BIOLOGY AND MANAGEMENT OF CHARCOAL ROT OF …prr.hec.gov.pk/jspui/bitstream/123456789/935/1/678S.pdf · BIOLOGY AND MANAGEMENT OF CHARCOAL ROT OF MUNGBEAN (Vigna radiata L.) Wilczek

BIOLOGY AND MANAGEMENT OF CHARCOAL ROT OF MUNGBEAN (Vigna radiata L.) Wilczek AND MASHBEAN

(Vigna mungo L.) Hepper

UMER IQBAL

04-arid-252

Department of Plant Pathology

Faculty of Crop and Food Sciences Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi Pakistan

2010

BIOLOGY AND MANAGEMENT OF CHARCOAL ROT OF MUNG BEAN (Vigna radiata L.) Wilczek AND MASH BEAN

(Vigna mungo L.) Hepper

by

Umer Iqbal

(04-arid-252)

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 2010

IN THE NAME OF

"ALLAH"

THE MOST MERCIFUL

THE MOST COMPASSIONATE

AND THE MOST BENEFICENT

CERTIFICATION

I hereby undertake that this research is an original one and neither part of this

thesis falls under plagiarism. If found the same, at any stage, I will be responsible for the

consequences.

Student’s Name: Umer Iqbal Signature: __________________

Registration No. 04 - arid - 252 Date: ______________________

Certified that the contents and form of thesis entitled “Biology and Management of Charcoal rot of Mungbean (Vigna radiata L.) Wilczek and Mashbean (Vigna mungo L.) Hepper” submitted by Mr. Umer Iqbal have been found satisfactory for the requirement of the degree. Supervisor: _________________________ (Dr. Tariq Mukhtar)

Co-Supervisor: ________________________

(Dr. Sheikh Muhammad Iqbal)

Member: _________________________ (Prof. Dr. Irfan Ul Haque)

Member: _________________________ (Prof. Dr. Muhammad Aslam) Chairperson: ______________________________ Dean: _____________________________________

Director, Advanced Studies: ________________________________

Dedicated To

The departed soul of my Affectionate Father

i

CONTENTS Page

List of Tables iv

List of Figures vi

List of Abbreviations vii

Acknowledgements ix

ABSTRACT 1

1. INTRODUCTION 3

2. REVIEW OF LITERATURE 7

2.1 TAXONOMY AND NOMENCLATURE 7

2.2 VARIABILITY AMONG ISOLATES 8

2.3 DISEASE CYCLE 10

2.4 SYMPTOMS 12

2.5 LOSSES CAUSED BY MACROPHOMINA PHASEOLINA 13

2.6 FACTORS AFFECTING THE INFECTION AND SEVERITY OF THE CHARCOAL ROT DISEASE

14

2.7 CHARCOAL ROT MANAGEMENT STRATEGIES 16

2.7.1 Biocontrol Agents 18

2.7.2 Antimycotic Plant Extracts 19

2.7.3 Fungicides 20

2.7.4 Host Plant Resistance 21

3 MATERIALS AND METHODS 23

3.1 FIELD SURVEY AND COLLECTION OF FUNGAL ISOLATES

23

3.2 PRESERVATION AND TRANSPORTATION OF SPECIMENS

23

3.2.1 Isolation, Purification and Identification of M. phaseolina

23

3.2.2 Storage of Pure Culture of M. phaseolina 24

3.2.3 Multiplication of M. phaseolina 24

3.3 COLLECTION OF PLANT MATERIALS 25

3.3.1 Preparation of Aqueous Plant Extracts 25

3.4 COLLECTION OF ANTAGONISTS 25

3.4.1 Biomass Production of Antagonists 29

3.5 COLLECTION OF FUNGICIDES 29

ii

3.6 COLLECTION OF GERMPLASM 29

3.7 PHYSIOCHEMICAL COMPOSITION OF THE EXPERIMENTAL SITE

31

3.8 METEREOLOGICAL DATA 31

3.9 CULTURE MEDIA USED 31

3.9.1 Corn Meal Agar 31

3.9.2 Potato Dextrose Agar 31

3.9.3 Malt Extract Agar 33

3.9.4 Chloroneb Mercury Rose Bengal Agar 33

3.9.5 Richard’s medium 33

3.9.6 Glucose Agar 34

3.10 DETERMINATION OF VARIABILITY AMONG ISOLATES OF M. PHASEOLINA

34

3.10.1 Morphological Variability 34

3.10.1.1 Radial Growth 34

3.10.1.2 Sclerotial Size 35

3.10.1.3 Sclerotial Weight 35

3.10.2 Pathogenic Variability 37

3.11 MANAGEMENT OF CHARCOAL ROT DISEASE 37

3.11.1 Evaluation of Different Antagonists for their Efficacy against Macrophomina phaseolina

37

3.11.1.1 In vitro Evaluation of Antagonists 37

3.11.1.2 Pot Culture Assay 38

3.11.2 Evaluation of Plant Extracts for their Effectiveness against Macrophomina phaseolina.

38

3.11.2.1 In vitro Evaluation of Plant Extracts 39


3.11.3 Evaluation of Different Fungicides for their effectiveness against Macrophomina phaseolina.

40

3.11.3.1 In vitro Evaluation of Fungicides 40


3.11.4 Evaluation of Mung and Mash germplasm for their Resistance against Charcoal rot Disease under Greenhouse and Field Conditions.

41

3.12 STATISTICAL ANALYSIS 41

iii

4. RESULTS 43

4.1 MORPHOLOGICAL VARIABILITY AMONG MACROPHOMINA PHASEOLINA ISOLATES

43

4.1.1 Radial Growth 43

4.1.2 Sclerotial Size 43

4.1.3 Sclerotial Weight 44

4.1.2 Pathogenic Variability among Macrophomina phaseolina Isolates

51

4.1.2.1 Pathogenic Variability among Mungbean 51

4.1.2.2 Pathogenic Variability among Mashbean 52

4.2 MANAGEMENT OF CHARCOAL ROT DISEASE 60

4.2.1 Inhibitory Effect of Antagonists against Macrophomina phaseolina

60

4.2.2 Effects of Antagonists on the Plant Survival of Mungbean and Mashbean

60

4.2.3 Effects of plant extracts on the growth of M. Phaseolina

64

4.2.4 Effects of Plant Extracts on the Plant Survival of Mungbean and Mashbean

64

4.2.5 Inhibitory Effect of Fungicides on the Radial Growth of M. phaseolina

72

4.2.6 Effect of Fungicides on Plant Survival of Mungbean and Mashbean

72

4.2.7 Host Plant Resistance against Macrophomina phaseolina

73

4.2.7.1 Mash Germplasm Screening 73

4.2.7.2 Mung Germplasm Screening 77

5. DISCUSSION 80

SUMMARY 89

LITERATURE CITED 92

iv

LIST OF TABLES

Table No. Page

3.1 Locations for the collection of Macrophomina phaseolina

isolates

26

3.2 List of antagonistic plants 28

3.3 List of fungicides used in the study 30

3.4 Physio-chemical analyses of the soil samples from experimental

site

32

3.5 Disease scoring scale (1-9) for the assessment of charcoal rot

disease

42

4.1 Morphological variations among different isolates of

Macrophomina phaseolina

45

4.1.1 Isolates categorized into three classes on the basis of radial

growth

47

4.1.2 Isolates categorized into three classes on the basis of size of

sclerotia

48

4.1.3 Isolates categorized into three classes on the basis of weight of

sclerotia

49

4.2 Differential response of selected mungbean cultivars against

various isolates of Macrophomina phaseolina

54

4.3 Differential response of selected mashbean cultivars against

various isolates of Macrophomina phaseolina

57

4.4 Effect of antagonists on the radial growth of Macrophomina

phaseolina

61

4.5 Effect of antagonists on plant survival of mungbean 62

4.6 Effect of antagonists on plant survival of mashbean 63

4.7 Effect of plant extracts on the growth of Macrophomina

phaseolina

66

4.8 Relationships between concentrations of plant extracts and 67

v

radial growth of M. phaseolina

4.9 Effect of plants extracts on the plant survival of mungbean 68

4.10 Relationships between concentrations of plant extracts and

mungbean plant survival

69

4.11 Effect of plants extracts on the plant survival of mashbean 70

4.12 Relationships between concentrations of plant extracts and

mash plant survival

71

4.13 In vitro radial growth inhibition of Macrophomina phaseolina 74

4.14 Effect of fungicides on plant survival of mungbean against charcoal rot (M. Phaseolina)

75

4.15 Effect of fungicides on plant survival of mashbean against

charcoal rot (M. Phaseolina)

76

4.16 Relative resistance/susceptibility of mashbean germplasm

against charcoal rot

78

4.17 Relative resistance/susceptibility of mungbean germplasm

against charcoal rot

79

vi

LIST OF FIGURES

Fig. No. Page

3.1 Average Precipitation, Temperature and RH % at NARC

during the summer season 2007-08.

36

4.1 Dendrogram derived from cluster analysis (UPGMA)

showing relationship among the 65 isolates of

Macrophomina phaseolina on the basis of morphological

characters collected from 14 districts of Punjab and KPK

provinces.

50

4.2 Dendrogram showing the clustering of the virulence of

Macrophomina phaseolina isolates on 3 Mungbean

cultivars

56

4.3 Dendrogram showing the clustering of the virulence of

Macrophomina phaseolina isolates on 3 Mashbean cultivars

59

vii

LIST OF ABREVATIONS

% Percent

µm micrometer

AFLP Amplified Fragment Length Polymorphism

CaCo3 Calcium Carbonate

cfu Colony forming units

cm Centimeter

CMA Corn Meal Agar

CMC Corboxy Methyl Cellulose

CMRA Chloroneb Mercury Rosebengal Agar

Cu Copper

cv Cultivar

DNA Deoxyribo Nucleic Acid

EC Electrical Conductivity

Et al and others

Fe Ferric

GOP Government of Pakistan

h Hour

IDM Integrated Disease Managment

k Potassium

Kg/Ha Kilogram per Hectare

KPK Khyber Pukhtoon Khaw

MEA Malt Extract Agar

MHC Moisture Holding Capacity

ml Milliliter

mm millimeter

Mn Manganese

N Nitrogen

NaOCl Sodium hypochlorite

NARC National Agricultural Research Center

viii

NB Nutrient Broth oC Centigrade

OM Organic Matter

P Phosphorus

PDA Potato Dextrose Agar

PDB Potato Dextrose Broth

PGRI Plant Genetic Resource Institute

ph Proportionate Hydrogen Ion

ppm Parts per Million

RAPD Random Amplified polymorphic DNA

RFLP Restriction Fragment Length Polymorphism

RH Relative Humidity

RM Richards Medium

SDW Sterilized Distilled Water

UPGMA Un-Weighted Pair Group Average

w/v Weight by Volume

WP Wettable Powder

Zn Zinc

ix

ACKNOWLEDGEMENTS

All praises and thanks for Almighty Allah, the most Merciful and

Beneficent whose bounteous blessings enabled me to complete this study and put it

in this form. I offer my humblest thanks to the Holly Prophet Muhammad (Peace

be upon him!) who is forever a torch of guidance and knowledge for the humanity

as a whole.

I feel highly privileged to express the deep sense of gratitude to my

supervisor Dr. Tariq Mukhtar, Associate Professor Department of Plant

Pathology, PMAS University of Arid Agriculture, Rawalpindi; under whose kind

supervision, sincere help and inspiring guidance the research work presented in this

dissertation was carried out.

Thanks are extended to the Supervisory Committee Members Dr. Sheikh

Muhammad Iqbal (Co- supervisor) Principal Scientific Officer, Crop Sciences

Institute, National Agricultural Research Center, Islamabad, Prof. Dr. Irfan Ul

Haque, Chairman, Department of Plant Pathology, PMAS, University of Arid

Agriculture, Rawalpindi Prof. Dr. Muhammad Aslam, Chairman, Department of

Entomology, PMAS, University of Arid Agriculture, Rawalpindi for their valuable

advice and invigorating encouragement during the course of present studies

The help extended by Dr. Ahmad Bakhsh Mahar, Chief Scientific Officer,

Hybrid Seed Programme, Dr. Muhammad Ashraf Zahid, Principal Scientific

Officer, Pulses Research Programme, Dr. Muhammad Zubair, Principal

Scientific Officer, Sugar Crops Research Programme, National Agricultural

x

Research Centre, Park Road, Islamabad is acknowledged here for their assistance in

composing of this manuscript. I also, wish to thanks my office colleagues, Shahid

Riaz Malik, Awais Rasool, Dr. Tariq Rafique, Zahid Mahmood, Muhammed

Asad Farooq, Dr. Rashid Saleem, Fahad Karim Awan and Rukhsana Afzal for

providing time to time assistance during the course of study.

Finally, my heartiest and sincere sense of gratitude to my affectionate

Mother and my uncle Prof. Dr. Muhammed Ashfaq, brothers, Dr. Izlal Ahmad,

Maj. Dr. Rehan Ahmad and Dr. Farrukh Iqbal, my Sister and other family

members for their prayers for my success. I appreciate the encouragement, which I

received from my cooperating Wife who inspired me from time to time to

accomplish this task and serving me intermittently, a cup of tea, while writing the

manuscript. Last but not least, I also thankful from core of heart to my amorous

sons, Hassan and Hamza, who missed a lot their “Baba” during thesis compiling

period.

(Umer Iqbal)

1

ABSTRACT

Charcoal rot, incited by Macrophomina phaseolina (Tassi) Goid, is a

serious disease of many crops, inflicting 100 % yield losses in Mung and Mash

bean under dry and hot conditions. Therefore, biology and management of the

disease was studied in 65 isolates of the fungus collected from 14 districts of

Punjab and Khyber Pukhtoon Khwa. Morphologically, the isolates differed

significantly in their growth behaviour, sclerotial size and weight. Sixteen isolates

developed fast growth, 11 were slow and the rest were intermediate. Nine isolates

produced large sized sclerotia, 26 small sized while the left over were intermediate.

Similarly 35 isolates produced high weight of sclerotia, 12 low and the rest were

intermediate in weight. Isolates collected form D.G.Khan, Chakwal and Bhakar

were found to be highly variable. All the isolates differed in variability in

aggressiveness. On the basis of infection, 23 isolates appeared to be highly virulent,

5 were least virulent and the remaining isolates showed intermediate response.

Biological agents, antagonistic plant materials and chemicals were tested against

the disease. All the test antagonists inhibited the growth of M. phaseolina

significantly, maximum of 79.63 % with T. harzianum and minimum of 58.14 %

with T. pseudokoningii over control. Antagonists also affected survival of mung

and mashbean plants significantly which was significantly higher at higher

concentrations of all the antagonists. Survival of mung and mash plants treated with

T. harzianum @ 2 x 108 was found maximum (83.33 and 80.0%) and minimum in

case of T. pseudokoningii. Similarly, all the test plants inhibited the growth of M.

phaseolina significantly, maximum in case of Carum lopticum (68.61%) and

2

Azadirachta indica (55.68%) the minimum (15.96%) inhibition with Nerium

indicum at 100% concentration. Percentage inhibition was significantly higher at

higher concentrations of all the plants as compared to other concentrations.

Survival of plants was also found to be maximum, where seeds were treated with

C. lopticum (83.33 and 76.66%) and A. indica (80.0 and 73.33%) at 100 %

concentration. Mentha piperita and Foeniculum vulgare showed minimum plant

survival (40 %) of mung and mash respectively. All fungicides inhibited the growth

of M. phaseolina significantly. Maximum efficacy was shown by Benomyl

(83.89%) and Carbendazim (79.11%) while Copperoxychloride showed the

minimum (23.57 %). The mung and mashbean germplasm varied greatly in

reaction to charcoal rot under inoculated conditions. In glass house studies, 14 out

of 100 mungbean accessions appeared to be highly resistant as against 34

accessions under field conditions. In case of Mash only 5 lines out of 100

accessions were found to be highly resistant in pot experiment as against 12

accessions in the field.

3

Chapter 1

INTRODUCTION

The food legumes, particularly the grains or pulses are important food stuff

in all tropical and subtropical countries. Pulses deal with those species of the plant

which belongs to the family fabaceae and sub-family faboidae. They constitute an

integral part of human diet as mature dry seeds and may also be used as immature

green seeds or as green pods with immature seed in it. They can be used for animals

in the form of hay and straw. The pulses have high protein contents (average 20-

26%). In addition to their value as food stuff, they are also important in cropping

systems because of their ability to produce nitrogen through nitrogen fixing

rhizobacteria resulting into an increase in the fertility of the soil and hence

ecomnomical as these can partially replace the expensive nitrogenous fertilizers.

The major pulses grown in Pakistan are chickpea and lentil in winter and mung and

mash bean in summer pulses.

Mungbean (Vigna radiata L.) Wilczek. is an ancient and well known

summer pulse crop of Pakistan. The economic product of mungbean is its seed,

which contains 22-24% of protein (Malik, 1994). Moreover, mungbean also

contains high amount of Vitamins A, B, C and niacin, and minerals such as

potassium, phosphorus and calcium, which are necessary for human body

(Rattanawongsa, 1993). Mungbean is digestible and does not cause flatulence as

many other legumes do. It is, therefore, a good substitute of animal protein and

forms a balanced diet when it is supplemented with cereals. The area sown under

4

this crop in the year 2007-08 was 245.9 thousand hectares in the country with a

toatal production of 177.7 thousand tones (GOP, 2008-09).

Mashbean (Vigna mungo L.) Hepper is also one of the commonly grown

pulse crops in many countries of the world. Its seed contains about 24% protein,

60% carbohydrates and 1.3% fats. Dry fodder (pod husk) is nutritive for milch

animals. It is also referred to as cover crop. Being legume crop, it fixes nitrogen to

an extent of 70-90 kg/ha and thus improves soil fertility. In Pakistan, it occupies an

area of over 32.5 thousand hectars with 17.3 thousand tones production (GOP,

2008-09). The crop is grown under a wide range of agro-ecological zones. The

average yield in Pakistan is very low as compared to its potential yield obtained in

many other countries.

