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ISOLATION OF HERBICIDAL CONSTITUENTS
FROM CULTURE FILTRATES OF DRECHSLERA
SPP. FOR THE MANAGEMENT OF SOME
NOXIOUS WEEDS OF WHEAT
MUHAMMAD AKBAR
INSTITUTE OF AGRICULTURAL SCIENCES,
UNIVERSITY OF THE PUNJAB,
LAHORE, PAKISTAN.
2012
ISOLATION OF HERBICIDAL CONSTITUENTS
FROM CULTURE FILTRATES OF DRECHSLERA
SPP. FOR THE MANAGEMENT OF SOME
NOXIOUS WEEDS OF WHEAT
By
Muhammad Akbar
A THESIS SUBMITTED FOR THE FULFILLMENT OF DEGREE OF
Doctor of Philosophy
in
Mycology & Plant Pathology
Supervisor DR. ARSHAD JAVAID
Co-supervisor
Dr. EJAZ AHMED
INSTITUTE OF AGRICULTURAL SCIENCES UNIVERSITY
OF THE PUNJAB, LAHORE
PAKISTAN
Certificate
This is to certify that the research work entitled “Isolation of Herbicidal Constituents
from Culture Filtrates of Drechslera spp. for the Management of Some Noxious Weeds of
Wheat” described in this thesis by Mr. Muhammad Akbar, is an original work of the author
and has been carried out under our direct supervision. We have personally gone through all the
data, results, materials reported in the manuscript and certify their correctness and authenticity.
We further certify that the material included in this thesis has not been used in part or full in a
manuscript already submitted or in the process of submission in partial or complete fulfillment
of the award of any degree from any institution. We also certify that the thesis has been
prepared under our supervision according to the prescribed format and we endorse its
evaluation for the award of Ph. D degree through the official procedures of the University of
the Punjab, Lahore, Pakistan.
Supervisor Co-supervisor
DR. ARSHAD JAVAID Dr. EJAZ AHMED
Assistant Professor Assistant Professor
Institute of Agricultural Sciences, Institute of Chemistry,
University of the Punjab, Lahore, University of the Punjab, Lahore,
Pakistan. Pakistan.
Dated: Dated:
Declaration Certificate
I hereby certify that the research work reported in this thesis entitled “ISOLATION OF
HERBICIDAL CONSTITUENTS FROM CULTURE FILTRATES OF DRECHSLERA SPP.
FOR THE MANAGEMENT OF SOME NOXIOUS WEEDS OF WHEAT” is an original work
carried out under the supervision of Dr. Arshad Javaid and co-supervision of Dr. Ejaz Ahmed.
I further certify that I have written this thesis independently and used no other aids and
resources than those indicated.
Muhammad Akbar Ph. D Scholar
Institute of Agricultural Sciences,
University of the Punjab,
Lahore, Pakistan.
Dated:
DEDICATIONS
To Allah Almighty for giving me the perseverance to carry this work to the end in spite of all
the hurdles.
To my beloved mother and father, I am highly indebted to you for your prayers, you both
could not live to see me prosperous. Allah may rest their souls in Heaven, Aameen.
ACKNOWLEDGMENTS All praises and thanks to the grace of Allah Almighty Who is the ultimate source of all
knowledge to mankind. He bestowed man with intellectual power and understanding and gave
him spiritual insight enabling him to discover his “Self” know his Creator through His wonders
and conquer nature. Bow in obeisance, I before my Lord, WHO bestows me to fortitude and
impetus to accomplish this task and elucidate a drop of already existing ocean of knowledge.
WHO made me reach at present pedestal of knowledge with quality of doing something
adventurous, novel, thrilling, sensational, and path bearing.
Next to all His Messenger Hazrat Muhammad (Peace Be upon Him) Who is an
eternal torch of guidance and fountain of knowledge for humanity. Who made mankind to get
out of depths of evil & darkness.
I give a sincere gratitude to my Ph. D supervisor, Dr. Arshad Javaid, Assistant
Professor, Institute of Agricultural Sciences and co-supervisor, Dr. Ejaz Ahmed, Assistant
Professor, Institute of Chemistry, University of the Punjab, Lahore, Pakistan for their personal
supervision, cordial co-operation and ever contribution, inspiring guidance, valuable
suggestions and sympathetic attitude in the preparation of this manuscript.
My thanks are due to Prof. Dr. M. Saleem Haider, Director, Institute of Agricultural
Sciences, University of the Punjab, Lahore, Pakistan for his valuable help towards the
completion of this research work.
I express my humblest thanks to compassionate dignified Prof. Dr. Rukhsana Bajwa,
ex-director Institute of Mycology and Plant Pathology, University of the Punjab, Lahore,
Pakistan for her motivating behavior and personal interest in the accomplishment of my Ph. D
degree.
My special and sincere thanks are due to Dr. Shakil Ahmed for his friendly co-
operation, sound advices, encouragement, motivation and valuable suggestions through- out
the course of this study.
It gives me immense pleasure to express my deep sense of gratitude to Prof. Dr. Phillip
Crews, Department of Chemistry & Biochemistry, University of California, Santa Cruz, USA
for providing lab. facilities to accomplish analyses of natural compounds.
Thanks are due to Haji Muhammad Ramzan Mayo and Mr. Akbar Ali Mayo, Dhing
Shah, District Qasur, for providing all facilities to carry out field experiment. Their unselfish
and honest passion is memorable. Thanks also to Dr. Javed Saleem and Dr. Muhammad Islam
for their guidance related to field experiment.
Higher Education Commission (HEC), Government of Pakistan needs a separate
mention as all this would have been impossible with out fellowships granted by HEC.
I am thankful to Federal Seed Certification Department, Lahore for providing certified
seeds of wheat varieties.
I am highly indebted to all my family members for their prayers, encouragements, deep
affections and patience. I deprived them of the love I owe them because of my studies.
Thanks are due to First Culture Bank of Pakistan for providing necessary fungal
cultures and Professor Dr. J. H. Mirza (Late), Dr. Uzma Bashir, Dr. Noureen Akhtar, Dr.
Irum Mukhtar and Miss Sobia, for helping me in the identification of fungal cultures.
Dr. Salik Nawaz Khan, I would say thanks for your guidance in research work and friendly
behavior.
Dr. Ghazala Nasim, I will always remember your appreciation and your efforts to aim for
excellence.
Thanks to Dr. Aamir Ali , Dr. Abdul Majid Khan, Dr. Akram Tariq Sial, Dr. Tariq
Riaz, Mr. Javaid Akram and Mr. Hassan Siddqi for helping me during all stages of my
thesis.
I will always remember honest contributions of Dr. Ahmed Ali Shahid, Dr. Safdar A.
