygamai strain M7491 within the clade
with strong 84% ML-BS support which means that it is more closely similar to this
strain. The F. nygamai isolates produced higher disease reactions and fell under
moderate and highly virulent categories.
The next two lineages i.e. F. commune and F. oxysporum diverged with
strong support (96% ML-BS) from F. nygamai and F. fujikuroi complex clades.
The phylogenetic analysis showed a sister group relationship of F. commune with
F. oxysporum showing 79% strong ML-BS value. This result is consistent with the
presence of high similarity in morphology of these two taxa. Little variation in
TEF-1α sequences was observed within isolates of F. commune isolated from same
as well as different geographical locations. This was seen in the resulted F.
commune clade divided into two sub-clades showing strong BS support (99%) with
each other. The first sub-clade consisted of three isolates viz. FWL11, FWB11 and
FWB4 isolated from Layyah and Bhakkar that showed 60% moderate ML-BS
value with F. commune strain NRRL28387. The second sub-clade of four isolates
133
viz. FWS11, FWB3, FWB9 and FWS13 from Bhakkar and Sialkot showed week
relationship (48% ML-BS) with two F. commune strains NRRL52764 and
NRRL52744. However, isolate FWS11 and strain NRRL52764 found to possess
more similarity with moderate support (64% ML-BS). The isolates under this clade
showed moderate to highly virulent disease reactions.
The major clade of F. oxysporum species included thirty one tested isolates
formed a strongly supported monophyletic group (ML-BS=99%) with the F.
oxysporum type strains and separated under two sub-clades. The first large sub-
clade was not strongly supported and received only moderate measures of support
with 69% ML-BS. Within this sub-clade, strongly to weekly supported nodes (ML-
BS=78 to 27%) were observed and included twenty nine isolates viz. FWS5,
FWG13, FWJ16, FWL9, FWL12, FWJ15, FWG1, FWJ14, FWJ35, FWJ8, FWJ11,
FWK2, FWK1, FWS7, FWL5, FWL7, FWS1, FWS3, FWS9, FWC15, FWC21,
FWB10, FWC22, FWC6, FWC5, FWC11, FWC10, FWC8 and FWL6. Among
these, the isolates FWS5, FWG13, FWJ16, FWL9, FWL12, FWJ15, FWG1,
FWJ14, FWJ35, FWJ8 and FWJ11 showed weekly (34% ML-BS) support with F.
oxysporum strains NRRL32158, FO-02911 and NRRL26871.
The only isolate FWK2 was resolved with moderate relationship with strain
JG22-5 (66% ML-BS) and also showed same value support with eleven isolates
(FWS5, FWG13, FWJ16, FWL9, FWL12, FWJ15, FWG1, FWJ14, FWJ35, FWJ8
and FWJ11). The isolates including FWK1, FWS7, FWL5, FWL7, FWS1, FWS3
and FWS9 grouped with the F. oxysporum strains 10-110, NRRL52736 and
NRRL25387 with 52% ML-BS value. Two isolates viz. FWC15 and FWC21
showed strong 84% ML-BS support branch withother, while 78% strong support
134
with NRRL20433. NRRL38271 was also observed with 78% ML-BS support with
the above isolates. The isolates FWB10, FWC22, FWC6, FWC5, FWC11, FWC10,
FWC8 and FWL6 showed week to moderate BS value supports (56 to 78%) with
the F. oxysporum strains NRRL32153, NRRL34936, NRRL52785, NRRL52787,
NRRL53121, NRRL43668, NRRL32156 and NRRL32154. The second small sub-
clade under F. oxysporum lineage included two isolates (FWJ2 and FWJ4). Both
the isolates showed strong (89% ML-BS) support with MUCL14162, while most
strong branch support (95% ML-BS) with NRRL25603. The isolates falling under
the clade of F. oxysporum showed avirulent to highly virulent wilt disease
reactions.
The maximum likelihood analysis based on TEF primers among the
identified thirteen most highly virulent isolates along with the reference sequences
is presented in Figure 4.22. The phylogenetic analysis resolved the isolates into
five different clades including F. oxysporum, F. nygamai, F. redolens, F. commune
and F. fujikuroi. This showed that TEF-1α region has sufficient sequence variation
which discriminated between the species prevalent in different geographical
locations. The most basal strongly supported branches represented the F.
beomiforme and F. concolor as the outgroups of the tree. The first lineage diverged
from the outgroup phylogeny comprised of F. redolens clade. Only one isolate
belonging to district Narowal was falling in the clade i.e. FWN2 that showed strong
100% monophyletic ML-BS with F. redolens strain NRRL22901. However, there
was weak support for the branches connecting with the other clades (ML-BS=66%,
66%, 18%). The next diverged nygamai clade included two isolates from Jhelum
and Layyah i.e. FWJ49 showed strong ML-BS support of 100% with F. nygamai
strain NRRL13488, while FWL2 showed 82% strong support with F. nygamai
135
Phylogenetic tree based on Maximum likelihood analysis generated from the translation elongation factor-1α gene sequences of 13 most highly virulent Fusarium isolates from lentil along with the sequences of Genbank accessions. F. beomiforme and F. concolor were used to root the tree. Maximum likelihood bootstrap values from 1000 maximum likelihood replications are indicated on the branches.
Figure 4.22:
136
strain NRRL13488. Both of these also showed moderate branch support (76% ML-
BS) with NRRL25486. None of the sequenced isolates fall under the F. fujikuroi
complex clade. F. commune and F. oxysporum were resolved as sister group clades
with the most identical morphological characters. Two isolates recovered from
Sialkot viz. FWS11 and FWS13 were differentiated as species of F. commune, thus
falling into a separate clade with the F. commune strain NRRL28387. This clade
showed monophyly and strongly supported with 95% ML-BS value. This showed
that both isolates can be distinguished as F. commune.
Similarly, the major clade of F. oxysporum species was strongly supported
with 82% ML-BS support and included eight tested isolates that grouped with the
F. oxysporum type strains. These isolates characterized as F. oxysporum exhibited
little variation in their sequences and belonged to different geographical locations
i.e. district Chakwal, Layyah, Khushab, Gujrat, Jhelum and Bhakkar. The clade
included two sub-clades; one sub-clade with two isolates FWL6 and FWB10. The
isolate FWL6 showed strong 99% ML-BS value with the F. oxysporum strain
MUCL14162, while FWB10 with moderate 59% value with these two. This means
that FWL6 is more similar to the type strain. In the second sub-clade, six isolates
with two type strains were falling. FWC15 was showing strongly supported branch
relationship (ML-BS=83%) with NRRL20433. Isolates FWL12, FWK2, FWL9,
FWG1 and FWJ35 were showing 72% moderate support with NRRL25387.
The phylogenetic analysis conducted on the basis of partial TEF-1α
sequences helped in differentiating the isolates at species level. The generated trees
displayed well-suported clades encompassing five species of Fusarium. The results
137
suggested and confirmed that the isolates from different localities with varied
morphological characters belonged to different species.
Fusarium is a highly diverse and complex group of species among fungi.
Use of morphological characteristics to identify and distinguish fungal species
especially within Fusarium species complex is considered very much difficult
(Summerell et al., 2003 and Leslie and Summerell, 2006). Therefore,
identifications based on DNA sequences are regarded essential to identify various
species within the Fusarium species complex. Accordingly, species differentiation
and phylogenetic relationships was performed using part of the TEF gene region on
a collection of isolates of Fusarium species recovered from lentils, sampled from
various locations of major lentil producing region of Punjab, Pakistan. Reference
sequences of closely related species were also included in the analysis. TEF-1α
gene region was sequenced to confirm the morphological identifications of the
study and also to identify the recovered unknown Fusarium isolates.
The TEF-1α gene region shows a high level of sequence polymorphism
among closely related species. Therefore, it is considered the marker of choice as a
single-locus identification tool in Fusarium (Geiser et al., 2004). The TEF PCR
made possible the characterization of fungal pathogens at a species level. The
phylogenetic analysis based on TEF PCR amplification showed that
morphologically identified Fusarium isolates associated with lentil wilt are
genetically diverse and falling under different species. This information is so much
important and useful in order to develop lentil wilt resistant varieties against the
highly virulent species of Fusarium prevailing in the country. All the obtained TEF
DNA sequences of the isolates were analyzed and compared with sequences in the
138
NCBI GenBank database and also in the FUSARIUM-ID database (Geiser et al.,
2004) for the determination of the species.
Results of the identification and confirmation of the 67 Fusarium isolates as
determined by the partial DNA sequence of their TEF-1α gene region (Table 4.5)
showed that these belonged to five different species within the genus Fusarium.
The isolates exhibited variable similarity percentage to the reference sequences in
the database. Based on morphological characterization and identification, isolates
were identified as species of Fusarium belonging to three different species
including F. oxysporum, F. commune, F. equiseti, while rest were designated as
Fusarium species. These isolates were grouped into 67 type isolates based on
similar morphology. Among 213, most of the isolates (168) were characterized as
F. oxysporum, however, DNA sequencing confirmed 105 of the 168 isolates to be
F. oxysporum and revealed that 63 of 168 isolates belonged to other species of
Fusarium i.e. F. redolens. The morphological characters of isolates of both species
viz. F. oxysporum and F. redolens were found similar in the study and therefore,
differentiation was difficult. This has been documented by Leslie and Summerell
(2006), who proposed that F. redolens is very much similar to F. oxysporum and
therefore, difficult to identify based on morphology.
The 17 isolates of F. commune identified based on unique morphological
characters viz. long monophialides alongwith polyphialides, obovoid micro-
conidia, 3-septate straight macro-conidia and pigmentation were also confirmed as
F. commune based on DNA sequencing and phylogenetic analysis. Similarly, based
on distinguished morphological characters viz. slightly curved shape of macro-
conidia, elongate apical and foot-shaped basal cells of macro-conidia, pyriform
139
Table 4.5: Identification of Fusarium isolates based on DNA sequencing of the translation elongation factor 1-α gene region.
No. Isolate ID
GenBank Accession No.
Location Morphological Identification
Sequence Based Identification
Closely Related Sequence/ Taxa
Identity (%)
1 FWC1 KR108311 Bangali Gujar-Chakwal F. equiseti F. equiseti NRRL45997 99% 2 FWC2 KR108312 Bangali Gujar-Chakwal F. equiseti F. equiseti Strain 286-09 99% 3 FWC4 KR108313 Bangali Gujar-Chakwal F. equiseti F. equiseti Strain 286-09 99% 4 FWC5 KR139797 Bangali Gujar-Chakwal F. oxysporum F. oxysporum NRRL52787 100% 5 FWC6 KR139798 Bangali Gujar-Chakwal F. oxysporum F. oxysporum NRRL52787 99% 6 FWC7 KR108314 Bangali Gujar-Chakwal F. equiseti F. equiseti NRRL43680 98% 7 FWC8 KR139799 Piplee-Chakwal F. oxysporum F. oxysporum NRRL43668 99% 8 FWC9 KR052170 Piplee-Chakwal F. oxysporum F. redolens NRRL54967 99% 9 FWC10 KR139800 Piplee-Chakwal F. oxysporum F. oxysporum NRRL43668 99% 10 FWC11 KR139801 Piplee-Chakwal F. oxysporum F. oxysporum NRRL53121 99% 11 FWC12 KR052163 Piplee-Chakwal F. oxysporum F. redolens NRRL25123 99% 12 FWC15 KR139802 Piplee-Chakwal F. oxysporum F. oxysporum NRRL32158 99% 13 FWC17 KR108315 Piplee-Chakwal F. equiseti F. equiseti NRRL34037 99% 14 FWC21 KR139803 Piplee-Chakwal F. oxysporum F. oxysporum NRRL32158 99% 15 FWC22 KR139804 Piplee-Chakwal F. oxysporum F. oxysporum NRRL52787 99% 16 FWC23 KR052164 Dhudial-Chakwal F. oxysporum F. redolens NRRL25123 99% 17 FWA1 KR052165 Tanazaya Dam-Attock F. oxysporum F. redolens FF-00411 99% 18 FWJ2 KR139805 Pindi Gujran-Jhelum F. oxysporum F. oxysporum MUCL14162 99% 19 FWJ4 KR139806 Pindi Gujran-Jhelum F. oxysporum F. oxysporum MUCL14162 99% 20 FWJ8 KR139807 Dhapai-Jhelum F. oxysporum F. oxysporum FO-02911 99% 21 FWJ11 KR139808 Dhapai-Jhelum F. oxysporum F. oxysporum FO-02911 99% 22 FWJ14 KR139809 Dhapai-Jhelum F. oxysporum F. oxysporum NRRL32158 99% 23 FWJ15 KR139810 Dhapai-Jhelum F. oxysporum F. oxysporum NRRL32158 99% 24 FWJ16 KR139811 Dhapai-Jhelum F. oxysporum F. oxysporum FO-02911 99% 25 FWJ17 KR052171 Dhapai-Jhelum F. oxysporum F. redolens NRRL25123 99%
Continued……
140
26 FWJ18 KR052173 Dhapai-Jhelum F. oxysporum F. redolens NRRL52814 99% 27 FWJ19 KR052174 Dhapai-Jhelum F. oxysporum F. redolens NRRL25123 99% 28 FWJ20 KR108316 Dhuman Khanpur-Jhelum F. equiseti F. equiseti NRRL45997 99% 29 FWJ26 KR108317 Dhuman Khanpur-Jhelum F. equiseti F. equiseti NRRL43680 99% 30 FWJ28 KR052166 Panjaion-Jhelum F. oxysporum F. redolens NRRL52814 99% 31 FWJ35 KR139812 Khaiwal-Jhelum F. oxysporum F. oxysporum NRRL26871 99% 32 FWJ47 KR108318 Dakhlee Karhan-Jhelum F. equiseti F. equiseti NRRL45997 99% 33 FWJ48 KR108319 Dakhlee Karhan-Jhelum F. equiseti F. equiseti NRRL45997 99% 34 FWJ49 KR061301 Dakhlee Karhan-Jhelum Fusarium sp. F. nygamai NRRL52708 100% 35 FWJ50 KR108320 Dakhlee Karhan-Jhelum F. equiseti F. equiseti NRRL45997 99% 36 FWJ53 KR108321 Dakhlee Karhan-Jhelum F. equiseti F. equiseti NRRL45997 99% 37 FWJ62 KR108322 Chanaal-Jhelum F. equiseti F. equiseti NRRL43680 99% 38 FWG1 KR139813 Jalalpur Jatan-Gujrat F. oxysporum F. oxysporum NRRL32158 99% 39 FWG13 KR139814 Lambray-Gujrat F. oxysporum F. oxysporum NRRL32158 99% 40 FWS1 KR139815 Pasrur-Sialkot F. oxysporum F. oxysporum NRRL52736 99% 41 FWS3 KR139816 Pasrur-Sialkot F. oxysporum F. oxysporum NRRL52736 99% 42 FWS5 KR139817 Pasrur-Sialkot F. oxysporum F. oxysporum NRRL32158 99% 43 FWS7 KR139818 Pasrur-Sialkot F. oxysporum F. oxysporum Strain 10-110 99% 44 FWS9 KR139819 Pasrur-Sialkot F. oxysporum F. oxysporum Strain 10-110 100% 45 FWS11 KR075925 Pasrur-Sialkot F. commune F. commune NRRL52764 100% 46 FWS13 KR075926 Pasrur-Sialkot F. commune F. commune NRRL52744 99% 47 FWN2 KR011716 Dongian-Narowal F. oxysporum F. redolens NRRL25123 100% 48 FWN4 KR052167 Dongian-Narowal F. oxysporum F. redolens NRRL52619 99% 49 FWN5 KR052168 Dongian-Narowal F. oxysporum F. redolens NRRL52619 99% 50 FWN6 KR052169 Dongian-Narowal F. oxysporum F. redolens NRRL25123 99% 51 FWM1 KR052172 Chashma-Mianwali F. oxysporum F. redolens NRRL25123 99% 52 FWL1 KR061303 Fateh Pur-Layyah Fusarium sp. F. nygamai Strain M7491 99% 53 FWL2 KR061302 Fateh Pur-Layyah Fusarium sp. F. nygamai NRRL52708 100% 54 FWL4 KR061304 Fateh Pur-Layyah Fusarium sp. F. nygamai BW-1537 100%
Continued……
141
55 FWL5 KR139820 Chowk Azam-Layyah F. oxysporum F. oxysporum NRRL32158 99% 56 FWL6 KR139821 Chowk Azam-Layyah F. oxysporum F. oxysporum NRRL32154 99% 57 FWL7 KR139822 Chowk Azam-Layyah F. oxysporum F. oxysporum NRRL52736 99% 58 FWL9 KR139823 Karoor-Layyah F. oxysporum F. oxysporum NRRL32158 99% 59 FWL11 KR075927 Karoor-Layyah F. commune F. commune NRRL28387 99% 60 FWL12 KP297995 Karoor-Layyah F. oxysporum F. oxysporum NRRL32158 99% 61 FWB3 KR075928 Mankera-Bhakkar F. commune F. commune NRRL52744 99% 62 FWB4 KR075929 Mankera-Bhakkar F. commune F. commune NRRL28387 99% 63 FWB9 KR075930 AZRI-Bhakkar F. commune F. commune NRRL52744 99% 64 FWB10 KR139824 AZRI-Bhakkar F. oxysporum F. oxysporum NRRL32153 100% 65 FWB11 KR075931 AZRI-Bhakkar F. commune F. commune NRRL28387 98% 66 FWK1 KR139825 Nurpur-Khushab F. oxysporum F. oxysporum NRRL32158 99% 67 FWK2 KR139826 Adhikot-Khushab F. oxysporum F. oxysporum Strain JG22-5 100%
142
micro-conidial shape, chlamydospores formation (singly, short chains and clusters),
creamy white colony color and pale brown pigmentation indicated 23 isolates as F.
equiseti, which were confirmed molecularly as F. equiseti. Moreover,
morphologically unidentified 5 isolates of Fusarium species were confirmed as F.
nygamai. The use of phylogenetic analysis in addition to morphological
characterization in this study greatly helped in the confirmation of recovered
pathogens at species level. This was supported by the concept given by Aoki et al.
(2003) who suggested that phylogenetic techniques help identify new species,
which is usually difficult and often impossible by using conventional
morphological characters. Likewise, Waalwijk et al. (2004) and Williams et al.
(2002) proposed that PCR provides immense advantage over conventional methods
and offers highly specific and accurate detection and identification of the pathogen.
Also, Mule et al. (2005) suggested that a comparison at the DNA sequence levels
offers accurate classification of fungal species. The findings of the study based on
TEF-1α region suggested that this coding gene is an efficient tool could be used as
a phylogenetic marker to distinguish Fusarium species as shown by Geiser et al.
(2004), Leslie et al. (2001) and Taylor et al. (2000), who proposed the use of TEF-
1α gene region as phylogenetic marker in various fungal species like Fusarium.
TEF has also been shown as a successful marker in F. oxysporum by O’Donnell et
al. (2009) and Baayen et al. (2000). A high level of polymorphism among the
analyzed sequences was found in the study. This was in accordance to Arif et al.
(2012), Nicolaisena et al. (2009) and Bogale et al. (2007).
The molecular data divulged the results of isolations conducted for retrieval
of wilt pathogens associated with plant mortality from major lentil producing
143
districts of Punjab, Pakistan. Perusal of Table 4.6 revealed the involvement and
relationship of five major species of Fusarium with lentil wilt disease viz. F.
oxysporum, F. nygamai, F. redolens, F. commune and F. equiseti. The maximum
association and recovery of F. oxysporum (49.29%) was seen, which was followed
by F. redolens (29.57%), 10.79% of F. equiseti, 7.98% of F. commune and 2.34%
of F. nygamai.
District-wise prevalence and association of a particular species of Fusarium
with the wilt disease was observed and revealed that out of the five prevailing
species, the highest associations was that of F. oxysporum with a range of 0.46-
21.12% (mean 10.79%), followed by F. redolens (range 1.87-14.55%, mean
8.21%), F. equiseti (4.22-6.57%, mean 5.39%) and F. commune (range 0.46-
6.10%, mean 3.28%), while lowest association was of F. nygamai (range 0.46-
1.87%, mean 1.16%) (Table 4.6, Figure 4.23). This data showed variable range of
causal pathogens of wilt infection in surveyed areas. Total pathogens associated
with plant mortality due to wilt ranged from 1.40-32.86% with maximum
pathogens recovery from district Jhelum (32.86%), while minimum from district
Khushab (1.40%). Recovered pathogens from district Jhelum (32.86%) included
species of F. oxysporum (21.12%), F. redolens (4.69%), F. nygamai (0.46%) and
F. equiseti (6.57%). Recovery from district Chakwal (14.55%) included species of
F. oxysporum (4.22%), F. redolens (6.10%) and F. equiseti (4.22%). District
Narowal (14.55%) showed total recovery of F. redolens (14.55%) species. District
Gujrat (12.20) involved species of F. oxysporum (12.20) only. From Layyah
(7.51%), three species viz. F. oxysporum (5.16%), F. nygamai (1.87%) and F.
commune (0.46%) were found. District Bhakkar (6.57%) included two species viz.
F. oxysporum (0.46%) and F. commune (6.10%). Likewise, district Sialkot (6.10%)
144
Table 4.6: Involvement of major Fusarium species in lentil wilt and plant mortality.
No. District Mean Disease Incidence
(%)
Association of Prevalent Fusarium species (%)
Total (%)
F. oxysporum F. redolens F. nygamai F. commune F. equiseti
1 Chakwal 27.5 4.22 6.10 - - 4.22 14.55 2 Attock 7.5 - 2.34 - - - 2.34 3 Jhelum 20 21.12 4.69 0.46 - 6.57 32.86 4 Gujrat 17.5 12.20 - - - - 12.20 5 Sialkot 7.5 4.69 - - 1.40 - 6.10 6 Narowal 25 - 14.55 - - - 14.55 7 Mianwali 11 - 1.87 - - - 1.87 8 Layyah 82.5 5.16 - 1.87 0.46 - 7.51 9 Bhakkar 55.5 0.46 - - 6.10 - 6.57 10 Khushab 22.5 1.40 - - - - 1.40
Total (%) 28 49.29 29.57 2.34 7.98 10.79 100
145
Occurrence and frequency percentage of five species of Fusarium associated with lentil wilt and plant mortality in districts of Punjab.