The low yield of mung and mash in Pakistan can be attributed to several

biotic and abiotic constraints. Among biotic factors, diseases are the most

destructive. The losses due to diseases to pulse crops have been estimated up to 44

percent, depending upon the crop variety (Bashir, 1988). Mung and mashbean are

attacked by about 26 diseases in the world (Charles, 1978). Among these, charcoal

rot caused by Macrophomina phaseolina (Tassi) Goid, is of prime importance in

reducing crop yield especially in arid regions of the world (Hoes, 1985).

This pathogen causes seedling blight; stem rot and pod rot and attacks more

than 500 plant hosts (Sinclair, 1982). About 67 hosts of this pathogen have been

reported from Pakistan (Mirza and Qureshi, 1982; Shahzad and Ghaffar, 1986;

Shahzad et al., 1988). Tropical crop plants are seriously affected by this pathogen

(Malaguti 1990). M. phaseolina is classified as a Deuteromycete which shows two

5

asexual sub-phases, a mycelial phase named as Rhizoctonia bataticola (Taub)

Butler (1925) and the other a pycnidial phase called M. phaseolina (Dhingra and

Sinclair, 1978). The fungus is classified in the Botryosphaeriaceae according to

recent phylogenetic data (Crous et al., 2006).

The fungus produces dark brown lesions on the epicotyls and hypocotyls of

seedlings and seedling death follows because of obstruction of xylem vessels and

wilting. In adult plants, the pathogen causes red to brown lesions on roots and

stems, and produces dark mycelia and black microsclerotia. The stem shows

longitudinal dark lesions and the plant becomes defoliated and wilted (Abawi and

Pastor-Corrales, 1990).

The asexual structures formed by the fungus are pycnidia and

microsclerotia. The black, 0.1–1 mm sized microsclerotia are formed in soil,

infected seeds or host tissues and constitute the primary inoculum source of the

pathogen (Bouhot, 1968; Dhingra and Sinclair, 1978; Abawi and Pastor-Corrales,

1990). They can survive up to 15 years depending on environmental conditions, and

whether or not the sclerotia are associated with host residues (Cook et al., 1973;

Papavizas, 1977; Short et al., 1980). Secondary dispersal by Pycnidiospores

produced on infected stem and leaf tissues (Ali and Dennis, 1992).

M. phaseolina is a heat tolerant pathogen since sclerotia could withstand a

temperature range of 60–65 ◦C (Bega and Smith, 1962; Mihail and Alcorn, 1984).

The evidence suggests that it is primarily a root inhabiting fungus and produces

tuber or cushion shaped 1-8 mm diameter black sclerotia. These sclerotia serve as a

6

primary means of survival (Smith, 1969; Kaiser et al, 1980). Charcoal rot causes

60% losses to the mung and mash crops (Deshkar et al., 1974).

In Pakistan charcoal rot causes colossal losses which warrant necessary

control measures. As, the information regarding the variability and management of

this pathogen on mung and mash bean is lacking in the country, so the studies with

following objectives were planned

• Determine the morphological and pathogenic variability among

Macrophomina phaseolina isolates prevalent in different mungbean and

mashbean areas of Punjab and Khyber Pukhtoon Khwa.

• Find out the virulent strains from different isolates of the pathogen.

• Evaluation of different biocontrol fungi, antagonistic plants and fungicides for

the management of the disease.

• Screening of mung and mashbean germplasm for the identification of

resistant sources against the pathogen.

7

Chapter 2

REVIEW OF LITERATURE

Pulses belonging to Vigna spp. are important summer crops of Pakistan

which are prone to several diseases. Among the fungal diseases, charcoal rot is a

common, widespread, destructive and economically important disease in mung and

mash, causing obstruction of xylem vessels and wilting leading to the plant death

(Abawi and Pastor-Corrales, 1990). Various aspects related to pathogen and its

management is reviewed below.

2.1 Taxonomy and Nomenclature

Macrophomina phaseolina (Tassi) Goid. ( Tiarosporella phaseolina (Tassi)

Vander Aa) is a soilborne plant pathogenic fungus. It belongs to the anamorphic

Ascomycetes and is characterized by the production of both pycnidia and sclerotia

in host tissues and culture media. The pycnidial state was initially named

Macrophoma phaseolina by Tassi in 1901 and Macrophoma phaseoli by Maublanc

in 1905. In 1927, Ashby maintained the name Macrophomina phaseoli, while

Goidanich (1947) proposed Macrophomina phaseolina. Tiarosporella phaseolina

(Tassi) Van der Aa was used in 1981 by Van der Aa to designate the species.

Mihail (1992) indicated that there is an unconfirmed report of a teleomorph named

Orbilia obscura (Ghosh et al., 1964) of M. phaseolina, but since then no further

evidence appeared for the telemorph state. The sclerotial state was described for the

first time by Halsted as Rhizoctonia bataticola (Taub.) Butler on Ipomoea batatas

in 1890. According to Dhingra and Sinclair (1978), the same fungus was isolated

from cowpea in India in 1912 by Shaw and was then named Sclerotium bataticola.

8

Recently Crous et al. (2006) demonstrated that although the telemorph is unknown,

M. phaseolina is a member of the family Botryosphaeriaceae. The authors pointed

out the differences between Tiarosporella and Macrophomina, which produces in

the pycnidia percurrently proliferating conidiogenous cells. The pycnidiospores are

ellipsoid to obovoid, and measure (16-)20-24(-32) × (6-)7-9(-11) µm. During the

sclerotial formation, 50–200 individual hyphal cells aggregate to give multicellular

bodies named microsclerotia. The microsclerotia are black and are variable in size

(50–150 µm) depending on the available nutrients of the substrate on which the

propagules are produced (Short and Wyllie, 1978).

2.2 Variability among isolates

Much work has been done to elucidate the variability in morphology,

physiology, pathogenicity, and genotype of M. phaseolina. Variation in cultural

characteristics and virulence to soybean has been reported in the U.S. (Dhingra and

Sinclair, 1973). According to Ahmed and Ahmed (1969), cultural characteristics

and growth rates of 8 different jute isolates of M. phaseolina appeared to be related

to their pathogenicity. Isolates with fast mycelial growth and abundant sclerotial

production were more pathogenic on clusterbean (Cyamopsis tetragonoloba)

seedlings than isolates growing more slowly (Purkayastha et al., 2004). Color of

cultures on PDA, ability to sporulate in infected host plants and pycnidial size also

have been reported to vary greatly (Dhingra and Sinclair, 1978; Adam, 1986).

Isolates of M. phaseolina obtained from resistant sorghum genotypes were more

pathogenic on susceptible sorghum than two other isolates originally obtained from

susceptible sorghum genotypes (Diourte et al., 1995). Tentative demonstration of

9

host preference of M. phaseolina isolates was done by Pearson et al. (1986; 1987a,

b). Comparing growth aptitude of more than 2000 isolates from 13 states in

medium containing potassium chlorate, the authors classified isolates of M.

phaseolina from maize as chlorate-resistant and those from soybean as chlorate-

sensitive. The growth of the last group was inhibited by chlorite produced in the

medium. In contrast, Zazzerni and Tosi (1989) reported that M. phaseolina isolates

from four host species varied widely in chlorate-utilization irrespective of their

original host and concluded that there was no evidence for host specialization

within M. phaseolina. Further evidence of lack of host specialization in M.

phaseolina was reported by Mihail and Taylor (1995). Their study on the

variability of 114 M. phaseolina isolates from different host species, soils and

continents clearly indicated that M. phaseolina is a heterogeneous species that

cannot be partitioned into distinct subspecies groups based on pathogenicity,

pycnidium production and chlorate utilization. Although Su et al. (2001) pointed

out that host specialization in M. phaseolina occurs with maize, no clear evidence

for the occurrence of formae speciales, subspecies or physiological races has been

reported so far.

Various recent studies were devoted to the genetic and pathogenic

variability of M. phaseolina. Significant advances in molecular detection and

differentiation of M. phaseolina isolates have been achieved using Restriction

Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA

(RAPD), and Amplified Fragment Length Polymorphism (AFLP) analysis (Mayek-

Pérez et al., 2001; Su et al., 2001; Purkayastha et al., 2006; Reyes-Franco et al.,

2006). So far, none of these methods have however been able to differentiate

10

isolates of M. phaseolina from specific hosts or geographic locations. The lack of a

strong correlation between genotype and geographical origin suggests a high

diversity level within and among the population of M. phaseolina (Jana et al.,

2005). Nevertheless, Jana et al. (2003) developed a single RAPD primer OPA-13

that distinguishes isolates of M. phaseolina from soybean, sesame, groundnut,

chickpea, cotton, common bean, okra, and 13 other hosts and this could be useful

as taxonomic marker for population studies.

2.3 Disease cycle

M. phaseolina causes seedling blight, root rot and root and stem rot of more

than 500 cultivated and wild plant species including economically important crops

as soybean, common bean, sorghum, maize, cotton, peanut, cowpea (Dhingra and

Sinclair, 1977; Bouhot, 1967, 1968; Adam, 1986; Gray et al., 1990; Hall, 1991;

Diourte et al., 1995). Softwood forest trees (Abies, Pinus, Pseudotsuga) (McCain

and Scharpf, 1989), fruit trees (Citrus spp., Cocoa nucifera, Coffea spp.) and weed

species (Songa and Hillocks, 1996) are also hosts of the pathogen. The fungus was

reported in North and South America, Asia, Africa and Europe. However, it is

economically more important in subtropical and tropical countries with a semi-arid

climate (Wrather et al., 1997; 2001).

M. phaseolina produces sclerotia in root and stem tissues of its hosts which

enable it to survive adverse environmental conditions (Cook et al., 1973; Meyer et

al., 1974; Short et al., 1980). In PDA, pycnidia are not produced except under some

specific incubation conditions (Gaetán et al., 2006) and only sometimes in host

crops (Mihail and Taylor, 1995), and their importance in the epidemiology of the

11

fungus likely depends on the host involved as well as the fungal isolate (Ahmed

and Ahmed, 1969). On cowpea, pycnidia are produced at the end of the rainy

season, but their epidemiological significance seems minor. On the contrary, in jute

crops, pycnidiospores produced on early infected stem and leaf tissues have been

reported to be responsible for secondary spread of the disease (Anonymous, 1940).

Microsclerotia in soil, infected seeds or host tissues serve as primary

inoculum (Bouhot, 1968; Dhingra and Sinclair, 1977; Abawi and Pastor-Corrales,

1990). Root exudates induce germination of microsclerotia and root infection of

hosts. The infective hyphae enter into the plant through root epidermal cells or

wounds. During the initial stages of pathogenesis, the mycelium penetrates the root

epidermis and is restricted primarily to the intercellular spaces of the cortex of the

primary roots. As a result, adjacent cells collapse and heavily infected plantlets may

die. At flower onset, the fungal hyphae grow intracellularly through the xylem and

form microsclerotia that plug the vessels (Short et al., 1978; Mayek-Pérez et al.,

2002) and disrupt host cells. The infected plants show necrotic lesions on stems,

branches, and peduncles. From pod peduncles, the fungus spreads to the pods and

invades grains. Heavily infected plants die prematurely due to the production of

fungal toxins e.g. phaseolinone (Bhattacharya et al., 1994) and production of fungal

tissue that plugs host vessels. In soybean, formation of microsclerotia is

conditioned by flowering and pod setting (Wyllie and Calvert, 1969) and may be

indicative of initiation of death of the host (Short and Wyllie, 1978). After plant

death, colonization by mycelia and formation of sclerotia in host tissue continue

until tissues are dry. The mycelium and microsclerotia produced in infected plant

material, including plant residues are the means of propagation of the pathogen.

12

Microsclerotia in soil, host root and stems are the main surviving propagules. After

decay of root and plant debris, microsclerotia are released into the soil. They are

distributed generally in clusters at the soil surface and are localized mainly at a

depth of 0–20 cm (Alabouvette, 1976; Mihail, 1989; Campbell and Van der Gaag,

1993). They can survive for 2–15 years depending on environmental conditions,

and whether or not the sclerotia are associated with host residue (Cook et al., 1973;

Papavizas, 1977; Short et al., 1980; Baird et al., 2003). Factors that adversely affect

the survival of these propagules include repeated freezing and thawing of soil, low

carbon: nitrogen ratios in soil and soil moisture content (Dhingra and Sinclair,

1974; Dhingra and Sinclair, 1975).

2.4 Symptoms

Disease symptoms are clearly visible from the time of emergence and can

be evaluated at various stages of development of the plant. After emergence,

symptoms can be observed on the cotyledons as brown to dark spots. However,

cotyledons remain on the plant for only a few days, especially when diseased. The

margins of the cotyledons become bright red, then beige or brown, and finally

brown to black. Often, they are covered with a grayish mycelium pad bearing

scattered sclerotia. This mycelium can be observed also inside entirely colonized

cotyledons. At the unifoliate leaf stage, typical symptoms are pinhead-size,

charcoal-colored spots which are mostly restricted to the hypocotyl section of the

stem, including its subterranean part. Infected spots may expand and develop into

large necrotic lesions, usually resulting in death of the plant. M. phaseolina can

also infect roots which show necrotic lesions (Bouhot, 1967; Adam, 1986). On

13

adult plants, M. phaseolina causes lesions on stems, spikes, pods and seeds. On

stems, lesions are beige and appear at the ramification point of the lateral secondary

branches. Colonized tissues become gray and covered with abundant minute black

punctuations. Initially these punctuations are immersed, becoming gradually more

prominent. From pod peduncles, the fungus spreads to the pods and invades grains.

However, necrotic lesions may appear anywhere on the pods. Infected green pods

are initially blue-green, and then turn brown to reddish. When infection occurs on

mature, dry pods, they become white to gray and are covered with locally or widely

distributed black bodies on the pod. The fungus penetrates the pod and grains,

inducing diverse symptoms depending on tissue infestation level. Early infested

grains abort or become empty and dry. The affected parts of the pod become

narrow or shrunken, deformed and thin when damaged grains are located at the pod

tip (Bouhot, 1965; 1967). The most striking symptom is the sudden wilting and

drying of the whole plant, most of the leaves remaining green. The stem and

branches are then covered with black bodies and give the charcoal or ashy

appearance of dead plants. Withering can be observed from seedling to maturing

stage and is the result of necrosis of roots, stems and mechanical plugging of xylem

vessels by microsclerotia, but also by toxin production, and enzymatic action (Chan

and Sackston, 1973; Kuti et al., 1997; Jones and Wang, 1997).

2.5 Losses caused by Macrophomina phaseolina

Yield losses as a result of infection caused by charcoal rot (M. phaseolina)

are difficult to assess in quantitative value (Cloud and rupe, 1994) as the affects of

the disease caused by this fungus can be quite subtle and may not be noticed.

14

Increase in yield on chickpea after controlling the disease was reported by Quaiser

et al (1986), in cumin (Lodha, 1995), maize (Begum et al, 1989) and peanut

(Sharma and Bhowmik, 1986), although the yield factor was probably associated

with the effects of other soil biota and the establishment of more plant stand.

In some crops, the yield losses caused by M. phaseolina may result from

plant death or lodging. In sorghum, lodging can occur at maturity as fungus

weakens the stem and microsclerotia form in the vascular tissues (Edmunds, 1964;

Odvody and Dunkle, 1979). Harvesting losses of more thsn 50 % can occur

(Frederiksen, 1986). Hiremath and Palakshappa (1994) reported 100% lodging in

sorghum genotypes and yield losses of up to 60 % were reported (Ayr, 1985) due to

charcoal rot disease.

There are several information sources on soybean losses due to charcoal rot

disease (Doupnok, 1993; Pratt and Wrather, 1998; Wrather et al, 1997 and Wyllie,

1988). Annual losses of 30-50 % in soybeans have been reported (Sharifi et al,

2005; Ramezani et al, 2007; Senthilkumar et al, 2009). Charcoal rot causes the

greatest or second greatest economic loss for soybean producers (Pratt and Wrather,

1998; Wrather et al, 2003).

2.6 Factors affecting the infection and severity of the charcoal rot disease

Root infection is affected by growth stage and environment. High root

infection can occur before reproductive development and is then associated with

hot and dry weather early in the growing season (Cloud and Rupe, 1994). M.

phaseolina can infect beans also under relatively dry conditions (Olaya and Abawi,

1993). However, there are also reports where a high moisture holding capacity (40–

15

50% MHC) resulted in greater M. phaseolina colonization on peanut (Husain and

Ghaffar, 1995). Agarwal and Goswani (1973) reported a significant synergistic

effect in soybean when the root-knot nematode Meloidogyne incognita preceded

infection by M. phaseolina by three weeks, and suggested that M. incognita

predisposes plants to the fungal infection, similar to the vascular pathogens

Fusarium oxysporum and Verticillium dahliae. In white clover, M. phaseolina also

tends to be associated with higher final densities of the plant pathogenic nematodes

Meloidogyne trifoliophila, Helicotylenchus dihystera and Heterodera trifolii (Zahid

et al., 2002). In contrast, in a pot experiment the simultaneous addition of M.

phsaeolina and Meloidogyne javanica resulted in reduced nematode galls, which

was ascribed to the effect of toxic metabolites on the nematode produced by the

fungus (Gupta and Mehta, 1989).