Anwar, Dr. Tehmina Anjum, Miss Shabnam Javed, Mrs. Ruqia Suleman, Mr. Noor
Zaman, Dr. Abdul Hanan, Dr. Asad Shabbir, Mrs. Saira Sroya, and Mr. Muhammad
Khalil Ahmed Khan.
Mr. Waheed Anwar, Mr. Aqeel Ahmed, my dear friends and colleagues, you both were
my computer mentors, I cannot pay your honesty and passion towards me.
Mr. Muhammad Aslam, Mr. Taufiq Asghar, Mr. Ehsan Zaidi, Mr. Khurram, Mr.
Amad, Mr. Irfan Ali, Mr. Irfan Mahmood, Mr. Ishtiaq Ahmed, Mrs. Aliya, Mr.
Muhammad Iqbal Shad, Mr. Sarfraz Nawaz, Mr. Ishfaq, Mr. Muhammad Akram, Mr.
Abid, Mr. Abdul Raffay, Mr. Amjad, Mrs. Shazia, Miss Faiza, Mr. Manzoor Ilahi, Mr.
Niaz, Chacha Sadiq, Mr. Abbas, Mr. Imran, Mr. Muhammad Nasir Shah, Mr. Asif and
Mr. Samsoon Masih, I say a big thank to all of you.
In addition, special thanks to all my friends, lab members and hostel fellows for their
kind cooperation, constructive criticism, and valuable suggestions during the progress of my
studies and research and in preparation of this manuscript. Thanks are also to the members of
the IAGS for their time devotion, synergistic help, cooperation and valuable input during my
studies and research.
Muhammad Akbar
Contents
Title Page #
Certificate
Acknowledgments
List of Abbreviations i
Summary iii
Chapter 1: Introduction 1.1. Importance of Wheat 1
1.2. Importance of Weeds 2
1.3. Management of Weeds 4
1.3.1. Mechanical Control 4
1.3.1.1. Tillage 4
1.3.1.2. Flooding 5
1.3.1.3. Fire 5
1.3.1.4. Hand Hoeing 6
1.3.1.5. Mulching 6
1.3.2. Cultural Methods 6
1.3.2.1. Competitive Crops and Cultivars 6
1.3.2.2. Crop Rotation 7
1.3.2.3. Increased Crop Density 7
1.3.2.4. Intercropping 8
1.3.2.5. Companion Cropping 8
1.3.3. Biological Weed Control 8
1.3.4. Chemical Method 11
1.3.5. Natural Products as Herbicides 14
1.3.5.1. Herbicides from Plants 14
1.3.5.2. Herbicides from Fungi 16
1.4. Genus Drechslera 19
1.5. Objectives 22
Chapter 2: Materials and Methods 23 2.1. Selection of Test Fungal Isolates 23
2.2. Single Spore Isolation 23
2.3. Selection of Weeds of Wheat 23
2.4. Selection of Test Wheat Varieties 24
2.5. Preparation of Culture Filtrates of the Test Fungi 24
2.6. Laboratory Screening Bioassays 24
2.7. Foliar Spray Bioassays 25
2.8. Field Trials 26
2.8.1. Field Preparation 26
2.8.2. Sowing of Seeds 26
2.8.3. Treatments and Experimental Layout 27
2.8.4. Schedule of Foliar Sprays 27
2.8.5. Harvesting and Data Collection 28
2.9. Statistical Analysis 28
2.10. Organic Solvent Extraction 28
2.11. Leaf Discs Bioassays with Crude Organic Fractions 29
2.12. Isolation of Compounds through Chromatographic Techniques 31
2.12.1. Thin Layer Chromatography 31
2.12.2. Preparative Thin Layer Chromatography 32
2.12.3. Reversed Phase High Performance Liquid Chromatography 32
2.13. Spectroscopic Analyses 32
2.14. Leaf Discs Bioassays with Purified Chromatographic Fractions 33
Chapter 3: Results 34 3.1. Laboratory Bioassays 34
3.1.1. Effect of Fungal Culture Filtrates on Germination 34
and Growth of C. album
3.1.1.1. Effect on Germination 34
3.1.1.2. Effect on Shoot Growth 34
3.1.1.3. Effect on Root Growth 35
3.1.2. Effect of Fungal Culture Filtrates on Germination 35
and Growth of R. dentatus
3.1.2.1. Effect on Germination 35
3.1.2.2. Effect on Shoot Growth 35
3.1.2.3. Effect on Root Growth 36
3.1.3. Effect of Fungal Culture Filtrates on Germination 36
and Growth of P. minor
3.1.3.1. Effect on Germination 36
3.1.3.2. Effect on Shoot Growth 36
3.1.3.3. Effect on Root Growth 37
3.1.4. Effect of Fungal Culture Filtrates on Germination 37
and Growth of A. fatua
3.1.4.1. Effect on Germination 37
3.1.4.2. Effect on Shoot Growth 37
3.1.4.3. Effect on Root Growth 38
3.1.5. Effect of Fungal Culture Filtrates on Germination 38
and Growth of Wheat
3.1.5.1. Wheat var. Inqlab 91 38
3.1.5.1.1. Effect on Germination 38
3.1.5.1.2. Effect on Shoot Growth 38
3.1.5.1.3. Effect on Root Growth 39
3.1.5.2. Wheat var. Sehar 2006 39
3.1.5.2.1. Effect on Germination 39
3.1.5.2.2. Effect on Shoot Growth 39
3.1.5.2.3. Effect on Root Growth 39
3.1.5.3. Wheat var. Uqab 2000 40
3.1.5.3.1. Effect on Germination 40
3.1.5.3.2. Effect on Shoot Growth 40
3.1.5.3.3. Effect on Root Growth 40
3.2. Foliar Spray Bioassays 52
3.2.1. Effect of Fungal Culture Filtrates on Growth of C. album 52
3.2.1.1. Effect on Shoot Growth 52
3.2.1.2. Effect on Root Growth 52
3.2.2. Effect of Fungal Culture Filtrates on Growth of R. dentatus 52
3.2.2.1. Effect on Shoot Growth 52
3.2.2.2. Effect on Root Growth 53
3.2.3. Effect of Fungal Culture Filtrates on Growth of P. minor 53
3.2.3.1. Effect on Shoot Growth 53
3.2.3.2. Effect on Root Growth 53
3.2.4. Effect of Fungal Culture Filtrates on Growth of A. fatua 54
3.2.4.1. Effect on Shoot Growth 54
3.2.4.2. Effect on Root Growth 54
3.2.5. Effect of Fungal Culture Filtrates on Growth of Wheat 54
3.2.5.1. Effect on Shoot Growth 54
3.2.5.2. Effect on Root Growth 55
3.3. Field Experiment 70
3.3.1. Effect of Fungal Culture Filtrates on Weed Biomass 70
3.3.2. Effect of Fungal Culture Filtrates on Wheat Growth and Yield 70
3.4. Leaf Discs Bioassays Using Crude Organic Fractions 76
3.5. Leaf Discs Bioassays Using Purified Chromatographic Fractions 76
3.6. Spectroscopic Data of Isolated Compounds 84
3.6.1. Compound 1, (Holadysenterine) 84
3.6.2. Compound 2, (Z)- docos-5-en-1-oic acid 85
Chapter 4: Discussion 86
Conclusion 94
Future Prospects 94
References 95
Appendices
List of Tables Title Page #
Chapters 3: Results Table 1 42
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Chenopodium album in laboratory bioassays.