Figure 4.23:
146
also showed recovery of same two species viz. F. oxysporum (4.69%) and F.
commune (1.40%). F. redolens alone was found associated with wilt disease in two
districts viz. Attock (2.34%) and Mianwali (1.87%). While, in district Khushab
(1.40%), only species of F. oxysporum was found responsible for wilt disease. The
above mentioned observations illustrated the plant mortality due to wilt disease and
the Fusarium pathogens involved at both seedling and adult stages of crop when
the wilted samples were collected from various locations and districts.
Above results showed that wilt disease incidence reported in each district
during survey and ascertained isolations confimed the association of different
Fusarium pathogens. The frequency percentage of major Fusarium pathogens
showed the maximum involvement of F. redolens in wilt disease incidence
recorded in district Chakwal (27.5% mean wilt incidence), which was followed by
F. oxysporum and F. equiseti. In district Mianwali (11%), Narowal (25%) and
Attock (7.5%), species of F. redolens was associated only and responsible for wilt
incidence recorded during survey. The maximum prevalence of this species in three
districts might occurred as a result of various factors, among which, host specificity
is a basic reason for any pathogen to occur in a particular area and it was observed
during survey that a common cultivar Masoor-2006 was found in three surveyed
districts. Similarly, due to the presence of conducive environmental conditions in
these districts, the maximum prevalence of this species might have occurred.
Likewise, high pathogenic ability and inoculum potential is also a reason for the
species to prevail in a particular area. In addition, host resistance plays an
important role in the development of infection and therefore, the host resistance
against this species might be expressed less as compared to other morphologically
147
similar species like F. oxysporum. This showed that under the availability of same
environmental conditions and amount of pathogen inoculum, the genetic makeup of
the plants also plays role in resistance reaction of the plants towards the inoculated
pathogens (Mohammadi et al., 2012). In district Jhelum (20%), the dominant
species was that of F. oxysporum followed by F. equiseti and F. redolens, while
least dominant was F. nygamai. Collectively, these four species were responsible
for wilt incidence in this district at both crop stages. In districts Gujrat (17.5%) and
Khushab (22.5%), F. oxysporum was the only prevalent species and involved in
causing plant mortality due to lentil wilt disease. In districts Bhakkar (55.5%) and
Sialkot (7.5%), F. oxysporum and F. commune were predominant and responsible
for varied range of wilt disease incidence. In Layyah (82.5%), three species viz. F.
oxysporum, F. nygamai and F. commune were found to be associated with recorded
wilt incidence.
In conclusion, the identified four new species of Fusarium viz. F. nygamai,
F. redolens, F. commune and F. equiseti in addition to F. oxysporum, are reported
from Pakistan as associated with lentil wilt disease. According to current
knowledge, no such information is available on the involvement of different fungal
pathogens in the vascular wilt disease of lentil from Pakistan. Therefore, this is the
first report of current prevalent species of Fusarium associated with lentil wilt. It
has been reported that vascular wilt of lentil is generally attributed to the most
devastating fungus F. oxysporum Schlecht. emend. Snyder & Hansen f. sp. lentis
Vasudeva and Srinivasan (Khare, 1981). However, various reports around the
world also showed that the wilt disease is incited by several other pathogenic
species of Fusarium and therefore, support this study.
148
Other reported species of Fusarium associated with lentil wilt-root rot
complex diseases included F. acuminatum Ell. and Ev., F. avenaceum (Fr.) Sacc.,
F. culmorum (W. G. Smith) Sacc., F. solani (Mart.) Appel and Wollenw. Emend.
Snyd. and Hans. (Burgess et al., 1988a), F. equiseti (Corda) Sacc. and F.
sporotrichioides Sherb. (Rauf and Banniza, 2007). F. avenaceum alongwith F.
orthoceros have also been reported as causal agents of lentil wilt from Argentina
by Kotova et al. (1965). In a study, Belabid et al. (2000) reported various Fusaria
including F. oxysporum, F. moniliforme and F. equiseti as associated with lentil
wilt in Algeria. Later, Chaudhary et al. (2006) showed the involvement of F.
oxysporum f. sp. lentis, Ralstonia bataticola, R. salani, F. solani and Sclerotium
rolfsii in lentil wilt-root rot complex in Uttar Pradesh and found responsible for 7%
mean plant mortality.
Similarly, more species were found to be associated with the wilt disease of
lentil. Like F. oxysporum, isolates of F. redolens Wollenw. [syn: F. oxysporum var.
redolens (Wr.) Gordon] are also responsible for causing several diseases including
wilts, seedling damping-off and cortical rot (Booth, 1971). In USA and Europe, F.
redolens has been reported as the causal agent of vascular wilt disease of lentil
(Riccioni et al., 2008). Likewise, wilting-like symptoms on chickpea produced by
F. redolens were reported in Lebanon, Morocco, Pakistan and Spain by Jimenez-
Fernandez et al. (2011). Similarly, frequent isolation of F. redolens from necrotic
and discolored root and crown tissues of chickpea, pea, lentil and durum wheat
have also been reported by Taheri et al. (2011) in Saskatchewan. F. nygamai
species has also been found to be associated with the rhizoplane of lentil plants in
149
Egypt by Abdel-Hafez et al. (2012).
It was also observed that maximum wilt incidence and isolations were noted
at flowering or adult plant stage as compared to the seedling stage. Various factors
including the stage of the crop, crop variety, weather including warm and dry
conditions later in the season favors the development of the inoculum in the soil.
Similar increase in infection caused by F. oxysporum f. sp. lentis with an increase
in lentil plant age was also examined by Kumar et al. (2004).
The genetic polymorphism has been reported in other fungi as a direct
record of genetic evolution (Sanders, 2002). In this study, the genetic diversity in
isolates identified by DNA sequencing showed the ability of Fusarium wilt
pathogens to get adapted under different climatic conditions prevailing in
geographical diverse regions. This information would be extremely helpful to
identify best management strategies. By considering this, the lentil breeding
programs should focus on host resistance against all the virulent races and strains
of wilt pathogen. In this way, vascular wilt disease can be managed effectively in
the future.
Understanding the ecology, behavior and species diversity of this soil-
borne, economically important and diverse complex of Fusarium pathogens is
extremely important for its worldwide occurence. The utilization of molecular
markers such as TEF for use in sequencing and phylogeny together with
morphological and pathogenicity data for the characterization of the Fusarium
isolates can help significantly improve the understanding of the variability found
within this important pathogen. The understanding of the evolutionary relationships
150
among the species of this genus and correct identification of species is significantly
imperative for the management of diseases globally.
4.7 MANAGEMENT OF FUSARIUM WILT
4.7.1 Management Through Host Plant Resistance
Screening of lentil germplasm comprised of 23 lines and cultivars was
conducted under screen house in pot experiment employing root dip method. The
disease parameters including disease severity index, disease incidence and yield
reduction were recorded. Disease parameters were noted based on typical wilt
symptoms started at varied days, mostly 15-20 days after inoculation. Initially,
disease symptoms started with the leaf distortion and some chlorosis. Later, more
chlorosis occurred on foliage, followed by necrosis and later plant stunting was
observed. Finally, the susceptible plants showed wilted growth with vascular
discoloration and finally death. Based on these symptoms and the assessment of
wilt incidence and severity index, screened lines and cultivars were characterized
into three groups of infection types as resistant (1-3), moderate (4-6) and
susceptible (7-9) using the 0-9 rating scale. None of the line or cultivar was found
immune (0) against the disease.
The data on disease incidence, severity index and yield reduction are
presented in Table 4.7 and Appendix 9. Significant variations in disease incidence,
severity index and yield reduction were observed, where disease incidence ranged
from 20 to 100%, disease severity index from 4.44 to 100%, while reduction in
yield was found 9.60 to 100% among the germplasm.
151
Table 4.7: Screening of lentil germplasm against Fusarium wilt.
No. Tested Germplasm Disease Severity Index* (%)
Disease Incidence (%)
Yield Reduction (%)
Infection Type
Inoculated Uninoculated Inoculated Unioculated Inoculated Unioculated (IT) 1 Markaz-09 5.92 j 0 j 26.67 c 0 d 24.94 e 0 g R** 2 Masoor-86 4.44 j 0 j 20 c 0 d 20.63 e 0 g R 3 Masoor-2006 5.92 j 0 j 26.67 c 0 d 11.69 f 0 g R 4 Punjab Masoor-00518 12.59 i 0 j 46.67 b 0 d 12.72 f 0 g R 5 Punjab Masoor-09 4.4 4j 0 j 20 c 0 d 9.60 f 0 g R 6 BL-2 22.22 h 0 j 40 b 0 d 41.2 d 0 g M*** 7 NL-1 30.37 g 0 j 46.67 b 0 d 61.94 c 0 g M 8 Mansehra-89 Bold seeded 99.26 ab 0j 100 a 0 d 100 a 0 g S**** 9 NL-2 91.85 cd 0 j 100 a 0 d 100 a 0 g S 10 NL-3 60.74 f 0 j 100 a 0 d 77.90 b 0 g S 11 NARC-08-2 93.33 bcd 0 j 100 a 0 d 100 a 0 g S 12 NARC-11-1 99.26 ab 0 j 100 a 0 d 100 a 0 g S 13 NARC-11-2 100 a 0 j 100 a 0 d 100 a 0 g S 14 NARC-11-3 100 a 0 j 100 a 0 d 100 a 0 g S 15 NARC-06-1 97.04 abc 0 j 100 a 0 d 100 a 0 g S 16 08504 89.62 d 0 j 100 a 0 d 100 a 0 g S 17 08505 99.26 ab 0 j 100 a 0 d 100 a 0 g S 18 09506 94.81 abcd 0 j 100 a 0 d 100 a 0 g S 19 01505 100 a 0 j 100 a 0 d 100 a 0 g S 20 03501 81.4 8e 0 j 100 a 0 d 100 a 0 g S 21 04533 90.36 d 0 j 100 a 0 d 100 a 0 g S 22 06513 100 a 0 j 100 a 0 d 100 a 0 g S 23 NARC-08-1
(Susceptible check) 100 a
0 j
100 d
0 d
100 a
0 g S
LSD value at α=0.05 5.93 8.73 4.40 - At α=0.05 level of significance means sharing same letters are non-significant. Data based on mean of three replications. *Disease severity percentage on the basis of 0-9 scale; **,*** and**** are Resistant, Moderately susceptible and Susceptible, respectively.
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The screening results showed that among 23 lentil germplasm lines and
cultivars, five viz. Markaz-09, Masoor-86, Masoor-2006, Punjab Masoor-00518
and Punjab Masoor-09 were found to be resistant against the disease and scored
between 1-3 on the rating scale. Two lines viz. BL-2 and NL-1 gave moderately
susceptible reaction falling between 4-5 scale range and the remaining lines
including Mansehra-89 bold seeded, NL-2, NL-3, NARC-08-2, NARC-11-1,
NARC-11-2, NARC-11-3, NARC-06-1, 08504, 08505, 09506, 01505, 03501,
04533, 06513 and NARC-08-1 (susceptible check) were found susceptible on 7-9
scale range. In pathogenicty test, NARC-08-1 showed similar results (100%
severity index, incidence and yield reduction), whereas, the cultivar Masoor-93
gave moderately susceptible reaction against the isolate (FWL12) with 66.66%
severity index, 100% incidence and 53.68% reduction in yield. The severity index
and incidence recorded in Masoor-93 was maximum than observed in two screened
moderately susceptible lines (BL-2 and NL-1), while yield reduction was found
intermediate of the two lines. The disease incidence in resistant cultivars ranged
from 20 to 46.67%, in the two moderately susceptible lines it was 40 and 46.67%,
while in susceptible lines, 100% incidence was recorded. The disease severity
index data based on 0-9 scale showed variation among the screened germplasm.
The severity index in resistant cultivars ranged from 4.44 to 12.59%, while in the
two moderately susceptible reaction lines it was 22.22 and 30.37%. On the other
hand, the susceptible lines showed 60.74 to 100% severity index.
The data recording on grain yield after harvest showed great variation
among the tested lines and cultivars. The data based on yield reduction showed that
the wilt disease significantly affects the grain yield. The susceptible lines were
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found completely dead before maturity and therefore, did not produced any yield
(100% reduction) except one line viz. NL-3 that showed 77.90% reduction in yield
compared to the control and susceptible check. This line produced few lentil seeds
that were found shrivelled. The two moderately susceptible lines viz. BL-2 and
NL-1 resulted in 41.2% and 61.94% grain yield reduction, respectively. The
resistant ones were found to produce sufficient yield with the reduction ranging
from 9.60 to 24.94%. This data showed a significant variation in seed yield
reduction among the tested lines and cultivars. The data based on seed weight
reduction showed that the wilt disease significantly affects the grain yield and
affects the quality of seed.
The pathogen F. oxysporum f. sp. lentis is responsible for severe disease
damage under hot and dry weather conditions (Bayaa and Erskine, 1998) with the
optimum temperature range suitable for disease development is 22–25 °C, thus
causing huge losses in areas with such conditions (Mohammadi et al., 2011). A
significant variation in host response to wilt disease was observed among the
germplasm tested under controlled screen house conditions. Similar findings were
achieved by Pouralibaba and Alaii (2004) through evaluation of thirty lentil
cultivars and accessions in field and glasshouse.
In this study, the pathogenic virulence showed different effects on different
germplasm tested viz. resistant, moderately susceptible and susceptible reactions.
This observation suggests that in the presence of similar environmental condition
and amount of inoculum provided, the genetic makeup of the plant also affects the
resistance reaction of the plants towards the fungus (Mohammadi et al., 2012). This
study revealed susceptibility of sixteen lines or cultivars with 100% wilted plants
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against the inoculated pathogen. On the contrary, Stoilova and Chavdarov (2006)
screened 32 lentil genotypes under greenhouse conditions and reported three
accessions susceptible with 45 and 50% of total wilted plants.
The root dip method employed in this study proved to be efficient and
reproducible in producing wilt disease and symptoms as demonstrated by Taheri et
al. (2010). Mohammadi et al. (2012) alo screened germplasm using the same root
dip method under green house and naturally infested field and found it effective in
producing disease symptoms. Disease parameters including percent wilt incidence
and percent severity index were noted based on typical wilt symptoms (Bowers and
Locke, 2000). The variation in incubation period occurred depending upon the
resistance reaction offered by different germplasm against the pathogen. Based on
a rating scale described by Bayaa et al. (1995), the screened lines and cultivars
were grouped into three categories as resistant, moderately susceptible and
susceptible, thus, helped in the identification of resistant sources. The susceptible
check used showed clear wilt symptoms that were visible after 15 days of
inoculation which increased with complete death later in the season. This was
useful in recording the onset of disease and greatly helped in characterizing the
other tested germplasm accurately.
The range of incidence and severity index noted in five resistant cultivars
i.e. 20 to 46.67% and 4.44 to 12.59%, respectively with the yield reduction ranged
from 9.60 to 24.94% suggested that these are an important source of resistance to
be exploited in breeding programs against the disease. However, on the other hand,
the susceptible lines produced zero yield. This data based on yield reduction
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showed that the wilt disease significantly affects the grain yield and deteriorates the
quality of seed. This suggests that higher the wilt incidence and severity, lower will
be the seed quantity and quality.
4.7.2 Biological Management
The biological control agents viz. Trichoderma harzianum and T. viridi
were used as soil treatments for the management of Fusarium lentil wilt. To
analyze the influence of both the microbes on pathogen, disease parameters viz.
disease severity index, incidence and yield reduction were scrutinized (Figure 4.24,
Appendix 10 and 11).
Results of the experiment revealed the effectiveness of both the treatments,
however, maximum control was recorded with T. harzianum as compared to T.
viridi (Figure 4.24). The treatment with T. harzianum showed significant difference
with control plants. The disease severity index with T. harzianum was found to be
8.9%, while disease incidence was 26.7% with 16.27% yield reduction. The
inoculated control was observed with 100% wilt severity, incidence and yield
reduction, whereas, uninoculated control was found with 0% severity, incidence
and grain yield reduction. On the other hand, treatment with T. viridi showed
17.8% severity index and 66.7% incidence with the yield reduction of 31.13%.
This data showed that T. harzianum was the best treatment for biological control of
Fusarium wilt without reducing grain yield.
The results confirmed that the inoculation of conidial suspension of T.
harzianum significantly (p ≤ 0.05) decreased the lentil wilt disease to 73.33%
wilted lentil plants as compared to inoculated control with 100% wilted and dead
156
Influence of antagonists on percent disease severity index, incidence and grain yield reduction. Different letters indicated on bars represent significant differences in wilt severity, incidence and yield reduction values (P<0.05).
Figure 4.24:
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plants during the early stage of the plants. This decrease in wilt incidence in
contrast to inoculated control is possibly due to an increase in the population
density of biocontrol agent in the potting mixture. No visible difference was noted
between the morphology and physiology of the uninoculated plants treated only
with water and microbial treated plants.
Biocontrol is the best and effective substitute, especially against soil-borne
pathogens such as Fusarium species. This method offers advantages such as
environment friendly, cost effective and extended plant protection (Gohel et al.,
2007). Among several antagonists used for biological management, the species
within the genus Trichoderma are used extensively as biocontrol agents against
soil- and seed-borne diseases, such as, Fusarium wilt (Etebarian, 2006). Within
genus Trichoderma, two species were utilized in this study and among which, T.
harzianum found the most efficient. The result was supported by Dubey et al.
(2007) who proposed the efficiency of this biocontrol agent for controlling
Fusarium wilt disease. In the present study, two species of Trichoderma were
employed against highly virulent isolate of Fusarium responsible for lentil wilt.
The results of the treatments suggested that both the microbes have the ability to
reduce the disease damage, however, T. harzianum was highly efficient in
controlling wilt disease and reducing severity of disease (8.9%). Similar results
were reported by Dolatabadi et al. (2012), who observed reduced disease severity
with increased plant height with the combination of T. harzianum+S. vermifera.
Similarly, Kumar et al. (2013a) also observed significant reduction in incidence
and maximum grain yield in field trials against lentil wilt with T.
harizanum+Pseudomonas fluorescence.
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Akrami et al. (2011) evaluated three isolates of Trichoderma viz. T1 (T.
harzianum), T2 (T. asperellum) and T3 (T. virens) alone and in combination
against Fusarium rot of lentil. The green house experiment showed more
effectiveness of isolates T1 and T2 isolates and their combination as compared to
other treatments. Disease severity was found to be reduced ranging from 20 to
44%, while dry weight increased from 23 to 52%. Various other studies have also
reported disease control using Trichoderma species, such as, Poddar et al. (2004)
reported decreased chickpea wilt incidence with isolate of T. harzianum.
Correspondingly, Siddiqui and Singh (2004) found maximum plant growth,
increased transpiration and decreased wilt disease index caused by F. oxysporum f.
sp. ciceris through treatment with T. harzianum. Later, Dubey et al. (2006) also
reported reduced wilt disease incidence in chickpea using Trichoderma species.
Likewise, in a study, Ghahfarokhi and Goltapeh (2010) reported Trichoderma
species, P. indica and S. vermifera as the most effective biocontrol agents against
take-all diseases of wheat caused by Gaeumannomyces graminis var. tritici .
4.7.3 Chemical Management
All the tested fungicides checked the growth of the pathogen, however,
Benomyl and Thiophanate methyl proved to be most effective both in vitro and in
vivo in managing the wilt disease. This reports that systemic fungicides found to be
superior in inhibiting the fungal mycelial growth in plates as well as in pot seed
treatment.
4.7.3.1 In vitro evaluation of fungicides
All the tested fungicides checked the growth of the pathogen at variable rate
in vitro with the mean reduction of 69.9%. The data on measurement of the radial
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growth (Table 4.8, Appendix 12) of the selected virulent isolate in plates revealed
that the systemic fungicides were found to be superior in inhibiting the fungal
mycelial growth in plates (Figure 4.25 and 4.26, Appendix 13 and 14).
Out of the four fungicides, Benomyl (76.6%) proved to be the most
effective against the highly virulent Fusarium isolate and significantly checked the
fungal growth at all concentrations used. It was followed by Thiophanate methyl
(73%) with little inhibition difference. The two non-systemic fungicides viz.
Captan and Dithane M-45 were found to be inferior in their efficacy against the
pathogen as compared to the two systemic fungicides. However, among the non-
systemic, Captan was however found to be more effective than Dithane M-45 that
showed least inhibitory response. Captan showed 67.8% mean mycelial growth
reduction, while Dithane M-45 showed 62.3% mean reduction. This reduction is
much lower than the reduction showed by systemic fungicides.
The data showed that inhibition in mycelial growth increased with the
increase in the concentration of the fungicides (Table 4.8, Figure 4.26, Appendix
13) as compared with the control i.e. 0% with 9.0±0.0 cm growth. At the lowest 10
ppm concentration, the highest inhibition of the growth was observed in case of
Benomyl (46.7%) with 4.8±0.10 cm growth, followed by Thiophanate methyl
(43%) with 5.1±0.06 cm growth. Captan showed 5.2±0.0 cm growth and 42.2%
reduction, while Dithane M-45 with 5.4±0.10 cm growth and 40% reduction.