Many studies have demonstrated the lack of consistent correlation between

the severity of host infection and charcoal rot incidence. Visible symptoms of the

disease in the field are most apparent under conditions that reduce plant vigor, such

as poor soil fertility (Sinclair and Backman, 1989), high seeding rates (Pearson et

al., 1984; Sinclair and Backman, 1989), low soil water content (Meyer et al., 1974;

Ali and Ghaffar, 1991; Sheikh and Ghaffar, 1979; Kendig et al., 2000), high

temperatures (Odvody and Dunkle, 1979; Mihail, 1989), and root injury (Canaday

et al., 1986). The timing of host reproduction is another factor that has a strong

influence on charcoal rot development. In Euphorbia lathyris, early flowering

plants succumb more rapidly to charcoal rot than later flowering ones (Mihail,

1989). In sorghum, post-flowering water-stressed plants showed more severe

charcoal rot symptoms than plants without water stress (Diourte et al., 1995). Initial

16

population density of sclerotia in soil was directly correlated with the severity of

charcoal rot of soybean and was inversely related to soybean yield (Short et al.,

1980). Mihail (1989) found that average symptom expression and mortality

increased with increasing soil temperature and that mortality increased markedly

after the soil temperature at 5 cm reached the range of 28–30 °C.

2.7 Charcoal rot management strategies

Most of the described control methods aim to reduce the number of

sclerotia in soil or to minimize the contact of the inoculum and the host.

Solarization (Grinstein et al., 1979; Katan et al., 1980; Pullman et al., 1981;

Usmani and Ghaffar, 1982), addition of organic amendments (Ghaffar et al., 1969;

Dhingra and Sinclair, 1975), maintenance of high soil moisture content (Dhingra

and Sinclair, 1975) and fumigation (Watanabe et al., 1970) have been suggested as

possible methods to manage soilborne pathogens. Solarization alone was not

effective at controlling M. phaseolina in forest (Old, 1981) and field (Mihail and

Alcorn, 1984) soils. Soil moisture content greatly affects the sensitivity of resting

structures to heat treatment (Lodha et al., 2003), and one summer irrigation was

sufficient to reduce the population of M. phaseolina by 25–42 % (Lodha and

Solanki, 1992; Lodha, 1995). Solarization of moistened soil further augmented this

reduction in the top soil, but many propagules survived at lower depths (Lodha and

Solanki, 1992). Amendments with nitrogen-enriched pearl millet residues

significantly reduced the population of M. phaseolina within 45 days by 94 %

(Sharma et al., 1995). Combined effects of amendments, irrigation and

polyethylene mulching resulted in the almost complete eradication of the

17

population of M. phaseolina (93–99 % reduction) at 0–30 cm soil depth within 15

days. A considerable reduction (75–95 %) was also achieved by natural heating of

irrigated soil for 15 days after amending with cruciferous residues (Lodha et al.,

1997). Cabbage residue incorporation in the soil without heating also reduced the

population of Fusarium oxysporum f. sp. conglutinans effectively (Ramirez-

Villapudua and Munneke, 1987). The effect was mainly attributed to toxic volatiles

such as mercaptan, dimethyl sulphid, and isothiocyanate formed during the

decomposition of the cabbage residues (Gamliel and Stapleton, 1993).

Tillage is a crucial cultural measure that could affect the inoculum potential

of soilborne pathogens. If the pathogen requires high inoculum density to infect

plants, then increased dispersal over the soil profile could reduce disease severity.

If, however, a low inoculum density is sufficient for infection, then dispersal may

aggravate incidence and severity (Olanya and Campbell, 1988). As low inoculum

densities are sufficient to cause charcoal rot, tillage can increase damage by M.

phaseolina especially when highly susceptible hosts such as Euphorbia lathyris are

cultivated, in which a soil sclerotial density < 1 microsclerotium per gram soil can

cause more than 90% plant mortality (Young and Alcorn, 1984). Tillage reduces

the stratification of organic residue on the surface, which in turn can influence soil

temperature and moisture (Campbell and Van der Gaag, 1993), soil chemistry

(Blevins et al., 1980), population of soil animals, and the structure of microbial

communities (Franchini et al., 2006). These changes in physical and biological

factors may in turn also affect disease incidence and severity of M. phaseolina.

18

Irrigation at any time during the cropping season reduces disease infection

in soybean (Kendig et al., 2000). Furthermore the type of irrigation can also affect

charcoal rot disease. The density of soil sclerotia and the number of diseased melon

plants was higher in drip irrigated plots than in furrow irrigated plots (Nischwitz et

al., 2004).

2.7.1 Biocontrol agents

Management strategies to control charcoal rot also include the use of

biocontrol agents to prevent host infection or to suppress the growth of the

pathogen (Ghaffar et al., 1969; Siddiqui and Mahmood, 1993). In jasmine,

Trichoderma harzianum and T. viride were effective against M. phaseolina (Alice

et al., 1996). T. harzianum and Pseudomonas fluorescens significantly reduced the

germination of sclerotia by 60 % in natural field soil (Srivastava et al., 1996).

Strains of Bradyrhizobium sp. and Rhizobium meliloti were reported to be

antagonistic against M. phaseolina and to have plant growth promoting properties

in groundnut (Arora et al., 2001; Deshwall et al., 2003). Blackgram seeds

inoculated with three Aspergillus spp showed significantly low disease intensity

against charcoal rot (Eswaran and Rahul, 2004). Three antagonists (Trichoderma

harzianum, Epicoccum nigrum and Paecilomyces lilacinus) significantly

suppressed M. phaseolina in vitro and in vivo by producing an inhibition zone

while T. harzianum suppressed them by overgrowing (Hashem, 2004). Adekunle et

al. (2001) treated cowpea seeds with three (Trichoderma spp.) and planted in soils

amended with M. phaseolina. He found significantly greater plant stands

percentage than the controls. In tomato and eggplant Saccharomyces cerevisiae the

19

biological control agent reduced disease percentage, improved root lengths and

fresh and dry weights (Attyia and Youssry, 2001). Seed treated with T. harzianum

at sowing were effective to provide a considerable reduction of the disease caused

by M. phaseolina in sesame (Pineda, 2001). Pan et al. (2001) screened the

antagonistic properties of 4 Gliocladium virens strains against M. phaseolina. All

the strains were highly antagonistic against M. phaseolina. Black gram seeds

treated with Trichoderma viride significantly reduced the sclerotial germination of

M. phaseolina (Rettinassababady et al. 2000). Aspergillus versicolor grown in soil-

compost medium was found to control infection caused by Macrophomina

phaseolina in jute (Bhattacharyya et al, 1985). The incidence of root rot in

blackgram was significantly reduced by 50 % when treated with Trichoderma spp.

alone or in combination with biofertilizer both under glass house and field

conditions (Indra and Gayathri, 2003). Soil amendment with Trichoderma

harzianum formulated on sugar cane bagasse at the rate of 10 % (w/w) of soil

shown a highly effect in reducing root rot incidence caused by M. phaseolina at

pre-emergence stage respectively ( Mohamedy et al, 2006). Soil application of talc

based formulation of T. harzianum, T. polysporum and T. viride effectively

controlled the root rot (M. phaseolina) of Egg plant under field condition

(Ramezani, 2008). Seed treatment both Trichoderma virens and Pseudomonas

fluorescens along with soil application supported the maximum plant stand and less

root rot incidence in pigeon pea (Lokesha and Benagi, 2007).

2.7.2 Antimycotic plant extracts

20

Several studies have dealt with the antimycotic effect of plant compounds

on M. phaseolina. The essential oil actinidine isolated from Nepeta clarkei was

effective in vitro against M. phaseolina (Saxena and Matela, 1997). Kazmi et al.

(1995) and Alice et al. (1996) reported that in vitro neem oil was more or equally

effective compared to benomyl and carbendazim. However, neem seed extracted

samples of different locations showed variable suppression of growth of the

pathogen. More effective inhibition of the growth of M. phaseolina was obtained

by aqueous extracts of Cymbogon citratus (Bankole and Adebanjo, 1995). Powder

of Datura fastulosa (Datura) was also reported to be effective against M.

phaseolina and Meloidogyne javanica infection in a pot experiment (Ehteshamul et

al., 1996). The aqueous extracts of Tephrosia candida and Boehmeria nivea could

well inhibit the formation of sclerotia of M. phaseolina (Anuradha et al. 2003).

Extracts of pulverized bark of Prosopis africana and leaves of Nauclea latifolia

100 % inhibited both radial mycelial growth and sclerotial formation of M.

phaseolina (Oluma et al. 2002). Datar (1999) found that aqueous extracts of

Polyalthia longifolia, Allium sativum and Parthenium hysterophorus were found

most effective in reducing mycelial growth of M. phaseolina. Neem leaf extract,

Marigold leaf extract and Garlic bulb extract at 5 % as seed treatments significantly

reduced the charcoal rot incidence and increased yield (Sinha and Sinha, 2004).

2.7.3 Fungicides

Researchers also have investigated the in vitro sensitivity of different

isolates of M. phaseolina to fungicides (Al-beldawi et al., 1973; Rama et al., 1981)

and the efficacy of fungicide application to seed and soil to reduce fungal

21

germination and infection (Kannaiyan et al., 1980; Alice et al., 1996). However,

until now, chemical control of M. phaseolina is difficult and neither profitable nor

advisable (Pearson et al., 1984). Bavistin 50 WP [carbendazim] was the most

effective against M. phaseolina, a major pathogen of mothbean (Rathore and

Rathore, 1999). Application of carbendazim in combination with Thiram resulted

in the highest seed germination percentage and lowest root rot incidence in

chickpea (Prajapati et al. 2003).

2.7.4 Host Plant Resistance

Sources of resistance to some soilborne pathogens have been identified, but

highly resistant cultivars are often not available for polyphagous and unspecialized

pathogens like M. phaseolina. Pastor-Corrales and Abawi (1988) reported some

selected bean lines with stable resistance to the fungus. Resistance in beans to M.

phaseolina has been associated with drought tolerance (Pastor-Corrales and Abawi,

1988). In common bean resistance to M. phaseolina is controlled by two dominant

complementary genes (Olaya et al., 1996). Grezes-Besset et al. (1996) reported

resistance to M. phaseolina in Ricinus persicus and incorporated this in cultivated

castor bean. The improved lines showed high seedling resistance to the disease.

Based on seed yield and the levels of lower stem and taproot colonization, Smith

and Carvil (1997) identified four resistant cultivars among 24 soybean cultivars

screened for resistance to M. phaseolina. Research on sources for resistance to M.

phaseolina in sorghum has led to breeding lines and cultivars with stable

performance. Resistance in sorghum was associated with delayed leaf senescence

(Ducan, 1984; Diourte et al., 1995). The stability of this resistance, however, was

22

influenced by water stress. In soybean, resistance factors to M. phaseolina do not

protect plants against infection, but more likely restrict the growth rate of the

fungus within plant tissues (Smith and Carvil, 1997). Reduced growth of the

pathogen within host tissues may be due to lower levels of the stress related free

amino acids proline and asparagine in resistant than in susceptible cultivars

(Pearson et al., 1987b).

Mung and mash cultivars highly resistant to M. phaseolina and adapted to

the different production areas with acceptable agronomic characteristics are not

available. Moderate levels of resistance were reported in India (Sohi and Rawal,

1983; Singh and Lodha, 1986; Mahabeer et al., 1995). Several cowpea varieties

and lines were screened for resistance to M. phaseolina in a disease nursery and pot

experiments. These accessions including lines with delayed leaf senescence and

drought resistant cultivars were susceptible to the pathogen (Adam, 1986; M.

Ndiaye unpublished). Nevertheless, better cowpea stands were attributed to

moderate resistance of one cultivar (Gaikwad and Sokhi, 1987). Higuera and Murty

(1987) screened 414 cowpea progenies of white and black seed types in a study to

verify the supposed resistance to M. phaseolina in cowpea black seed types. The

authors concluded that seed color had no influence on resistance, but that

significant variation in resistance to charcoal rot exists among cowpea accessions

which could be used in breeding programs.

23

Chapter 3

MATERIALS AND METHODS

3.1 FIELD SURVEY AND COLLECTION OF FUNGAL ISOLATES

During 2005-06, surveys were conducted in 14 major mung and mash

producing areas of Punjab and Khyber Pakhtoon Khwa (KPK) provinces of

Pakistan (Table-3.1). Samples of stems bearing microsclerotia of Macrophomina

phaseolina and characteristic symptoms of charcoal rot were collected from the

infected plants from farmers’ fields and research institutes and designated.

Depending on the number of diseased foci, 3–5 plants were sampled per field.

3.2 PRESERVATION AND TRANSPORTATION OF SPECIMENS

The diseased samples were first packed in paper bags and then in 15×20-cm

polyethylene bags, labelled, brought to the lab and stored at 4oC until processed for

identification.

3.2.1 Isolation, Purification and Identification of M. Phaseolina

For isolation of M. phaseolina, the samples were cut into small pieces (5-10

mm long), surface sterilized with 1 % sodium hypochlorite for 2 minutes and then

rinsed twice in sterilized distilled water. The pieces were placed on Chloroneb

Mercury Rose Bengal Agar (CMRA) medium (Meyer et al., 1973) in petridishes

and incubated in dark at 25 ± 1oC for 6 days. A small portion of the fastest growing

colony of M. phaseolina was taken from the periphery of 90 mm diameter petri

dish and spread on petridishes containing glucose agar medium (glucose, 20 g;

agar, 20 g and water, 1000 ml) and incubated in dark at 25 ± 1oC for 6 days. A

24

small portion of the colony having sclerotia was taken into a drop of sterilized

water and agitated with a sterilized needle to separate the sclerotia from the

mycelia. Sclerotia were then transferred to 90 mm dia. petridishes containing

CMRA medium. Colonies appearing from single sclerotium were again transferred

to CMRA medium containing petriplates, incubated and identified on the basis of

standard key (Barnett and Hunter, 1972).

3.2.2 Storage of Pure Culture of M. phaseolina

The purified culture (5 mm disc) from each isolate growing on PDA was

transferred to 10 ml culture tubes and incubated in dark at 25±1°C for 6 days, until

the surface of PDA was covered with dense sclerotial layer of the fungal culture.

The culture tubes were labeled and stored at 4oC.

3.2.3 Multiplication of M. phaseolina

Seeds of rice were moistened (1 g rice seeds: 1 ml water) and placed in

conical flasks. The mouth of each flask was plugged with cotton wool and wrapped

in aluminum foil and autoclaved at 15 psi at 121°C for 3 h. After cooling for 12 h,

the flasks were again autoclaved at 121°C for another 3 h. On cooling, the seeds

were inoculated with a 5 mm diameter mycelial plug from a 7-day old culture of M.

phaseolina and incubated at 25±1°C for 15 days. The flasks were shaken at

alternate days for uniform colonization of the grains. The inoculum was stored at

4°C untill used in the field. For confirmation 0f the fungus, the colonized rice seeds

were plated on PDA plates and incubated at 25±1°C for 5 days. The plates were

examined under stereoscope for mycelial growth.

25

3.3 COLLECTION OF PLANT MATERIALS

Leaves or seeds or flowers of twenty different antagonistic plants (Table-

3.2) used in the study were collected from different locations of Pakistan.

3.3.1 Preparation of Aqueous Extracts

Leaves or seeds or flowers of test plants were surface sterilized for 2

minutes in 70% ethanol. Samples were then rinsed twice in sterilized distilled

water, dried under room temperature for 21 days and ground to powder separately.

For preparation of aqueous extracts of crushed dry sample of each plant was soaked

in sterilzed distilled water at 1:1 w/v, vigorously stirred and left for 24 hrs. The

suspensions were passed separately through 4 ply muslin cloth, filtered through

Whatman’s filter paper No.41. The filterates were further passed through Millipore

filter of 0.2 µm pore size to avoid the bacterial contamination and stored at 4°C

until use. The extracts thus obtained were arbitrarily termed as ‘S’ (100 %). Further

dilutions (75%, 50% and 25%) were prepared by the addition of requisite amount

of distilled water.

3.4 COLLECTION OF ANTAGONISTS

Seven antagonistic fungi viz, Trichoderma harzianum, T. hamatum, T.

koningi, T. pseudokoningi, T. viride, T. aureoviride and Gliocladium virens used in

the experiment were collected from the First Fungal Culture Bank (FFCB), The

University of Punjab, Lahore.