Table 2 42
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Rumex dentatus in laboratory bioassays.
Table 3 45
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Phalaris minor in laboratory bioassays.
Table 4 45
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Avena fatua in laboratory bioassays.
Table 5 48
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of wheat var. Inqlab 91 in laboratory bioassays.
Table 6 48
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of wheat var. Sehar 2006 in laboratory
bioassays.
Table 7 48
Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of wheat var. Uqab 2000 in laboratory
bioassays.
Table 8 72
Effect of foliar spray of herbicide bromoxynil+MCPA and culture filtrates of four
Drechslera spp. on biomass of Rumex dentatus.
Table 9 78
Leaf discs bioassays using crude organic fractions on punctured leaf surface.
Table 10 79
Leaf discs bioassays using crude organic fractions on unpunctured leaf surface.
Table 11 81
Leaf discs bioassays using purified chromatographic fractions from chloroform
fraction of D. australiensis on punctured leaf surface.
Table 12 82
Leaf discs bioassays using purified chromatographic fractions from ethyl acetate
fraction of D. australiensis on punctured leaf surface.
List of Figures Title Page #
Chapters 3: Results Fig. 1 56
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Chenopodium album plants.
Fig. 2 58
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Rumex dentatus plants.
Fig. 3 60
Effect of foliar spray of culture filtrates of Drechslera spp. on growth of 1-week and 2-
week old Phalaris minor plants.
Fig. 4 62
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Avena fatua plants.
Fig. 5 64
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old plants of wheat var. Inqlab 91.
Fig. 6 66
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old plants of wheat var. Sehar 2006.
Fig. 7 68
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old plants of wheat var. Uqab 2000.
Fig. 8 73
Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA and
culture filtrates of four Drechslera spp. on different growth parameters of field grown
wheat.
Fig. 9 74
Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA and
culture filtrates of four Drechslera spp. on grain yield and 100 grains weight of field
grown wheat.
Fig. 10 84
Chemical structure of holadysenterine
Fig. 11 85
Chemical structure of (Z)- docos-5-en-1-oic acid
List of Plates Title Page #
Chapters 2: Materials and Methods Plate 1 30
Scheme for leaf discs bioassays using crude organic fractions.
Chapters 3: Results Plate 2 43
Effect of culture filtrates of four Drechslera species on germination and growth of C.
album in laboratory bioassays.
Plate 3 44
Effect of culture filtrates of four Drechslera species on germination and growth of R.
dentatus in laboratory bioassays.
Plate 4 46
Effect of culture filtrates of four Drechslera species on germination and growth of P.
minor in laboratory bioassays.
Plate 5 47
Effect of culture filtrates of four Drechslera species on germination and growth of A.
fatua in laboratory bioassays.
Plate 6 49
Effect of culture filtrates of four Drechslera species on germination and growth of
Inqlab 91 in laboratory bioassays.
Plate 7 50
Effect of culture filtrates of four Drechslera species on germination and growth of
Sehar 2006 in laboratory bioassays.
Plate 8 51
Effect of culture filtrates of four Drechslera species on germination and growth of
Uqab 2000 in laboratory bioassays.
Plate 9 57
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Chenopodium album plants
Plate 10 59
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Rumex dentatus plants.
Plate 11 61
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Phalaris minor plants.
Plate 12 63
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old Avena fatua plants.
Plate 13 65
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old wheat var. Inqlab 91 plants.
Plate 14 67
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old wheat var. Sehar 2006 plants.
Plate 15 69
Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-week
and 2-week old wheat var. Uqab 2000 plants.
Plate 16 75
Effect of foliar spray of recommended (RD) and half dose (HD) of
Bromoxynil+MCPA and culture filtrates of four Drechslera spp. on field grown weed
and wheat.
Plate 17 80
Effect of crude chloroform (A) and ethyl acetate (B) fraction of culture filtrate of
Drechslera australiensis on punctured leaf discs of Rumex dentatus.
Plate 18 83
Effect of 2,4-D and chromatographic fractions(A), (C), (D), (F)and (H) of culture
filtrate of Drechslera australiensis on punctured leaf discs of Rumex dentatus.
i
List of Abbreviations
kg ha-1
Kilogram per hectare
GR Glyphosate-resistant
µg-1
Per microgram
AAL Alternaria alternata
ppm Parts per million
2,4-D 2, 4- dichlorophenoxyacetic acid
mM Milli molar
µM Micro molar
M Molar
mL Milliliter
Monocot. Monocotyledon
Dicot. Dicotyledon
P cm-2
Pascal per square centimeter
rpm Revolutions per minute
cm Centi meter
CRD Completely randomized design
g Gram
mg kg-1
Milligram per kilogram
NPK Nitrogen phosphorous potassium
RCBD Randomized complete block design
cm Centi meter
mg Milli gram
µg µl-1
Micro gram per microliter
µl Microliter
TLC Thin Layer chromatography
PTLC Preparative Thin Layer Chromatography
RPHPLC Reversed Phase High Performance Liquid Chromatography
UV Ultra violet
Rf Retention factor
DI Deionized
MS Mass Spectrometry
EIMS Electron Impact Mass Spectroscopy
HREIMS High Resolution Electron Impact Mass Spectroscopy
NMR Nuclear Magnetic Resonance
MHz Mega Hertz
1D One Dimensional
2D Two Dimensional 1NMR Hydrogen NMR
13NMR Carbon NMR
COSY Correlation Spectroscopy
HMBC Heteronuclear Multiple Bond Correlation
Conc. Concentration
mm Millimeter
ii
wt. Weight
FCF Fungal Culture Filtrates
var. Variety
CF Culture filtrate
FD Full dose
HD Half dose
g plot-1
Gram per plot
RD Recommended dose
m.p. Melting point
cm-1
per centimeter
Calcd. calculated
iii
Summary
Wheat (Triticum aestivum L.) is a major crop of Pakistan and is regarded as the
staple food of people of the country. The average grain yield in Pakistan is very low as
compared to yield potential possessed by most of the wheat cultivars. One of the most
important reasons for the low yield of the wheat in the country is the infestation of weeds.