Each fungicide showed more decreased fungal growth and it continued to
reduce with the increase in concentration. Similarly, at 20, 30 and 50 ppm
concentrations, each fungicide showed intermediate reduction response between
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Table 4.8: Effect of fungicides at different concentrations on mycelia radial growth of Fusarium isolate. Fungicides
Mycelial Radial Growth* (7days) (cm)
10ppm 20ppm 30ppm 50ppm 100ppm Mean Dithane M-45 5.4±0.10 3.6±0.06 3.3±0.12 2.7±0.12 2.0±0.0 3.4±1.27 Captan 5.2±0.0 3.2±0.06 2.3±0.10 2.0±0.06 1.8±0.06 2.9±1.39 Benomyl 4.8±0.10 2.6±0.0 1.2±0.0 1.0±0.06 0.9±0.10 2.1±1.66 Thiophanate M 5.1±0.06 2.8±0.0 1.7±0.10 1.5±0.06 1.1±0.12 2.4±1.61 Control 9.0±0.0 9.0±0.0 9.0±0.0 9.0±0.0 9.0±0.0 9.0±0.0 Mean 5.1±0.25 3.1±0.44 2.1±0.90 1.8±0.73 1.5±0.53 2.7±0.56 LSD value at α=0.05: Fungicide = 0.04; Concentration = 0.04; Interraction = 0.10
At α=0.05 level of significance means sharing same letters are non-significant. *Mean of three replications
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Mycelial radial growth in MEA plates treated with fungicides at 30 ppm concentration.
Figure 4.25:
162
Mycelial growth inhibition (%) at five concentrations. Different letters indicated on bars represent significant differences in inhibition values (P<0.05).
Figure 4.26:
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minimum and maximum ranges. Such as, Benomyl was superior in maximum
inhibition and minimum radial growth at all three concentrations (71.1, 86.7,
88.5% inhibition; 2.6±0.0, 1.2±0.0, 1.0±0.06 cm radial growth) followed by
Thiophanate methyl (68.9, 81.1, 83.7%; 2.8±0.0, 1.7±0.10, 1.5±0.06 cm). The
remaining two non-systemic fungicides viz. Captan and Dithane M-45 showed
inferior response at 20, 30 and 50 ppm concentrations i.e. 64.1, 74.4, 78.1%
(3.2±0.06, 2.3±0.10, 2.0±0.06 cm) and 60.4, 63.0, 70.4% (3.6±0.06, 3.3±0.12,
2.7±0.12 cm), respectively.
Best fungus control was observed at highest fungicidal concentration (100
ppm) at which all the fungicides greatly inhibited the growth of the fungus. At this
concentration, Benomyl showed the maximum growth reduction percentage and
minimum growth i.e. 90% and 0.9±0.10 cm as compared to the rest of the three
fungicides. Thiophanate methyl (88.1%; 1.1±0.12 cm) showed the intermediate
reduction response. Both non-systemic fungicides viz. Captan and Dithane M-45
showed their maximum efficacy and highest inhibition, however, inferior reduction
response compared to systemic fungicides at highest concentration (100 ppm) viz.
80.4 (1.8±0.06 cm) and 77.8% (2±0.0 cm), respectively.
The results suggested that systemic fungicides are more effective in
reducing the fungal growth as compared to the non-systemic fungicides. However,
in a study Kasyap et al. (2008) found much reduced fungal growth with non-
systemic fungicide such as Captan (88.3%). On the contrary, in this study, highest
inhibition (90%) was obtained with Benomyl at maximum 100 ppm concentration,
which means that slight increase in concentration would completely inhibit the
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fungal growth. Likewise, in a similar study, Sharma et al. (2002) found no mycelial
growth i.e. complete 100% inhibition of Fusarium oxysporum f. sp. lini , a causal
agent of linseed wilt with Benomyl at three concentration (500, 1000 and 1500
ppm. Similarly, Ayub (2001) tested eleven fungicides and reported Benomyl,
Folicar and Derosal, as the most effective against mycelial growth of Fusarium wilt
of chickpea. Later, Christian et al. (2007) tested five fungicides and found
Benomyl with highest inhibition of fungus along with Cabindazim and Captan. The
intermediate response was obtained with Thiophanate methyl (88.1%) and Captan
(80.4%). At all concentrations checked in the study, Dithane M-45 showed least
77.8% inhibitory response with 2.0±0.0 cm radial growth. Comparatively, Singh et
al. (2010) found 66% inhibition with 28 mm diameter at 200 ppm concentration of
Dithane M-45. Also, De et al. (2003) reported complete inhibition of growth of
pathogen by Dithane M-45 (mancozeb) i.e 0.25%. Likewise, Dabbas et al. (2008)
found complete inhibition of F. oxysporum f. sp. pisi at 200 ppm concentration.
4.7.3.2 In vivo evaluation of fungicides
Based on the best efficacy of Benomyl and Thiophanate methyl in vitro,
these two were used in in vivo seed treatment under green house conditions. The
disease parameters including seed germination, disease severity index, disease
incidence and yield reduction were calculated (Figure 4.27, Appendix 15 and 16).
The fungicidal seeds treatments with Benomyl and Thiophanate methyl
were effective for seeds germination (100%) and resulted in improved germination
than control. The results confirmed that both the fungicides significantly (p ≤ 0.05)
reduced the wilt disease to 86.67 to 93.33% wilted plants as compared to the
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Percent disease severity index, incidence and grain yield reduction. Different letters indicated on bars represent significant differences in wilt severity, incidence and yield reduction values (P<0.05).
Figure 4.27:
166
inoculated control (0%) with the maximum control provided by Benomyl (Figure
4.27). These results suggested that both fungicides were efficient in reducing the
wilt incidence and severity index measured on a 0-9 scale compared to the
inoculated check. However, the fungicidal treatment did not significantly increase
the yield component compared to uninoculated check and produced varied grain
yield. The seed treatment proved Benomyl as superior in reducing the wilt
incidence, severity and yield reduction compared to Thiophanate methyl. The lentil
plants germinated with Benomyl treated seeds produced only 6.7% wilt incidence
and 1.5% wilt severity index, which showed the best effectiveness of Benomyl
against the most highly virulent Fusarium pathogen. On the other hand,
Thiophanate methyl also showed effective wilt control next to Benomyl. The
Thiophanate methyl treated seeds showed 13.3% incidence and 3% severity index.
The fungicidal seed treatments did not increased the yield component
compared to uninoculated check but significantly minimized the yield reduction.
The data on yield (Figure 4.27, Appendix 15) after harvest showed a considerable
difference from untreated check with 100% yield reduction. The Benomyl
treatment resulted in 17.16% yield reduction, while Thiophanate methyl treatment
produced 22.47% reduction in yield. This data showed that fungicidal seed
treatment with Benomyl significantly minimized the reduction compared to
Thiophanate methyl, which was statistically different from the inoculated control.
Figure 4.27 and Appendix 15 showed statistically significant difference in yield
reduction with Benomyl and Thiophanate methyl treatments viz. 17.16% and
22.47%, respectively, from the inoculated check (100%). Therefore, from the
above findings, it was concluded that wilt incidence and severity had some
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negative effect on lentil seed yield, suggesting that an increase in wilt incidence
and severity, there will be reduction in seed weight.
The seed treatment suggested that the systemic fungicides viz. Benomyl and
Thiophanate methyl used in this study are efficient in controlling lentil wilt and
reducing the damage with the best control provided by Benomyl. These results
were in accordance with the Garkoti et al. (2013) who observed Benomyl with
1.0% wilt incidence and 15.1 gm 1000-seed weight. However, the first lentil seed
treatment conducted by Kovacikova (1970) showed that Captan effectively
controlled wilt disease (0.2%) through dry seed treatment. The germination of
seeds observed with treated seeds was found improved with both fungicides.
Similar results were obtained by Thakur et al. (2002) who reported highest
chickpea seeds germination with bavistin (91.6%), Benomyl (83.3%) and Captan
(75%). Andrabi et al. (2011) also reported increased seed germination when treated
with Carbendazim (71.24%), Carbendazim plus Mancozeb (62.21%) and
Mancozeb (61.46%).
Fusarium wilt of lentil majorly caused by Fusarium oxysporum f. sp. lentis
is a significant production constraint in Pakistan and there are very limited or no
resistance sources and control options against the disease. In this study, chemical
seed treatment, biological control agents and resistant lentil germplasm identified
can be used as a recommendation for managing wilt disease. These reported
management options can be practised under different geographical areas of the
country. The five cultivars viz. Markaz-09, Masoor-86, Masoor-2006, Punjab
Masoor-00518 and Punjab Masoor-09 found resistant against wilt can be used by
the farmers. Growing wilt resistant varieties is essential to control the disease
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caused by various pathogens in Punjab. Sources of resistance identified and
reported in this study could be used for production in this major lentil region. Also,
these can be utilized in lentil breeding programs to further enhance the productivity
and resistance by producing new wilt resistant varieties. The utilization of highly
effective biocontrol agent Trichoderma harzianum in managing wilt would be an
effective and environmentally safe method. The identified effective fungicides can
also be used by the farmers for reducing wilt damage through dry seed treatment.
Following this approach, seed as well as soil-borne pathogen inoculum can
possibly be eliminated. Also, the information regarding the application of these
fungicides along with the biological control agents can be used as a component in
integrated disease management of lentil wilt.
Lentil wilt disease is one of the important production constraints in
Pakistan. Therefore, the objectives of this study were to investigate the incidence
and distribution of Fusarium wilt in lentil producing areas of Punjab, morpho-
molecular characterization of Fusarium isolates recovered from wilted lentil plants
and the management of wilt disease through host plant resistance, biological
control agents and chemicals. This study provides a comprehensive report on the
investigation of lentil wilt status in Punjab, Pakistan and diversity of Fusarium
species prevalent in major lentil producing region of the country and involved in
lentil wilt disease. As wilt disease surveys are needed in order to understand the
distribution and incidence of this disease on lentil and for formulating effective
disease management strategy. Therefore, this report helped in assessing the true
relative risk of this disease in Pakistan and in employing timely and necessary
management strategies against the disease. The results clearly identified the areas
with 100% wilt prevalence with varying levels of incidence (7.5 to 82.5%). The
169
information provided in this effort can be utilized for the development of disease
maps and for the improvement of lentil crop in Pakistan.
Fusarium species are cosmopolitan which are found in soils around the
world and are economically important plant pathogens. Various Fusarium species
have been identified to be associated with the wilt disease around the world.
However, no report has been found from Pakistan in context with the involvement
of different Fusarium species, their biology, ecology and evolution. This research
study addressed the biology and evolution of pathogenic and nonpathogenic
species of Fusarium recovered from lentil from various districts of the country.
Significant amount of diversity was found among the isolates based on morpho-
molecular and pathogenic characterization. This confirmed that the causal agent of
Fusarium wilt of lentils belonged to different species within the genus. The
characterization and phylogenetic analysis revealed five different species of
Fusarium as associated with the wilt disease and therefore, reported for the first
time from Pakistan. The results of the dissertation have implications for
understanding the diversity in Fusarium species among their morphology, genetics
and pathogenicity. The genus Fusarium is composed of phylogenetically diverse
species, of which, several are responsible for causing lentil wilt, which is evident
from the diversity of isolates found in only one lentil field involved in disease.
Characterization of Fusarium isolates has practical importance for a successful
lentil breeding program against this pathogen. A high degree of morpho-molecular
and pathogenic variation found in Fusarium isolates supports using different
isolates from different districts and locations of the country in pathogenicity testing
and thus it would help in a selection procedure of lentil breeding program for
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developing a resistant cultivar for our country. The results of the pathogenicity test
suggested that any isolate of the identified species of Fusarium is potentially able
to cause wilt disease on lentils. Thus, the development of wilt resistant lentil
cultivars should involve a wide variety of Fusarium isolates.
Management of the disease is very much important so as to control the
disease in time because it could be a future threat to lentil cultivation in Pakistan.
Various microbial agents are present that serve as an efficient biological
management tool against wilt disease. However, it is important to identify the best
antagonistic activity of biocontrol agents by testing them against the prevalent
isolates of Fusarium responsible for current wilt disease damage in the country.
Considering this, the present study provides information on the best efficiency of T.
harzianum against the most virulent Fusarium isolate. In the same way, a number
of fungicides are available but there is limited or no current information available
on the effective fungicides to manage the wilt disease. Therefore, considering the
limited research work on the management of lentil wilt through chemicals this
current research study was conducted to evaluate the available fungicides, which
would be helpful in managing the wilt disease.
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SUMMARY
In Pakistan, lentil is the second highly grown winter season legume crop
next to chickpea in terms of quality and quantity. It is grown on an area of 30.8
thousand hectare annually, out of this, 24 thousand hectare (77.41%) is planted in
Punjab province comprising of Sialkot, Narowal, Gujrat, Rawalpindi, Jhelum,
Chakwal and Thal districts, where two-third of the area is sown under rain-fed
conditions. In Pakistan, the production of lentil is much lower than main lentil
producing countries such as Canada (1.5 million metric tonnes), as about 15
thousand tonnes yield was recorded during 2012 in the country. Susceptibility to
diseases is one of the main production constraints to the lentil crop. The crop is
vulnerable to a number of diseases, which adversely affects seed yield and its
quality. Among them, the most significant and serious soil-borne threat is the
occurrence of vascular wilt disease.
Vascular wilt of lentil is caused by several species of Fusarium but the most
devastating fungus is Fusarium oxysporum Schlect. ex. Fr. belonging to the order
Hypocreales of class Ascomycetes with unknown sexual stage. Wilt is found in all
major lentil growing areas of Pakistan and could be visualized at both seedling and
adult stages of plant growth. The disease is responsible for huge losses each year in
Pakistan, yet, there is a scarcity and lack of literature and information regarding its
occurrence, distribution, losses and species involved. Also, effective management
options against the current prevalent pathogens responsible for wilt disease are
lacking. Consequently, comprehensive studies pertaining to wilt incidence,
distribution, biology of prevalent species of Fusarium wilt pathogen and disease
171
172
management need to be addressed. Therefore, the study was planned keeping in
view the national interests to avoid future losses caused by lentil wilt.
A two year field survey data (2011-12 and 2012-13) and laboratory
isolations ascertained various isolates of Fusarium as associated with lentil wilt.
Disease was found widespread with 100% prevalence in all major lentil growing
districts of Punjab viz. Bhakkar, Layyah, Mianwali, Khushab, Sialkot, Narowal,
Chakwal, Attock, Gujrat and Jhelum. The mean wilt incidence was found 28% with
varied incidence percentages ranging from 7.5-82.5% observed in various districts
at two plant stages. Maximum mean wilt incidence was found at adult plant stage
(32.4%) than at seedling (23.05%). During both survey years maximum disease
incidence was recorded in district Layyah (85%, 80%; mean 82.5%), while
minimum in districts Attock (10%, 5%; 7.5%) and Sialkot (10%, 5%; 7.5%).
The isolations from wilted samples collected during survey ascertained the
involvement of Fusarium pathogens in wilt incidence identified in the fields. Two
hundred and thirteen Fusarium isolates were recovered from wilted samples that
showed significant morphological variations among each other. Tentatively, the
isolates were identified as species of F. oxysporum, F. commune, F. equiseti and
few were designated as Fusarium species based on their close morphological
similarities reported in identification keys. The isolates were grouped on the basis
of similar morphology into sixty seven type isolates for subsequent study.
To confirm and characterize the virulence of the identified isolates, in vitro
pathogenicity testing through root dip method using line NARC-08-1 and cultivar
Masoor-93 showed excellent production of wilt symptoms for pathogenic
173
characterization using parameters viz. disease severity index, incidence and yield.
Based on 0-9 rating scale, the results showed high pathogenic variability on two
different germplasm used i.e. isolates showed avirulent to highly virulent response
on NARC-08-1 with 0 to 100% wilt incidence and disease severity index, while
avirulent to moderately virulent on Masoor-93 with 0 to 100% incidence and 0 to
66.66% severity index.
The isolates were grouped as highly virulent (13 isolates, 19.40%),
moderately virulent (41, 61.19%), low virulent (8, 11.94%) and avirulent (5,
7.46%) based on their reaction to most susceptible line NARC-08-1. While,
reaction to cultivar Masoor-93, the isolates were characterized as moderately
virulent (24 isolates, 35.82%), low virulent (16, 23.88%) and avirulent (27,
40.30%). The study on the effect of wilt disease on lentil grain yield showed that
disease is responsible for statistically significant (p ≤ 0.05) reduction in yield on
both germplasm tested compared to the control. The reduction in harvested yield of
NARC-08-1 ranged from 11.86 to 100 %, while reduction observed on Masoor-93
ranged from 6.47 to 53.68%. The results of the experiment revealed that isolates of
different Fusarium species are highly pathogenic and involved in causing wilt
infection with huge loss in grain yield. The experiment greatly helped in
identifying the most virulent isolates responsible for severe wilt disease.
To confirm and identify the Fusarium isolates at species level and resolve
phylogenetic relationship between them, these were subjected to molecular
characterization employing translation elongation factor TEF-1α primers viz. ef1
and ef2. The amplification of TEF-1α gene region by PCR amplified a single DNA
fragment of size 700bp in each of the isolates. Further, the sequencing and
174
phylogenetic analysis resulted in sufficient resolution and resolved the isolates into
various clades differentiating into five distinct species. The identified species
included F. oxysporum, F. redolens, F. nygamai, F. commune and F. equiseti. This
data supported the morphological variation found among the isolates and divulged
the association of these identified species in wilt disease incidence in the major
lentil producing districts of Punjab, Pakistan.
The concluded data based on disease assessment through survey, isolations,
morphological and molecular characterizations revealed district-wise prevalence
and association of a particular species of Fusarium with the wilt disease. It
suggested that out of the five prevalent species, the highest associations was that of
F. oxysporum with a range of 0.46-21.12% (mean 10.79%), followed by F.
redolens (range 1.87-14.55%, mean 8.21%), F. equiseti (4.22-6.57%, mean 5.39%)
and F. commune (range 0.46-6.10%, mean 3.28%), while lowest association was of
F. nygamai (range 0.46-1.87%, mean 1.16%). As reported, highest incidence in
district Layyah (82.5%), three species viz. F. oxysporum, F. nygamai and F.
commune were found to be associated with recorded wilt incidence. While, lowest
incidence recorded at two districts viz. Attock (7.5%) and Sialkot (7.5%), species
of F. redolens was associated and responsible for wilt incidence in Attock and F.
oxysporum and F. commune were predominant and responsible for varied range of
wilt disease incidence.
Further, the characterized most virulent F. oxysporum isolate FWL12
(GenBank accession number KP297995) among the highly virulent isolates was
selected for use in management trials against the disease. Three trails viz. screening
for host resistance, biological and chemical management were conducted to
175
identify the best and effective management strategy against the highly virulent
pathogen responsible for the most devastating wilt disease. Management of disease
through host plant resistance employing a collection of germplasm viz. cultivars,
varieties and lines was conducted using root dip inoculation method. The results
revealed resistance in five cultivars viz. Markaz-09, Masoor-86, Masoor-2006,
Punjab Masoor-00518, Punjab Masoor-09 against the inoculated pathogen, which
was significantly different (p ≤ 0.05) from control treatments and other germplasm
in terms of disease parameters viz. incidence (20 to 46.67%), severity (4.44 to
12.59%) and yield reduction (9.60 to 24.94%).
The biological management employing biocontrol agents viz. T. harzianum
and T. viridi revealed maximum efficiency of T. harzianum in reducing lentil wilt
disease. The treatment with T. harzianum showed significant difference with
control plants. The disease severity index based on 0-9 scale was found to be 8.9%,
while disease incidence was 26.7% with 16.27% yield reduction. The inoculated
control was observed with 100% wilt severity, incidence and yield reduction,
whereas, uninoculated control was found with 0% severity, incidence and yield
reduction. The results confirmed that the inoculation of conidial suspension of T.
harzianum significantly (p ≤ 0.05) decreased the lentil wilt disease to 73.33%
wilted lentil plants as compared to inoculated control with 100% wilted and dead
plants during the early stage of the plants. The study proved T. harzianum as the
best and effective antagonist against Fusarium species.
Chemical management involved the in vitro evaluation of four fungicides
including Benomyl, Thiophanate methyl, Captan and Dithane M-45 using poison
food technique and the in vivo evaluation of two best effective fungicides viz.
176
Benomyl and Thiophanate methyl as seed treatment against the disease. The in
vitro evaluation was conducted at five concentrations viz. 10, 20, 30, 50 and 100
ppm with the maximum inhibition obtained at 100 ppm, while minimum at 10 ppm.
The four fungicides checked the growth of the pathogen at variable rate with the
mean reduction of 69.9%. However, the data on measured radial growth revealed
that systemic fungicides (Benomyl and Thiophanate methyl) were superior in
inhibiting the fungal mycelial growth than non-systemic (Captan and Dithane M-
45). Benomyl (76.6%) proved the most effective in checking the fungal growth
followed by Thiophanate methyl (73%), Captan (67.8%) and Dithane M-45
(62.3%). At 100 ppm, systemic fungicides greatly inhibited the growth i.e. 90%
with 0.9±0.10 cm radial growth (Benomyl) and 88.1% with 1.1±0.12 cm
(Thiophanate methyl). Both non-systemic fungicides viz. Captan and Dithane M-45
showed their maximum efficacy and highest inhibition at highest concentration
(100 ppm) used viz. 80.4 (1.8±0.06 cm) and 77.8% (2±0.0 cm), respectively.
In vivo seed treatment in pot experiment under green house conditions
revealed effectiveness of both fungicides in controlling the disease through analysis
of disease parameters including seed germination, disease incidence, disease
severity index and yield. The results confirmed that both the fungicides
significantly (p ≤ 0.05) reduced the wilt disease to 86.67 to 93.33% as compared to
the inoculated control (0%). Benomyl was superior resulted in 6.7% incidence and
1.5% severity index compared to Thiophanate methyl with 13.3% incidence and
3% severity index. The seed germination in treatments was 100% and found
improved than control. The fungicidal treatments did not increased the yield
component compared to uninoculated check, however, significantly minimized the
yield reduction. Compared with the inoculated check (100%), both treatments
177
showed statistically significant difference in yield reduction 17.16% and 22.47%,
respectively. The results suggested that both fungicides were efficient in managing
the disease and reducing the damage.