26

Table 3.1 Locations for the collection of M. phaseolina isolates

Isolates District Location MP-1 Jatli MP-2 Kallar saidan MP-3

Rawalpindi Mandra

MP-4 Vehowa MP-5 Kot Haibat MP-6 Kot Chutta MP-7

D.G.Khan

Mangrotha MP-8 Thatha Gurmani MP-9 Mehmood kot MP-10 Kot addu MP-11

M. Garh

Seetpur MP-12 Piplan MP-13 Wanbhachran MP-14 Wattakhel MP-15 Hernoli MP-16

Mianwali

Shahbazkhel MP-17 Mankera MP-18 Kalurkot MP-19 Gauharwala MP-20 Basti Shah Alam MP-21

Bhakkar

AZRI, Bhakkar MP-22 Fatehpur MP-23 Chaubara MP-24 Kot Sultan MP-25 Karor MP-26 Paharpur MP-27 Dullaywala MP-28

Layyah

Kharewala MP-29 AARI, Faisalabad MP-30 NIAB, Faisalabad MP-31 Thikriwala MP-32

Faisalabad

Ramdawali MP-33 Mansoorwali

27

MP-34 Naina Kot MP-35 Chajwal MP-36

Narowal Noor Kot

MP-37 Sahowali MP-38 Pejokey MP-39 Muradpur MP-40 Sambrial MP-41

Sialkot

Bhagowal MP-42 Balkasar MP-43 Dhudial MP-44 Bhagwal MP-45

Chakwal

BARI, Chakwal MP-46 NARC, Islamabad MP-47 Chakshahzad MP-48 Tarnol MP-49

Islamabad

Kuri MP-50 Abba Khel MP-51 Band Kurai MP-52 Bigwani MP-53 Kot Jai MP-54 Malana MP-55

D.I.Khan

Yarik MP-56 Ahmad Wala MP-57 Shadi Khel MP-58 Shan Wala MP-59

Kohat

Gumbat MP-60 Ghori Wala MP-61 Sarai Gambila MP-62 Sarai Naurang MP-63 Domeli MP-64 Nurar MP-65

Bannu

Mira Khel

28

Table 3.2 List of antagonistic plants

Common Name

Botanical Name Location of collection

Plant parts used

1 Neem Azadirachta indica D.G.Khan leaves

2 Bhang Cannabis sativa Islamabad leaves

3 Olive Olea europaea Murree leaves

4 Mint Mentha piperita Islamabad leaves

5 Bakain Melia azedarach D.G.Khan leaves

6 Kaner Nerium indicum Rawalpindi leaves

7 Ajwain Carum lopticum Islamabad seed

8 Aak Calotropis procera Bhawalpur leaves

9 Niazboo Ocimum americanum Islamabad seed

10 Sweet fennel Foeniculum vulgare Islamabad seed

11 Shisham Dalbergia sissoo D.G.Khan leaves

12 Fenugreek Trigonella foenumgraecum Islamabad leaves

13 Amaltas Cassia fistula Rawalpindi leaves

14 Soye Anethum graveolens Islamabad seed

15 Black seed Nigella sativa Islamabad seed

16 Chamomile Matricaria chammomilla Islamabad flower

17 Senna Cassia angustifolia Islamabad flower

18 Parthenium Parthenium hysterophorus Islamabad leaves

19 Hena Lawsonia inermis Rawalpindi leaves

20 Tobacco Nicotiana tabacum Jampur leaves

29

3.4.1 Biomass Production of Antagonists

For biomass production of antagonists Richard’s medium was used

(Harman, 1991). The medium (100 ml) was poured in 250 ml Erlenmeyer flasks,

autoclaved at 15 psi for 30 minutes, inoculated with two scoops of 10 mm diameter

taken from a 7 days old culture of each antagonist on PDA and incubated at

25±1°C for 15 days. For making slurry the broth along with biomass of each

antagonist was homogenized using a homogenizer for 30 seconds. The propagules

of each fungal slurry were enumerated using a haemocytometer. Based on these

counts, the concentrations (2×104, 2×106 and 2×108 propagules/ml) were achieved

by the addition of requisite amount of distilled water to the slurry of fungal

antagonist. Two ml of gelatin as an adhesive was added to eight ml of fungal

suspension of each concentration of each antagonist giving desired concentration of

2×104, 2×106 and 2×108 propagules/ml.

3.5 COLLECTION OF FUNGICIDES

Fungicides given in Table 3.3 were purchased from the local market for

their evaluation against M. Phaseolina.

3.6 COLLECTION OF GERMPLASM

Seeds of one hundred accessions, each of mung and mash were collected

from Gene Bank of Plant Genetic Resource Institute (PGRI), NARC, Islamabad for

the identification of resistant sources.

30

Table 3.3 List of fungicides used in the study

Fungicide Chemical Name Formulation Manufacturer

Antracol Propineb 70 WP Bayer (Pvt) Ltd.

Trimiltox- Copper+ 41 WP Syngenta

Forte Mancozeb

Dithane M-45 Mancozeb 80 WP Rohm & Hass Ltd.

Derosal Carbendazim 50 WP Bayer (Pvt) Ltd.

Captan Captan 50 WP ICI (Pvt) Ltd.

Benlate Benomyl 50 WP Du Pont

Ridomil Gold Matalaxyl+Mancozeb 68 WP Syngenta

Cobox Copper oxychloride 50 WP Pak Agro

Daconil Chlorothalonil 75 WP Syngenta

31

3.7 PHYSICOCHEMICAL COMPOSITION OF THE EXPERIMENTAL SITE

Soil samples were collected from 0-15 and 15-30 cm depths prior to sowing

of the crop and got analysed for some of the soil properties and different elements.

Which are given in Table (3.4).

3.8 METEOROLOGICAL DATA

The trial on screening of germplasm for the identification of resistance was

conducted at Pulses Pathology Research Area, NARC, Islamabad during the

summer season 2007. The average annual precipitation, temperature and relative

humidity recorded during the period have been shown in Fig 3.1

3.9 CULTURE MEDIA USED

Following media were used for isolation, purification and multiplication of

pathogenic fungi and antagonists.

3.9.1 Corn Meal Agar (CMA)

Corn meal = 20 g

Agar = 20 g

Dextrose = 20 g

Water = 1000 ml

3.9.2 Potato Dextrose Agar (PDA)

Potato starch = 20 g

Dextrose = 20 g

Agar = 20 g

32

Table 3.4 Physio-chemical analyses of the soil samples from experimental site

Parameter Depth

(inches)

Unit Value

pH 0-15 - 8.2

15-30 - 8.3

EC 0-15 dS/m 0.22

15-30 dS/m 0.18

CaCO3 0-15 % 5.1

15-30 % 5.4

OM 0-15 % 1.3

15-30 % 1.2

NO3-N 0-15 mg/kg 2.1

15-30 mg/kg 1.7

NaHCO3-P 0-15 mg/kg 3.6

15-30 mg/kg 3.4

K 0-15 mg/kg 49

15-30 mg/kg 45

Zn 0-15 mg/kg 0.28

15-30 mg/kg 0.20

Cu 0-15 mg/kg 0.52

15-30 mg/kg 0.50

Fe 0-15 mg/kg 8.6

15-30 mg/kg 7.0

Mn 0-15 mg/kg 10.8

15-30 mg/kg 10.2

33

Water = 1000 ml

3.9.3 Malt Extract Agar (MEA)

Malt extract = 20 g

Peptone = 1 g

Agar = 20 g

Dextrose = 20 g

Water = 1000 ml

3.9.4 Chloroneb Mercury Rose Bengal Agar (CMRA)

Chloroneb = 300 mg

Hg Cl = 90 mg

Streptomycin = 40 mg

Penicillin = 60 mg

Agar = 200 mg

Water = 1000 ml

3.9.5 Richard’s medium (RM)

KNO3 = 10 g

KH2PO4 = 5 g

MgSO4 = 1.3 g

Sucrose = 8 g

FeCl3 = 20 mg

Water = 1000 ml

34

3.9.6 Glucose Agar (GA)

Dextrose = 20 g

Agar = 20 g

Water = 1000 ml

3.10 DETERMINATION OF VARIABILITY AMONG ISOLATES OF M.

PHASEOLINA.

Variability among 65 isolates of M. phaseolina was studied on the basis of

morphological characteristics and their pathogenicity.

3.10.1 Morphological Variability

Morphological variability was based on radial growth, sclerotial size and

weight.

3.10.1.1 Radial growth

For studying variability in radial growth, the isolates were grown on Potato

Dextrose Agar. Fifteen ml of autoclaved PDA was poured in 90 mm diameter

petriplates and allowed to solidify. Five millimeter plug from the actively growing

culture of each isolate of the fungus was placed in the center of PDA plates

separately and incubated at 25±1°C for five days. Each isolate was replicated five

times. After stipulated period, the growth of each isolate was measured in terms of

colony diameter with the help of verniarcalipers and their means were computed.

35

On the basis of radial growth, the isolates were categorized as fast (> 80 mm),

medium (61-80 mm) and slow (< 61 mm) growing.

3.10.1.2 Sclerotial size

For measuring sclerotial size, slides from seven days old pure culture of M.

Phaseolina isolates were prepared and examined under microscope. Sizes of ten

randomly selected sclerotia were measured using ocular micrometer and their

means were found. These isolates were classified as large (>25µm), medium (21-25

µm) and small (<21 µm) sized.

3.10.1.3 Sclerotial Weight

In order to measure the dry weight of sclertia, each isolate of the fungus was

cultured on Potato Dextrose Broth (PDB). Hundred milliliter of broth in 250 ml

Erlenmeyer flasks was sterilized and inoculated with a 5 mm diameter scoop of

each isolate on cooloig with five replicates. The flasks were then incubated at

25±1°C for 15 days. The sclerotia were then filtered through Whatman,s filter paper

No.41, wrapped in aluminium foil and oven dried at 45°C for 24 hrs. The sclerotia

of each isolate were weighed using electric balance and grouped as high (> 0.15

mg), medium (0.11-0.15 mg) and low (< 0.11 mg) weight.

36

0

5

10

15

20

25

30

35

40

Jan Feb Mar Apr May June July Aug Sep Oct Nov Dec0

50

100

150

200

250

300

350

400Mini.TempMax.Temp.R.H%Rainfall(mm)

Fig 3.1 Average Precipitation, Temperature and R.H% at NARC during the summer season 2007.

37

3.10.2 Pathogenic Variability

The pathogenicity of 65 isolates was studied on each of three cultivars of

mung (NM-92, NM-51 and AEM-96) and mash (Mash-88, Mash-2 and Mash-97) in

the glasshouse in a split plot design with cultivars as main plots and the isolates as

subplots. Each treatment was repeated three times. Seeds were disinfested by

immersing them in 2.5% NaOCl for 5 min, rinsed in sterilized water and air dried.

Before sowing, an indigenous Rhizobium spp. was used for coating surface

disinfected seeds.

Ten rhizobium coated seeds of each of three cultivars of mung and mash

were sown in 1-L pots containing 800 gm soil infested with each isolates of M.

phaseolina @ 2 gm/Kg soil. Pots without inoculum served as controls. The pots

were then placed on a tray and incubated in a glasshouse at 30 ± 2°C. Disease

severity incited by each isolate on each cultivar was assessed after 20 days of

germination using the disease rating scale developed by Abawi and Pastor (1990).

3.11 MANAGEMENT OF CHARCOAL ROT DISEASE

3.11.1 Evaluation of different antagonists for their efficacy against M.

Phaseolina.

3.11.1.1 In vitro evaluation of antagonists

Seven biocontrol agents viz, Trichoderma harzianum, T. hamatum, T. koningi, T.

pseudokoningi, T. viride T. aureoviride and Gliocladium virens were tested for their

antagonistic activity against M. phaseolina in vitro by dual culture technique

38

(Morton and Stroube, 1955). Petridishes (90 mm dia.) containing 15 ml of

autoclaved PDA were inoculated with 5-mm diameter mycelial discs each of 7-day-

old culture of the pathogen (MP-7) and antagonists at eqi distance from the

periphery and incubated for 6 days at 25±1°C in an incubator. Petriplates without

antagonists served as control. Each treatment was replicated five times. After 6 days

the radial growth of the pathogen was measured. Percent inhibition of radial growth

of M. phaseolina was calculated by applying the formula (Vincent, 1927).

Colony growth in Control–Colony growth in treated

% Growth inhibition= ----------------------------------------------------------------- x 100

Colony growth in Control

3.11.1.2 Pot culture assay

To evaluate the efficacy of antagonists in pots, ten seeds each of mung (NM-

92) and mash (Mash-98) were sown in pots containing 800 gm soil infested with

rice seeds colonized with M. phaseolina (MP-7) @ 2 gm/Kg soil. Each treatment

was replicated five times. The pots were placed in a glasshouse at 30oC in

completely randomized design (CRD). Data on percentage germination was

recorded after 20 days, and percentage increase or decrease over control was

calculated.

3.11.2 Evaluation of plant extracts for their effictiveness against M. Phaseolina.

Aqueous extracts of twenty different plant species were used for their

evaluation against phytopathogenic fungi M. phaseolina in vitro and pot culture

assay.

39

3.11.2.1 In vitro evaluation of plant extracts

For in vitro evaluation of aqueous plant extracts poisoned food technique

(Nene and Thapliyal, 1993) was used. Potato dextrose agar (PDA) medium was

used in the study. Twenty five ml of sterile extract from each concentration of test

plants was mixed with 175 ml potato dextrose agar medium amended with

streptomycin and carefully agitated to allow for proper mixing of extract and media.

15ml of aliquots of the amended media were dispensed into 90mm petridishes.

Once the amended agar had solidified, 5mm discs from the actively growing edge

of a 5 days old colony of M. phaseolina (MP-7) on PDA was placed in the center of

each plate and incubated at (25±1°C) for 6 days. The medium with Inoculum disc

but without any extract served as control. Each treatment was replicated five times.

The in vitro tests were carried out to measure the effects of the leaf extracts on

radial growth of the fungi. The inhibition of mycelial growth was determined by the

following formulae.

Control (area) – Treatment (area) % Mycelial inhibition = ------------------------------------------------------- x 100

Control (area)


Seeds each of mung (NM-92) and mash (Mash-98) were surface sterilized

for 10 min in 5% commercial sodium hypochlorite solution, washed in sterile

distilled water and air dried. The seeds were then soaked in different solutions of

botanical extracts for 30 min and then air dried for 3-4 hours in laminar flow

chamber. Control seeds treated with sterile distilled water were planted in soils

40

amended with rice seeds colonized with M. phaseolina @ 2gm/kg soil. Ten seeds

were planted in each pot. Each treatment was replicated five times. Data on

percentage germination/plant survival was recorded after 20 days.

3.11.3 Evaluation of Different Fungicides for their Effectiveness against M.

phaseolina.

Nine fungicides were tested for their efficiency to retard the fungal growth

of M. phaseolina in vitro and through pot culture assay.

3.11.3.1 In vitro evaluation of fungicides

The effectivity of the test fungicides was tested by using poisoned food

technique (Nene and Thapliyal, 1993). Requisite quantity of active ingredient of

each fungicide was mixed in autoclaved PDA to obtain the required concentrations

of 50, 100 and 150 ppm. Poisoned medium was then poured into each sterilized 90

mm dia sterilized petriplate, allowed to solidify and proceded as described in

section 3.11.2.1


For testing the effectivity of fungicides against M. phaseolina (MP-7) in

pots, surface sterilized seeds each of mung (NM-92) and mashbean (Mash-98) were

treated @ 1, 2 and 3 gm of a.i of each fungicide per kg of seed as slurry method.

Control seeds were treated with sterile distilled water. Ten seeds in five replications

were sown in sterile pots containing a mixture of soil and sand at the rate of 1: 1 (v:

v) amended with the rice seeds colonized with M. phaseolina @ 2gm/kg soil. The

41

pots were kept in growth rooms at 30 oC. Data on percentage germination/plant

survival was recorded after 20 days.

3.11.4 Evaluation of Mung and Mash Germplasm for their Resistance against Charcoal rot Disease under Greenhouse and Field Conditions.

One hundred accessions/lines each of mung and mash was screened for their

resistance against charcoal rot. Screening was done under artificial inoculation of

M. phaseolina in green house as well as in the field conditions. Field plots were

inoculated @ 2g of rice seeds infected with M. phaseolina per meter of row. Each

test entry was sown in 4m row length and plant to plant distance was maintained

30cm. In the green house 2-3 rice seeds infected with M. phaseolina are placed

around each seed of mung and mash in pots and covered with clean soil. Ten seeds

were planted in each pot having five replications. Data on disease severity was

recorded at maturity using disease ratting scale developed by Pastor Corrales and

Abawi (1988).

3.12 Statistical Analysis

All the lab experiments were repeated twice. Since there were no

discrepancies in the mean values of all the corresponding treatments of the repeated

experiments, the data of both the trials were amalgamated before statistical analysis.

Data were subject to Analysis of Variance (ANOVA) and means were compared by

Duncan’s Multiple Range Test (DMRT) at P=0.005, using Mstat C software.

Regression equations were drawn in Microsoft Excel 2003. Isolates were clustered

using Un-Paired Group Mean Average (UPGMA) method within the Statistica

version 6.1.

42

Table 3.5 Disease scoring scale (1-9) for the assessment of charcoal rot disease

Score Disease reaction Symptoms

1 Highly resistant (HR) No visible symptoms.

3 Resistant (R) Lesions are limited to cotyledonary tissues.

5 Tolerant (T) Lesions have progressed from cotyledons to about

2 cm of stem tissues.

7 Susceptible (S) Lesions are extensive on stem and branches.

9 Highly Susceptible (HS) Most of the stem and growing points are infected.

A considerable amount of Sclerotia are produced.

43

Chapter 4

RESULTS

4.1 MORPHOLOGICAL VARIABILITY AMONG MACROPHOMINA

PHASEOLINA ISOLATES

Significant variations have been observed in the morphology among 65

isolates of M. phaseolina in terms of radial growth, sclerotial size and weight.

4.1.1 Radial growth

Significant differences among 65 isolates of M. phaseolina collected from

different districts were observed on the basis of radial growth (F = 11.75; df = 64,

130; P < 0.001). The individual average radial growth of 65 isolates of M.

phaseolina ranged from 43.67 to 87.17 mm observed after 5 days of incubation.

Maximum colony diameter of 87.17 and 86.67 mm were observed in case of isolate

MP-7 (D.G.khan) and MP-26 (Layyah) proving to be the fast growing, while

isolates MP-29, MP-30, MP-31 and MP-34 showed minimum radial growth and

were rated as slow growing with average growth less than 55 mm. The rests of the

isolates showed intermediate growth (Table 4.1, Column 2). On the basis of growth

the isolates were classified as Fast, Intermediate and Slow growing (Table 4.1.1).

4.1.2 Sclerotial size

Significant variations were also observed among these isolates regarding

size of their sclerotia (F = 3.53; df = 64, 130; P < 0.001). Maximum sclerotial size

was observed in case of isolates MP-20 and MP-3 showing 29.00 and 27.33 µm

diameter, while the isolates MP-39, MP-38 and MP-45 were found to be the

smallest in size. The individual average sclerotial size of isolates ranged from

44

17.00-29.00 µm which are given in (Table 4.1, Column 3). On the basis of size, the

isolates were categorized as large, medium and small sized and shown in Table

4.1.2.

4.1.3 Sclerotial weight

Sclerotial weight was another parameter considered for variability. The

analysis of variance showed significant variability in sclerotial weight among the

isolates (F = 6.07; df = 64, 130; P < 0.001). The scrutiny of the data of sclerotial

weight given in (Table 4.1, Column 2) showed that MP-20, MP-23, MP-24 and

MP-52 produced maximum sclerotia giving maximum weight above 0.20 gm. The

lowest sclerotial production was found in isolate MP-46 with average weight of

0.087 g. The remaining isolates were intermediate in sclerotial weight. The isolates

were then grouped as high, medium and low weight as shown in Table 4.1.3.