Up to 45 weed species have been reported from different wheat growing areas of
Pakistan. Use of synthetic herbicides is believed to be the most effective strategy for the
management of weeds in wheat fields. However, indiscriminate use of these agro-
chemicals leads to environmental and health problems on one hand and evolution of
herbicide resistance in weeds on the other hand. The alternative to these synthetic agro-
chemicals is the use of natural products isolated from plants and fungi or their synthetic
analogues. The present study was, therefore, designed to evaluate the herbicidal potential
of culture filtrates of four Drechslera species viz. D. australiensis (Bugnicourt)
Subramanian & Jain., D. biseptata (Sacc. & Roum.) Richardson & Fraser, D. hawaiiensis
M.B. Ellis, and D. holmii (Luttr.) Subramanian & Jain, against four problematic weeds of
wheat namely Chenopodium album L., Rumex dentatus L. (dicotyledonous), Avena fatua
L. and Phalaris minor Retz. (monocotyledonous), and identification of active herbicidal
constituents through various chromatographic and spectroscopic techniques.
Culture filtrates of the four test Drechslera species were prepared by incubating
these fungi in M-1-D broth. In laboratory bioassays, seeds of the four selected weed
species and the three wheat varieties were exposed to original (100%) and diluted (50%)
fungal culture filtrates in Petri plates of 9-cm diameter. Culture filtrates of all the four
Drechslera spp. exhibited herbicidal activity against all the four test weeds. However, the
activity varied with the fungal species, concentration of the culture filtrates and the target
weed species. R. dentatus was found to be the most susceptible weed species. Original
culture filtrates of various Drechslera spp. significantly reduced germination, shoot
length and biomass as well as root length and biomass of R. dentatus by 12–56%, 67–
85%, 68–88%, 69–94% and 63–88% respectively. In general, culture filtrates of D.
australiensis exhibited the best herbicidal activity followed by culture filtrates of D.
hawaiiensis. Germination as well as root and shoot growth of the three test wheat
varieties was also adversely affected by culture filtrates of the various Drechslera
iv
species, however, wheat showed less susceptibility to the application of fungal culture
filtrates as compared to the target weed species especially R. dentatus.
In foliar spray bioassays, pot grown 1-week and 2-week old plants of the test
weed species and the three wheat varieties were sprayed with original Drechslera culture
filtrates four times with intervals of 4 days. Growth response of various weeds species to
fungal culture filtrates was highly variable in these bioassays. R. dentatus was found to be
the most susceptible weed species. Culture filtrates of all the four Drechslera species
significantly reduced shoot length and shoot biomass of 1-week old R. dentatus by 23–
42% and 54–60%, respectively, over control. The effect of foliar spray was more
pronounced in case of 1-week than in 2-week old plants. Culture filtrates of D.
australiensis exhibited the highest herbicidal activity against this weed species followed
by culture filtrates of D. hawaiiensis. The effect of foliar spray of fungal culture filtrates
was generally nonsignificant on the growth of other three target weed species and wheat
varieties.
The most susceptible weed species R. dentatus was selected for field trials. R.
dentatus was grown in field plots in 1:1 ratio with a wheat variety Sehar 2006. Over all
twelve treatments were made to assess the effect of culture filtrates of four Drechslera
spp. and a commercial synthetic herbicide on different growth parameters of the weed
and wheat. Culture filtrates of D. australiensis proved to be highly effective causing 58%
reduction in weed biomass over weedy check with subsequent increase of 27% in grain
yield of wheat.
In laboratory, pot and field studies, metabolites of D. australiensis exhibited the
best herbicidal activity against R. dentatus. This fungal species was thus selected for
isolation of active herbicidal ingredients. Culture filtrates of this fungal species were
successively extracted with n-hexane, chloroform, ethyl acetate and n-butanol followed
by evaporation in a rotary evaporator under reduced pressure. Solutions of different
concentrations of these crude extracts were prepared and were applied to wounded and
non-wounded leaf discs of R. dentatus. A positive reaction was indicated by the
appearance of a necrotic spot. Chloroform fraction exhibited the best herbicidal activity.
Six chemical constituents from this fraction were separated through Thin Layer
Chromatography (TLC), and the compounds were purified by Preparative Thin Layer
v
Chromatography (PTLC), followed by Reversed Phase High Performance Liquid
Chromatography (RPHPLC). Herbicidal activities of these isolated constituents were
evaluated by leaf discs bioassays. Two of the six isolated compounds exhibited the best
herbicidal activity. These compounds were identified as holadysenterine and (Z)-docos-5-
en-1-oic acid. through various spectroscopic techniques viz. Electron Impact Mass
Spectroscopy (EIMS), High Resolution Electron Impact Mass Spectroscopy (HREIMS)
and One Dimensional and Two Dimensional Nuclear Magnetic Resonance Spectroscopy
(1D and 2D NMR).
Results of the present study suggest that the metabolites of the test Drechslera
species possess herbicidal activity. The herbicidal activity of these metabolites varies
with test Drechslera species as well as the target weed species. Metabolites of D.
australiensis were found the most effective natural herbicides against broad-leaf weed R.
dentatus. All the test wheat varieties were found resistant to these metabolites. Further
studies are required to use structures of the two isolated herbicidal constituents as
analogues for the preparation of eco-friendly herbicides for the management of R.
dentatus.
1
Chapter 1
Introduction
1.2. Importance of Wheat
Wheat (Triticum aestivum L.), family Poaceae, is a globally pivotal cereal crop
with respect to area and production (Ashrafi et al. 2009). It is grown under irrigated as
well as rain-fed conditions worldwide (Zhao et al. 2009). It is regarded as the staple food
of Pakistan. Due to the presence of characteristic protein called gluten, wheat is widely
used as the principal cereal for the making of bread (Van Der Borght et al. 2005). Wheat
grains are rich in bioactive compounds which provide nutritional benefits to humans
(Loladze 2002). It contains carbohydrates as maltose, fructose, glucose, raffinose,
sucrose, starch and fructan (Högy et al. 2011). In addition to protein gluten, wheat also
contains minerals including macro-elements as K, Ca, Mg, P, S and Na; micro-elements
as Fe, Co, Se, Zn,, Cu, Mn, Cr, Mo and Ni and trace elements as Al, B, Cd, Pb and Si.
In Pakistan, wheat is cultivated as a winter crop on a huge area. It occupied an
area of 8666 thousands hectares during the year 2011–2012 having an average grain yield
equals to 2714 kg ha-1
and total production 23515 thousands tonnes (Anonymous 2012),
which is too less when compared to per hectare yield of advanced countries of the world,
inspite of the fact that most of its cultivars possess much higher yield potential. One of
the major reason for this low yield is weed infestation. In some studies, these weeds have
been investigated to incur yield losses from 10–83%, depending upon type of weed as
well as wheat cultivar, when grown in 1:1 ratio under experimental conditions (Siddiqui
et al. 2010; Anjum and Bajwa 2010).