This study provided an overall current status of wilt pathogen in Punjab and
high lightened the areas under current high risk of its spread where by adopting
timely management strategies we can avoid any wilt epidemic in the future. The
findings also showed that this crop being a poor man’s meat needs to be focused
regarding increase in its production in the country because its cultivation is
continuously reducing in lentil regions as a result of diseases and shiftment of lentil
areas to other crops. The results of this dissertation based on phylogenetic analysis
have implications for understanding the genetic diversity, as well as evolution of
pathogenicity of the identified Fusarium species. The identified four virulent and
morpho-molecularly diverse species of Fusarium viz. F. redolens, F. nygamai, F.
commune and F. equiseti are reported for the first time on lentils in Pakistan. This
information on identified virulent and genetically diverse isolates of these species
should be considered by the plant breeders for developing resistant lentil varieties.
This would be helpful in escalating the lentil production through the development
of wilt resistant lentil varieties, thus, providing ultimate benefit to farmers. Also,
the reported resistant cultivars should also be used in breeding program. Moreover,
the addition of T. harzianum in soil and seed treatment with Benomyl and
Thiophanate methyl may reduce the devastating effect of widely occurring
Fusarium wilt pathogen, enhance yield and provide more economic return to the
lentil grower.
178
CONCLUSION AND RECOMMENDATIONS
• Lentil Fusarium wilt is prevalent in major lentil growing areas of Punjab, therefore,
it should be timely addressed, as the presence of inoculum under suitable
environment can cause severe epidemics in the future.
• Area under lentil is continuously reducing, which is an alarming situation for lentil
crop production in Punjab region and it needs to be focused immediately.
• Four new virulent Fusarium species in addition to F. oxysporum viz. F. redolens,
F. equiseti, F. commune and F. nygamai are reported for the first time from
Pakistan as associated with lentil vascular wilt. Highly virulent isolates of
Fusarium should be used in developing resistant lentil varieties.
• Five lentil cultivars viz. Markaz-09, Masoor-86, Masoor-2006, Punjab Masoor-
00518 and Punjab Masoor-09 are resistant against F. oxysporum.
• Soil application with T. harzianum at the time of sowing is an efficient control
measure for devastating F. oxysporum, which should be incorporated to manage the
disease without imposing negative effects on environment.
• Seed treatment with Benomyl and Thiophanate methyl exhibited high efficacy in
reducing disease severity. Seed treatment with either of these chemicals may be
incorporated in integrated disease management program, which ultimately will
reduce seed as well as soil-borne inoculum of the pathogen.
179
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Appendix 1: List of districts and locations visited for wilt disease along with percent disease prevalence, incidence and isolates recovered.
No. District Area/ Location
Disease Prevalence (%)
Disease Incidence (%)
Isolates recovered (ID/ range) 2011-12 2012-13 2011-12 2012-13
1 Chakwal Bangali Gujar 100 100 5 10 FWC1-7 Piplee 100 100 90 50 FWC8-22 Dhudial 100 100 10 20 FWC23-28 BARI 100 100 15 20 FWC29-31 2 Attock Tanazaya Dam 100 100 10 4 FWA1-2 Khaur 100 100 15 6 FWA3-4 Jand 100 100 5 5 FWA5 3 Jhelum Pindi Gujran 100 100 5 5 FWJ1-7 Dhapai 100 100 15 10 FWJ8-19 Dhuman Khanpur 100 100 10 20 FWJ20-27 Panjaion 100 100 20 25 FWJ28-34 Khaiwal 100 100 25 35 FWJ35-46 Dakhlee Karhan 100 100 5 20 FWJ47-53 Morha Skeiha 100 100 30 25 FWJ54-61 Chanaal 100 100 50 20 FWJ62-70 4 Gujrat Jalalpur jatan 100 100 20 5 FWG1-3 Shergarh (Dolat Nagar) 100 100 25 15 FWG4-8 Sombre 100 - 15 - FWG9 Naseera 100 - 25 - FWG10-11 Bhaddar 100 - 30 - FWG12 Lambray 100 - 35 - FWG13-26 5 Sialkot Pasrur road, Field 1 100 100 14 2 FWS1-3 Field 2 100 100 12 8 FWS4-7 Chowinda 100 100 4 5 FWS8-13 6 Narowal Dongian 100 100 15 20 FWN1-15 Behble 100 100 25 30 FWN16-21 Zafarwal Road 100 100 10 10 FWN22-23 Mureed Ke Road 100 100 50 40 FWN24-31 7 Mianwali Chashma 100 100 10 16 FWM1 Piplan 100 100 16 4 FWM2 Harnoli 100 - 10 - FWM3-4 8 Layyah Fateh Pur 100 100 80 90 FWL1-4 Chowk Azam 100 100 85 70 FWL5-8 ARI, Karoor 100 100 90 80 FWL9-16 9 Bhakkar Mankera 100 100 60 60 FWB1-4 Garh Morr 100 100 50 60 FWB5-7 AZRI 100 100 55 48 FWB8-11 Darya Khan 100 - 55 - FWB12-14 10 Khushab Nurpur 100 100 20 10 FWK1 Adhikot 100 100 30 20 FWK2 Hassan Pur Tiwana 100 100 25 30 FWK3
- = No field present.
206
Appendix 2: Morphological characterization of Fusarium isolates: Size of micro-conidia.
No. Isolate ID No.
Reading 1
Reading 2
Reading 3
Reading 4
Reading 5
Mean (µm)
1 FWC1 6.0x2.0 8.0x2.5 8.0x2.0 9.0x2.0 10.0x2.0 8.2x2.1 2 FWC2 10.0x3.0 10.0x2.0 8.0x3.0 10.0x2.5 10.0x3.0 9.6x2.7 3 FWC3 5.0x2.0 3.5x2.0 9.0x4.0 7.0x5.0 8.0x5.0 6.5x3.6 4 FWC4 7.0x2.0 7.0x1.5 6.0x2.0 6.0x2.0 6.0x2.0 6.4x1.9 5 FWC5 5.0x2.0 5.0x2.0 5.0x2.0 3.5x2.0 4.0x2.0 4.5x2.0 6 FWC6 6.0x2.0 5.0x2.0 6.0x3.0 7.0x2.0 5.0x2.0 5.8x2.2 7 FWC7 5.0x2.0 6.0x2.0 5.0x2.0 5.0x2.0 7.0x2.0 5.6x2.0 8 FWC8 6.0x3.0 5.0x2.5 8.0x3.0 5.5x2.5 10.0x3.0 6.9x2.8 9 FWC9 7.0x2.0 8.0x2.5 6.0x2.5 8.0x3.0 7.0x2.5 7.2x2.5 10 FWC10 8.0x2.5 9.0x3.0 10.0x3.0 10.0x3.0 7.0x3.0 8.8x2.9 11 FWC11 5.0x3.0 6.0x2.0 4.0x2.0 6.0x2.0 6.0x2.0 5.4x2.2 12 FWC12 10.0x4.0 9.0x3.0 8.0x3.0 8.0x2.5 7.0x2.5 8.4x3.0 13 FWC13 6.0x2.5 8.0x2.5 6.0x2.0 10.0x2.5 7.0x3.0 7.4x2.5 14 FWC14 8.0x2.5 6.0x2.5 8.0x3.0 10.0x2.0 7.0x2.0 7.8x2.4 15 FWC15 6.0x2.5 7.0x3.0 7.0x3.0 8.0x3.0 6.0x2.5 6.8x2.8 16 FWC16 8.0x2.5 7.0x2.5 6.0x2.0 10.0x2.0 8.0x2.5 7.8x2.3 17 FWC17 8.0x3.0 10.0x3.0 6.0x2.5 7.0x2.5 8.0x3.0 7.8x2.8 18 FWC18 7.0x2.0 8.0x3.0 10.0x2.5 7.0x2.5 6.0x2.0 7.6x2.4 19 FWC19 6.0x2.0 8.0x2.5 8.0x2.5 8.0x3.0 7.0x2.0 7.4x2.4 20 FWC20 8.0x3.0 7.0x2.5 8.0x2.5 6.0x2.0 7.0x2.5 7.2x2.5 21 FWC21 9.0x3.0 10.0x3.0 6.0x2.5 8.0x3.0 8.0x3.0 8.2x2.9 22 FWC22 7.0x2.5 6.0x2.5 8.0x2.5 6.0x2.5 6.0x2.5 6.6x2.5 23 FWC23 8.5x3.0 9.0x3.0 9.0x3.5 10.0x3.5 8.0x3.0 8.9x3.2 24 FWC24 7.0x 2.5 10.0x4.0 9.0x4.0 8.0x3.5 7.0x3.0 8.2x3.4 25 FWC25 5.0x2.5 5.0x2.5 8.0x3.0 6.0x2.5 8.0x2.5 6.4x2.6 26 FWC26 5.0x2.5 9.0x3.5 10.0x4.0 8.5x3.0 5.0x2.0 7.5x2.8 27 FWC27 6.0x2.5 6.0x2.0 7.0x2.0 5.0x3.0 10.0x3.5 6.8x2.6 28 FWC28 8.0x3.5 10.0x4.0 8.0x3.5 7.0x3.5 6.0x3.0 7.8x3.5 29 FWC29 8.0x3.0 7.0x3.0 5.0x2.0 5.0x2.5 6.0x2.5 6.2x2.6 30 FWC30 5.0x2.5 6.0x2.5 7.0x3.0 9.0x3.5 6.0x2.5 6.6x2.8 31 FWC31 9.0x3.5 8.0x3.0 5.0x2.0 5.0x2.5 6.0x2.75 6.6x2.75 32 FWA1 5.0x3.0 5.0x2.0 8.5x3.0 10.0x4.0 9.0x3.5 7.5x3.1 33 FWA2 8.0x3.5 7.5x3.5 8.0x4.0 6.0x2.5 8.0x3.0 7.5x3.3 34 FWA3 8.0x2.5 7.0x2.5 5.0x2.5 6.0x2.5 6.0x2.5 6.4x2.5 35 FWA4 6.75x2.5 5.0x3.0 8.0x2.5 7.0x2.5 6.0x2.0 6.55x2.5 36 FWA5 10.0x4.0 6.0x2.0 8.0x3.0 7.0x3.0 7.0x3.0 7.6x3.0 37 FWJ1 8.0x2.5 7.0x2.5 6.0x2.0 5.0x2.0 6.0x2.0 6.4x2.2 38 FWJ2 7.0x2.0 10.0x2.0 8.0x2.0 4.5x2.0 6.0x2.0 7.1x2.0 39 FWJ3 6.0x2.5 6.0x2.0 8.0x2.0 7.0x2.0 7.0x2.0 6.8x2.1 40 FWJ4 6.0x2.0 5.0x2.0 5.0x2.0 6.0x2.5 5.0x2.0 5.4x2.1 41 FWJ5 5.0x2.5 4.0x2.5 4.0x2.5 4.0x2.5 5.0x2.5 4.4x2.5 42 FWJ6 6.0x2.5 5.0x2.5 4.0x2.5 5.0x2.5 5.0x2.5 5.0x2.5 43 FWJ7 5.0x3.0 4.0x2.5 5.0x2.5 4.0x2.5 5.0x2.5 4.6x2.6 44 FWJ8 5.5x4.0 5.0x2.0 5.0x2.5 5.0x2.0 5.0x2.0 5.1x2.5 45 FWJ9 7.0x3.0 6.0x2.5 5.5x2.5 5.0x2.5 6.0x2.75 5.9x2.65 46 FWJ10 6.0x2.5 5.0x2.0 6.0x2.5 7.0x3.0 5.0x2.5 5.8x2.5 47 FWJ11 5.0x2.0 5.5x2.5 6.0x2.75 5.0x1.75 5.0x1.25 5.3x2.05 48 FWJ12 7.0x3.0 8.0x3.0 6.0x2.5 6.0x2.75 6.0x2.5 6.6x2.75 49 FWJ13 8.0x2.5 6.0x2.0 6.0x2.5 7.0x2.5 6.0x2.0 6.6x2.3 50 FWJ14 7.0x2.0 8.0x2.0 8.0x2.0 7.0x2.0 6.0x2.0 7.2x2.0
Continued……
207
51 FWJ15 6.0x2.5 8.0x2.5 5.0x2.5 6.0x2.5 8.0x2.5 6.6x2.5 52 FWJ16 8.0x2.5 7.0x2.0 6.0x2.0 7.0x2.0 7.0x2.0 7.0x2.1 53 FWJ17 6.0x2.0 5.0x2.5 8.0x2.5 6.0x2.0 6.0x2.0 6.2x2.2 54 FWJ18 7.0x2.75 8.0x3.0 6.0x2.5 6.0x2.5 6.0x2.5 6.6x2.65 55 FWJ19 8.0x2.0 6.0x2.0 8.0x2.5 6.0x2.5 8.0x2.0 7.2x2.2 56 FWJ20 8.0x2.5 7.0x2.5 6.0x2.0 5.0x2.0 6.0x2.5 6.4x2.3 57 FWJ21 5.0x2.5 7.5x2.5 5.0x2.0 7.5x2.5 5.0x1.75 6.0x2.25 58 FWJ22 6.0x2.5 7.0x3.0 5.0x2.5 8.0x3.0 6.0x2.5 6.4x2.7 59 FWJ23 7.0x3.0 8.0x3.25 6.0x2.5 7.0x3.0 6.0x2.5 6.8x2.85 60 FWJ24 5.0x2.75 6.0x2.5 5.5x2.5 6.0x2.5 7.0x3.0 5.9x2.65 61 FWJ25 6.0x2.5 7.0x2.5 8.0x3.0 6.0x2.5 7.0x2.5 6.8x2.6 62 FWJ26 5.0x2.5 6.0x2.5 6.0x2.5 5.0x2.5 6.0x2.5 5.6x2.5 63 FWJ27 7.0x2.75 6.0x2.5 7.0x3.0 8.0x3.0 6.0x2.5 6.8x2.75 64 FWJ28 8.0x3.0 10.0x3.5 7.0x3.0 7.0x3.0 8.0x3.0 8.0x3.1 65 FWJ29 9.0x3.5 8.0x3.0 10.0x3.5 7.0x2.75 8.0x3.0 8.4x3.15 66 FWJ30 6.0x2.5 7.0x3.0 6.0x2.75 6.0x2.5 6.0x2.5 6.2x2.65 67 FWJ31 7.0x3.0 8.0x3.0 7.0x3.0 9.0x3.0 7.0x3.0 7.6x3.0 68 FWJ32 5.0x2.5 6.0x2.5 9.0x3.0 6.0x2.5 6.0x2.5 6.4x2.6 69 FWJ33 8.0x3.0 5.0x2.5 5.0x2.5 6.0x2.5 8.0x3.0 6.4x2.7 70 FWJ34 6.0x3.0 7.0x3.0 7.0x3.0 8.0x3.0 8.0x3.0 7.2x3.0 71 FWJ35 5.0x3.0 7.0x2.0 6.0x2.5 8.0x2.0 6.0x3.0 6.4x2.5 72 FWJ36 4.0x3.0 5.0x2.0 7.0x2.0 5.0x2.0 6.0x2.5 5.4x2.3 73 FWJ37 5.0x2.0 6.0x2.0 5.0x3.0 6.0x2.0 4.0x2.0 5.2x2.2 74 FWJ38 4.0x3.0 6.0x2.5 3.0x2.0 4.0x2.0 6.0x2.0 4.6x2.3 75 FWJ39 6.0x2.0 5.0x2.0 5.0x2.0 6.0x2.5 7.0x2.0 5.8x2.1 76 FWJ40 5.0x2.5 6.0x2.0 6.0x2.0 5.0x2.0 5.0x2.0 5.4x2.1 77 FWJ41 6.0x2.5 6.0x3.0 6.0x2.0 5.0x2.0 6.0x2.5 5.8x2.4 78 FWJ42 6.0x3.0 5.0x2.0 6.0x3.0 4.0x3.0 5.0x2.0 5.2x2.6 79 FWJ43 5.0x2.0 5.0x2.0 6.0x2.0 4.0x2.0 5.0x2.0 5.0x2.0 80 FWJ44 6.0x2.0 6.0x2.5 6.0x2.0 8.0x2.5 5.0x2.0 6.2x2.2 81 FWJ45 5.0x3.0 5.0x2.5 8.0x3.0 4.0x2.0 5.0x2.0 5.4x2.5 82 FWJ46 7.0x3.0 6.0x3.0 6.0x2.5 8.0x3.0 5.0x2.0 6.4x2.7 83 FWJ47 10.0x2.5 10.0x3.0 7.5x2.5 7.5x2.5 7.0x2.5 8.4x2.6 84 FWJ48 8.0x3.0 9.0x3.5 7.0x2.5 6.0x2.5 7.0x2.5 7.4x2.8 85 FWJ49 10.0x3.0 10.0x2.5 7.0x3.0 7.5x2.5 8.0x3.0 8.5x2.8 86 FWJ50 7.5x2.5 8.0x3.0 6.0x2.5 9.0x3.0 8.0x3.0 7.7x2.8 87 FWJ51 8.0x3.0 7.0x2.5 6.0x2.5 6.0x2.5 6.0x2.5 6.6x2.6 88 FWJ52 5.0x2.5 6.0x2.75 7.0x3.0 8.0x3.0 10.0x3.0 7.2x2.85 89 FWJ53 7.0x3.0 8.0x3.0 10.0x3.0 10.0x3.0 8.0x3.0 8.6x3.0 90 FWJ54 8.0x3.0 10.0x4.0 6.0x3.0 6.0x3.0 7.0x3.0 7.4x3.2 91 FWJ55 6.0x2.5 7.0x3.0 8.0x2.0 6.0x2.0 6.0x2.0 6.6x2.3 92 FWJ56 8.0x3.0 7.0x2.5 5.0x2.0 5.0x2.5 6.0x3.0 6.2x2.6 93 FWJ57 10.0x3.0 8.0x3.5 6.0x2.5 7.0x2.0 5.0x2.5 7.2x2.7 94 FWJ58 5.0x2.5 8.0x3.0 8.0x2.5 10.0x4.0 8.0x3.5 7.8x3.1 95 FWJ59 5.0x2.0 10.0x3.0 7.0x3.0 8.0x3.0 5.0x2.0 7.0x2.6 96 FWJ60 6.0x2.5 5.0x2.0 5.0x2.0 7.0x3.0 5.0x2.5 5.6x2.4 97 FWJ61 10.0x3.5 8.0x2.5 5.0x2.0 5.0x2.0 5.0x2.0 6.6x2.4 98 FWJ62 6.0x3.5 7.0x2.5 8.0x2.5 8.0x2.5 4.5x2.0 6.7x2.6 99 FWJ63 5.0x2.0 8.0x3.0 5.0x2.0 9.0x3.0 5.0x2.0 6.4x2.4 100 FWJ64 4.5x2.0 9.0x2.5 6.0x2.5 6.0x2.0 3.0x2.0 5.7x2.2 101 FWJ65 8.0x3.0 5.0x2.0 6.0x2.0 5.0x2.5 3.0x2.0 5.4x2.3 102 FWJ66 8.0x2.0 8.0x3.0 6.0x2.5 6.0x2.0 6.0x2.0 6.8x2.3 103 FWJ67 7.0x2.5 8.0x3.0 8.0x3.0 5.0x2.5 6.0x2.0 6.8x2.6 104 FWJ68 5.0x2.5 5.0x2.5 8.0x3.0 6.0x2.0 8.0x2.5 6.4x2.5 105 FWJ69 8.0x3.0 8.0x2.0 6.0x2.0 5.0x2.0 5.0x2.5 6.4x2.3