The cluster analysis of 65 isolates of 14 districts on an average basis of

three morphological parameters (Radial growth, sclerotial size and sclerotial

weight) is shown in Fig4.1. In the dendogram, three main clusters were

distinguished, at a linkage distance of around 50 %. The first cluster comprised 8

isolates of Faisalabad and M. Garh districts, the second cluster comprised 13

isolates of Chakwal, Bhakkar and D. G. Khan districts and the third cluster

consisted of 44 isolates of the remaining districts. The isolates belonging to the

districts Chakwal, Bhakhar and D.G.khan showed optimum growth performance,

while the isolates belonging to M. Garh and Faisalabad exhibited poor growth

performance. The isolates in the third group were found intermediate in their

growth performance (Figure-4.1).

45

Table 4.1 Morphological variations among different isolates of M. phaseolina ISOLATES SCLEROTIAL WEIGHT SCLEROTIAL SIZE RADIAL GROWTH

1 2 3 4

MP-1 0.1000 H-L 26.67A-D 63.17I-R

MP-2 0.1233 E-L 23.50B-L 65.33I-P

MP-3 0.1000 H-L 27.33AB 75.17A-I

MP-4 0.1300D-L 19.67G-O 71.67F-M

MP-5 0.1500A-K 20.67F-O 72.33E-M

MP-6 0.1633A-H 20.33F-O 78.00A-H

MP-7 0.1500A-K 24.17A-J 87.17A

MP-8 0.1100G-L 20.83F-O 32.00T

MP-9 0.1300D-L 24.67A-H 52.83Q-S

MP-10 0.1300D-L 23.00B-M 83.00A-F

MP-11 0.1500A-K 19.67G-O 54.67O-S

MP-12 0.1633A-H 18.17L-O 64.33I-R

MP-13 0.1500A-K 21.17E-O 68.33H-N

MP-14 0.1300D-L 19.33H-O 84.67A-E

MP-15 0.1367C-L 19.00I-O 74.50B-K

MP-16 0.1433B-L 21.83C-O 68.00H-N

MP-17 0.1800A-F 24.50A-I 82.17A-G

MP-18 0.1900A-D 26.83A-C 74.83A-J

MP-19 0.1900A-D 21.17E-O 85.33A-C

MP-20 0.2100A 29.00A 62.50J-R

MP-21 0.1700A-G 20.33F-O 78.00A-H

MP-22 0.1900A-D 20.17F-O 71.83F-M

MP-23 0.2033AB 17.50M-O 65.17I-Q

MP-24 0.2000A-C 22.50B-O 64.00I-R

MP-25 0.1800A-F 22.67B-N 66.33H-O

MP-26 0.1700A-G 23.33B-L 86.67AB

MP-27 0.1800A-F 21.67C-O 82.83A-F

MP-28 0.1733A-G 17.33NO 43.83S

MP-29 0.1333D-L 18.00L-O 43.67S

MP-30 0.1333D-L 22.67B-N 44.00S

MP-31 0.1500A-K 26.33A-E 53.83P-S

MP-32 0.1167F-L 19.83G-O 83.33A-F

MP-33 0.1167F-L 17.67M-O 84.17A-F

46

MP-34 0.1000H-L 21.83C-O 53.17P-S

MP-35 0.1267D-L 24.67A-H 72.50D-M

MP-36 0.1000H-L 22.17B-O 61.50M-R

MP-37 0.1300D-L 19.33H-O 52.33RS

MP-38 0.08667KL 17.50M-O 73.00C-M

MP-39 0.0933I-L 17.00O 67.33H-N

MP-40 0.1000H-L 25.00A-G 74.17B-L

MP-41 0.1233E-L 23.50B-L 71.83F-M

MP-42 0.1333D-L 24.33A-J 73.17C-M

MP-43 0.1200E-L 18.83J-O 81.33A-G

MP-44 0.1233E-L 19.00I-O 85.00A-D

MP-45 0.1333D-L 22.50B-O 86.17AB

MP-46 0.0800L 17.50M-O 84.83A-E

MP-47 0.08667KL 21.33D-O 65.17I-Q

MP-48 0.1000H-L 18.50K-O 81.17A-G

MP-49 0.0900J-L 22.33B-O 64.17I-R

MP-50 0.1800A-F 20.33F-O 81.83A-G

MP-51 0.1667A-G 21.67C-O 56.50N-R

MP-52 0.2033AB 25.67A-F 78.00A-H

MP-53 0.1767A-F 23.50B-L 67.17H-N

MP-54 0.1900A-D 23.50B-L 78.50A-H

MP-55 0.2000A-C 24.17A-J 63.83I-R

MP-56 0.1567A-I 27.50AB 62.17K-R

MP-57 0.1833A-E 22.83B-N 71.83F-M

MP-58 0.1500A-K 24.00A-K 74.17B-L

MP-59 0.1567A-I 26.67A-D 78.17A-H

MP-60 0.1833A-E 19.33H-O 61.17M-R

MP-61 0.1800A-F 19.67G-O 69.67G-M

MP-62 0.1667A-G 21.50C-O 84.50A-E

MP-63 0.1500A-K 24.17A-J 61.67L-R

MP-64 0.1533A-J 19.83G-O 78.67A-H

MP-65 0.1567A-I 24.17A-J 54.50O-S

LSD 0.052 4.325 10.04

47

Table 4.1.1 Isolates categorized into three classes on the basis of radial

growth

S. No Category Number Isolates

1 Fast growing

> 80 mm

16 MP-7, MP-10, MP-14, MP-17, MP-19, MP-26, MP-27, MP-32, MP-33, MP-43, MP-44, MP-45, MP-46, MP-48, MP-50, MP-62

2 Medium growing

61-80 mm

38 MP-1, MP-2, MP-3, MP-4, MP-5, MP-6, MP-12, MP-13, MP-15, MP-16, MP-18, MP-20, MP-21, MP-22, MP-23, MP-24, MP-25, MP-35, MP-36, MP-38, MP-39, MP-40, MP-41, MP-42, MP-47, MP-49, MP-52, MP-53, MP-54, MP-55, MP-56, MP-57, MP-58, MP-59, MP-60, MP-61, MP-63, MP-64

3 low growing

< 61 mm

11 MP-8, MP-9, MP-11, MP-28, MP-29, MP-30, MP-31, MP-34, MP-37, MP-51, MP-65

48

Table 4.1.2 Isolates categorized into three classes on the basis of size of

sclerotia


1 Large

>25 um

9 MP-1, MP-3, MP-18, MP-20, MP-31, MP-40, MP-52, MP-56, MP-59

2 Medium

21-25 um

30 MP-2, MP-7, MP-9, MP-10, MP-13, MP-16, MP-17, MP-19, MP-24, MP-25, MP-26, MP-27, MP-30, MP-34, MP-35, MP-36, MP-41, MP-42, MP-45, MP-47, MP-49, MP-51, MP-53, MP-54, MP-55, MP-57, MP-58, MP-62, MP-63, MP-65

3 Small

< 21 um

26 MP-4, MP-5, MP-6, MP-8, MP-11, MP-12, MP-14, MP-15, MP-21, MP-22, MP-23, MP-28, MP-29, MP-32, MP-33, MP-37, MP-38, MP-39, MP-43, MP-44, MP-46, MP-48, MP-50, MP-60, MP-61, MP-64

49

Table 4.1.3 Isolates categorized into three classes on the basis of weight of

sclerotia


1 High

>0.15 mg

35 MP-5, MP-6, MP-7, MP-11, MP-12, MP-13,MP-17, MP-18, MP-19, MP-20, MP-21, MP-22, MP-23, MP-24, MP-25, MP-26, MP-27, MP-28, MP-31, MP-50, MP-51, MP-52, MP-53, MP-54, MP-55, MP-56, MP-57, MP-58, MP-59, MP-60, MP-61, MP-62, MP-63, MP-64, MP-65

2 Medium

0.11-0.15 mg

18 MP-2, MP-4, MP-9, MP-10, MP-14, MP-15, MP-16, MP-29, MP-30, MP-32, MP-33, MP-35, MP-37, MP-41, MP-42, MP-43, MP-44, MP-45

3 Low

< 0.11 mg

12 MP-1, MP-3, MP-8, MP-34, MP-36, MP-38, MP-39, MP-40, MP-46, MP-47, MP-48, MP-49

50

FaisalabadM.Garh

ChakwalBhakkar

D.G.KhanIslamabad

MianwaliBannu

NarowalSialkot

LayyahKohat

D.I.KhanRawalpindi

0

2

4

6

8

10

12

14

16

18

Link

age

Dis

tanc

e

Figure 4.1 Dendrogram derived from cluster analysis (UPGMA) showing relationship among the 65 isolates of M. phaseolina

on the basis of morphological characters collected from 14 districts of Punjab and KPK provinces.

51

4.1.2 Pathogenic Variability among M. Phaseolina Isolates

4.1.2.1 Pathogenic Variability among mungbean

Highly significant differences were observed among isolates, varieties and

their interactions. Significant variations in pathogenicity were found among sixty

five isolates of the fungus (F=34.31; DF=64, 388; P< 0.001) when tested against

three mungbean cultivars (NM-92, NM-51 and AEM-96) which also varied in

response to the isolates (F=52.049; DF=64, 388; P< 0.001). Six isolates viz, MP-7,

MP-13, MP-18, MP-48, MP-56 and MP-64 were found highly virulent against NM-

92 with mean disease severity scoring of 7.3. Five isolates viz, MP-8, MP-10, MP-

26, MP-35 and MP-60 were found to be least virulent in their reaction with average

disease score ranging between 2.3 to 3.7 showing that the cultivar is resistant

against these isolates (Table 4.2, Column 4).

Similarly nine isolates viz, MP-5, MP-16, MP-17, MP-22, MP-33, MP-37,

MP-44, MP-56 and MP-63 were detected to be virulent against NM-51 with

average disease score ranging from 7.0 to 7.7, while six isolates viz, MP-11, MP-

30, MP-31, MP-38, MP-53 and MP-60 exhibited least pathogenic reaction against

NM-51 with disease severity ranging from 2.0-3.0 and the remaining isolates

proved to be intermediate in their pathogenicity (Table 4.2 Coulmn 3).

Seventeen isolates appeared to be highly virulent towards AEM-96 as these

gave disease score above 7, while six isolates viz, MP-8, MP-25, MP-30, MP-31,

MP-35, MP-36 and MP-41 with average disease score ranged up to 3 proved to be

least virulent. The rest of the isolates found to be moderately virulent as shown in

52

(Table 4.2 Coulmn 4).

The Cluster analysis on the basis of pathogencity is shown in Fig 4.2. Sixty

five isolates were categorized into five clusters on the basis of 52ashbean52city

against three 52ashbean cultivars. Ten fungal isolates, of districts placed in cluster

2 and 3 were appeared to be least virulent. Whereas, eight isolates of diverse origin

in cluster 5 proved to be highly virulent for their virulence against 52ashbean

cultivars. The remaining isolates falling in cluster 3 and 4 were regarded as

moderately virulent.

4.1.2.2 Pathogenic Variability among mashbean

The analysis of variance showed highly significant pathogenic differences

among 65 isolates of M. phaseolina (F = 33.92; df = 64, 388; P < 0.001). against

mashhbean cultivars. Significant variations were also observed among cultivars (F

= 79.01; df = 2, 388; P < 0.001) in their response towards isolates. In Mash-88,

twenty three isolates with average disease score 7.0 and above were found to be

highly virulent. The other isolates exhibited intermediate reaction in relation to

pathogenicitycity against the cultivar and regarded as moderately virulent Table

4.3, Column 2.

Similarly, five isolates namely MP-16, MP-17, MP-25, MP-44 and MP-50

showed highly virulent reaction against Mash-2 with average disease score 7.0 and

above, While the remaining isolates were found to be moderately virulent as shown

in Table-4.3, Column 3. On the other hand, 48 isolates were found to be highly

virulent towards Mash-97 on the basis of disease score 7 and above, While the rest

of the isolates appeared moderately virulent (Table 4.3 Column 4).

53

Five clusters of 65 isolates of M. phaseolina against three 53ashbean

cultivars showed that the isolate MP-9 in cluster-1 and MP-8, MP-10, MP-29 and

MP-30 in cluster-2 were found least aggressive in their reaction against three mash

cultivars, while 12 isolates of different origins were found to be more aggressive,

were in cluster 4 and 5, while the remaining were placed in cluster 3 (Fig 4.3).

54

Table 4.2 Differential response of selected mungbean cultivars against various isolates of M. phaseolina

ISOLATES NM-92 NM-51 AEM-96

1 2 3 4

MP-1 5.7DEFG 5.7DEFG 5.7DEFG

MP-2 7.0ABC 4.7GHIJ 7.3AB

MP-3 5.7DEFG 5.3EFGH 7.0ABC

MP-4 6.7ABCD 7.0ABC 7.3AB

MP-5 6.0CDEF 7.7A 7.3AB

MP-6 6.3BCDE 5.7DEFG 6.0CDEF

MP-7 7.3AB 6.7ABCD 7.0ABC

MP-8 2.7MN 5.7DEFG 3.0LMN

MP-9 4.3HIJK 5.3EFGH 4.7GHIJ

MP-10 2.3MN 5.7DEFG 5.0FGHI

MP-11 5.7DEFG 2.7MN 4.3HIJK

MP-12 6.0CDEF 4.7GHIJ 5.7DEFG

MP-13 7.3AB 6.0CDEF 4.3HIJK

MP-14 4.7GHIJ 7.0ABC 5.3EFGH

MP-15 4.3HIJK 5.0FGHI 7.0ABC

MP-16 6.3BCDE 7.3AB 5.7DEFG

MP-17 7.0ABC 7.3AB 6.0CDEF

MP-18 7.3AB 5.7DEFG 5.7DEFG

MP-19 5.7DEFG 6.0CDEF 4.3HIJK

MP-20 4.3HIJK 6.3BCDE 7.3AB

MP-21 4.7GHIJ 7.0ABC 5.3EFGH

MP-22 6.0CDEF 7.3AB 4.7GHIJ

MP-23 5.3EFGH 5.7DEFG 7.7A

MP-24 4.7GHIJ 5.0FGHI 5.7DEFG

MP-25 5.7DEFG 5.3EFGH 2.7MN

MP-26 2.7MN 6.7ABCD 7.3AB

MP-27 3.3KLM 7.0ABC 7.7A

MP-28 5.3EFGH 4.3HIJK 5.7DEFG

MP-29 6.0CDEF 4.0IJK 7.0ABC

MP-30 5.3EFGH 2.3MN 3.0LMN

MP-31 4.3HIJK 2.0N 3.3KLM

55

MP-32 2.3MN 4.7GHIJ 4.7GHIJ

MP-33 5.7DEFG 7.3AB 6.7ABCD

MP-34 5.3EFGH 4.3HIJK 7.3AB

MP-35 3.3KLM 4.7GHIJ 2.3MN

MP-36 5.3EFGH 4.3HIJK 2.7MN

MP-37 5.7DEFG 7.3AB 7.0ABC

MP-38 4.3HIJK 3.0LMN 5.3EFGH

MP-39 6.0CDEF 5.7DEFG 6.7ABCD

MP-40 2.7MN 4.0IJK 5.7DEFG

MP-41 7.0ABC 5.3EFGH 3.3KLM

MP-42 5.7DEFG 7.0ABC 7.3AB

MP-43 6.7ABCD 5.7DEFG 7.0ABC

MP-44 6.0CDEF 7.7A 7.7A

MP-45 4.7GHIJ 7.0ABC 5.7DEFG

MP-46 5.7DEFG 4.3HIJK 2.7MN

MP-47 5.3EFGH 4.7GHIJ 5.7DEFG

MP-48 7.3AB 6.0CDEF 7.0ABC

MP-49 5.3EFGH 4.0IJK 5.7DEFG

MP-50 5.7DEFG 5.7DEFG 7.3AB

MP-51 5.3EFGH 7.0ABC 7.7A

MP-52 4.7GHIJ 4.3HIJK 5.3EFGH

MP-53 5.7DEFG 2.7MN 7.3AB

MP-54 4.3HIJK 4.0IJK 5.7DEFG

MP-55 7.0ABC 5.3EFGH 7.3AB

MP-56 7.3AB 7.3AB 6.0CDEF

MP-57 5.7DEFG 5.3EFGH 7.7A

MP-58 4.7GHIJ 5.7DEFG 7.0ABC

MP-59 5.7DEFG 4.3HIJK 5.3EFGH

MP-60 3.7JKL 2.3MN 6.7ABCD

MP-61 4.3HIJK 4.7GHIJ 7.3AB

MP-62 5.3EFGH 5.7DEFG 5.7DEFG

MP-63 6.7ABCD 7.3AB 4.7GHIJ

MP-64 7.3AB 5.3EFGH 7.7A

MP-65 5.7DEFG 4.0IJK 5.3EFGH

56

MP-

46M

P-36

MP-

25M

P-41

MP-

13M

P-60

MP-

38M

P-31

MP-

30M

P-11

MP-

40M

P-32

MP-

10M

P-35

MP-

8M

P-27

MP-

26M

P-45

MP-

21M

P-14

MP-

19M

P-9

MP-

63M

P-22

MP-

56M

P-17

MP-

16M

P-51

MP-

42M

P-37

MP-

33M

P-44

MP-

5M

P-7

MP-

4M

P-53

MP-

34M

P-29

MP-

58M

P-20

MP-

61M

P-15

MP-

57M

P-23

MP-

39M

P-50

MP-

3M

P-48

MP-

43M

P-64

MP-

55M

P-2

MP-

54M

P-52

MP-

24M

P-65

MP-

59M

P-47

MP-

49M

P-28

MP-

12M

P-18

MP-

6M

P-62

MP-

1

0

1

2

3

4

5Li

nkag

e D

ista

nce

Fig- 4.2 Dendrogram showing the clustering of the virulence of M. phaseolina isolates on 3 Mungbean cultivars.