1.2. Importance of Weeds
Weeds are plants which grow out of their proper places and whose virtues have
not yet been discovered (Kazi et al. 2007). These are regarded as the most undesirable,
aggressive and noxious element of world's vegetation. Weeds are unwanted plants, which
wrought noteworthy reduction in the yield of crop plants to variable extent depending
2
upon type and severity of infestation of weed species, type and density of crop plants,
environmental factors and soil fertility level etc. (Ahmadvand et al. 2009; Armin and
Asghripour 2011; Chauhan et al. 2012). Weeds compete with the crop plants by
occupying the space, which would otherwise be available to the crop plants (Wright et al.
2001). Weeds are also extremely likely to be competing for other sources such as water,
light and nutrients (Rajcan et al. 2001; Blackshaw et al. 2005; Erbs et al. 2009).
Water requirement for the growth of weeds is primarily of interest from the stand-
point of competition with the crop plant for the available moisture. It has been reported
that black mustard (Brassica nigra L.) transpires about four times more water than a crop
plant (Thakur 1984). In areas of low rainfall, enormous cover of weeds prevents a large
proportion of the rain falling in moderate shower from reaching the ground at all.
Moreover, weeds exert effect on nearby crop plants through uptake of water and the
intensity of this influence depends on relative rooting depths of the weed and the crop
plant (Soffe 2011).
Nutrition is another important factor that promotes plant growth. However, in
weed-crop competition, generally application of nutrients benefits weeds more than the
crop plants because of greater ability of weeds to accumulate mineral elements.
Tollennaar et al. (1997) found that under reduced nitrogen conditions, maize (Zea mays
L.) yield was reduced to 47% due to weeds infestation. Holm (1971) reported that weeds
contained approximately twice the nitrogen, 1.6 times phosphorus, 3.5 times potassium,
7.6 times calcium and 3.3 times magnesium as compared to that of corn. In other studies,
application of nitrogen fertilizers favored wild oat (Avena fatua L.) and green foxtail
[Setaria viridis (L.) Beauv.] over wheat, indicating that the addition of nitrogen profits
weeds more than crop plants (Peterson and Nalewaja 1992). It has been discovered that
there exists a correlation between density of weeds and protein content of wheat grains.
About one and half wild oat plants per meter square reduce protein content of wheat grain
by 1% (Khan 2008). Iqbal and Wright (1999) investigated the competitive ability of
lamb's quarters (Chenopodium album L.), wild mustard (Sinapis arvensis L.) and
littleseed canarygrass (Phalaris minor Retz.), in relation to wheat crop. Although there
was no effect of weed density on wheat plant height but there was significant decrease in
uptake of total nitrogen by wheat plants, dry biomass and grain yield. More over, weed
3
competition in wheat significantly effect number of tillers per square meter as well as
number of grains per spike (Chaudhary et al. 2008; Siddiqui et al. 2010). Growth habit of
some weeds, for example, orange eye butterflybush (Buddleja davidii Franch.) profits
them in behaving as strong competitors for light allowing lesser and lesser light to reach
crop plant resulting in frailty of crop plant (Richardson et al. 1996). Some weeds
intercept light due to their greater height than crop plants. e.g. wild oat reduces light
penetration and ultimately growth of wheat by being taller than the wheat plants (Cudeny
et al. 1991). Similarly, velvetleaf (Abutilon theophrasti Medik.) intercepts light due to its
greater height than soybean [Glycine max (L.) Merril.] (Akey et al.1990). Studies have
shown that weed canopy architecture perspectives especially plant height, location of
branches and leaf area determine the impact of interspecific light competition resulting in
low yield of crop plants (Naseri et al. 2012).
Inhibitory effects of weeds on crop plants through the release of phytochemicals
are also well documented. Reduction of 14–19% in the yield of soybean by the extracts of
dried residues of several weed species including C. album, red-root amaranth
(Amaranthus retroflexus L.) and A. theophrasti has been reported (Bhowmik and Doll
1992). Similar cases of reduction in crop yield by allelopathic weeds include: Purple
nutsedge (Cyperus rotundus L.) and Indian shot (Canna indica L.) in rice (Oryza sativa
L.), C. album and A. retroflexus in safflower (Carthamus tinctorius L.), Dogbane (Rhazya
stricta Decne.) on maize and crabgrass (Digitaria horizontalis Willd.) on dry bean
(Phaseolus vulgaris L.), and turnip (Brassica rapa L.) and soybean (Javaid et al. 2007;
Lin et al. 2009; Rezaie and Yarnia 2009; Khan et al. 2011; Teixeira et al. 2011).
Infestation of weeds is among the major causes of low yield of wheat that
harnesses most of the moisture and nutrients. Wheat crop faces both monocot. and dicot.
weeds infestation. Siddiqui and Bajwa (2001) and Qureshi and Bhatti (2001) have
reported 45 types of weeds from wheat growing regions of Pakistan. In these studies, P.
minor, wild oat, burclover (Medicago polymorpha L.), lesser swinecress [Coronopus
didymus (L.) Sm.], small meliot (Melilotus parviflora Desf.), toothed dock (Rumex
dentatus L.) and C. album appeared to be the most frequently occurring and densely
populated weeds. Yield losses due to these weeds in different wheat cultivars were
estimated as 20-60% (Siddiqui et al. 2010). The wheat yield losses by these weeds has
4
been recorded up to 80% taking into account various environmental factors, weed type
and its density, and also wheat density and cultivar (Khera et al. 1995; García-Martín et
al. 2007; Qasem 2007). Thus weed management has become a serious peril to wheat
yield as weeds not only reduce yield of the crop but also deteriorate quality of the
produce in many cases (Memon et al. 2003).
1.3. Management of Weeds
At present weeds are being considered to be the major cause of suboptimal crop
yield throughout the world despite centuries of efforts in their management (Oerke 2006).
Weed management in organic agriculture uses preventive methods that include different
strategies like intercropping, cover crops, green manure and mulches. Roots of
allelopathic plants release compounds in the soil that are toxic to weeds (Campiglia et al.
2009; Isik et al. 2009; Flower et al. 2012). However suppressive effect on weeds is
influenced by type of species, seeding rate and method, planting date, decomposition
time of plant residues and weather (Yalcin and Cakir 2006; Ortiz-Monasterio and Lobell
2007; Kalinova 2010; Chauhan et al. 2011). Several methods of the weed management
are in vogue such as cultural and mechanical, biological, chemical and management
through natural products.