Continued……
208
106 FWJ70 6.0x2.0 5.0x2.5 5.0x2.0 8.0x3.0 7.0x3.0 6.2x2.5 107 FWG1 5.0x3.0 5.0x2.0 8.0x3.0 9.0x3.0 10.0x3.0 7.4x2.8 108 FWG2 5.0x2.0 7.0x3.0 5.0x2.0 6.0x2.5 6.0x2.5 5.8x2.4 109 FWG3 4.0x2.0 8.0x3.0 5.0x3.0 5.0x3.0 8.0x3.0 6.0x2.8 110 FWG4 8.0x3.0 5.0x2.0 5.0x3.0 6.0x2.5 6.0x2.5 6.0x2.6 111 FWG5 6.0x3.0 5.0x2.5 8.0x3.0 6.0x3.0 5.0x3.0 6.0x2.9 112 FWG6 6.0x2.5 7.0x3.0 8.0x3.0 5.0x2.0 5.0x2.0 6.2x2.5 113 FWG7 7.0x3.0 6.0x2.5 5.0x2.0 6.0x2.5 5.0x2.5 5.8x2.5 114 FWG8 6.0x2.5 6.0x2.0 5.0x2.0 8.0x3.0 5.0x2.5 6.0x2.4 115 FWG9 5.0x3.0 5.0x3.0 6.0x3.0 8.0x3.0 5.0x3.0 5.8x3.0 116 FWG10 7.0x3.0 8.0x3.0 6.0x3.0 5.0x2.5 5.0x2.5 6.2x2.8 117 FWG11 5.0x2.0 6.0x2.0 5.0x2.5 8.0x3.0 10.0x3.0 6.8x2.5 118 FWG12 6.0x2.5 6.0x3.0 6.0x3.0 8.0x3.0 5.0x2.0 6.2x2.7 119 FWG13 6.0x2.0 5.0x2.0 4.0x2.5 4.0x2.5 9.0x3.0 5.6x2.4 120 FWG14 8.0x2.0 6.0x2.5 6.0x2.0 5.0x2.0 4.0x2.5 5.8x2.2 121 FWG15 6.0x2.5 6.0x2.5 8.0x2.5 6.0x3.0 4.0x2.5 6.0x2.6 122 FWG16 5.0x2.5 4.0x2.5 6.0x2.0 6.0x2.5 4.0x2.5 5.0x2.4 123 FWG17 6.0x2.5 6.0x2.5 5.0x2.5 5.0x2.5 5.0x2.5 5.4x2.5 124 FWG18 4.0x2.0 6.0x2.5 5.0x2.0 5.0x2.5 8.0x2.5 5.6x2.3 125 FWG19 9.0x3.0 8.0x2.5 6.0x2.5 6.0x2.5 5.0x2.0 6.8x2.5 126 FWG20 7.0x3.0 6.0x2.5 6.0x2.5 4.0x2.0 5.0x2.5 5.6x2.5 127 FWG21 5.0x2.5 7.0x3.0 8.0x3.0 6.0x3.0 6.0x2.5 6.4x2.8 128 FWG22 6.0x2.5 8.0x2.75 5.0x2.0 5.0x2.5 7.0x3.0 6.2x2.55 129 FWG23 6.0x2.5 5.0x2.5 5.0x2.5 5.0x2.5 6.0x2.5 5.4x2.5 130 FWG24 4.0x3.0 6.0x2.5 7.0x3.0 6.0x2.5 5.0x2.5 5.6x2.7 131 FWG25 8.0x3.0 6.0x2.5 6.0x3.0 5.0x2.5 5.0x2.0 6.0x2.6 132 FWG26 5.0x2.5 4.0x2.0 5.0x2.5 8.0x3.0 5.0x2.75 5.4x2.55 133 FWS1 7.0x3.0 8.0x3.0 6.0x2.5 9.0x3.0 10.0x3.0 8.0x2.9 134 FWS2 5.0x2.5 5.0x2.5 6.0x2.5 3.5x2.5 4.5x2.5 4.8x2.5 135 FWS3 5.0x2.5 5.0x2.5 5.0x2.5 5.0x2.5 6.0x2.5 5.2x2.5 136 FWS4 7.0x3.0 6.0x2.5 8.0x3.0 5.0x2.0 5.0x2.5 6.2x2.6 137 FWS5 4.5x2.5 6.0x2.5 5.0x2.5 6.0x2.5 5.0x2.5 5.3x2.5 138 FWS6 6.0x2.5 3.5x2.5 4.5x2.0 5.0x2.5 5.0x2.5 4.8x2.4 139 FWS7 7.0x3.0 7.0x3.0 6.0x2.5 6.0x2.5 6.0x2.5 6.4x2.7 140 FWS8 5.0x2.5 4.0x2.5 5.0x2.75 6.0x2.5 4.0x2.0 4.8x2.45 141 FWS9 9.0x3.0 5.0x2.5 5.0x2.5 5.0x2.5 5.0x2.5 5.8x2.6 142 FWS10 6.0x2.5 5.0x2.5 5.0x2.5 4.0x2.5 6.0x2.5 5.2x2.5 143 FWS11 7.0x3.0 10.0x3.0 6.0x2.5 5.0x2.5 4.0x2.0 6.4x2.6 144 FWS12 5.0x2.5 6.0x2.75 7.0x3.0 5.0x2.5 5.0x2.5 5.6x2.65 145 FWS13 6.0x2.5 5.0x2.5 8.0x3.0 5.0x2.5 5.0x2.5 5.8x2.6 146 FWN1 6.0x2.5 5.0x2.5 5.0x2.5 6.0x2.5 5.0x2.5 5.4x2.5 147 FWN2 7.0x2.5 5.0x2.0 5.0x2.0 5.0x2.0 6.0x2.0 5.6x2.1 148 FWN3 5.0x2.5 5.0x2.5 6.0x2.5 5.0x2.75 6.0x2.5 5.4x2.55 149 FWN4 4.0x2.0 5.0x2.0 6.0x2.5 5.0x2.0 6.0x2.5 5.2x2.2 150 FWN5 7.0x2.0 7.0x2.0 5.0x2.0 5.0x2.0 5.0x2.0 5.8x2.0 151 FWN6 5.0x2.5 4.0x2.0 4.0x2.0 6.0x2.5 5.0x2.0 4.8x2.2 152 FWN7 6.0x2.0 7.0x3.0 5.0x2.25 5.0x2.0 5.0x2.0 5.6x2.25 153 FWN8 5.0x2.5 5.0x2.75 5.0x2.5 5.0x3.0 5.0x3.0 5.0x2.75 154 FWN9 3.5x2.0 5.0x2.5 6.25x2.5 5.0x2.5 5.0x2.0 4.95x2.3 155 FWN10 5.0x2.5 5.0x2.5 3.0x1.5 5.0x1.25 2.5x1.25 4.1x1.8 156 FWN11 6.0x2.5 5.0x2.5 7.0x2.5 6.0x2.5 8.0x3.0 6.4x2.6 157 FWN12 5.0x2.0 8.0x3.0 4.0x2.0 6.0x2.5 5.0x2.5 5.6x2.4 158 FWN13 5.0x2.5 5.0x2.5 6.0x2.5 7.0x2.75 6.0x2.5 5.8x2.55 159 FWN14 6.0x2.5 5.0x2.0 5.0x2.5 5.0x2.5 4.0x2.0 5.0x2.3 160 FWN15 5.0x2.0 5.0x2.0 7.0x3.0 6.0x2.5 6.0x2.5 5.8x2.4
Continued……
209
161 FWN16 2.5x2.0 2.0x1.5 4.0x3.0 5.0x3.0 4.0x3.0 3.5x 2.5 162 FWN17 5.0x2.5 3.5x2.5 6.0x2.5 4.0x2.0 5.0x3.0 4.7x2.5 163 FWN18 4.0x2.5 8.0x3.0 5.0x3.0 6.0x3.0 5.0x2.5 5.6x2.8 164 FWN19 7.0x3.0 8.0x3.0 8.0x3.0 8.0x3.0 7.0x3.0 7.6x3.0 165 FWN20 3.5x2.5 4.5x2.0 5.0x2.5 5.0x2.5 5.0x2.5 4.6x2.4 166 FWN21 4.0x2.0 6.0x2.5 5.0x2.75 4.0x2.5 4.0x2.0 4.6x2.35 167 FWN22 7.0x3.0 8.0x3.5 6.0x2.5 5.0x2.5 5.0x2.5 6.2x2.8 168 FWN23 5.0x3.0 5.0x2.5 7.0x3.0 4.0x2.0 4.0x2.0 5.0x2.5 169 FWN24 6.0x2.5 8.0x3.0 6.0x3.0 5.5x3.0 6.0x3.0 6.3x2.9 170 FWN25 5.0x3.0 4.0x2.0 4.0x2.0 6.0x3.0 5.0x3.0 4.8x2.6 171 FWN26 7.0x3.0 5.0x2.5 8.0x3.0 5.0x2.5 5.0x3.0 6.0x2.8 172 FWN27 5.0x3.0 8.0x3.0 6.0x3.0 7.0x3.0 7.0x3.0 6.6x3.0 173 FWN28 6.0x3.0 5.0x2.5 5.0x2.5 6.0x2.5 5.0x2.5 5.4x2.6 174 FWN29 4.0x2.5 7.0x3.0 5.0x2.5 5.5x2.5 6.0x3.0 5.5x2.7 175 FWN30 6.0x2.5 5.0x2.5 4.0x2.0 6.0x2.5 4.0x2.0 5.0x2.3 176 FWN31 8.0x3.0 6.0x2.75 7.0x3.0 7.0x3.0 6.0x2.5 6.8x2.85 177 FWM1 6.0x2.0 6.0x2.5 8.0x3.0 7.0x3.0 5.0x2.0 6.4x2.5 178 FWM2 9.0x3.0 8.0x3.0 5.0x2.5 5.0x2.0 5.0x2.0 6.4x2.5 179 FWM3 4.5x2.0 5.0x2.5 5.0x2.5 8.0x3.0 6.0x2.5 5.7x2.5 180 FWM4 6.0x2.5 5.0x2.5 6.0x2.5 8.0x3.0 6.0x2.5 6.2x2.6 181 FWL1 5.0x2.0 4.0x2.0 4.0x2.5 3.0x2.0 5.0x1.25 4.2x1.95 182 FWL2 5.0x2.5 4.0x2.0 4.0x2.0 5.0x2.5 6.0x2.5 4.8x2.3 183 FWL3 7.0x3.0 5.0x2.5 6.0x3.0 5.0x2.0 5.0x3.0 5.6x2.7 184 FWL4 5.0x1.25 3.0x1.25 5.0x2.5 3.5x1.25 3.5x2.5 4.0x1.75 185 FWL5 3.0x1.5 5.0x1.5 3.5x2.0 3.5x2.5 5.0x1.25 4.0x1.75 186 FWL6 5.0x2.5 5.0x2.5 5.0x2.0 5.0x2.5 5.0x2.5 5.0x2.4 187 FWL7 4.0x2.0 6x2.0 7.0x2.5 6.0x2.0 6.0x2.0 5.8x2.1 188 FWL8 4.0x1.75 4.0x1.75 4.0x1.75 4.0x1.75 4.0x1.75 4.0x1.75 189 FWL9 5.0x2.0 5.0x1.25 3.0x2.5 3.5x1.5 3.5x1.5 4.0x1.75 190 FWL10 4.0x2.0 5.0x2.0 6.0x2.0 9.0x2.0 6.0x2.0 6.0x2.0 191 FWL11 6.25x2.75 5.0x3.0 5.0x2.75 4.5x3.0 4.5x3.0 5.05x2.9 192 FWL12 4.0x2.0 5.0x2.0 5.0x1.25 3.0x2.0 4.0x2.5 4.2x1.95 193 FWL13 5.0x2.5 5.0x2.5 6.0x3.0 5.0x3.0 6.0x3.0 5.4x2.8 194 FWL14 5.0x3.0 6.0x2.5 7.0x3.0 5.0x3.0 5.0x3.0 5.6x2.9 195 FWL15 6.0x2.5 6.0x3.0 5.0x2.5 5.0x2.5 5.0x3.0 5.4x2.7 196 FWL16 7.0x3.0 7.0x3.0 6.0x3.0 5.0x2.5 5.0x2.5 6.0x2.8 197 FWB1 6.0x2.0 4.0x1.5 6.5x2.0 4.0x2.0 5.0x2.0 5.1x1.9 198 FWB2 5.0x2.5 5.0x2.0 5.0x3.0 7.0x2.5 5.0x2.5 5.4x2.5 199 FWB3 6.0x2.5 6.0x2.0 3.0x1.5 3.5x1.5 8.0x2.0 5.3x1.9 200 FWB4 5.0x2.5 4.5x1.5 5.5x2.0 6.0x2.5 4.0x1.5 5.0x2.0 201 FWB5 8.0x2.5 6.0x2.0 5.0x2.0 5.0x2.0 6.0x2.5 6.0x2.2 202 FWB6 5.0x2.5 5.0x2.5 3.75x2.5 5.0x2.5 6.25x2.5 5.0x2.5 203 FWB7 4.5x1.5 5.0x2.0 3.5x2.0 6.0x2.0 6.0x2.0 5.0x1.9 204 FWB8 4.0x2.5 5.0x3.0 6.0x2.5 3.5x2.0 5.0x2.5 4.7x2.5 205 FWB9 6.5x3.5 5.0x3.0 7.0x3.0 5.0x3.0 8.0x3.0 6.3x3.1 206 FWB10 7.0x3.0 5.0x3.5 7.0x2.5 7.0x3.0 7.0x2.5 6.6x2.9 207 FWB11 5.5x3.5 7.0x3.5 6.0x3.5 5.0x2.5 7.0x3.5 6.1x3.3 208 FWB12 5.5x2.0 6.0x2.75 4.5x2.0 6.0x2.0 3.0x1.25 5.0x2.0 209 FWB13 5.0x2.5 5.0x2.0 4.5x3.0 5.0x1.25 5.5x1.25 5.0x2.0 210 FWB14 6.0x2.5 5.0x2.0 4.0x2.0 6.0x2.0 6.0x2.0 5.4x2.1 211 FWK1 5.0x2.5 5.0x2.5 6.0x2.5 8.0x2.5 5.0x2.5 5.8x2.5 212 FWK2 7.0x3.0 7.0x3.0 6.0x2.0 5.0x2.0 5.0x2.5 6.0x2.5 213 FWK3 6.0x2.0 6.0x2.5 6.0x2.5 6.0x2.0 8.0x3.0 6.4x2.4
210
Appendix 3: Morphological characterization of Fusarium isolates: Size of macro-conidia. No. Isolate
ID No. Reading 1
Reading 2
Reading 3
Reading 4
Reading 5
Mean (µm)
1 FWC1 18.0x2.5 20.0x2.5 24.0x2.5 18.0x2.5 16.0x2.5 19.2x2.5 2 FWC2 38.0x4.0 48.0x4.5 32.0x5.0 42.0x4.5 50.0x5.0 42.0x4.6 3 FWC3 40.0x4.0 25.0x4.0 36.0x4.0 26.0x4.0 16.0x3.0 28.6x3.8 4 FWC4 14.0x2.5 15.0x3.0 15.0x2.5 12.0x2.0 15.0x3.0 14.2x2.6 5 FWC5 16.0x2.5 12.0x2.5 10.0x2.0 15.0x2.5 16.0x2.5 13.8x2.4 6 FWC6 20.0x3.0 16.0x3.0 15.0x3.0 18.0x3.0 20.0x3.0 17.8x3.0 7 FWC7 16.0x3.0 18.0x3.0 12.0x2.0 12.0x2.0 15.0x3.0 14.6x2.6 8 FWC8 24.0x6.0 17.0x4.0 18.0x3.0 20.0x3.0 20.0x3.0 19.8x3.8 9 FWC9 14.0x2.5 20.0x2.5 16.0x2.5 24.0x3.0 15.0x2.5 17.8x2.6 10 FWC10 30.0x4.0 24.0x3.5 22.0x3.0 16.0x3.0 20.0x3.0 22.4x3.3 11 FWC11 25.0x4.0 24.0x4.0 30.0x4.5 20.0x3.5 24.0x4.0 24.6x4.0 12 FWC12 16.0x2.5 10.0x2.0 16.0x3.0 25.0x4.0 11.0x2.0 15.6x2.7 13 FWC13 28.0x3.0 26.0x2.5 30.0x3.5 30.0x3.5 8.0x2.5 24.4x3.0 14 FWC14 30.0x3.5 35.0x4.0 25.0x4.0 8.0x2.5 30.0x4.0 25.6x3.6 15 FWC15 32.0x4.0 26.0x3.0 28.0x3.0 22.0x3.0 23.0x3.0 26.2x3.2 16 FWC16 10.0x3.0 25.0x5.0 30.0x3.0 32.0x4.0 28.0x3.5 25.0x3.7 17 FWC17 12.0x2.5 36.0x4.0 23.0x3.0 18.0x3.0 14.0x2.5 20.6x3.0 18 FWC18 30.0x3.5 35.0x4.0 26.0x4.0 10.0x3.0 8.0x3.0 21.8x3.5 19 FWC19 28.0x3.0 35.0x4.0 30.0x3.5 26.0x2.5 10.0x2.5 25.8x3.1 20 FWC20 30.0x3.5 28.0x2.5 30.0x4.0 26.0x2.5 12.0x2.5 25.2x3.0 21 FWC21 28.0x3.0 36.0x4.0 22.0x3.0 32.0x4.0 30.0x4.0 29.6x3.6 22 FWC22 16.0x2.5 28.0x3.5 29.0x3.5 34.0x4.0 18.0x2.5 25.0x3.2 23 FWC23 26.0x3.0 24.0x3.0 26.0x3.0 30.0x4.0 26.0x3.0 26.4x3.2 24 FWC24 31.0x3.0 25.0x3.0 27.0x3.0 26.0x3.0 30.0x3.0 27.8x3.0 25 FWC25 26.0x3.0 25.0x3.0 23.0x3.0 18.0x3.0 21.0x3.0 22.6x3.0 26 FWC26 30.0x4.0 25.0x4.0 20.0x3.0 26.0x4.0 22.0x3.0 24.6x3.6 27 FWC27 31.0x4.0 34.0x4.0 32.0x4.0 26.0x4.0 22.0x4.0 29.0x4.0 28 FWC28 28.0x4.0 18.0x2.5 30.0x4.0 20.0x3.0 22.0x3.0 23.6x3.3 29 FWC29 30.0x4.0 26.0x4.0 15.0x2.5 18.0x3.0 20.0x3.0 21.8x3.3 30 FWC30 16.0x2.5 30.0x4.0 34.0x4.0 20.0x3.0 26.0x4.0 25.2x3.5 31 FWC31 24.0x3.0 27.0x4.0 25.0x4.0 30.0x4.0 34.0x4.0 28.0x3.8 32 FWA1 18.0x2.5 12.0x2.5 16.0x2.5 27.0x3.5 24.0x3.0 19.4x2.8 33 FWA2 28.0x4.0 34.0x4.0 24.0x3.0 25.0x4.0 33.0x4.0 28.8x3.8 34 FWA3 35.0x4.5 28.0x4.0 32.0x5.0 26.0x4.0 20.0x3.5 28.2x4.2 35 FWA4 18.0x3.0 15.0x3.0 18.0x3.0 28.0x3.5 20.0x3.0 19.8x3.1 36 FWA5 30.0x4.0 26.0x4.0 18.0x2.5 18.0x2.5 15.0x3.0 21.4x3.2 37 FWJ1 20.0x3.0 16.0x2.5 22.0x3.0 18.0x2.5 18.0x3.0 18.8x2.8 38 FWJ2 14.0x2.0 18.0x2.5 22.0x3.0 20.0x3.0 16.0x2.5 18.0x2.6 39 FWJ3 11.0x2.5 10.0x2.0 11.0x2.0 18.0x2.5 16.0x2.5 13.2x2.3 40 FWJ4 16.0x3.0 18.0x3.0 12.0x2.5 14.0x2.5 16.0x3.0 15.2x2.8 41 FWJ5 12.0x2.5 11.0x2.0 22.0x3.0 18.0x2.5 12.0x2.5 15.0x2.5 42 FWJ6 18.0x3.0 16.0x2.5 16.0x3.0 22.0x3.0 15.0x3.0 17.4x2.9 43 FWJ7 20.0x3.0 18.0x3.0 22.0x3.0 20.0x3.0 20.0x3.0 20.0x3.0 44 FWJ8 12.5x2.5 10.0x2.5 12.5x2.5 11.0x2.5 15.0x2.5 12.2x2.5 45 FWJ9 28.0x3.5 18.0x2.5 10.0x2.5 11.0x2.5 11.0x2.5 15.6x2.7 46 FWJ10 18.0x2.5 20.0x3.0 14.0x2.0 20.0x2.5 20.0x3.0 18.4x2.6 47 FWJ11 13.0x2.5 12.0x2.0 10.0x3.0 10.0x2.0 10.0x2.5 11.0x2.4 48 FWJ12 14.0x2.5 11.0x2.0 14.0x2.0 12.0x2.0 22.0x2.5 14.6x2.2 49 FWJ13 12.0x2.5 28.0x3.0 22.0x2.5 14.0x2.5 11.0x2.0 17.4x2.5 50 FWJ14 26.0x2.0 23.0x2.0 15.0x2.5 14.0x2.5 20.0x2.5 19.6x2.3
Continued……
211