57

Table- 4.3 Differential response of selected mashbean cultivars against various isolates of M. phaseolina

ISOLATES Mash-88 Mash-2 Mash-97

1 2 3 4

MP-1 7.0 EFG 5.7 HIJ 7.3 DEF

MP-2 8.3 ABC 5.7 HIJ 6.7 EFG

MP-3 7.7CDE 6.3FGH 7.0EFG

MP-4 7.0EFG 7.7CDE 8.7AB

MP-5 6.7 EFG 7.7CDE 7.0EFG

MP-6 8.7AB 7.0EFG 7.7CDE

MP-7 9.0A 7.3DEF 6.3FGH

MP-8 5.7HIJ 4.3LMN 5.0JKL

MP-9 3.7NO 5.7HIJ 3.3O

MP-10 6.0GHI 4.3LMN 4.3LMN

MP-11 6.3FGH 5.3IJK 7.0EFG

MP-12 7.0EFG 5.3IJK 7.3DEF

MP-13 8.7AB 7.3DEF 5.7HIJ

MP-14 6.0GHI 7.3DEF 7.0EFG

MP-15 5.3IJK 5.7HIJ 7.7CDE

MP-16 7.7CDE 9.0A 7.0EFG

MP-17 7.3DEF 9.0A 7.3DEF

MP-18 8.0BCD 7.3DEF 7.0EFG

MP-19 6.7EFG 7.0EFG 8.7AB

MP-20 5.3IJK 6.3FGH 7.0EFG

MP-21 6.0GHI 7.0EFG 9.0A

MP-22 7.0EFG 7.7CDE 9.0A

MP-23 5.7HIJ 7.0EFG 7.3DEF

MP-24 5.3IJK 7.3DEF 7.0EFG

MP-25 6.3FGH 9.0A 7.7CDE

MP-26 5.7HIJ 6.7EFG 7.7CDE

MP-27 4.7KLM 7.0EFG 7.0EFG

MP-28 6.0GHI 7.7CDE 7.3DEF

MP-29 6.3FGH 4.3LMN 4.3LMN

MP-30 6.3FGH 3.3O 4.0MNO

MP-31 4.3LMN 5.7HIJ 7.0EFG

58

MP-32 5.0JKL 5.3IJK 6.3FGH

MP-33 4.3LMN 5.0JKL 5.7HIJ

MP-34 5.3IJK 6.0GHI 5.7HIJ

MP-35 6.7EFG 7.0EFG 6.3FGH

MP-36 3.7NO 5.7HIJ 6.7EFG

MP-37 6.7EFG 5.3IJK 7.0EFG

MP-38 4.7KLM 4.3LMN 7.7CDE

MP-39 7.3DEF 6.3FGH 7.3DEF

MP-40 6.0GHI 5.7HIJ 7.3DEF

MP-41 4.7KLM 6.0GHI 5.7HIJ

MP-42 7.0EFG 7.3DEF 8.3ABC

MP-43 9.0A 7.7CDE 7.3DEF

MP-44 7.3DEF 9.0A 7.3DEF

MP-45 9.0A 7.3DEF 7.0EFG

MP-46 6.7EFG 5.3IJK 7.0EFG

MP-47 7.3DEF 6.0GHI 7.3DEF

MP-48 7.7CDE 6.3FGH 7.0EFG

MP-49 7.0EFG 5.7HIJ 7.3DEF

MP-50 6.7EFG 8.0BCD 9.0A

MP-51 6.3FGH 7.3DEF 7.7CDE

MP-52 7.0EFG 5.7HIJ 6.3FGH

MP-53 6.0GHI 5.7HIJ 7.7CDE

MP-54 6.3FGH 7.0EFG 8.3ABC

MP-55 7.7CDE 7.7CDE 7.3DEF

MP-56 9.0A 7.0EFG 7.3DEF

MP-57 6.7EFG 7.7CDE 9.0A

MP-58 5.7HIJ 7.3DEF 7.0EFG

MP-59 6.7EFG 7.0EFG 9.0A

MP-60 6.7EFG 7.7CDE 7.0EFG

MP-61 5.3IJK 7.0EFG 8.7AB

MP-62 7.0EFG 7.7CDE 6.3FGH

MP-63 4.7KLM 7.0EFG 7.3DEF

MP-64 5.7HIJ 7.3DEF 7.0EFG

MP-65 6.3FGH 7.7CDE 6.7EFG

59

MP-

9M

P-30

MP-

29M

P-10

MP-

8M

P-38

MP-

41M

P-34

MP-

33M

P-32

MP-

36M

P-31

MP-

53M

P-40

MP-

15M

P-63

MP-

27M

P-20

MP-

26M

P-24

MP-

23M

P-64

MP-

58M

P-14

MP-

25M

P-44

MP-

17M

P-16

MP-

62M

P-35

MP-

51M

P-28

MP-

65M

P-60

MP-

5M

P-61

MP-

21M

P-42

MP-

54M

P-59

MP-

19M

P-57

MP-

50M

P-22

MP-

4M

P-13

MP-

7M

P-55

MP-

18M

P-56

MP-

45M

P-43

MP-

6M

P-47

MP-

39M

P-48

MP-

3M

P-2

MP-

52M

P-46

MP-

37M

P-11

MP-

12M

P-49

MP-

1

0

1

2

3

4

5Li

nkag

e D

ista

nce

Fig- 4.3 Dendrogram showing the clustering of the virulence of M. phaseolina isolates on 3 Mashbean cultivars.

60

4.2 MANAGEMENT OF CHARCOAL ROT DISEASE

4.2.1 Inhibitory effect of antagonists against M. phaseolina

All the antagonists significantly retarded the growth of M. phaseolina in

vitro (F=21.525; df= 6, 14; P< 0.001). Maximum reduction (79.63%) was observed

with T. harzianum while T. pseudokoningii caused the minimum decrease (58.14%)

in growth of all the test antagonists. The inhibition in growth caused by all the

antagonists is given in Table 4.4.

4.2.2 Effects of antagonists on the plant survival of mungbean and

mashbean

Antagonists exhibited significant variations in their efficacy (F=18.088;

df=6, 14; P< 0.001) on plant survival of mungbean when used as seed treatment.

Maximum average plant survival was obtained with T. harzianum followed by T.

hamatum and T. viride while T. pseudokoningii gave the poorest germination of all

the test biocontrol agents. Maximum individual germination of 86.67% was

achieved with T. harzianum at a concentration of 2x108 (propagules/ml), while the

minimum (33.33%) was recorded with T. pseudokoningii at 2x104. Individual

germination of mung with all the antagonists at three concentrations is given in

Table 4.5.

Concentrations also had significant effects on plant survival being

maximum at the highest concentration. The plant survival decreased as the

concentrations of the antagonists decreased. The antagonists gave almost the

similar kind of results in case of mash and are shown in Table 4.6.

61

Table 4.4 Effect of antagonists on the radial growth of M. phaseolina

S.No Treatments % growth inhibition

over control

1 T. harzianum 79.63

2 T. hamatum 76.30

3 T. viridae 73.70

4 T. aueroviride 68.89

5 T. koningii 64.81

6 T. pseudokoningii 58.14

7 G. virens 62.22

LSD Value = 4.62 aValues are the means of the five replicate samples bFigures following ± are standard errors

62

Table 4.5 Effect of antagonists on plant survival of mungbean

Increase in plant survival over control

at

S.No

Antagonist

2×104 2×106 2×108 Average

1 T. harzianum 63.33 ± 3.33 73.33 ± 3.33 86.67 ± 3.33 74.44

2 T. hamatum 60.00 ± 0.00 70.00 ± 0.00 76.67 ± 3.33 68.89

3 T. viride 56.67 ± 3.33 63.33 ± 3.33 70.00 ± 0.00 63.33

4 T. aueroviride 53.33 ± 3.33 60.00 ± 0.00 66.67 ± 3.33 60.00

5 T. koningii 43.33 ± 3.33 53.33 ± 3.33 56.67 ± 3.33 51.11

6 T. pseudokoningii 33.33 ± 3.33 40.00 ± 5.77 43.33 ± 3.33 38.86

7 G. virens 40.00 ± 5.77 43.33 ± 3.33 50.00 ± 5.77 44.44

LSD Value for average values = 5.96 aValues are the means of the five replicate samples bFigures following ± are standard errors

63

Table 4.6 Effect of antagonists on plant survival of mashbean

Increase in plant survival over control

at

S.No

Antagonist

2×104 2×106 2×108 Average

1 T. harzianum 63.33 ± 3.33 70.00 ± 5.77 80.00 ± 5.77 71.11

2 T. hamatum 60.00 ± 5.77 66.67 ± 3.33 73.33 ± 3.33 66.66

3 T. viride 56.67 ± 3.33 60.00 ± 0.00 66.67 ± 3.33 61.11

4 T. aueroviride 46.67 ± 3.33 56.67 ± 3.33 63.33 ± 3.33 55.56

5 T. koningii 43.33 ± 3.33 46.67 ± 3.33 56.67 ± 3.33 48.89

6 T. pseudokoningii 30.00 ± 0.00 43.33 ± 3.33 50.00 ± 0.00 41.11

7 G. virens 33.33 ± 3.33 46.67 ± 3.33 53.33 ± 3.33 44.44

LSD Value = 8.94 aValues are the means of the five replicate samples bFigures following ± are standard errors

64

4.2.3 Effects of plant extracts on the growth of M. Phaseolina

The aqueous extracts of all the tested plants significantly suppressed the

growth of M. phaseolina (F=165.80; df=19,120; P> 0.001). Of all the plants, C.

lopticum proved to be the most effective in suppressing the growth of the pathogen

at all concentrations followed by A. indica and N. sativa as against N. indicum, F.

vulgare and T. foenumgraecum which appeared to be least effective. The individual

growth inhibitions at four concentrations of test plants are given in Table 4.7.

Similarly, concentrations also had significant inhibitory effect on the growth

(F=1362.89; df= 3, 120; P> 0.001), being the maximum at 100 % concentration of

the extracts. As the concentration of extracts lowered, the magnitude of inhibition

of growth of the fungus also decreased significantly. A direct relationship between

concentrations and growth inhibitions was observed in case of all the test plants as

shown in Table 4.7.1.

4.2.4 Effects of plant extracts on plant survival of mungbean and mashbean

All the plant extracts when used as seed treatment significantly enhanced plant

germination in terms of plant survival. Of all the test plants, C. lopticum showed

maximum increase in survival of mungbean plants over control followed by A.

indica and N. sativa. On the other hand, N. indicum, F. vulgare and T.

foenumgraecum were appeared the least effective in reducing the damage of the

pathogen. The maximum individual survival (83.52 %) was achieved with 100 %

concentration of C. lopticum as against the minimum of 20 % obtained with L.

inermis at 25 % concentration. The individual percent increases of plant survival at

65

four concentrations of the test plants are given in Table 4.8. Significant effect of

concentrations was also observed. Maximum plant increase in survival was

recorded at 100% concentration of extracts. The effect of the plant extracts

diminished as the concentration decreased. The effect of concentrations was found

to be directly proportional to plant survival and these relationships have been

shown by regression equations given in Table 4.8.1. A similar pattern was observed

in case of mashbean. The individual increases in plant survivals and relationships

between the concentrations and plant survivals are given in Table 4.9, 4.9.1.

66

Table 4.7 Effect of plant extracts on the growth of M. phaseolina

Plant Extract % inhibition over control

25 % 50 % 75 % 100 % Average

Azadirachta indica 43.33±0.58 44.44±0.58 58.89±1.16 76.11±1.04 55.69

Cannabis sativa 14.44±1.16 17.59±1.01 25.19±0.88 31.11±0.58 22.08

Olea europaea 36.30±0.33 7.96±2.17 48.15±1.48 64.07±2.03 46.62

Mentha Piperita 28.33±1.44 30.37±1.45 37.41±2.03 39.26±2.33 33.84

Melia azadirachta 24.44±1.16 26.85±0.73 38.70±1.30 49.44±0.76 34.86

Nerium indicum 8.15±1.20 11.11±1.16 17.22±1.32 27.41±0.88 15.97

Carum Lopticum 57.41±0.73 62.59±2.03 70.93±1.01 83.52±1.92 68.61

Calotropis procera 30.74±0.88 31.67±0.76 41.67±1.32 47.04±0.88 37.78

Ocimum americanum 30.19±2.05 32.22±2.08 49.07±1.30 54.81±1.45 41.57

Foeniculum vulgare 7.04±1.20 9.44±1.61 18.15±1.45 31.11±1.15 16.44

Dalbergia sissoo 17.04±1.45 18.70±1.18 29.81±1.01 38.15±1.45 25.93

Trigonella foenumgraecum 7.78±1.15 8.70±0.44 21.48±1.20 32.04±1.17 17.50

Cassia fistula 26.85±1.64 32.96±1.45 38.70±1.01 44.44±1.73 35.74

Anethum graveolens 10.74±0.88 14.44±1.15 31.85±1.45 44.26±1.17 25.32

Nigella sativa 48.33±2.36 52.04±1.74 60.56±1.61 70.37±0.88 57.82

Matricharria chammomilla 17.78±1.15 21.48±1.45 28.15±1.45 39.44±1.44 26.71

Cassia angustifolia 27.04±1.59 30.93±1.17 42.04±1.01 55.19±0.33 38.80

Parthenium hysterophorus 32.22±0.58 32.22±1.73 40.56±1.44 49.44±0.76 38.61

Lawsonia inermis 30.37±2.03 38.89±1.15 44.07±1.20 50.74±0.88 41.02

Nicotiana tabacum 18.52±2.03 25.19±1.45 28.15±0.88 38.52±1.45 27.59

LSD Value= 2.83 aValues are the means of the five replicate samples bFigures following ± are standard errors

67

Table 4.7.1 Relationships between concentrations of plant extracts and radial

growth of M. phaseolina

Plant Extract Regression equation R2

Azadirachta indica y = 45.111x + 27.500 0.9003

Cannabis sativa y = 23.037x + 7.685 0.9776

Olea europaea y = 37.407x + 23.241 0.8951

Mentha Piperita y = 15.926x + 23.889 0.9385

Melia azadirachta y = 34.74x + 13.148 0.9427

Nerium indicum y = 25.556x + 0.0006 0.9398

Carum Lopticum y = 34.667x + 46.944 0.9646

Calotropis procera y = 23.555x + 23.056 0.9237

Ocimum americanum y = 36.296x + 18.889 0.9175

Foeniculum vulgare y = 32.371x + -3.7967 0.9211

Dalbergia sissoo y = 29.778x + 7.3144 0.9372

Trigonella foenumgraecum y = 34.222x + -3.8889 0.9171

Cassia fistula y = 23.407x + 21.112 0.9998

Anethum graveolens y = 47.184x + 4.1661 0.9502

Nigella sativa y = 29.852x + 39.167 0.9655

Matricharria chammomilla y = 28.667x + 8.7961 0.9464

Cassia angustifolia y = 38.223x + 14.907 0.9525

Parthenium hysterophorus y = 24.00x + 23.611 0.8877

Lawsonia inermis y = 26.52x + 24.444 0.9909

Nicotiana tabacum y = 25.185x + 11.852 0.9538

68

Table 4.8 Effect of plants extracts on the plant survival of mungbean

Plant Extract % increase over control

25 % 50 % 75 % 100 % Average


Cannabis sativa 30.00±0.58 33.33±0.33 40.00±0.00 40.00±0.58 35.83

Olea europaea 36.67±0.33 36.67±0.33 46.67±0.00 60.00±0.58 45.00

Mentha Piperita 33.33±0.33 23.33±0.33 33.33±0.33 40.00±0.58 32.50


Nerium indicum 36.67±0.33 30.00±0.00 40.00±0.00 43.33±0.33 35.00

Carum Lopticum 46.67±0.33 50.00±0.00 63.33±0.33 83.33±0.33 60.83






Cassia fistula 26.67±0.00 26.67±0.33 33.33±0.33 43.33±0.33 32.50


Nigella sativa 46.67±0.33 53.33±0.33 60.00±0.58 76.67±0.33 59.17







69

Table 4.8.1 Relationships between concentrations of plant extracts and mungbean plant survival


Azadirachta indica Y = 41.336x + 36.665 0.9468

Cannabis sativa Y = 14.668x + 26.665 0.8963

Olea europaea Y = 31.996x + 25.005 0.8727

Mentha Piperita Y = 20.004x + 19.995 0.8824

Melia azadirachta Y = 23.992x + 18.34 0.9000

Nerium indicum Y = 23.992x + 20.005 0.9529

Carum Lopticum Y = 49.324x + 30.005 0.9157

Calotropis procera Y = 34.664x + 16.67 0.9134

Ocimum americanum Y = 45.328x + 18.335 0.9966

Foeniculum vulgare Y = 32.008x + 13.33 0.9601

Dalbergia sissoo Y = 32.008x + 14.995 0.9931

Trigonella foenumgraecum Y = 35.992x + 16.67 0.9918

Cassia fistula Y = 22.656x + 18.34 0.8626

Anethum graveolens Y = 31.996x + 18.335 0.9931

Nigella sativa Y = 38.668x + 35.00 0.9396

Matricharria chammomilla Y = 22.66x + 20.005 0.9796

Cassia angustifolia Y = 53.332x + 15.00 0.899

Parthenium hysterophorus Y = 37.324x + 15.005 0.9561

Lawsonia inermis Y = 28.00x + 11.665 0.9692

Nicotiana tabacum Y = 29.332x + 18.335 0.9308

70

Table 4.9 Effect of plants extracts on the plant survival of mashbean

Plant Extract % increase over control

25 % 50 % 75 % 100 % Average


Cannabis sativa 33.33±0.33 36.67±0.33 43.33±0.33 50.00±0.58 40.83

Olea europaea 40.00±0.58 43.33±0.33 50.00±0.58 56.67±0.33 47.50

Mentha Piperita 23.33±0.33 26.67±0.33 36.67±0.33 46.67±0.33 33.33


Nerium indicum 23.33±0.33 33.33±0.33 43.33±0.33 46.67±0.33 36.67

Carum Lopticum 36.67±0.33 40.00±0.58 60.00±0.58 70.00±0.58 51.67






Cassia fistula 26.67±0.33 30.00±0.58 40.00±0.58 46.67±0.33 35.83


Nigella sativa 43.33±0.33 50.00±0.00 60.00±0.58 76.67±0.33 57.50







71

Table 4.9.1 Relationships between concentrations of plant extracts and

mashbean plant survival.