1.3.1. Mechanical Control
It involves the removal of weeds by various tools/implements including tillage,
flooding, fire, hand hoeing, pulling and mulching.
1.3.1.1. Tillage
The principle of this method is simply to turn the weeds under or bring them to
the soil surface where they die on account of desiccation (Swanton et al. 1999). This
method also affects the nature and extent of weed populations (Blackshaw et al. 1994).
This practice can be employed before and after planting. Tillage before planting results in
germination of weed seeds and subsequent destruction of seedlings during soil
preparation (Dirk 2007). However the behavior of weeds and their interaction with crops
5
is a complex phenomenon which is still under investigation. Weed species having
photoblastic germination tend to be more problematic in conservation agriculture. Also,
in the absence of tillage, perennial weeds may also become more problematic (Chauhan
et al. 2012).
1.3.1.2. Flooding
The principle of this technique lies in promoting weed seed decay and
germination. For this tactic to be useful, seeds of weeds should be submerged for an
extended period of time (Rao 2000). For example, weedy rice (Oryza nivara S.D. Sharma
& Shastry) is a noxious weed of cultivated rice. Infestations of weedy rice have been
recorded to have spread to 40-75% of the total of rice cultivation region in European
countries (Ferrero 2003). In a field experiment, weedy rice plant density declined
significantly by the application of winter flooding. Here flooding caused more than 95%
decrease in the number of viable weed seeds when compared to fields which were left dry
between rice crops (Fogliatto 2010). Winter flooding is a common management practice
in America, where rice fields are flooded in autumn following rice harvest until the
spring before tillage operations (Van Groenigen et al. 2003).
1.3.1.3. Fire
Use of conventional fire has long been witnessed to control unwanted vegetation.
Nowadays a modified technique of fire known as flame cultivation is used on very small
scale. In this method, fire is used for selective control of weeds in crop rows. But it
requires great care as it can damage the crop as well as having negative effects on soil
(Tu et al. 2001). Gleadow and Narayan (2007) carried out a study in Australia where a
weed sweet pittosporum (Pittosporum undulatum Vent.) has colonized into many
habitats, causing a serious damage to structural diversity and floristic composition due to
its competitive ability that created an environment conducive to its own progeny and
deleterious for other plant species. They concluded that high temperatures associated with
wildfires are enough to disrupt the invasion cycle of test plant species.
6
1.3.1.4. Hand Hoeing
This method has proven to be very effective to eradicate annual and biennial
weeds as it not only eradicates weeds but also improves soil aeration due to stirring.
However, this method is less effective in case of perennial weeds. In spite of all its
advantages, it is not considered to be cost effective (Tu et al. 2001). Besides, there are
reports that hand hoeing is less effective when compared with chemical control (Subhan
et al. 2004).
1.3.1.5. Mulching
Organic mulching is a strategy in which at least 30% of the surface of soil is
covered by plant material. It conserves the soil, improves the soil ecology, stabilizes and
enhances crop yield and improves various environmental factors. Although mulching
practices are no solutions but they represent technological options that integrate
conservation and productivity considerations (Erenstein 2003). Mulching has smothering
effect on weeds by casting shadow on weed plants, which results in little photosynthesis
resulting in frail weed plants offering lesser competition to main crop. Hiltbrunner et al.
(2007) in a field experiment concluded that legumes can be used to suppress weeds in
wheat field. Due to high cost, it is considered cost-effective only in case of high value
crops like tea and coffee. For example, guatemala grass (Tripsacum laxum Nash.) is used
as mulching in tea fields (Rao 2000). However, mechanical methods are not feasible
where weeds resemble morphologically to crop. e.g. wild oat (Avena ludoviciana
Durieu.) and P. minor mimic wheat before flowering. Also, mechanical weed control is
not cost-effective and becomes difficult in broadcast sown wheat.
1.3.2. Cultural Methods
1.3.2.1. Competitive Crops and Cultivars
Under field conditions, both weeds and crop compete with each other for same
resources (Turk and Tawaha 2003). So if those cultivars of crops are planted that have
7
high vigor and able to grow more rapidly as compared to weeds, significant losses due to
these weeds can be prevented (Corre-Hellou et al. 2011). In this regard, competitiveness
of the crop seems to be important in weed behaviour. For example, pea (Pisum sativum
L.), a weak competitor, had much higher biomass at harvest compared with oats and
winter wheat (Lundkvist et al. 2008). More rapidly growing crops cause stifling effect on
the growth of weeds by casting its shadow as well as excluding weeds out of competition
by utilizing resources like nutrients, sunlight, moisture and carbon dioxide (Aldrich and
Kremer 1997; Radicetti et al. 2012).
1.3.2.2. Crop Rotation
Monocrop culture practiced for an extended period of time in a particular area
helps to establish associated weeds of that particular crop in that area. Management of
weeds through crop rotation is effective because changing patterns of disturbance
diversifies selection pressure that prevents the proliferation of weed species well suited to
the practices associated with a monoculture crop. For example, wild mustard populations
can be reduced by selective treatment of small grain grown in rotation with row crops
(Turk and Tawaha 2003). Crop rotations have also been reported to break disease cycle,
improve nitrogen fixation and water-use efficiency (Ryan et al. 2008). Thus wheat-
legume rotation system has proven to be the best in certain regions as it results in the
highest yield and protein content of wheat crop. Also this treatment does not need
fertilizers to achieve better crop yield and is considered to be more sustainable system for
low rainfall zones (Galantini et al. 2000).
1.3.2.3. Increased Crop Density
The principle of this practice lies in the fact that greater number of crop plants in
the field offers more competition to weeds on account of their smothering effects as well
as competition for limited resources. This technique uses enhanced seed rate and narrow
inter and intra row spacing. Also there exists optimum seed rate for obtaining higher
grain yield. However, such practices need to be optimized in a particular area
(Ahmadvand et al. 2009; Marwat et al. 2011).
8
1.3.2.4. Intercropping
Intercropping is considered to be an efficient tool for better land use efficiency
and weed suppression. As an example, wheat and bean (Vicia faba L.) were grown as
intercrops. Regarding weed suppression, intercrops were more effective than wheat sole
crops (Eskandari 2011). In another study, pea-barley intercrops have shown to lessen the
weed biomass when compared with the pea and barley sole crops (Corre-Hellou et al.
2011).
1.3.2.5. Companion Cropping
Judicious use of living mulches is very important factor as it requires adapted
seeding rate and technique as well as type of main and cover crop. In a study, living
mulches belonging to four different genotypes of legumes were used in order to tap its
full potential in winter wheat crop. It was found that legumes producing more dry matter
namely birdsfoot trefoil (Lotus corniculatus L.) and white clover (Trifolium repens L.)
and controlled weeds better than species producing less dry matter such as subclover
(Trifolium subterraneum L.) and strong-spined medick (Medicago truncatula Gaertner)
(Hiltbrunner et al. 2007).