51 FWJ15 10.0x2.0 8.0x2.0 10.0x2.0 12.0x2.0 14.0x2.0 10.8x2.0 52 FWJ16 30.0x3.0 22.0x2.5 11.0x2.0 16.0x2.0 18.0x2.5 19.4x2.4 53 FWJ17 16.0x2.5 14.0x2.0 11.0x2.0 18.0x2.5 20.0x2.5 15.8x2.3 54 FWJ18 17.0x2.5 26.0x2.5 22.0x2.5 10.0x2.0 10.0x2.0 17.0x2.3 55 FWJ19 12.0x2.0 16.0x2.5 22.0x2.5 15.0x2.0 18.0x2.5 16.6x2.3 56 FWJ20 26.0x3.0 42.0x4.5 50.0x5.0 26.0x3.5 16.0x3.0 32.0x3.8 57 FWJ21 30.0x3.0 30.0x3.0 26.0x3.0 25.0x3.0 30.0x3.0 28.2x3.0 58 FWJ22 48.0x5.0 36.0x4.5 20.0x2.5 26.0x3.0 24.0x3.0 30.8x3.6 59 FWJ23 22.0x2.5 30.0x3.0 26.0x2.5 30.0x3.0 30.0x3.0 27.6x2.8 60 FWJ24 16.0x2.5 25.0x3.0 11.0x2.5 22.0x2.5 26.0x3.0 20.0x2.7 61 FWJ25 15.0x2.5 30.0x3.0 26.0x3.0 16.0x2.5 25.0x3.0 22.4x2.8 62 FWJ26 42.0x4.0 11.0x2.5 28.0x3.0 22.0x2.5 16.0x2.5 23.8x2.9 63 FWJ27 36.0x4.0 25.0x3.0 15.0x2.5 16.0x2.5 13.0x2.5 21.0x2.9 64 FWJ28 14.0x2.5 10.0x2.5 23.0x3.0 36.0x3.5 16.0x2.5 19.8x2.8 65 FWJ29 16.0x2.5 22.0x3.0 26.0x3.0 24.0x3.0 22.0x3.0 22.0x2.9 66 FWJ30 20.0x3.0 28.0x3.0 11.0x2.5 16.0x2.5 16.0x2.5 18.2x2.7 67 FWJ31 18.0x2.5 18.0x2.5 10.0x2.0 20.0x2.5 18.0x2.5 16.8x2.4 68 FWJ32 17.0x2.5 22.0x2.5 31.0x3.0 16.0x2.5 18.0x2.5 20.8x2.6 69 FWJ33 18.0x2.5 20.0x2.5 27.0x3.0 10.0x2.5 15.0x2.5 18.0x2.6 70 FWJ34 14.0x2.5 25.0x3.0 12.0x2.5 18.0x2.5 16.0x2.5 17.0x2.6 71 FWJ35 24.0x3.0 38.0x3.5 16.0x2.5 30.0x3.0 22.0x3.0 26.0x3.0 72 FWJ36 16.0x2.5 9.0x2.5 10.0x2.5 12.0x2.5 16.0x2.5 12.6x2.5 73 FWJ37 15.0x2.5 22.0x3.0 16.0x2.5 20.0x2.5 15.0x2.5 17.6x2.6 74 FWJ38 16.0x2.5 17.0x2.5 18.5x2.5 20.0x2.5 20.0x2.5 18.3x2.5 75 FWJ39 14.5x2.5 20.0x2.5 17.0x2.5 25.5x2.5 25.0x2.5 20.4x2.5 76 FWJ40 18.0x2.5 32.0x3.5 23.0x2.5 18.0x2.5 18.0x2.5 21.8x2.7 77 FWJ41 14.0x2.5 10.0x2.25 16.0x2.5 26.0x2.75 18.0x2.5 16.8x2.5 78 FWJ42 27.0x2.5 18.0x2.5 15.0x2.5 15.5x2.5 20.0x2.5 19.1x2.5 79 FWJ43 18.0x2.5 20.0x2.5 20.0x2.5 26.0x2.75 20.0x2.5 20.8x2.55 80 FWJ44 15.0x2.5 20.0x2.5 25.0x2.5 16.0x2.5 20.0x2.5 19.2x2.5 81 FWJ45 33.0x3.5 16.0x2.5 22.0x3.0 20.0x3.0 19.5x3.0 22.1x3.0 82 FWJ46 14.0x2.5 10.0x2.0 18.0x2.5 20.0x3.0 20.0x3.0 16.4x2.6 83 FWJ47 18.5x2.5 15.0x2.5 15.0x2.5 20.0x2.5 16.0x2.5 16.9x2.5 84 FWJ48 15.0x2.5 17.0x2.75 22.0x3.0 27.0x3.0 12.0x2.5 18.6x2.75 85 FWJ49 11.0x2.0 15.0x3.0 10.0x3.0 15.0x4.0 15.0x3.5 13.2x3.1 86 FWJ50 18.0x2.5 22.0x3.0 26.0x3.0 20.0x3.0 24.0x3.0 22.0x2.9 87 FWJ51 20.0x2.5 19.0x2.5 20.0x2.5 26.0x3.0 22.0x2.5 21.4x2.6 88 FWJ52 26.0x3.0 22.0x3.0 28.0x3.0 18.0x2.5 22.0x3.0 23.2x2.9 89 FWJ53 16.0x2.5 27.0x3.0 18.0x2.5 20.0x3.0 26.0x3.0 21.4x2.8 90 FWJ54 16.0x3.0 17.5x4.0 11.0x3.0 19.0x4.0 17.0x4.0 16.1x3.6 91 FWJ55 15.0x2.5 21.0x3.5 16.0x3.0 20.0x3.0 16.0x3.0 17.6x3.0 92 FWJ56 22.0x3.0 18.0x2.75 12.5x2.5 22.0x3.0 20.0x3.0 18.9x2.85 93 FWJ57 25.0x3.0 18.0x3.0 12.0x2.5 16.0x3.0 15.0x3.0 17.2x2.9 94 FWJ58 18.0x3.0 16.0x2.5 18.0x3.0 20.0x3.0 18.0x3.0 18.0x2.9 95 FWJ59 14.0x2.5 15.0x2.5 26.0x3.0 18.0x3.0 15.0x2.5 17.6x2.7 96 FWJ60 15.5x2.5 18.0x2.5 22.0x3.0 21.0x3.0 18.0x2.5 18.9x2.7 97 FWJ61 18.0x2.5 24.0x3.0 22.0x3.0 22.0x3.0 18.0x2.5 20.8x2.8 98 FWJ62 17.0x2.5 15.0x2.5 12.0x2.5 12.0x2.5 20.0x3.0 15.2x2.6 99 FWJ63 18.0x3.0 12.0x2.5 20.0x2.5 26.0x3.0 12.5x2.5 17.7x2.7 100 FWJ64 15.0x2.5 17.0x2.5 17.5x2.5 15.0x2.5 18.0x2.5 16.5x2.5 101 FWJ65 11.5x2.5 22.0x3.0 24.0x3.0 16.0x2.5 20.0x3.0 18.7x2.8 102 FWJ66 32.0x3.5 18.0x3.0 20.0x2.5 14.0x2.5 18.0x2.75 20.4x2.85 103 FWJ67 20.0x2.75 28.0x3.5 11.0x2.5 17.0x2.5 21.0x2.5 19.4x2.75 104 FWJ68 22.0x3.0 24.0x3.0 16.0x2.5 18.0x2.5 32.0x3.5 22.4x2.9 105 FWJ69 12.0x2.5 21.0x2.5 30.0x3.5 20.0x2.5 12.0x2.5 19.0x2.7
Continued……
212
106 FWJ70 17.5x2.5 18.0x2.5 17.0x2.5 21.0x2.5 11.0x2.5 16.9x2.5 107 FWG1 20.0x3.0 15.0x2.5 16.0x3.0 18.0x3.0 12.0x2.5 16.2x2.8 108 FWG2 18.0x2.5 12.0x3.0 18.0x3.0 18.0x2.5 16.0x2.5 16.4x2.7 109 FWG3 18.5x3.5 17.0x3.0 22.0x3.0 24.0x3.0 20.0x3.0 20.3x3.1 110 FWG4 14.0x2.5 20.0x3.0 20.0x3.0 16.0x2.5 15.0x2.5 17.0x2.7 111 FWG5 30.0x3.5 14.0x2.5 16.0x2.5 18.0x3.0 22.0x3.0 20.0x2.9 112 FWG6 12.0x2.5 18.0x2.5 15.0x2.5 18.0x2.5 17.0x2.5 16.0x2.5 113 FWG7 16.0x2.5 26.0x3.0 16.0x2.75 11.0x2.5 20.0x2.5 17.8x2.65 114 FWG8 23.0x2.75 20.0x2.5 16.0x2.5 26.0x3.0 30.0x3.0 23.0x2.75 115 FWG9 14.0x2.5 16.0x2.5 12.0x2.5 12.0x2.5 18.0x2.5 14.4x2.5 116 FWG10 28.0x4.0 14.0x2.5 18.0x3.0 22.0x3.0 20.0x3.0 20.4x3.1 117 FWG11 12.0x2.75 16.0x2.5 12.0x2.5 28.0x3.5 24.0x3.0 18.4x2.85 118 FWG12 20.0x3.0 22.0x3.0 16.0x2.5 15.0x2.5 30.0x3.5 20.6x2.9 119 FWG13 16.0x3.5 18.0x4.0 21.0x4.0 18.0x3.5 24.0x4.0 19.4x3.8 120 FWG14 18.0x4.0 17.0x3.5 20.0x3.0 16.0x3.5 22.0x3.0 18.6x3.4 121 FWG15 19.0x4.0 16.0x3.5 30.0x4.0 18.0x3.5 15.0x2.5 19.6x3.5 122 FWG16 18.0x3.5 14.0x3.0 26.0x4.0 20.0x3.0 22.0x3.5 20.0x3.4 123 FWG17 12.0x2.5 18.0x4.0 18.0x4.0 21.0x4.0 25.0x4.0 18.8x3.7 124 FWG18 20.0x3.0 16.0x2.5 24.0x3.5 16.0x3.0 20.0x3.0 19.2x3.0 125 FWG19 15.0x2.5 18.0x4.0 20.0x3.0 16.0x3.5 26.0x4.0 19.0x3.4 126 FWG20 20.0x3.0 16.0x3.5 16.0x3.5 28.4x4.0 15.0x2.5 19.0x3.3 127 FWG21 22.0x4.0 12.0x2.5 15.0x2.5 26.0x4.0 12.0x2.5 17.4x3.1 128 FWG22 30.0x4.0 22.0x3.5 18.0x3.0 16.0x3.0 14.0x2.5 20.0x3.2 129 FWG23 12.0x2.5 15.0x2.5 20.0x3.0 21.0x4.0 20.0x3.0 17.6x3.0 130 FWG24 20.0x3.0 18.0x3.0 12.0x2.5 22.0x3.5 25.0x4.0 19.4x3.2 131 FWG25 15.0x2.5 21.0x3.5 22.0x3.0 15.0x2.5 15.0x2.5 17.6x2.8 132 FWG26 14.0x2.5 16.0x2.5 15.0x2.5 30.0x4.0 22.0x4.0 19.4x3.1 133 FWS1 28.0x3.0 14.0x2.5 22.0x3.0 20.0x3.0 19.0x3.0 20.6x2.9 134 FWS2 10.0x2.5 22.0x2.5 18.0x2.5 21.0x2.5 20.0x2.5 18.2x2.5 135 FWS3 30.0x3.0 16.0x2.5 26.0x3.0 14.0x2.5 10.0x2.5 19.2x2.7 136 FWS4 13.0x2.5 22.0x3.0 20.0x2.5 16.0x2.5 15.0x2.5 17.2x2.6 137 FWS5 18.0x2.5 20.0x2.5 18.0x2.5 17.5x2.5 20.0x2.5 18.7x2.5 138 FWS6 15.0x2.5 16.0x2.5 23.0x2.5 16.0x2.5 16.0x2.5 17.2x2.5 139 FWS7 28.0x3.0 16.0x2.5 18.0x2.5 16.0x2.5 16.0x2.5 18.8x2.6 140 FWS8 19.0x2.5 22.5x2.5 15.0x2.5 16.0x2.75 20.0x2.5 18.5x2.55 141 FWS9 20.0x2.5 27.0x3.0 18.0x2.5 20.0x2.5 20.0x2.5 21.0x2.6 142 FWS10 11.0x2.5 12.0x2.5 23.0x2.5 16.0x2.5 18.0x2.5 16.0x2.5 143 FWS11 20.0x2.5 28.0x3.0 22.0x3.0 15.0x2.5 15.5x2.5 20.1x2.7 144 FWS12 18.0x2.5 10.0x2.5 11.0x2.5 10.0x2.5 20.0x2.5 13.8x2.5 145 FWS13 22.0x3.0 16.0x2.5 10.0x2.5 18.0x2.5 16.0x2.5 16.4x2.6 146 FWN1 17.0x5.0 22.0x5.0 16.0x4.5 11.0x3.0 14.0x3.0 16.0x4.1 147 FWN2 22.0x3.0 18.0x2.5 20.0x3.0 22.0x3.0 15.0x2.5 19.4x2.8 148 FWN3 15.0x2.5 15.0x2.5 22.0x3.0 15.0x2.5 16.0x2.5 16.6x2.6 149 FWN4 16.0x3.0 17.5x3.0 11.0x2.5 8.0x2.5 24.0x3.0 15.3x2.8 150 FWN5 20.0x3.0 18.0x2.0 22.0x3.0 20.0x3.0 15.0x2.5 19.0x2.7 151 FWN6 9.0x2.5 18.0x2.5 26.0x3.0 10.0x2.5 16.0x2.5 15.8x2.6 152 FWN7 12.5x2.5 10.0x2.5 10.0x2.5 15.0x2.5 12.0x2.5 11.9x2.5 153 FWN8 15.0x2.0 15.0x2.0 16.5x2.5 15.0x2.5 14.5x2.5 15.2x2.3 154 FWN9 7.0x2.5 10.0x2.5 12.5x2.5 15.0x2.5 15.0x2.5 11.9x2.5 155 FWN10 10.0x2.5 10.0x2.5 22.5x7.5 12.5x2.5 10.0x2.5 13.0x3.5 156 FWN11 22.0x3.0 11.5x2.5 16.0x2.5 18.0x2.5 20.0x2.5 17.5x2.6 157 FWN12 25.0x3.0 12.0x2.5 10.0x2.5 10.0x2.5 12.0x2.5 13.8x2.6 158 FWN13 10.0x2.5 10.0x2.5 15.0x2.5 26.0x3.0 14.5x2.5 15.1x2.6 159 FWN14 12.0x2.5 14.0x2.5 10.0x2.5 12.5x2.5 12.0x2.5 12.1x2.5 160 FWN15 16.0x2.5 20.0x3.0 26.0x3.0 10.0x2.5 12.0x2.5 16.8x2.7
Continued……
213
161 FWN16 30.0x3.5 12.0x2.5 22.0x2.75 18.0x2.5 12.0x2.5 18.8x2.75 162 FWN17 20.0x3.0 18.0x3.0 26.0x3.0 12.0x2.5 18.0x3.0 18.8x2.9 163 FWN18 10.0x2.5 30.0x3.0 18.0x2.5 20.0x2.5 17.0x2.5 19.0x2.6 164 FWN19 16.0x2.5 18.0x2.5 16.0x2.5 28.0x3.0 11.0x2.5 17.8x2.6 165 FWN20 24.0x3.0 12.0x2.5 11.5x2.5 20.0x3.0 12.0x2.5 15.9x2.7 166 FWN21 12.0x2.5 13.0x2.5 16.0x2.5 12.0x2.5 12.0x2.5 13.0x2.5 167 FWN22 14.0x2.5 33.0x3.0 26.0x3.0 16.0x2.5 12.0x2.5 20.2x2.7 168 FWN23 22.0x3.0 20.0x3.0 16.0x2.5 16.5x2.5 11.0x2.5 17.1x2.7 169 FWN24 13.0x2.5 20.0x2.5 18.0x2.5 20.0x2.5 21.0x2.5 18.4x2.5 170 FWN25 11.0x2.5 28.0x3.5 16.0x2.5 15.0x2.5 20.0x2.5 18.0x2.5 171 FWN26 10.0x2.25 14.0x2.5 17.5x2.5 12.0x2.5 10.0x2.5 12.7x2.35 172 FWN27 29.0x3.0 12.0x2.5 18.0x2.5 17.0x2.5 12.0x2.25 17.6x2.55 173 FWN28 11.0x2.5 20.0x2.5 26.0x3.0 16.0x2.5 12.0x2.5 17.0x2.6 174 FWN29 18.0x2.5 20.0x2.5 12.0x2.75 16.0x2.5 16.0x2.5 16.4x2.55 175 FWN30 15.0x2.5 16.0x2.5 32.0x3.0 20.0x2.5 16.0x2.5 19.8x2.6 176 FWN31 12.0x2.5 16.0x2.5 18.0x2.5 10.0x2.5 10.0x2.5 13.2x2.5 177 FWM1 22.0x3.0 18.0x3.0 21.0x2.5 15.0x2.5 16.0x2.5 18.4x2.7 178 FWM2 20.0x3.0 14.0x2.5 11.0x2.5 24.0x3.0 12.0x2.5 16.2x2.7 179 FWM3 30.0x3.5 17.0x2.5 20.0x2.5 17.0x2.5 15.0x2.5 19.8x2.7 180 FWM4 16.0x2.5 22.0x2.5 24.0x3.0 20.0x2.5 12.0x2.5 18.8x2.6 181 FWL1 10.0x3.0 10.5x2.5 15.0x2.5 12.5x2.0 17.0x2.0 13.0x2.4 182 FWL2 22.0x3.5 15.5x2.5 12.0x2.5 12.0x2.5 10.0x2.5 14.3x2.7 183 FWL3 15.0x3.0 12.0x3.0 12.0x3.0 20.0x3.0 16.0x3.0 15.0x3.0 184 FWL4 10.0x2.0 15.0x2.5 7.5x2.0 10.0x2.0 10.0x2.0 10.5x2.1 185 FWL5 10.0x2.0 10.0x2.5 12.5x2.0 8.5x2.5 10.0x2.0 10.2x2.2 186 FWL6 10.0x3.0 7.5x2.5 8.0x2.5 7.5x2.0 7.0x2.5 8.0x2.5 187 FWL7 15.0x3.0 20.0x3.0 12.0x3.0 14.0x3.0 20.0x3.0 16.2x3.0 188 FWL8 8.5x2.0 10.0x1.5 11.5x2.5 10.0x2.0 10.0x2.0 10.0x2.0 189 FWL9 10.0x2.0 8.5x2.0 12.5x2.75 8.5x2.25 10.5x2.25 10.0x2.25 190 FWL10 20.0x3.0 18.0x3.0 11.0x2.5 26.0x3.5 20.0x3.0 19.0x3.0 191 FWL11 10.0x2.5 10.5x3.0 12.5x3.5 8.5x3.5 8.5x2.5 10.0x3.0 192 FWL12 10.0x3.0 15.0x2.5 17.0x2.0 12.5x2.0 10.5x2.5 13.0x2.4 193 FWL13 12.0x3.0 12.0x3.0 12.0x3.0 16.0x3.0 15.0x3.0 13.4x3.0 194 FWL14 15.0x3.0 16.0x3.0 15.0x3.0 12.0x2.5 12.0x3.0 14.0x2.9 195 FWL15 12.0x3.0 14.0x3.0 20.0x3.0 18.0x3.0 20.0x3.0 16.8x3.0 196 FWL16 18.0x3.0 15.0x2.5 16.0x3.0 16.0x3.5 20.0x3.0 17.0x3.0 197 FWB1 15.0x3.0 12.0x2.0 15.0x3.0 16.0x3.0 18.0x3.0 15.2x2.8 198 FWB2 12.5x2.5 15.0x2.5 12.5x2.5 10.0x2.5 10.0x2.5 12.0x2.5 199 FWB3 16.0x2.0 18.0x2.5 15.0x2.0 10.0x2.0 12.0x2.0 14.2x2.1 200 FWB4 10.0x2.0 10.0x2.0 10.0x2.0 7.5x2.0 15.0x2.0 10.5x2.0 201 FWB5 12.5x2.5 15.0x2.5 15.0x2.5 12.0x2.5 16.0x2.5 14.1x2.5 202 FWB6 15.0x2.5 12.5x2.5 10.0x2.5 12.5x2.5 10.0x2.5 12.0x2.5 203 FWB7 15.0x3.0 12.0x2.0 16.0x2.5 12.0x2.5 15.0x3.0 14.0x2.6 204 FWB8 10.0x3.0 8.0x2.5 12.0x2.5 10.0x2.5 10.0x2.5 10.0x2.6 205 FWB9 10.0x2.5 16.0x2.5 26.0x3.0 30.0x3.0 12.0x2.5 18.8x2.7 206 FWB10 20.0x3.0 18.0x3.0 22.0x3.0 15.0x3.0 20.0x3.0 19.0x3.0 207 FWB11 10.0x2.5 11.0x2.5 18.0x2.5 22.0x3.0 12.0x2.5 14.6x2.6 208 FWB12 10.0x2.5 10.0x2.5 8.0x2.5 12.0x2.5 10.0x2.5 10.0x2.5 209 FWB13 12.0x2.5 10.0x2.5 12.5x2.5 7.5x2.5 10.0x2.5 10.4x2.5 210 FWB14 18.0x3.0 12.0x2.5 15.0x2.5 15.0x2.0 12.0x3.0 14.4x2.6 211 FWK1 26.0x3.0 15.0x2.5 20.0x2.5 18.0x2.5 15.0x2.5 18.8x2.6 212 FWK2 21.0x2.75 20.0x2.5 17.5x2.5 20.0x2.5 20.0x2.5 19.7x2.55 213 FWK3 17.0x2.5 12.0x2.5 18.0x2.5 12.0x2.5 20.0x3.0 15.8x2.6
214
Appendix 4: Morphological characterization of Fusarium isolates: Diameter of chlamydospores.
No. Isolate ID No.