Azadirachta indica y= 37.324x + 35.005 0.9561

Cannabis sativa y= 22.668x + 26.665 0.9797

Olea europaea y= 22.672x + 33.33 0.9796

Mentha Piperita y= 32.008x + 13.33 0.9601

Melia azadirachta y= 22.66x + 21.67 0.9796

Nerium indicum y= 32.008x + 16.66 0.9601

Carum Lopticum y= 44.008x + 29.995 0.9595

Calotropis procera y= 32.008x + 13.33 0.9601

Ocimum americanum y= 34.672x + 23.33 0.9137

Foeniculum vulgare y= 28.00x + 13.335 0.9692

Dalbergia sissoo y= 21.328x + 21.67 0.9847

Trigonella foenumgraecum y= 38.668x + 11.665 0.9398

Cassia fistula y= 28.00x + 18.335 0.9692

Anethum graveolens y= 32.008x + 14.995 0.9931

Nigella sativa y= 47.996x + 21.67 0.9391

Matricharria chammomilla y= 31.996x + 13.335 0.7577

Cassia angustifolia y= 31.996x + 26.67 0.96

Parthenium hysterophorus y= 28.00x + 21.665 0.9692

Lawsonia inermis y= 30.668x + 13.33 0.8601

Nicotiana tabacum y= 26.668x + 13.33 0.8334

72

4.2.5 Inhibitory effect of fungicides on the radial growth of M. phaseolina

Highly significant inhibitory effects of fungicides (F = 380.036; df = 8, 18;

P < 0.001) and their concentrations (F = 594.695; df = 2, 36; P < 0.001) were

recorded on the growth of M. phaseolina. The interaction between fungicides and

their concentration was also found to be highly significant (F = 13.097; df = 16, 36;

P < 0.001).

All the fungicides caused significant inhibition of the fungus over control.

Maximum individual inhibition of the growth of the fungus was recorded by

Benomyl (83.89%) followed by Carbendazim (79.11%) at a concentration of 150

ppm. Copperoxychloride at a concentration of 50 ppm gave the minimum

inhibition (12.50%). The individual inhibitions caused by the fungicides are given

in the Table 4.10. Concentrations also had significant inhibitory effects on the

growth of the fungus. All the fungicides caused maximum inhibition of the growth

of the fungus at a concentration of 150 ppm.

With the decrease in the concentration, the inhibition in the growth also

decreased. The inhibition of growth was found to be directly proportional to the

concentration.

4.2.6 Effect of fungicides on plant survival of mungbean and mashbean

Fungicides also affected significantly the plant survival of mungbean (F =

13.760; df = 8, 18; P < 0.001) and mashbean (F = 27.620; df = 8, 18; P < 0.001)

over control. Maximum plant survival was observed where the seeds were treated

with Benomyl followed by Carbendazim. However, Copper + Mancozeb and

73

Copperoxychloride treated seeds gave the minimum germination and survival of

plants. Doses also had a significant effect on the germination and plant survival.

Maximum germination and survival was recorded where the seeds were treated

with a concentration of 150 ppm and minimum was recorded in case of 50 ppm

concentration. The individual germinations and survivals of mung and mash are

shown in Table 4.11 and 4.12 respectively.

It was also observed that higher concentrations of all the fungicides were

significantly better as compared to other concentrations. With the decrease in the

concentration the germination and survival decreased significantly showing a direct

relationship between concentrations and plant survival. Benomyl at 150 ppm

concentration showed highest rate of plant survival 76.67% whereas Carbendazim

and Propineb on same concentration exhibited 66.67 and 63.33% plant survival

respectively. Other fungicides also showed enhanced effect on 150 ppm as

compared to other concentrations but having minimum plant survival rate (40-

56%). Copperoxychloride remained behind in its effectiveness in terms of disease

incidence and plant survival (Table 4.11 and 4.12). All chemicals were significant

on all concentrations by statistical analysis.

4.2.7 Host Plant Resistance against M. phaseolina

4.2.7.1 Mash germplasm screening

The mash genotypes showed differences in their response to charcoal rot in

terms of disease severity both in greenhouse and field conditions. Out of one

hundred accessions, five genotypes (013468, 013477, 013663, 013667 and 013668)

were found to be highly resistant, whereas 11 resistant, 30 tolerant, 23 susceptible.

74

Table 4.13 In Vitro radial growth inhibition of M. Phaseolina

% growth inhibition at Fungicides

50ppm 100ppm 150ppm Average

Propineb 41.11 ± 1.61

50.33 ± 1.88 61.44 ± 1.36 50.96

Copper+Mancozeb

8.33 ± 1.26

26.11 ± 1.80

53.56 ± 1.17

29.33

Mancozeb

21.44 ± 0.60

30.89 ± 1.01

56.11 ± 1.15

36.15

Carbendazim 63.33 ± 1.61

73.00 ± 1.30

79.11 ± 1.30

71.81

Captan 19.22 ± 2.17

31.67 ± 1.04

41.44 ± 1.17

30.78

Benomyl 66.89 ± 1.45

78.67 ± 1.59

83.89 ± 1.15

76.48

Meatalaxyl+Mancozeb 42.56 ± 1.01

51.44 ± 1.45

57.78 ± 0.87

50.59

Copper oxychloride 12.56 ± 0.93

23.89 ± 0.00

34.22 ± 1.01

23.56

Chlorothalonil 25.89 ± 1.30

34.44 ± 1.44

53.33 ± 2.75

37.89


75

Table 4.14 Effect of fungicides on plant survival of mungbean against


Plant survival at Fungicides


Propineb 43.33 ± 0.33

50.00 ± 0.58 63.33 ± 0.33

52.22

Copper+Mancozeb

26.67 ± 0.33

33.33 ± 0.33

43.33 ± 0.33

34.44

Mancozeb

30.00 ± 0.00

36.67 ± 0.33

46.67 ± 0.33

37.78


53.33 ± 0.33

66.67 ± 0.33

55.56

Captan 30.00 ± 0.58

36.67 ± 0.33

46.67 ± 0.33

37.78

Benomyl 50.00 ± 0.00

56.67 ± 0.33

76.67 ± 0.33

61.11


50.00 ± 0.00

56.67 ± 0.33

50.00


33.33 ± 0.33

46.67 ± 0.33

35.56


46.67 ± 0.33

56.67 ± 0.33

47.78

LSD Value=2.26 aValues are the means of the five replicate samples bFigures following ± are standard errors

76

Table 4.15 Effect of fungicides on plant survival of mashbean against


Plant survival at Fungicides


Propineb 40.33 ± 0.00

50.00 ± 0.58 63.33 ± 0.33

51.22

Copper+Mancozeb

20.00 ± 0.00

33.33 ± 0.33

43.33 ± 0.33

32.22

Mancozeb

23.33 ± 0.33

36.67 ± 0.33

46.67 ± 0.33

35.56


53.33 ± 0.33

66.67 ± 0.33

54.44

Captan 26.67 ± 0.33

36.67 ± 0.33

46.67 ± 0.33

36.67

Benomyl 46.67 ± 0.33

56.67 ± 0.33

76.67 ± 0.33

60.00


50.00 ± 0.00

56.67 ± 0.33

47.78


33.33 ± 0.33

46.67 ± 0.33

33.33


46.67 ± 0.33

56.67 ± 0.33

45.56


77

and rest of the genotypes were highly susceptible in greenhouse as against the

disease response under field conditions showed that out of 100 genotypes, 12

genotypes, 013664, 013663, 013662, 013655, 013654, 013652, 013651, 013643,

013482, 013468, 013454 and 013453 appeared as resistant with disease score ‘1’

were found highly resistant, whereas seventeen were resistant, twenty five tolerant,

16 genotypes were susceptible and 30 acted as highly susceptible lines as against

12, 17, 25, 16 and 30 genotypes respectively under field conditions as shown in

Table 4.13.

4.2.7.2 Mung germplasm screening

Mung genotypes also showed variations in their resistance/susceptibility to

charcoal rot disease under greenhouse and field conditions. Out of one hundred

accessions, fourteen (013987, 013992, 014026, 014033, 014062, 014098, 014218,

014219, 014245, 014253, 014255, 014256, 014257 and 014258) appeared to be

highly resistant, 18 resistant, 35 tolerant, 16 susceptible and rest of the genotypes

were highly susceptible.

Under field conditions, disease reaction was different from that appeared in

greenhouse. In field screening, 34 genotypes behaved as highly resistant, 30 as

resistant and 23 genotypes appeared tolerant. Eight genotypes were found

susceptible and only 5 were highly susceptible as shown in Table 4.14.

78

Table-4.16 Relative resistance/susceptibility of mashbean germplasm against charcoal rot

Disease Reaction Greenhouse Field

Highly resistant (HR)

013468,013477,013663,013667, 013668

013664,013663, 013662, 013655, 013654, 013652, 013651, 013643, 013482, 013468, 013454, 013453

Resistant (R)

013676,013680,013672,13669, 013662,013655,13652,013651, 013645, 013643, 013492

013672, 013668, 013667, 013660, 013658, 013657, 013656, 013653, 013648, 013630, 013507, 013506, 013493, 013487, 013477, 013469, 013462

Tolerant (T)

013453,013462,013482,013487, 013488,013493,013506,013507, 013570,013576,013592,013620, 013625,013626,013627,013628, 013629,013630,013648,013653, 013654,013656,013674,013675, 013677,013697,013699,013664, 013660, 013658

013492, 013503, 013505, 013511, 013568, 013615, 013616, 013618, 013625, 013626, 013628, 013629, 013634, 013640, 013645, 013669, 013671, 013673, 013674, 013675, 013676, 013677, 013680, 013696, 013699

Susceptible (S)

013454,013469,013470,013503, 013511,013563,013564,013568, 013590,013591,013600,013615, 013616,013619,013624,013640, 013657,013671,013673,013687, 013694, 013696, 013700

013703, 013700, 013697, 013687, 013686, 013684, 013670, 013627, 013622, 013620, 013600, 013570, 013562, 013550, 013488, 013470

Highly susceptible (HS)

013703,013705,013707,013710, 013711,013712,013713,013714, 013715,013716,013719,013695, 013693,013692,013690,013686, 013684,013670,013639,013636, 013634,013623,013622,013618, 013562,013561,013554,013553, 013551,013550, 013505

013705,013707,013710,013711, 013712,013713,013714,013715, 013716,013719,013690,013692 013693,013694,013695,013636, 013639,013619,013623,013624, 013551,013553,013554,013561, 013564,013563,013592,013591, 013590, 013576

79

Table-4.17 Relative resistance/susceptibility of mungbean germplasm against charcoal rot

Disease Reaction Greenhouse Field

Highly resistant (HR)

013987,013992,014026,014033,014062,014098,014218,014219,014245,014253,014255,014256,014257,014258

013987,013992,014026,014033,014062,014098,014149,014173,014184,014218,014219,014220,014222,014223,014239,014240,014241,014243,014245,014253,014255,014256,014257,014258,014259,014262,014263,014293,014294,014297,014307,014308,014309,014310

Resistant (R)

014149,014173,014184,014222,014233,014234,014235,014241,014243,014250,014293,014294,014297,014306,014307,014308,014314,014315

013953,013954,013956,013961,013962,014075,014089,014119,014121,014123,014125,014148,014213,014224,014233,014234,014235,014237,014247,014249,014250,014270,014277,014288,014290,014306,014312,014313,014314,014315

Tolerant (T)

013953,013954,013971,014044, 014063,014064,014073,014075,014119,014121,014123,014125, 014213,014220,014223,014232, 014237,014239,014240,014247,014249,014259,014262,014263,014270,014277,014287,014288, 014290,014292,014303,014304,014311,014312,014313

013955,013971,014035,014044,014063,014064,014073,014133,014221,014232,014251,014252,014274,014275,014276,014287,014292,014298,014301,014302,014303,014304,014311

Susceptible (S)

013955,013956,013961,013962,014035,014089,014148,014224,014251,014252,014275,014276,014301,014302,014309,014310,

014043,014225,014226,014278,014279, 014280,014281,014283,

Highly susceptible (HS)

014043,014133,014221,014225,014226,014227,014228,014274, 014278,014279,014280,014281,014283,014284,014285,014286,014298

014227,014228,014284,014285,014286

80

Chapter 5

DISCUSSION

Macrophomina phaseolina, a soil as well as seed-borne fungus, induces

charcoal rot in different crops including pulses i.e. mash and mung. In the present

studies, 65 isolates of M. phaseolina belonging to different regions of Punjab and

Khyber Pukhtoon Khwa (KPK) showed variations in different morphological traits

such as radial growth, sclerotial size and weight as well as in pathogenecity. The

isolates also varied in their pathogenicity. Morphological variability has also been

reported by many workers in terms of growth, colour, pycnidium production and

chlorate sensitivity among different isolates of M. phaseolina on different hosts

(Dhingra and Sinclair, 1973,1978; Pearson et al., 1986; Adam, 1986; Atiq et al.,

2001; Riaz et al., 2007) which corroborated our findings.similarly variations in

morphology and pathogenicity among M. phaseolina isolates taken from different

hosts as well as from different parts of the same host has also been observed by

Fernandaz et al. (2006). However, in the present studies, no relationship was found

among the morphological characters and pathogenicity of the isolates. Among the

highly virulent isolates of M. phaseolina viz, MP-7, MP-13, MP-18, MP-48, MP-56

and MP-64 against mung, not all the isolates were fast growing (radial growth > 80

mm) large sized (> 25 µm) or high weight (> 0.15 mg). Of these highly virulent

isolates, two (MP-7 and MP-48) were the fast, and the remaining 4 were medium

growing. Similarly, isolates MP-18 and MP-26 produced large sized, MP-7 and

MP-13 medium sized and MP-48 and MP-64 small sized sclerotia. Likewise, MP-7,

MP-13, MP18 and MP-64 were high and MP-48 was low weight. Similar pattern

81

was observed in moderately and least virulent isolates. Confirmatory and

contradictory findings in this regard have also been reported by others. A close

linkage between virulence and growth was reported by Rayner (1991). Purkayastha

et al. (2004) also found relationship between morphological variations and

pathogenicity. On the other hand, Dhingra and Sinclair (1978) and Fernandaz et al.

(2006) reported that pathogenicity has no relation with size and weight of sclerotia.

The pathogenic fungus, M. phaseolina, has a broad host range and exits in

two asexual forms which maintain its survival better (Dhingra and Sinclair, 1978;

Cloud and Rupe, 1991; Mihail and Taylor, 1992). Some workers also related

variability with the phenomena of host specialization in M. phaseolina. Su et al.

(2001) found host specialization in maize on the basis of pathogenic, genetic and

physiological differences. Similarly, Cloud and Rupe (1991) analyzed host

specialization in soyabean. This mechanism takes long time to establish with in a

specific host. Mihail and Taylor (1995) suggested that due to heterogenic nature of

M. phaseolina, categorization into distinct sub groups based upon pathogenicity and

morphology could not take place. Pathogenesis along with genetic diversity plays a

specific role in host plant resistance. Isolates having morphological similarity are

not necessarily identical genetically, they might have some differences. The

variable genetic pattern contributes for variation in morphology and pathogenesis,

which has been confirmed by using different molecular tools (Fuhlbohm, 1997;

Mayek-Parez et al., 2001; Alvaro et al., 2003; Jana et al., 2003; Rayes et al., 2006;

Rajkumar et al., 2007 and Allaghebandzadeh et al., 2008). As the pathogen has no

sexual phase, genetic diversity is produced either by fusion of vegetative cells or by

82

para-sexual recombination between nuclear genes (Carlile, 1996). Greatest genetic

variability makes survival of fungus better in nature (Rajkumar et al., 2007).

It is quite evident that variability in morphology, physiology, genetics,

pathogenicity etc. is imperative for the fungus to have better adaptation in response

to diversified environmental behavior. It also leads to host-plant resistance,

development of resistant varieties of different crops against disease and

implementation of new disease controlling strategies (Mayek-Parez et al., 2001;

Purkayastha et al., 2006).