Although cultural and mechanical practices are equally effective yet large
numbers of farmers are not very well trained in this regard (Thomas et al. 1999). Also
these methods become more effective when used in an integrated form (O’Donovan et al.
2001; Derksen et al. 2002). Under such circumstances weed control through chemical
herbicides has become the most popular method among the farmers.
1.3.3. Biological Weed Control
Biological control strategies are utilized in a classical (Kurose et al. 2012),
augmentative (Vorsino et al. 2012) or inundative mode (Gerber et al. 2011). Classical
biological control by means of plant pathogens has been used in many agro climatic areas
to control exotic weeds. The concept of this approach is simple to use: Discover effective
and highly host-specific agents from the weed's native geographic range, confirm their
biosafety and effectiveness, and introduce them into regions where the weed has been
9
newly invaded and needs control (Charudattan and Dinoor 2000; Cripps et al. 2011).
Augmentative approaches on the other hand depend on the release of additional numbers
of a natural enemy when too few are present to control a pest effectively (Lv et al. 2011).
While inundative approach depends on the release of large numbers of biological control
agents to control a pest when there is very low risk of the biological control agent to
spread or establish it permanently. In this approach, application may be made to induce
disease epidemics or to act as microbial pesticides (Williams et al. 2003; Fernando et al.
2010). Biological control of weeds using phyto-pathogens started in the 1970s when a
small number of noxious weeds were controlled by different strategies. Since then,
scientists are evaluating different strategies in a hope to solve some of the most
intractable weed problems (Charudattan and Dinoor 2000). In selecting classical
biological control agents for landscape-level suppression of weeds, prior evaluation of
their biosafety together with their effectiveness is of utmost importance.
A bioherbicide is defined as a plant pathogen used as a weed-control agent
through inundative and repeated applications of its inoculum. Bioherbicides provide
envovironment friendly, non-chemical method to control a number of weeds (Saxena and
Pandey 2002). Inspite of huge research work done on microbial herbicides, only few
bioherbicides have been registered so far. As for example, DeVine composed of isolate of
Phytophthora palmivora Butl.; Collego and BioMal, both based on Colletotrichum
gloeosporioides (Penz.) Penz. & Sacc.; Dr. BioSedge based on the Puccinia canaliculata
Arthur; CAMPERICO based on bacterium Xanthomonas; and Stumpout based on a
basidiomycete Cylindrobasidium. These have been used to control stranglervine
[Morrenia odorata (Hooker & Arnott) lindley], northern jointvetch, [Morrenia odorata
(Hooker & Arnott) lindley], round-leaved mallow (Malva pusilla Smith) and bluegrass
(Poa annua L.) (Yongqang 1998; Charudattan 2000; Charudattan and Dinoor 2000).
However, mass production of bioherbicides is difficult, and because of their specific
requirement for action conditions, these products are not world-famous and did not bring
significant economic benefits (TeBeest et al. 1992; Makowski 1993). There are many
other examples of bioherbicides from microbes. For example, mixture of Drechslera
gigantea Heald & Wolf, Exserohilum longirostratum (Subramanian) Sivanesan, and
Exserohilum rostratum (Drechsler) Leonard et Suggs, have also been described to control
10
many grassy weeds with 100% disease incidence having no injurious effects on crops
(Chandramohan and Charudattan 2001). C. rotundus and yellow nutsedge (Cyperus
esculentus L.) are serious weeds in Florida and in many other parts of the world.
Dactylaria higginsii (Luttr.) M.B. Ellis is a promising fungal bioherbicide candidate for
these weeds (Shabana et al. 2010a). The fungal pathogen Microsphaeropsis amaranthi
(Schwein.) Kuntze has been considered as a promising bioherbicide for the control of
waterhemp (Amaranthus tuberculatus Sauer) (Shabana et al. 2010b). The bacterium
Pseudomonas fluorescens (Flügge) Migula, has potential for biocontrol of S. viridis and
a large number of grassy weed species (Pedras et al. 2003; Banowetz et al. 2009).
Deleterious rhizobacteria have been shown to affect the invasive species cheatgrass
(Bromus tectorum L.) and can likely serve as biological control agents (Dooley and
Beckstead 2010). However, limited commercial interest, complexities in production,
assurance of efficacy and shelf-life of inoculum are serious limitations that have led to
the abandonment of several promising plant pathogens for the biocontrol of weeds
(Charudattan and Dinoor 2000). Lack of host specificity of potential biocontrol agent is a
major criterion that leads to ultimate rejection of proposed biocontrol agent (Yobo et al.
2009).
Biological control of weeds using insects as enemies has also been widely studied
successfully in many parts of the world (Manrique et al. 2008; Hough-Goldstein et al.
2009; Myint et al. 2012). For example, some floating invasive plants of major importance
have been controlled by biological agents such as American weevil (Stenopelmus
rufinasus Gyllenhal) controlled mosquito fern (Azolla filiculoides Lam.). Similarly,
species of Neochetina weevils controlled water hyacinth [Eichhornia crassipes (Mart).
Solms] and Salvinia weevil (Cyrtobagous salviniae Calder & Sands) controlled giant
salvinia (Salvinia molesta Mitchell) (McConnachie et al. 2004; Hill and McConnachie
2009; Coetzee et al. 2009; Julien et al. 2009). Deploying multiple biological control
agents where they can partition the target resources in space and/or time has been proven
to be more effective, e.g. in Australia, individual and combined effects of the rust fungus
Puccinia myrsiphylli (Thuem.) Wint. and a leafhopper (Zygina sp.) for biocontrol of a
weed, bridal creeper [Asparagus asparagoides (L.) Druce] have been investigated. In this
11
study, synergistic effect of both biological control agents was evident (Turner et al.
2010).
Classical biocontrol has not been a world-famous approach of weed management
in intensively managed crops due to the slowness of the classical biocontrol process
relative to the short duration of the cropping season. The frequent disruptions associated
with cropping practices can also have adverse effects on classical biocontrol agents
(Charudattan and Dinoor 2000). Also a biological control agent for a weed developed for
one country has proven to be ineffective against the same weed in another country
(Balciunas 2007). Depending on one’s point of view, this biological control approach has
been quite successful or wrought with limitations (Charudattan and Dinoor 2000). Recent
investigations point out certain unavoidable non target effects of biological control agents
due to complexities involved in the interactions of bio-control agents and target species
(Pearson and Callaway 2005). Also all introduced bio-control agents incur effects on
nontarget species (Delfosse 2005). So, there has been considerable debate on risks
associated with biological control, e.g. non-target impacts of bio-control agents (Barratt
et al. 2010).