Reading 1
Reading 2
Reading 3
Reading 4
Reading 5
Mean (µm)
1 FWC1 9 10 15 16 12 12.4 2 FWC2 6 6 10 8 8 7.6 3 FWC3 11 13 10 8 7 9.8 4 FWC4 6 8 10 8 10 8.4 5 FWC5 8 8 8 9 10 8.6 6 FWC6 8 9 8 8 10 8.6 7 FWC7 10 11 8 9 8 9.2 8 FWC8 10 7 6 8 6 7.4 9 FWC9 9 10 12 9 9 9.8 10 FWC10 6 8 8 8 9 7.8 11 FWC11 10 9 16 10 12 11.4 12 FWC12 18 20 10 9 18 15.0 13 FWC13 8 6 6 8 8 7.2 14 FWC14 6 5 9 8 8 7.2 15 FWC15 12 16 12 20 10 14.0 16 FWC16 8 6 9 6 6 7.0 17 FWC17 10 15 9 7 6 9.4 18 FWC18 9 8 8 9 6 8.0 19 FWC19 8 7 6 8 9 7.6 20 FWC20 8 6 7 7 6 6.8 21 FWC21 10 16 9 7 18 12.0 22 FWC22 17 9 12 10 15 12.6 23 FWC23 12 13 14 10 10 11.8 24 FWC24 12 14 11 12 12 12.2 25 FWC25 11 8 15 14 10 11.6 26 FWC26 11 12 10 13 11 11.4 27 FWC27 8 14 15 10 10 11.4 28 FWC28 8 20 15 10 13 13.2 29 FWC29 7 15 18 12 10 12.4 30 FWC30 12 8 16 10 9 11.0 31 FWC31 10 8 15 8 10 10.2 32 FWA1 11 12 10 12 13 11.6 33 FWA2 10 8 12 9 7 9.2 34 FWA3 13 14 12 10 10 11.8 35 FWA4 18 15 6 8 8 11.0 36 FWA5 10 12 15 15 8 12.0 37 FWJ1 18 9 10 20 18 15.0 38 FWJ2 16 8 10 10 10 10.8 39 FWJ3 10 8 6 7 8 7.8 40 FWJ4 12 10 9 12 9 10.4 41 FWJ5 8 9 9 10 9 9.0 42 FWJ6 11 10 12 9 9 10.2 43 FWJ7 9 11 12 10 12 10.8 44 FWJ8 8 8 8 10 14 9.6 45 FWJ9 12 15 10 11 8 11.2 46 FWJ10 9 20 8 11 8 11.2 47 FWJ11 10 11 10 12 12 11.0 48 FWJ12 18 16 12 10 14 14.0 49 FWJ13 12 12 10 8 10 10.4 50 FWJ14 11 14 12 10 10 11.4
Continued……
215
51 FWJ15 16 10 7 9 18 12.0 52 FWJ16 8 8 10 11 10 9.4 53 FWJ17 22 20 20 15 12 17.8 54 FWJ18 10 10 8 8 12 9.6 55 FWJ19 14 12 12 11 10 11.8 56 FWJ20 8 8 8 5 8 7.4 57 FWJ21 18 12 14 10 10 12.8 58 FWJ22 9 9 8 5 8 7.8 59 FWJ23 11 10 9 7 9 9.2 60 FWJ24 9 8 7 8 5 7.4 61 FWJ25 10 9 10 8 8 9.0 62 FWJ26 12 14 8 7 8 9.8 63 FWJ27 7 8 8 9 8 8.0 64 FWJ28 12 12 10 8 8 10.0 65 FWJ29 18 8 10 11 12 11.8 66 FWJ30 11 12 14 9 9 11.0 67 FWJ31 10 10 12 10 10 10.4 68 FWJ32 8 8 10 7 5 7.6 69 FWJ33 6 8 12 12 12 10.0 70 FWJ34 8 8 8 9 14 9.4 71 FWJ35 9 6 7 6 8 7.2 72 FWJ36 14 12 8 10 8 10.4 73 FWJ37 10 9 9 18 10 11.2 74 FWJ38 8 12 14 10 18 12.4 75 FWJ39 12 15 8 10 10 11.0 76 FWJ40 13 16 15 20 8 14.4 77 FWJ41 16 20 12 11 10 13.8 78 FWJ42 18 14 8 12 13 13.0 79 FWJ43 12 9 12 9 10 10.4 80 FWJ44 20 22 10 12 10 14.8 81 FWJ45 8 10 8 14 9 9.8 82 FWJ46 12 12 13 7 8 10.4 83 FWJ47 9 6 10 9 12 9.2 84 FWJ48 18 16 8 12 7 12.2 85 FWJ49 10 9 11 10 8 9.6 86 FWJ50 15 16 12 12 7 12.4 87 FWJ51 10 20 14 10 6 12.0 88 FWJ52 5 6 8 8 8 7.0 89 FWJ53 10 8 10 12 10 10.0 90 FWJ54 20 18 14 7 6 13.0 91 FWJ55 5 8 10 10 8 8.2 92 FWJ56 20 20 10 6 8 12.8 93 FWJ57 12 12 8 12 12 11.2 94 FWJ58 5 5 7 8 14 7.8 95 FWJ59 15 20 12 14 10 14.2 96 FWJ60 8 10 14 10 8 10.0 97 FWJ61 6 8 9 12 12 9.4 98 FWJ62 5 7 6 11 12 8.2 99 FWJ63 12 20 16 12 10 14.0 100 FWJ64 8 10 9 8 14 9.8 101 FWJ65 8 12 14 20 15 13.8 102 FWJ66 12 13 7 8 10 10.0 103 FWJ67 13 12 13 20 10 13.6 104 FWJ68 10 10 12 15 9 11.2 105 FWJ69 8 9 6 12 8 8.6
Continued……
216
106 FWJ70 11 12 8 8 15 10.8 107 FWG1 24 10 15 14 10 14.6 108 FWG2 16 15 12 12 8 12.6 109 FWG3 18 18 8 12 12 13.6 110 FWG4 8 9 8 10 8 8.6 111 FWG5 12 15 12 12 10 12.2 112 FWG6 14 22 14 12 16 15.6 113 FWG7 5 9 8 8 8 7.6 114 FWG8 12 8 7 12 10 9.8 115 FWG9 20 18 14 8 6 13.2 116 FWG10 6 18 10 10 8 10.4 117 FWG11 20 20 10 10 10 14.0 118 FWG12 8 12 8 9 8 9.0 119 FWG13 7 8 15 10 8 9.6 120 FWG14 8 9 12 12 15 11.2 121 FWG15 5 8 8 8 6 7.0 122 FWG16 22 10 8 7 12 11.8 123 FWG17 12 14 8 9 8 10.2 124 FWG18 16 11 10 9 12 11.6 125 FWG19 10 10 10 8 6 8.8 126 FWG20 12 18 18 20 8 15.2 127 FWG21 10 10 8 10 12 10.0 128 FWG22 12 12 12 12 8 11.2 129 FWG23 6 14 15 16 20 14.2 130 FWG24 10 9 10 8 10 9.4 131 FWG25 15 8 8 7 6 8.8 132 FWG26 9 20 12 12 12 13.0 133 FWS1 11 10 9 8 9 9.4 134 FWS2 13 12 11 12 12 12.0 135 FWS3 8 8 10 6 10 8.4 136 FWS4 9 8 8 10 8 8.6 137 FWS5 10 22 11 18 9 14.0 138 FWS6 7 10 5 8 8 7.6 139 FWS7 12 12 12 5 8 9.8 140 FWS8 9 8 8 8 10 8.6 141 FWS9 10 9 11 8 8 9.2 142 FWS10 20 15 10 10 10 13.0 143 FWS11 8 9 5 8 8 7.6 144 FWS12 10 12 18 22 10 14.4 145 FWS13 9 8 12 10 8 9.4 146 FWN1 6 10 8 8 8 8.0 147 FWN2 9 9 8 7 7 8.0 148 FWN3 9 20 10 12 10 12.2 149 FWN4 8 12 8 6 8 8.4 150 FWN5 7 8 10 9 9 8.6 151 FWN6 6 5 12 10 8 8.2 152 FWN7 14 12 10 9 9 10.8 153 FWN8 10 10 9 8 10 9.4 154 FWN9 12 8 8 8 8 8.8 155 FWN10 7 8 9 9 10 8.6 156 FWN11 14 10 12 10 10 11.2 157 FWN12 9 12 12 11 10 10.8 158 FWN13 8 6 8 8 10 8.0 159 FWN14 12 22 5 10 10 11.8 160 FWN15 6 8 8 8 8 7.6
Continued……
217
161 FWN16 12 12 11 12 10 11.4 162 FWN17 20 11 10 7 9 11.4 163 FWN18 6 8 10 12 8 8.8 164 FWN19 18 12 12 10 10 12.4 165 FWN20 6 15 8 6 6 8.2 166 FWN21 10 10 10 9 9 9.6 167 FWN22 24 18 16 12 15 17.0 168 FWN23 18 12 12 16 10 13.6 169 FWN24 12 15 8 8 9 10.4 170 FWN25 10 10 10 7 10 9.4 171 FWN26 8 5 8 10 8 7.8 172 FWN27 12 7 8 8 11 9.2 173 FWN28 20 8 9 5 6 9.6 174 FWN29 10 8 10 10 10 9.6 175 FWN30 8 6 8 8 8 7.6 176 FWN31 14 20 10 10 8 12.4 177 FWM1 14 12 10 14 15 13.0 178 FWM2 14 11 15 10 15 13.0 179 FWM3 9 14 10 8 13 10.8 180 FWM4 16 18 10 8 10 12.4 181 FWL1 10 11 10 9 9 9.8 182 FWL2 7 8 10 9 8 8.4 183 FWL3 10 12 10 10 12 10.8 184 FWL4 9 9 8 7 6 7.8 185 FWL5 9 9 10 9 9 9.2 186 FWL6 6 7 6 8 8 7.0 187 FWL7 16 10 9 8 10 10.6 188 FWL8 8 8 9 10 9 8.8 189 FWL9 8 9 9 8 10 8.8 190 FWL10 10 8 8 7 6 7.8 191 FWL11 12 12 10 9 9 10.4 192 FWL12 9 9 8 10 9 9.0 193 FWL13 12 10 9 9 11 10.2 194 FWL14 12 12 8 11 10 10.6 195 FWL15 12 12 11 12 10 11.4 196 FWL16 10 10 10 10 8 9.6 197 FWB1 12 9 12 12 13 11.6 198 FWB2 6 7 8 8 10 7.8 199 FWB3 9 10 10 9 12 10.0 200 FWB4 10 9 12 10 10 10.2 201 FWB5 12 10 10 11 10 10.6 202 FWB6 6 6 7 8 10 7.4 203 FWB7 12 10 9 9 9 9.8 204 FWB8 6 8 9 8 8 7.8 205 FWB9 12 12 11 12 10 11.4 206 FWB10 8 8 10 10 8 8.8 207 FWB11 10 10 14 12 11 11.4 208 FWB12 12 12 10 11 10 11.0 209 FWB13 10 11 9 12 12 10.8 210 FWB14 10 9 18 20 18 15.0 211 FWK1 6 12 14 20 8 12.0 212 FWK2 18 14 12 15 10 13.8 213 FWK3 21 15 16 8 10 14.0
218
Appendix 5: Morphological characterization of Fusarium isolates: Interseptal distance.
No. Isolate ID No.
Reading 1
Reading 2
Reading 3
Reading 4
Reading 5
Mean (µm)
1 FWC1 18 22 10 25 20 19.0 2 FWC2 15 32 26 30 29 26.4 3 FWC3 9 13 24 15 10 14.2 4 FWC4 10 19 21 13 20 16.6 5 FWC5 17 14 6 10 8 11.0 6 FWC6 20 16 27 25 30 23.6 7 FWC7 30 14 33 14 17 21.6 8 FWC8 25 13 18 7 20 16.6 9 FWC9 12 10 10 14 18 12.8 10 FWC10 10 7 8 16 10 10.2 11 FWC11 11 5 4 10 9 7.8 12 FWC12 22 14 15 14 15 16.0 13 FWC13 10 10 10 9 8 9.4 14 FWC14 16 12 12 10 8 11.6 15 FWC15 18 20 18 26 15 19.4 16 FWC16 29 22 15 16 10 18.4 17 FWC17 6 10.5 22 28 10 15.3 18 FWC18 32 9 9 10 8 13.6 19 FWC19 16 18 16 21 15 17.2 20 FWC20 12 12 12 10 16 12.4 21 FWC21 19.5 26 20 20 20 21.1 22 FWC22 14 16 11 23 22 17.2 23 FWC23 22 10 10 12 15 13.8 24 FWC24 20 18 10 28 30 21.2 25 FWC25 25 16 12 10 28 18.2 26 FWC26 25 30 16 20 20 22.2 27 FWC27 10 24 32 22 21 21.8 28 FWC28 12 8 19 7 12 11.6 29 FWC29 21 16 26 10 8 16.2 30 FWC30 7 19 22 24 15 17.4 31 FWC31 18 11 16 8 16 13.8 32 FWA1 16 20 15 20 22 18.6 33 FWA2 18 18 21 20 10 17.4 34 FWA3 8 10 10 12 10 10.0 35 FWA4 22 13 18 16 20 17.8 36 FWA5 18 14 10 20 12 14.8 37 FWJ1 10 20 26 22 15 18.6 38 FWJ2 20 18 22 8 10 15.6 39 FWJ3 7 15 10 8 13 10.6 40 FWJ4 15 15 15 20 15 16.0 41 FWJ5 10 16 20 12 24 16.4 42 FWJ6 10 14 10 20 20 14.8 43 FWJ7 8 10 13 6 10 9.4 44 FWJ8 16 20 22 12 18 17.6 45 FWJ9 26 10 23 18 14 18.2 46 FWJ10 15 10 12 12 19 13.6 47 FWJ11 5 22 8 10 10 11.0 48 FWJ12 16 18 18 24 22 19.6 49 FWJ13 32 6 14 14 20 17.2 50 FWJ14 14 12 21.5 12 12 14.3
Continued……
219
51 FWJ15 23 8 10 26 21 17.6 52 FWJ16 17 20 9 34 10 18.0 53 FWJ17 30 26 32 20 12 24.0 54 FWJ18 10 12 8 9 12 10.2 55 FWJ19 15 11 42 30 10 21.6 56 FWJ20 10 10 25 12 22 15.8 57 FWJ21 14 12 12 15 12 13.0 58 FWJ22 18 15 8 20 12 14.6 59 FWJ23 22 25 17 12 12 17.6 60 FWJ24 30 15 16 4 10 15.0 61 FWJ25 10 14 20 8 16 13.6 62 FWJ26 8 20 18 12 15 14.6 63 FWJ27 10 20 13 8 10 12.2 64 FWJ28 12 12 22 12 12 14.0 65 FWJ29 14 28 20 12 10 16.8 66 FWJ30 21 18 16 16 14 17.0 67 FWJ31 9 10 16 12 20 13.4 68 FWJ32 15 8 7 10 25 13.0 69 FWJ33 16 18 24 20 18 19.2 70 FWJ34 12 10 9 10 10 10.2 71 FWJ35 10 12 11 10 10 10.6 72 FWJ36 18 17 8 9 12 12.8 73 FWJ37 18 9 15 10 11 12.6 74 FWJ38 20 13 10 10 8 12.2 75 FWJ39 18 15 8 20 12 14.6 76 FWJ40 24 12 15 15 11 15.4 77 FWJ41 18 30 20 8 22 19.6 78 FWJ42 17 25 8 16 20 17.2 79 FWJ43 19 20 15 23 28 21.0 80 FWJ44 14 32 15 20 12 18.6 81 FWJ45 20 25 22 11 18 19.2 82 FWJ46 18 15 10 12 12 13.4 83 FWJ47 30 16 27 8 10 18.2 84 FWJ48 26 18 16 30 22 22.4 85 FWJ49 16 10 10 28 15 15.8 86 FWJ50 18 15 22 16 20 18.2 87 FWJ51 30 10 13 22 15 18.0 88 FWJ52 19 17 24 16 30 21.2 89 FWJ53 32 21 12 8 10 16.6 90 FWJ54 16 10 15 21 20 16.4 91 FWJ55 10 20 28 30 8 19.2 92 FWJ56 32 23 26 10 10 20.2 93 FWJ57 8 10 11 28 16 14.6 94 FWJ58 24 20 22 35 38 27.8 95 FWJ59 12 26 28 14 10 18.0 96 FWJ60 15 18 30 12 22 19.4 97 FWJ61 11 16 10 24 10 14.2 98 FWJ62 34 11 25 12 19 20.2 99 FWJ63 8 23 22 10 12 15.0 100 FWJ64 20 22 15 12 12 16.2 101 FWJ65 25 22 12 10 8 15.4 102 FWJ66 15 16 18 22 20 18.2 103 FWJ67 18 18 19 20 10 17.0 104 FWJ68 12 22 20 10 8 14.4 105 FWJ69 18 22 20 10 18 17.6
Continued……
220
106 FWJ70 19 18 26 22 28 22.6 107 FWG1 45 28 38 20 24 31.0 108 FWG2 40 15 22 24 48 29.8 109 FWG3 11 12 20 19 14 15.2 110 FWG4 15 10 10 14 10 11.8 111 FWG5 22 24 20 18 20 20.8 112 FWG6 29 17 22 22 16 21.2 113 FWG7 10 30 24 15 24 20.6 114 FWG8 16 16 23 18 10 16.6 115 FWG9 22 23 15 11 10 16.2 116 FWG10 39 23 18 18 22 24.0 117 FWG11 25 28 20 22 11 21.2 118 FWG12 20 35 17 28 20 24.0 119 FWG13 15 10 12 14 10 12.2 120 FWG14 11 19 20 20 10 16.0 121 FWG15 18 20 10 12 12 14.4 122 FWG16 10 11 16 16 8 12.2 123 FWG17 12 12 14 22 20 16.0 124 FWG18 23 8 22 12 10 15.0 125 FWG19 10 10 12 8 18 11.6 126 FWG20 16 10 20 12 10 13.6 127 FWG21 15 18 16 22 22 18.6 128 FWG22 14 10 10 10 23 13.4 129 FWG23 17 20 20 19 20 19.2 130 FWG24 24 22 18 18 12 18.8 131 FWG25 20 11 12 8 22 14.6 132 FWG26 15 17 23 10 10 15.0 133 FWS1 10 20 10 11 10 12.2 134 FWS2 8 9 15 23 20 15.0 135 FWS3 16 7 7 14 16 12.0 136 FWS4 32 12 28 18 20 22.0 137 FWS5 14 18 22 24 28 21.2 138 FWS6 38 12 19 8 10 17.4 139 FWS7 18 14 12 12 12 13.6 140 FWS8 10 10 6 15 12 10.6 141 FWS9 21 22 15 18 10 17.2 142 FWS10 8 8 5 9.5 10 8.1 143 FWS11 12 20 20 12 8 14.4 144 FWS12 24 15 15 10 10 14.8 145 FWS13 10 8 22 13 22 15.0 146 FWN1 16 22 12 10 12 14.4 147 FWN2 6 20 13 10 14 12.6 148 FWN3 11 11 20 12 22 15.2 149 FWN4 10 4 6 8 20 9.6 150 FWN5 24 9 7 5.5 10 11.1 151 FWN6 12 20 6 10 10 11.6 152 FWN7 10 5 5 6 5 6.2 153 FWN8 16 9 9 5 10 9.8 154 FWN9 22 19 15 6 18 16.0 155 FWN10 12 12 20 15 15 14.8 156 FWN11 16 25 22 12 10 17.0 157 FWN12 4 12 10 10 10 9.2 158 FWN13 18 15 16 15 8 14.4 159 FWN14 8 7 12 10 16 10.6 160 FWN15 10 14 13 9 8 10.8
Continued……
221
161 FWN16 23 12 21 26 28 22.0 162 FWN17 30 32 26 15 29 26.4 163 FWN18 15 8 16 16 36 18.2 164 FWN19 20 24 12 15 15 17.2 165 FWN20 33 22 14 11 12 18.4 166 FWN21 10 9 34 10 10 14.6 167 FWN22 18 17.5 15 15 15 16.1 168 FWN23 22 25 8 7 11 14.6 169 FWN24 12 11 17 12 12 12.8 170 FWN25 18 20 20 20 10 17.6 171 FWN26 7 15 10 22 16 14.0 172 FWN27 30 22 32 12 18 22.8 173 FWN28 15 12 10 20 9 13.2 174 FWN29 22 24 12 12 18 17.6 175 FWN30 10 8 15 9 9 10.2 176 FWN31 33 24 18 18 6 19.8 177 FWM1 10 12 22 12 20 15.2 178 FWM2 19 18 20 8 8 14.6 179 FWM3 10 17 13 10 12 12.4 180 FWM4 8 11 16 12 12 11.8 181 FWL1 16 9 16 18 20 15.8 182 FWL2 11 20 13.5 14 12 14.1 183 FWL3 20 20 11 9 8 13.6 184 FWL4 10 6 4 10 5 7.0 185 FWL5 16 15.5 16 5 7 11.9 186 FWL6 4 22 8 8 9 10.2 187 FWL7 6 8 11 15 8 9.6 188 FWL8 28 16 10 20 3 15.4 189 FWL9 10 10 10 23 5 11.6 190 FWL10 7 6 4 20 16 10.6 191 FWL11 12 11.5 12 12 12 11.9 192 FWL12 10 18 14 29 7 15.6 193 FWL13 16 9 8 7 10 10.0 194 FWL14 12 12 15 12 24 15.0 195 FWL15 10 20 21 20 10 16.2 196 FWL16 7.5 12 18 19 24 16.1 197 FWB1 21 7 4 12 12 11.2 198 FWB2 16 10 6 8 7 9.4 199 FWB3 26 18 22 24 15 21.0 200 FWB4 10 6 3 5 4 5.6 201 FWB5 8 6 10 8 8 8.0 202 FWB6 26 10 10 10 10 13.2 203 FWB7 11 15 5.5 18 30 15.9 204 FWB8 4 9 6 6 10 7.0 205 FWB9 10 10 6 4 5 7.0 206 FWB10 10 10 19 25 7 14.2 207 FWB11 6 5 5 8 9 6.6 208 FWB12 6 6 4 8 9 6.6 209 FWB13 4 10 6 6 4 6.0 210 FWB14 12 21 9 8 8 11.6 211 FWK1 12 20 23 18 8 16.2 212 FWK2 17 19 15 22 20 18.6 213 FWK3 8 18 12 10 15 12.6
222
Appendix 6: Mean percent disease severity index: NARC-08-1 and Masoor-93: Pathogenicity test.
No.