Biological agents such as fungi, bacteria etc. are considered as an important

tool for controlling both seed and soil borne phytopathogenic fungi for their safety

to environment, animals and human beings and ipso facto can replace chemicals,

which produce drastic effects on targeted as well as non-targeted organisms

(Papavizas., 1985 and Benitez et al., 2004)

Seven antagonistic fungi tested in this dissertation significantly reduced the

growth of M. phaseolina and improved the survival and germination of mung and

mashbean plants. Trichoderma species have been considered effective against a

plethora of pathogens. Alice et al, (1996) found that T. harzianum and T. viridae

effective against M. phaseolina infecting Jasmine. Hasamedin (2008) observed the

inhibition of root rot pathogen of egg plant by T. harzianum by 18.20%. Similarly,

Etebarian (2006) found that different Trichoderma spp such as T. harzianum (M), T.

harzianum (T39) and T. virens (DAR 74290) completely retarded M. phaseolina

growth inciting charcoal stem rot in melon. In addition, efficacy of other bio-agents

i.e. T.virens, Pseudomonas flouresences and Bacillus spp against M. phaseolina is

83

also documented (Lokesha and Benagi, 2007; Bealriz et al., 2009). Many soil borne

pathogens including Rhizoctonia solani, Pythium ultimum, Fusarium moniliforme

and Sclerotium rolfsii have aloso been controlled by T. harzianum (Hadar et al.,

1979; Mukhopadhyay et al., 1986; Papavizas and Lewis, 1989; Methew and Gupta,

1998; Rajappan and Ramaraj, 1999). Antagonistic activity of biological agents

might be attributed to parasitism, production of lytic enzymes such as chitinases and

glucanases (Chet, 1987, Woo et al., 2002, Compant et al., 2005) which degraded

the β-glucans, chitin and polysaccharides, responsible for fungal cell wall rigidness

(Gupta et al., 1995, Howell, 2003) or by antibiotic production such as glioviridin

and gliotoxin (Di Pietro et al., 1993) or by competition or rhizosphere competence.

In dual culture technique, Trichoderma hyphae hyper-parasitized the pathogen and

coagulated its protoplasm leading to shrinkage, granulated and vacuolated

protoplasm of the pathogen (Weindling, 1932; Inbar et al., 1996; Pandy and

Upadhyay, 2000). Suppression of fungal growth by antagonists might be due to

overgrowth forming inhibition zone (Hashem, 2004).

Dressing of mung and mash seeds with three different concentrations of

antagonists in greenhouse decreased the incidence of charcoal rot caused by M.

phaseolina and enhanced germination. The improvement in germination might be

due to multiplication of antagonists on seed surface which prevented the fungal

entry into seeds by instantly colonizing the roots (Chao et al., 1986; Rajuchander et

al., 1998). The enhanced germination might also be due to triggering of host plant

cell by antagonists to synthesize growth hormones and toxic substances in large

quantities which inhibited pathogenic fungi (Chakrabarty and Purkayastha, 1984;

Sheng, 1993, Hendelsman and Eric, 1996). Similar results by Pineda (2001) in

84

sesame, Malik and Dawar (2003) in chickpea and mashbean also confirmed our

findings.

Dressing of cowpea seeds with different Trichoderma spp. gave maximum

plant stand (Adekunle et al., 2001). Dawar et al. (2008) found a decrease in root rot

infection and enhanced plant height and weight in T. harzianum coated seeds of

okra and sunflower. Hussain et al. (1990) found that seed treatment with T.

harzianum, G. virens, Paecilomyces lilacinus or Streptomyces spp. reduced

Macrophomina infection in sunflower and mungbean. Several fungi such as M.

Phaseolina, F. semitecum, F. moniliforme and F. solani infecting cowpea,

horsegram, black and green gram have also been effectively controlled by seed

treatment with conidial suspension of T. harzianum (Krishna et al., 2003).

Rettinasbabady et al. (2000) found that T.viridae treated blackgram seeds

decreased sclerotial formation of M. Phaseolina. Similarly, the same fungus

effectively controlled R. bataticola in vitro (Kaswate et al., 2003). Seed treatment

and soil application with T.virens reduced root rot incidence in pigeon pea (Lokesha

and Benagi, 2007). Seed treatment with both T. harzianum and T. viridae also

reduced root rot incidence and increased growth in mungbean plants (Ashraf et al.,

2006) and hence regarded as a suitable method for controlling seed and soil borne

fungi (Chang and Kommedahl, 1968).

Aqueous extracts of twenty test plants significantly inhibited growth of M.

phaseolina in vitro and improved seedling emergence when tested in pots.

Fungicidal activities of antagonistic plants against pathogenic fungi are well

documented. A. indica has shown efficacy against F. solani, C. lunata and R.

85

bataticola on brinjal and sunflower (Hussain et al., 2000; Joseph et al., 2008).

Ahmed et al. (2002) reported the efficacy of A. indica against Bipolaris oryzae

under in vitro conditions. Likewise, F. oxysporum was effectively controlled by A.

indica extract (Taiga et al., 2008). Similarly, Singh et al. (1980) reported the

fungicidal activity of A. indica oil and extract against soil-borne pathogens viz. R.

solani, F. oxysporum f.sp.ciceri, S. rolfsii and S. sclerotium. Dwivedi and Shukla

(2000) found that 100% aqueous extracts of A. indica completely inhibited the

germination of Fusarium spp. M. phaseolina and Phytrophthora palmivora was

controlled by A. indica extract (Tasleem et al., 1998). The reduction in fungal

growth might be due to the presence of antifungal compounds in plant extracts

including glycoside, steroid, saponin, medicagenic acid, 3-O-B-D glycopyranoside,

(3-GleMA) ajone, Tannins, Sesquiterpenes, lactones, terpenoids and phobol esters

(Johnson and Nunley, 2000; Tiwari and Singh, 2004). In C. Lopticum, the inhibitory

effect was probably due to the presence of active chemical component, thymol.

Effectiveness of C. lopticum against Aspergillus, Fusarium, Penicillium and

Curvularia spp. has been reported by Singh et al. (2004). Similarly A. indica

contains nimolicinal, isolimolicinalide, azadirachtin, azadirachtol, nimilinone,

nimbocinal, nimbocinone (Tewari, 1992) which can suppress pathogenic fungi.

Strong inhibitory action of L. inermis was reported by Yasmin et al. (2008). Taiga

et al. (2008) also reported the fungitoxicity of tobacco against many fungi.

Aqueous and ethanolic extract of Alchemia cardifolia, Cassia alata and

Moringa oleifera suppressed the growth of F. verticilliodes and M. phaseolina

(Enikuomehin and Oyedeji, 2010). Capparis decidua, Lantana kamara and Tridax

procumbexs were found effective against F. oxysporum (Sharma and Kumar, 2009).

86

Varaprasad et al. (2009) also suggested strong activity of Terminalia chebula

against M. phaseolina.

In Mungbean and mashbean, germination increased as a result of treatment

with plant extracts. The enhanced germination might be attributed the deposition of

chemical compounds around seed surface and prevented penetration of the

pathogen. These chemicals might have caused lyses of sclerotia and triggered plant

growth hormones which resulted in increased germination and decreased disease

incidence. Hasan et al. (2005) reported that seed treatment with A. indica gave

99.33% seed germination in wheat. Similarly, seed treatment with garlic extract

against Colletolachum corchori, M. phaseolina, Botrydioiploida theobromae,

Fusarium spp, Penicillium spp, A. niger and A. flavus resulted in upto 77.50 %

increase in germination of jute (Islam et al., 2001). Also, seed dressing with

caraway and peppermint before sowing controled the root rot disease (Mougy and

Al-habeb, 2009). It was concluded that natural products have strong fungicidal

activity and can be applied for the control of different soil-borne diseases.

Benomyl has successfully controlled many diseases of different crops as leaf

spot in sugar beet (Kalaoglanidis et al., 2003), rice blast (Kamelwass-rao, 1976),

scab and powdery mildew of apples, cucurbits and strawberries (Scot et al., 1979).

Marley and Genga (2004) found that benomyl reduced the mycelial growth of

Stenocarpella maydis in vitro. It also inhibited the growth of F. oxysporum (El-

Tobshy et al., 1981). Mamza et al (2010) reported that benomyl along with thiram

and tricyclazole suppressed growth of F. pallideroseum isolated from castor. Khan

and Khan (2006) found that both benomyl and carbendazim inhibited 100%

mycelial growth of M. phaseolina. Carbendazim also inhibited the growth and

87

sclerotial production of M. phaseolina (Suryawashi et al., 2008). Similarly, seed

dressing with fungicides enhanced seedling emergence and reduced mortality rate in

legumes (Muthomi et al., 2007).

A number of mechanisms are involved in the suppression and inhibition of

pathogens by fungicides. It was found from the present investigation that fungicides

significantly caused reduction in growth of M. phaseolina and enhanced

germination of mung and mash. Fungicides act by binding with b-tubulin polymers

of pathogens which play a key role in nuclear division and result in inhibition of

polymerizing activity of microtubules. These also cause hindernce in different

regulatory cellular activities including mitosis, meiosis and cell shape maintenance

etc. (Nene and Thapliyal, 1993). Similarly, Carbendazim inactivates tubulin

function of pathogen necessary for their maintenance and growth (Butlers et al.,

1995).

Charcoal rot is a devastating disease of mung and mashbean in Pakistan that

may cause up to 100% yield losses under epidemic condition. The host plant

resistance is the most feasible and economical measure to reduce yield losses due to

this disease. Identification of resistant sources is an important pre requisite for

initiation of breeding program aimed to develop resistant varieties.

Our study revealed a considerable variability among mung and mashbean

germplasm for resistance against this disease both under field and greenhouse

conditions. Large number of genotypes was found resistant in the field than

greenhouse. The high disease development in greenhouse was due to highly

88

conducive environment for the pathogen. Miklas et al (1998) also reported that

mash genotypes showed resistance at seedling stage under higher disease pressure.

The high disease incidence under greenhouse might also be due to the use of

sterilized soil, resulting into poor interaction between the pathogen and the soil

microbiota, further reducing the chances of disease escape. On the contrary, there

was sufficient microflora in the field which competed with the pathogen, and much

more variations in the field micro environment as compared to that of greenhouse.

Pastor-Corrales and Abawi (1988) and Songa et al. (1997) observed that under

greenhouse conditions the disease severity increased.

Pastor-Corrales and Abawi (1998) screened 53 accessions of bean against

M. phaseolina and found 22 accessions as resistant, 15 tolerant and the rest as

susceptible. Ashraf et al. (2005) reported that out of fifty six chickpea lines only

one genotype was resistant to charcoal rot and the remaining were susceptible. Iqbal

et al. (2002) also reported resistant sources in mashbean against charcoal rot.

The highly resistant genotypes which showed resistance to M. phaseolina

both under greenhouse and field conditions may have resistant genes which could

be exploited to develop disease resistant varieties against charcoal rot through

hybridization.

89

SUMMARY

Mung (Vigna radiata L.) and mash (Vigna mungo L.) belonging to

family fabaceae, sub family faboidae are important summer pulse crops of

Pakistan. They are important component of human diet and animal feed as straw

and hay. The average production of mung and mash is very low in Pakistan as

compared to other countries. There are many limiting factors which are responsible

for low yield. Among the biotic factors, diseases are very important, and charcoal

rot caused by Macrophonima phaseolina is by far the most important contributing

significantly to low yield. The information on the variability and management of

this fungus on mung and mash is lacking in Pakistan. Therefore, present studies

were planned to investgate the biology and management of this pathogen. Plant

samples were collected from the different localities of Punjab and KPK to

determine morphological and pathogenic variability among various isolates of M.

phaseolina. Investigations were also made for the management of the pathogen

with biocontrol fungi, plant extracts and fungicides. Local germplasm of the test

crops were also screened to identify the resistant sources.

Significant variations were found in morphology of various isolates of the

fungus. On the basis of morphological characters, out of 65 isolates, 16 were found

to be fast growing catrgorized on the basis of radial growth (>80 mm), 9 produced

large sized (>25 µm) and 35 isolates produced high weight (>0.15 mg) of sclerotia.

All the isolates showed significant variability in aggressiveness against mung and

mash varities. Twenty three isolates were found to be highly virulent while five

90

were least virulent and the remaining isolates showed intermediate virulence

towards mung and mashbean.

All the antagonistic fungi significantly retarded the growth of M.

phaseolina. Maximum inhibition was observed in case of Trichoderma harzianum

(79.63 %) while T. pseudokoningii showed the minimum (58.14 %) inhibition over

control. These antagonists also significantly increased plant survival of mung and

mash at higher concentrations as compared to others. Seeds treated with T.

harzianum @ 2 x 108 gave 86.67% and 80% increase in plant survival of mung and

mash respectively. Minimum increase in plant survival was obtained with T.

pseudokoningii.

Aqueous extracts of all the tested plants, significantly inhibited the growth

of M. phaseolina. Maximum inhibition was observed in case of Carum Lopticum

(83.52%) and Azadirachta indica (76.11%) while Nerium indicum gave the

minimum (27.41%) inhibition at 100% concentration over control. Survival of

mung and mash plants was found to be the maximum when seeds were treated with

C. lopticum and A. indica at 100 % concentration. On the other hand, Mentha

piperita and F. vulgare showed minimum increase in plant survival.

Similarly, all the fungicides inhibited the growth of M. phaseolina and

increased plant survival significantly. The maximum inhibition was observed with

Benomyl (83.89 %) and Carbendazim (79.11%) while Copperoxychloride gave the

minimum (34.22 %) inhibition at 150 ppm concentration over control. Plant

survival was found to be the maximum in mung and mash when seeds were treated

91

with Benomyl @ 3 gm a.i/Kg of seed and the minimum when seeds were treated

with Copperoxychloride @ 1 gm a.i/Kg of seed.

Germplasm accessions of mung and mash showed different levels of

resistance under field and greenhouse conditions. Out of one hundred mungbean

accessions, 14 genotypes were highly resistant under green house conditions, while

in the field 34 accessions were found highly resistant. In case of mashbean, 5

accessions in greenhouse and 12 under field conditions were found highly resistant

against charcoal rot.

Major Findings

• The isolates collected from different localities varied significantly on the

basis of morphological characters and pathogenecity.

• No relationship was found among the morphological characteristics (growth,

size and weight of sclerotia) with the pathogenecity.

• 23 isolates appeared to be highly virulent on mung and mash cultivars.

• T. harzianum was found to be the most effective antagonistic agent in

reducing the growth of fungus and increasing plant survival.

• C. lopticum and A. indica exhibited significant inhibitory effect against the

pathogen and increased plant survival.

• Benomyl and Carbendazim significantly inhibited growth of the pathogen

and enhaced plant survival.

• Five accessions of mash and 14 of mung were found highly resistant against

charcoal rot disease under greenhouse as against 12 and 34 under field

conditions.

92

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Appendices Appendices 1: Analysis of variance (ANOVA) for three morphological

traits in 65 isolates of Macrophomina phaseolina. Mean Squares Source of variation

Degrees of Freedom

Sclerotium weight (g)

Sclerotial size (µm)

Radial growth (mm)

Isolates 64 0.004** 25.299** 453.756** Error 130 0.001 7.168 38.626 CV % 16.45 12.21 8.90 ** Highly Significant at P >0.01

Appendices 2: Two factor analysis of variance (ANOVA) for the reaction of 65 isolates of Macrophomina phaseolina to 3 Mungbean cultivars

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Isolates 64 574.735 8.980 34.313 0.000 Varieties 2 27.244 13.622 52.049 0.000 Isolates x Varieties 128 569.644 4.450 17.005 0.000 Error 388 101.545 0.262

Coefficient of Variation: 9.22%

Appendices 3: Two factor analysis of variance (ANOVA) for the reaction of 65 isolates of Macrophomina phaseolina to 3 Mashbean cultivars

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Isolates 64 487.976 7.625 33.922 0.000 Varieties 2 35.518 17.759 79.010 0.000 Isolates x Varieties 128 368.926 2.882 12.823 0.000 Error 388 87.210 0.225

Coefficient of Variation: 7.06%

122

Appendices 4: Analysis of variance (ANOVA) for the reaction of 7 antagonists on the radial growth of Macrophomina phaseolina

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Antagonists 6 897.905 149.651 21.525 0.000 Error 14 97.333 6.952

Coefficient of Variation: 9.48% Appendices 5: Analysis of variance (ANOVA) for the reaction of 7

antagonists with 3 concentrations on the plant survival of mashbean

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Antagonists 6 6955.556 1159.259 20.287 0.000 Error 14 800.000 57.143 Concentrations 2 2593.651 1296.825 45.389 0.000 Antagonists x Concentrations

12 206.349 17.196 0.602 0.000

Error 28 800.000 28.571 Coefficient of Variation: 9.62% Appendices 6: Analysis of variance (ANOVA) for the reaction of 7

antagonists with 3 concentrations on the plant survival of mungbean

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Antagonists 6 9130.159 1521.693 18.088 0.000 Error 14 1177.778 84.127 Concentrations 2 2146.032 1073.016 84.500 0.000 Antagonists x Concentrations

12 231.746 19.312 1.521 0.000

Error 28 355.556 12.698 Coefficient of Variation: 6.22%

123

Appendices 7: Two factor analysis of variance (ANOVA) for the reaction of 20 plant extracts with 4 concentrations on the Radial Growth of Macrophomina Phaseolina

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Extracts 19 36995.608 1947.137 165.802 0.000 Error 40 469.750 11.744 Concentrations 3 15023.025 5007.675 1362.890 0.000 Extracts x Concentrations

57 1299.933 22.806 6.207 0.000

Error 120 440.917 3.674 Coefficient of Variation: 3.30% Appendices 8: Two factor analysis of variance (ANOVA) for the

reaction of 20 plant extracts with 4 concentrations on the plant survival of Mashbean

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability


57 1663.750 29.189 0.737 0.000


reaction of 20 plant extracts with 4 concentrations on the plant survival of Mungbean

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability


57 2840.417 49.832 1.458 0.0432


124

Appendices 10: Two factor analysis of variance (ANOVA) for the reaction of 9 fungicides with 3 concentrations on the radial growth of Macrophomina phaseolina

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability

Fungicides 8 20484.432 2560.554 380.036 0.000 Error 18 121.278 6.738 Concentrations 2 6521.451 3260.725 594.695 0.000 Fungicides x Concentrations

16 1184.994 71.812 13.097 0.000


reaction of 9 fungicides with 3 concentrations on the plant survival of Mungbean

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability


16 227.160 14.198 0.742 0.000


reaction of 9 fungicides with 3 concentrations on the plant survival of Mashbean

Source of Variation

Degrees of Freedom

Sum of Squares

Mean Squares

F-Value Probability


16 103.704 6.481 0.262 0.000


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