Isolate ID
Disease Severity Index (%) NARC-08-1 Masoor-93
Rep 1 Rep 2 Rep 3 Mean Rep 1 Rep 2 Rep 3 Mean 1 FWC1 0 0 0 0 0 0 0 0 2 FWC2 0 0 0 0 0 0 0 0 3 FWC4 0 0 0 0 0 0 0 0 4 FWC5 55.5 51.1 53.3 53.3 6.66 2.22 2.22 3.7 5 FWC6 55.55 55.55 53.33 54.81 11.11 11.11 11.11 11.11 6 FWC7 0 0 0 0 0 0 0 0 7 FWC8 55.55 55.55 55.55 55.55 0 0 0 0 8 FWC9 22.22 22.22 22.22 22.22 0 0 0 0 9 FWC10 0 0 0 0 0 0 0 0 10 FWC11 62.22 62.22 55.55 60.00 2.22 0 2.22 1.48 11 FWC12 62.22 66.66 64.44 64.44 55.55 55.55 55.55 55.55 12 FWC15 86.66 88.88 88.88 88.14 66.66 66.66 64.44 65.92 13 FWC17 55.55 51.11 51.11 52.59 22.22 22.22 22.22 22.22 14 FWC21 44.44 44.44 46.66 45.18 0 0 0 0 15 FWC22 51.11 55.55 55.55 54.07 55.55 57.77 57.77 57.03 16 FWC23 57.77 60 53.33 57.03 2.22 2.22 2.22 2.22 17 FWA1 55.55 55.55 55.55 55.55 0 0 0 0 18 FWJ2 60 60 60 60 22.22 13.33 13.33 16.29 19 FWJ4 62.22 66.66 62.22 63.7 64.44 66.66 66.66 65.92 20 FWJ8 62.22 64.44 66.66 64.44 44.44 46.66 46.66 45.92 21 FWJ11 66.66 66.66 66.66 66.66 0 0 0 0 22 FWJ14 44.44 46.66 44.44 45.18 48.88 48.88 46.66 48.14 23 FWJ15 44.44 48.88 44.44 45.92 0 0 0 0 24 FWJ16 62.22 57.77 55.55 58.51 0 0 0 0 25 FWJ17 28.88 33.33 28.88 30.36 0 0 0 0 26 FWJ18 44.44 48.88 48.88 47.4 4.44 2.22 4.44 3.7 27 FWJ19 55.55 55.55 51.11 54.07 11.11 11.11 13.33 11.85 28 FWJ20 66.66 66.66 66.66 66.66 44.44 51.11 51.11 48.89 29 FWJ26 46.66 46.66 44.44 45.92 13.33 11.11 13.33 12.59 30 FWJ28 55.55 55.55 55.55 55.55 0 0 0 0 31 FWJ35 100 100 100 100 51.11 44.44 53.33 49.63 32 FWJ47 22.22 20 22.22 21.48 0 0 0 0 33 FWJ48 20 22.22 20 20.74 4.44 4.44 8.88 5.92 34 FWJ49 88.88 88.88 91.11 89.62 55.55 53.33 53.33 54.07 35 FWJ50 11.11 11.11 11.11 11.11 0 0 0 0 36 FWJ53 24.44 22.22 22.22 22.96 0 0 0 0 37 FWJ62 22.22 22.22 20 21.48 0 0 0 0 38 FWG1 91.11 88.88 88.88 89.62 62.22 55.55 55.55 57.77 39 FWG13 51.11 44.44 44.44 46.66 13.33 11.11 13.33 12.59 40 FWS1 44.44 44.44 44.44 44.44 2.22 2.22 2.22 2.22 41 FWS3 66.66 66.66 66.66 66.66 2.22 2.22 2.22 2.22 42 FWS5 55.55 51.11 53.33 53.33 51.11 51.11 44.44 48.89 43 FWS7 66.66 66.66 66.66 66.66 44.44 44.44 44.44 44.44 44 FWS9 60 57.77 60 59.26 0 0 0 0 45 FWS11 100 100 100 100 66.66 64.44 66.66 65.92 46 FWS13 100 100 100 100 22.22 33.33 33.33 29.63 47 FWN2 100 100 100 100 55.55 55.55 55.55 55.55 48 FWN4 55.55 51.11 55.55 54.07 2.22 2.22 2.22 2.22 49 FWN5 62.22 64.44 66.66 64.44 0 0 0 0
Continued……
223
50 FWN6 55.55 53.33 55.55 54.81 0 2.22 0 0.74 51 FWM1 24.44 24.44 20 22.96 0 0 0 0 52 FWL1 66.66 66.66 66.66 66.66 62.22 62.22 55.55 60.00 53 FWL2 88.88 91.11 91.11 90.37 66.66 66.66 66.66 66.66 54 FWL4 53.33 55.55 55.55 54.81 44.44 44.44 44.44 44.44 55 FWL5 62.22 66.66 66.66 65.18 0 0 0 0 56 FWL6 100 100 100 100 44.44 44.44 44.44 44.44 57 FWL7 55.55 55.55 55.55 55.55 0 0 0 0 58 FWL9 100 100 100 100 64.44 64.44 62.22 63.7 59 FWL11 44.44 44.44 44.44 44.44 0 0 0 0 60 FWL12 100 100 100 100 66.66 66.66 66.66 66.66 61 FWB3 51.11 44.44 44.44 46.66 0 0 0 0 62 FWB4 53.33 55.55 44.44 51.11 0 0 0 0 63 FWB9 44.44 44.44 51.11 46.66 0 0 0 0 64 FWB10 100 100 100 100 55.55 55.55 51.11 54.07 65 FWB11 44.44 44.44 44.44 44.44 55.55 55.55 55.55 55.55 66 FWK1 62.22 62.22 55.55 60.00 2.22 0 2.22 1.48 67 FWK2 88.88 88.88 91.11 89.62 44.44 44.44 44.44 44.44 68 Control 0 0 0 0 0 0 0 0
224
Appendix 7: Mean percent disease incidence: NARC-08-1 and Masoor-93: Pathogenicity test.
No.
Isolate ID
Disease Incidence (%) NARC-08-1 Masoor-93
Rep 1 Rep 2 Rep 3 Mean Rep 1 Rep 2 Rep 3 Mean 1 FWC1 0 0 0 0 0 0 0 0 2 FWC2 0 0 0 0 0 0 0 0 3 FWC4 0 0 0 0 0 0 0 0 4 FWC5 100 100 100 100 20 20 40 26.67 5 FWC6 100 100 100 100 100 100 100 100 6 FWC7 0 0 0 0 0 0 0 0 7 FWC8 100 100 100 100 0 0 0 0 8 FWC9 100 100 100 100 0 0 0 0 9 FWC10 0 0 0 0 0 0 0 0 10 FWC11 100 100 100 100 20 0 20 13.33 11 FWC12 100 100 100 100 100 100 100 100 12 FWC15 100 100 100 100 100 100 100 100 13 FWC17 100 100 100 100 20 20 0 13.33 14 FWC21 100 100 100 100 0 0 0 0 15 FWC22 100 100 100 100 100 80 80 86.7 16 FWC23 100 100 100 100 20 20 20 20 17 FWA1 100 100 100 100 0 0 0 0 18 FWJ2 100 100 100 100 100 100 100 100 19 FWJ4 100 100 100 100 100 100 100 100 20 FWJ8 100 100 100 100 100 100 100 100 21 FWJ11 100 100 100 100 0 0 0 0 22 FWJ14 100 100 100 100 100 100 100 100 23 FWJ15 100 100 100 100 0 0 0 0 24 FWJ16 100 100 100 100 0 0 0 0 25 FWJ17 100 100 100 100 0 0 0 0 26 FWJ18 100 100 100 100 20 20 20 20 27 FWJ19 100 100 100 100 20 20 40 26.67 28 FWJ20 100 100 100 100 100 100 100 100 29 FWJ26 100 100 100 100 20 20 20 20 30 FWJ28 100 100 100 100 0 0 0 0 31 FWJ35 100 100 100 100 100 100 100 100 32 FWJ47 100 100 100 100 0 0 0 0 33 FWJ48 100 100 100 100 20 20 20 20 34 FWJ49 100 100 100 100 100 100 100 100 35 FWJ50 100 100 100 100 0 0 0 0 36 FWJ53 100 100 100 100 0 0 0 0 37 FWJ62 100 100 100 100 0 0 0 0 38 FWG1 100 100 100 100 100 100 100 100 39 FWG13 100 100 100 100 40 20 40 33.33 40 FWS1 100 100 100 100 20 20 20 20 41 FWS3 100 100 100 100 20 20 20 20 42 FWS5 100 100 100 100 80 80 100 86.7 43 FWS7 100 100 100 100 100 100 100 100 44 FWS9 100 100 100 100 0 0 0 0 45 FWS11 100 100 100 100 100 100 100 100 46 FWS13 100 100 100 100 100 100 100 100 47 FWN2 100 100 100 100 100 100 100 100 48 FWN4 100 100 100 100 40 20 20 26.67 49 FWN5 100 100 100 100 0 0 0 0
Continued……
225
50 FWN6 100 100 100 100 0 20 0 6.67 51 FWM1 100 100 100 100 0 0 0 0 52 FWL1 100 100 100 100 100 100 100 100 53 FWL2 100 100 100 100 100 100 100 100 54 FWL4 100 100 100 100 100 100 100 100 55 FWL5 100 100 100 100 0 0 0 0 56 FWL6 100 100 100 100 100 100 100 100 57 FWL7 100 100 100 100 0 0 0 0 58 FWL9 100 100 100 100 100 100 100 100 59 FWL11 100 100 100 100 0 0 0 0 60 FWL12 100 100 100 100 100 100 100 100 61 FWB3 100 100 100 100 0 0 0 0 62 FWB4 100 100 100 100 0 0 0 0 63 FWB9 100 100 100 100 0 0 0 0 64 FWB10 100 100 100 100 100 100 100 100 65 FWB11 100 100 100 100 100 100 100 100 66 FWK1 100 100 100 100 40 0 20 20 67 FWK2 100 100 100 100 100 100 100 100 68 Control 0 0 0 0 0 0 0 0
226
Appendix 8: Mean grain yield reduction: NARC-08-1 and Masoor-93: Pathogenicity test.
No.
Isolate ID
Yield Reduction (%) NARC-08-1 Masoor-93
Rep 1 Rep 2 Rep 3 Mean Rep 1 Rep 2 Rep 3 Mean 1 FWC1 11.45 18.09 6.05 11.86 27.57 27.61 27.42 27.53 2 FWC2 14.90 11.02 26.53 17.48 14.84 23.7 18.62 19.05 3 FWC4 30.64 29.28 31.18 30.37 27.85 24.65 31.99 28.16 4 FWC5 52.27 53.78 49.09 51.71 16.81 23.9 39.72 26.81 5 FWC6 44.19 56.83 42.04 47.69 16.17 30.91 31.85 26.31 6 FWC7 15.74 17.52 16.09 16.45 16.60 17.17 23.86 19.21 7 FWC8 42.25 56.25 43.28 47.26 2.25 6.67 10.48 6.47 8 FWC9 14.05 20.89 18.99 17.98 17.30 23.43 15.12 18.62 9 FWC10 14.81 23.03 20.15 19.33 20.53 18.92 25.4 21.62 10 FWC11 48.48 47.70 31.67 42.62 34.39 37.64 31.99 34.67 11 FWC12 45.2 46.38 38.23 43.27 42.19 43.7 41.67 42.52 12 FWC15 100 100 100 100 49.93 46.73 44.69 47.12 13 FWC17 44.95 47.78 45.52 46.08 37.83 37.51 38.58 37.97 14 FWC21 48.48 31.74 32.75 37.66 16.60 12.79 30.24 19.88 15 FWC22 56.06 46.05 47.60 49.90 42.48 36.7 39.72 39.63 16 FWC23 46.8 46.88 50.91 48.20 30.73 37.98 35.35 34.69 17 FWA1 45.11 44.82 45.61 45.18 18.21 16.16 23.72 19.36 18 FWJ2 44.44 44.74 56.72 48.63 35.23 36.43 38.37 36.68 19 FWJ4 55.3 53.37 50.17 52.95 39.31 38.45 36.16 37.97 20 FWJ8 49.41 47.04 50.91 49.12 41.42 38.05 42.2 40.56 21 FWJ11 50.76 36.35 48.51 45.21 14.42 28.89 25.27 22.86 22 FWJ14 49.75 43.33 31.76 41.61 49.02 47.21 44.76 47.00 23 FWJ15 56.99 43.17 47.10 49.09 23.49 22.42 25 23.64 24 FWJ16 54.12 45.89 49 49.67 23.63 27.2 32.26 27.70 25 FWJ17 17.34 19.9 25.12 20.79 11.25 17.71 34.74 21.23 26 FWJ18 45.52 44.74 42.62 44.29 28.12 22.22 26.9 25.75 27 FWJ19 46 44.44 46.8 45.75 23.98 24.4 33.86 27.41 28 FWJ20 46.72 49.01 49.25 48.33 49.79 47.27 51.81 49.62 29 FWJ26 37.37 44.08 41.04 40.83 28.27 28.62 27.76 28.22 30 FWJ28 47.39 66.37 50 54.59 11.25 20.27 30.24 20.59 31 FWJ35 100 100 100 100 45.15 46.26 48.12 46.51 32 FWJ47 24.41 21.38 17.33 21.04 18.28 31.85 20.16 23.43 33 FWJ48 32.58 32.65 31.67 32.30 28.13 28.41 28.43 28.32 34 FWJ49 100 100 100 100 42.26 44.38 45.50 44.05 35 FWJ50 20.45 21.38 17.00 19.61 19.41 13.8 16.53 16.58 36 FWJ53 23.23 25.74 26.45 25.14 15.26 21.75 24.66 20.56 37 FWJ62 26.68 26.40 24.79 25.96 22.57 19.12 20.50 20.73 38 FWG1 100 100 100 100 42.33 46.26 52.02 46.87 39 FWG13 49.92 55.43 41.87 49.07 24.61 31.85 34 30.15 40 FWS1 56.73 50.49 37.48 48.23 26.02 27.27 31.05 28.11 41 FWS3 70.12 62.91 65.92 66.32 23.91 24.71 28.36 25.66 42 FWS5 57.41 44.82 56.55 52.93 42.97 46.8 45.50 45.09 43 FWS7 44.36 40.71 42.87 42.65 44.87 43.43 47.58 45.29 44 FWS9 54.55 47.37 54.81 52.24 9.63 15.08 26.68 17.13 45 FWS11 100 100 100 100 51.76 46.67 56.72 51.72 46 FWS13 100 100 100 100 46.91 51.31 48.72 48.98 47 FWN2 100 100 100 100 49.09 44.78 50.27 48.05 48 FWN4 53.87 42.68 57.46 51.34 24.33 30.98 34 29.77 49 FWN5 53.37 58.55 57.63 56.52 28.9 14.21 17.14 20.08
Continued……
227
50 FWN6 43.52 39.97 41.79 41.76 30.66 31.45 27.02 29.71 51 FWM1 19.61 20.97 16.00 18.86 21.24 24.44 26 23.89 52 FWL1 53.87 50.58 52.99 52.48 38.68 49.23 51.34 46.42 53 FWL2 100 100 100 100 40.86 45.45 41.40 42.57 54 FWL4 50.17 52.06 51.82 51.35 39.94 47.21 42.2 43.12 55 FWL5 68.52 66.86 67.83 67.74 14.56 17.1 17.74 16.47 56 FWL6 100 100 100 100 39.94 47.21 42.2 43.12 57 FWL7 42.17 44.08 56.14 47.46 21.8 23.64 25.13 23.52 58 FWL9 100 100 100 100 44.44 47.61 47.58 46.54 59 FWL11 48.32 48.85 48.76 48.64 17.01 16.50 13.44 15.65 60 FWL12 100 100 100 100 53.87 53.67 53.49 53.68 61 FWB3 50.51 49.34 49.5 49.78 9.42 21.68 22.98 18.03 62 FWB4 61.03 42.85 54.73 52.87 10.69 17.17 13.71 13.86 63 FWB9 68.01 62.5 59.95 63.49 12.80 17.64 17.34 15.93 64 FWB10 100 100 100 100 36.92 55.82 41.40 44.71 65 FWB11 54.38 50 50.83 51.74 44.73 39.93 51.28 45.31 66 FWK1 44.36 56.99 58.37 53.24 39.94 31.58 20.56 30.69 67 FWK2 100 100 100 100 47.26 47.61 45.43 46.77 68 Control 0 0 0 0 0 0 0 0
228
Appendix 9: Mean percent disease severity index, incidence and yield reduction: Screening for host resistance.
No. Lentil Germplasm
Disease Severity index (%) Disease Incidence (%) Yield Reduction (%)
Rep 1
Rep 2
Rep 3
Mean Rep 1
Rep 2
Rep 3
Mean Rep 1
Rep 2
Rep 3
Mean
1 Markaz-09 8.88 4.44 4.44 5.92 40 20 20 26.67 15.75 31.7 27.41 24.95 2 Masoor-86 4.44 4.44 4.44 4.44 20 20 20 20 20.43 10.47 30.91 20.60 3 Masoor-2006 4.44 4.44 8.88 5.92 20 20 40 26.67 12.73 15.78 6.55 11.69 4 Punjab M-00518 8.88 8.88 20 12.59 40 40 60 46.67 13.1 7.15 17.92 12.72 5 Punjab M-09 4.44 4.44 4.44 4.44 20 20 20 20 16.46 5.25 7.1 9.60 6 BL-2 11.11 33.33 22.22 22.22 20 60 40 40 43.69 33.33 46.58 41.2 7 NL-1 40 13.33 37.77 30.37 60 20 60 46.67 61.55 62.74 61.54 61.94 8 NL-2 91.11 88.88 95.55 91.85 100 100 100 100 100 100 100 100 9 NL-3 53.33 62.22 66.66 60.74 100 100 100 100 83.45 77.9 72.36 77.90 10 Mansehra-89 100 100 97.77 99.26 100 100 100 100 100 100 100 100 11 NARC-08-2 93.33 86.66 100 93.33 100 100 100 100 100 100 100 100 12 NARC-11-1 100 97.77 100 99.26 100 100 100 100 100 100 100 100 13 NARC-11-2 100 100 100 100 100 100 100 100 100 100 100 100 14 NARC-11-3 100 100 100 100 100 100 100 100 100 100 100 100 15 NARC-06-1 100 91.11 100 97.04 100 100 100 100 100 100 100 100 16 08504 86.66 95.55 86.66 89.62 100 100 100 100 100 100 100 100 17 08505 97.77 100 100 99.26 100 100 100 100 100 100 100 100 18 09506 100 95.55 88.88 94.81 100 100 100 100 100 100 100 100 19 01505 100 100 100 100 100 100 100 100 100 100 100 100 20 03501 84.44 80 80 81.48 100 100 100 100 100 100 100 100 21 04533 95.55 88.88 86.66 90.36 100 100 100 100 100 100 100 100 22 06513 100 100 100 100 100 100 100 100 100 100 100 100 23 NARC-08-1 100 100 100 100 100 100 100 100 100 100 100 100
229
Appendix 10: Influence of antagonists on disease severity index, incidence and grain yield reduction.
Treatment Disease Severity Index*
(%)
Disease Incidence*
(%)
Yield Reduction*
(%) T. harzianum
8.9 c 26.7 c 16.27 c
T. viridi
17.8 b 66.7 b 31.13 b
Inoculated Control
100 a 100 a 100 a
Uninoculated Control
0 d 0 d 0 d
LSD value at α=0.05 4.18 15.37 3.33 At α=0.05 level of significance means sharing same letters are non-significant. *Mean of three replications.
230
Appendix 11: Biological management: Disease severity index, incidence and grain yield reduction.
Treatment Disease Severity Index (%)
Disease Incidence (%)
Yield Reduction (%)
R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean
T. harzianum 6.66 6.66 13.33 8.9 20 20 40 26.7 16.23 17.42 15.15 16.27
T. viridi 17.77 20 15.55 17.8 80 60 60 66.7 32.61 27.29 33.48 31.13
Inoculated Control 100 100 100 100.0 100 100 100 100 100 100 100 100
Uninoculated Control 0 0 0 0 0 0 0 0 0 0 0 0
231
Appendix 12: Mycelial radial growth at different concentrations of fungicides.
Fungicide
Mycelial Radial growth (cm)
10ppm 20ppm 30ppm 50ppm 100ppm
R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean
Dithane M-45 5.4 5.3 5.5 5.4 3.6 3.6 3.5 3.6 3.2 3.4 3.4 3.3 2.8 2.6 2.6 2.7 2.0 2.0 2.0 2.0
Captan 5.2 5.2 5.2 5.2 3.2 3.3 3.2 3.2 2.2 2.3 2.4 2.3 1.9 2.0 2.0 2.0 1.7 1.8 1.8 1.8
Benomyl 4.8 4.9 4.7 4.8 2.6 2.6 2.6 2.6 1.2 1.2 1.2 1.2 1.0 1.1 1.0 1.0 0.8 1.0 0.9 0.9
Thiophanate Methyl 5.1 5.1 5.2 5.1 2.8 2.8 2.8 2.8 1.6 1.8 1.7 1.7 1.5 1.4 1.5 1.5 1.0 1.0 1.2 1.1
Control 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0
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Appendix 13: Effect of fungicides at different concentrations on percent growth inhibition of Fusarium isolate.
Fungicides
Mycelial Growth Inhibition*
(%)
10ppm 20ppm 30ppm 50ppm 100ppm Mean
Dithane M-45 40.0 n 60.4 k 63.0 j 70.4 h 77.8 f 62.3 b
Captan 42.2 m 64.1 j 74.4 g 78.1 f 80.4 e 67.8 ab
Benomyl 46.7 l 71.1 h 86.7 c 88.5 b 90.0 a 76.6 a
Thiophanate Methyl 43.0 m 68.9 i 81.1 e 83.7 d 88.1 b 73.0 ab
Control 0 o 0 o 0 o 0 o 0 o 0 c
Mean 43.0 d 66.1 c 76.3 b 80.2 ab 84.1 a 69.9
LSD value at α=0.05: Fungicide = 11.53; Concentration = 5.15; Interraction = 1.35
At α=0.05 level of significance means sharing same letters are non-significant. *Mean of three replications.
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Appendix 14: Percent mycelial growth inhibition at different concentrations of fungicides.
Fungicide
Mycelial Growth Inhibition (%)
10ppm 20ppm 30ppm 50ppm 100ppm
R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean
Dithane M-45 40.00 41.11 38.88 40.00 60 60 61.11 60.37 64.44 62.22 62.22 62.96 68.88 71.11 71.11 70.37 77.77 77.77 77.77 77.77
Captan 42.22 42.22 42.22 42.22 64.44 63.33 64.44 64.07 75.55 74.44 73.33 74.44 78.88 77.77 77.77 78.14 81.11 80 80 80.37
Benomyl 46.66 45.55 47.77 46.66 71.11 71.11 71.11 71.11 86.66 86.66 86.66 86.66 88.88 87.77 88.88 88.51 91.11 88.88 90 90.00
Thiophanate Methyl 43.33 43.33 42.22 42.96 68.88 68.88 68.88 68.88 82.22 80 81.11 81.11 83.33 84.44 83.33 83.70 88.88 88.88 86.66 88.14
Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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Appendix 15: Effect of fungicidal seed treatment on disease severity index, incidence and grain yield reduction.
Fungicide Disease Severity Index* (%)
Disease Incidence* (%)
Yield Reduction* (%)
Benomyl
1.5 b 6.7 b 17.16 a±1.41
Thiophanate Methyl
3.0 b 13.3 b 22.47 b±1.25
Inoculated Control
100 a 100 a 100 d±0
Uninoculated Control
0 b 0 b 0 d±0
LSD value at α=0.05 5.53 24.3 1.77 At α=0.05 level of significance means sharing same letters are non-significant. *Mean of three replications.
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Appendix 16: Fungicidal seed treatment: Disease severity index, incidence and grain yield reduction.
Fungicide Disease Severity Index (%)
Disease Incidence (%)
Yield Reduction (%)
R1 R2 R3 Mean R1 R2 R3 Mean R1 R2 R3 Mean
Benomyl 0 4.44 0 1.5 0 20 0 6.7 15.62 18.4 17.46 17.16
Thiophanate Methyl 8.88 0 0 3.0 40 0 0 13.3 23.9 21.6 21.9 22.47
Inoculated Control 100 100 97.77 99.3 100 100 100 100 100 100 100 100
Uninoculated Control 0 0 0 0 0 0 0 0 0 0 0 0