1.3.4. Chemical Method
To combat weeds, chemical herbicides have been a major breakthrough. At
present, control of weeds by the application of herbicides is considered to be the most
effective method of controlling weeds. So herbicides have become the basis for weed
control in intensive agriculture (Rüegg et al. 2007). Chemical weed control is preferred
because it is cost-effective as well as it offers no physical damage to the crop that
happens during hand hoeing and tillage. Moreover, the control is more effective as the
weeds even within the rows are killed which otherwise escape invariably during
mechanical control, on account of morphological mimicry to wheat. Different
acetamides, aliphatics, arsenicals, benzamides and sulfonylureas are being used as
herbicides (Verstraeten et al. 2002).
There are many successful attempts of weed management through chemical
herbicides (Jordan et al. 2009; Krutz et al. 2009; Hulting et al. 2012). Sometimes
herbicidal mixtures are used because none of the herbicides alone controls a wide enough
12
range of weeds to be suitable in all circumstances. It is, therefore, desirable to mix
herbicides to get the best fit for the weeds in the crop. Various chemical herbicides such
as Topic (clodinafop-propargyl), Puma Super (fenoxaprop-p-ethyl), Affinity
(carnfentrazone ethyl + isoproturon), Buctril Super (bromoxynil octanovate +
heptanovate ester), imidazolinone herbicides, imazapyr, imazapyr plus imazapic, and
imazapyr plus imazamox etc. are very effective in controlling weeds of wheat fields in
various parts of the world (Bibi et al. 2005, 2008; Cheema et al. 2006; Usman et al. 2010;
Kleemann et al. 2009). However efficacy of different herbicides to control weeds in
wheat varies and in some cases it is site dependant (Zand et al. 2010).
Although synthetic herbicides are every effective in the management of weeds,
however, indiscriminate use of these agrochemicals have created a number of problems.
Due to frequent use of herbicides, there have been dramatic increase in the frequency and
diversity of weed biotypes that are herbicide-resistant, that poses a threat to the
sustainability of agriculture worldwide (Yuan et al. 2007; Llewellyn et al. 2009). For
example, overwhelming evolution of resistance of a number of weeds including wild oats
to various chemical herbicides have been reported (Vila-Aiub et al. 2005; Singh et al.
2012). More than 200 distinct weed biotypes that are resistant to various herbicides, have
evolved world over (Devine and Shukla 2000). Furthermore, many herbicides are very
toxic to some sensitive crops and may cause severe crop injury in some cases as in
cotton, corn, many vegetable crops and wheat (Kadir and Charudattan, 2000; Usman et
al. 2010; Sikkema et al. 2007). Since herbicides have also shown to affect crops so in this
scenario herbicide resistant crops are being developed. e.g. glyphosate-resistant crops like
soybean, cotton (Gossypium hirsutum L.) and maize have been widely adopted in USA
(Sankula 2006). Due to the development of glyphosate-resistant crops, many farmers
depend solely on glyphosate for weed control and its recurrent use is the main cause for
the development of herbicide resistant weeds (Holt 1992). Also, in recent years, the use
of chemicals is becoming more restrictive due to public awareness regarding ill effects of
all the chemical herbicides (Marin et al. 2003; Rial-Otero et al. 2005). The herbicides
used to boost agricultural food production may not only combat pests and weeds but also
present toxic properties and cause genetic aberrations into exposed fauna and flora (Losi-
Guembarovski et al. 2004). The herbicides have a long residence time in the atmosphere
13
and come to earth in the form of wet and dry depositions (Waite et al. 2005). There are
evidences that herbicides present in the atmosphere induce intracellular overproduction of
reactive oxygen species and herbicide induced oxidative stress disturbing the
photosynthesis of target plants e.g. in wheat and tilapia (Oreochromis niloticus L.), thus
damaging plant cells (Peixoto et al. 2006; Song et al. 2006, 2007; Wang and Zhou 2006).
Phenylurea herbicides are used world over, that often pollute surface and groundwater in
concentrations exceeding the limiting value of 0.1 µg-l for drinking water (Badawi et al.
2009). Herbicides are also known to affect aquatic life. For example, in green alga
Raphidocelis subcapitata (Korsh) Nygaard et al., a reduction in photosynthesis has been
reported. The most toxic herbicides documented in this regard include, atrazine,
ametryme, chlorotoluron, cyanazine, isoproturon and diuron (Ma et al. 2006). Natural
waters have been investigated to contain complex mixtures of herbicides as well as
herbicide breakdown products as contaminants potentially posing a threat to marine
communities through chemical interactions (Magnusson et al. 2010). In some
experiments, application of herbicides have shown to effect aquatic plants (Huiyun et al.
2009; Vervliet-Scheebaum et al. 2010). Frequent use of herbicides has led to weed
adaptation via the selection of resistance mechanisms enabling weed plants to withstand
herbicide application in at least 194 weed species worldwide (Heap 2008; Heap 2010;
Johnson et al. 2009; Knezevic et al. 2010). For example, Wimmera ryegrass (Lolium
rigidum Gaudin) is the most prevalent and noxious grassy weed of winter cereals in
Spain. Due to frequent use of herbicides to control this weed, its populations are evolving
herbicide resistance (Loureiro et al. 2010). This herbicide resistance has posed serious
concerns for agriculture because it disrupts herbicide-based weed eradication and also
because alternative strategies of controlling weeds have proven to be less effective (Bond
and Grundy 2001; Bastiaans et al. 2008). Moreover, due to greater water solubility, high
polarity and heat stability, it is difficult to fade away them from the atmosphere (Bonnet
et al. 2008; Rashid et al. 2010).
In addition to their harmful effects on environment, high cost associated with the
use of herbicides is a limiting factor in the profitability of crop production (Partridge et
al. 2006). Agricultural producers cannot give up their chemical tools for weed control
until research provides them with workable alternatives (Quimby et al. 2002). Moreover,
14
at many places, the use of chemical herbicides is severely restricted or mostly banned
(Vurro et al. 2012). Hence, there is dire need of alternate eco-friendly, cost effective and
bioefficaceous methods of weed control. In this scenario, there have been numerous
efforts to control invasive weeds by natural products from plants and microbes (Van
Driesche et al. 2010; Javaid 2010; Aliferis and Jabaji 2011; Berner and Bruckart 2012).
1.3.5. Natural Products as Herbicides
1.3.5.1. Herbicides from Plants
Chemical interactions between and among both plants and microorganisms
through release of biologically active chemical compounds into the environment is
known as allelopathy. Allelopathic potential of certain weeds and crop species that exists
in nature can influence the growth and distribution of associated weed species and the
yield of desired crops. By virtue of this, allelopathy has been harnessed successfully in
biocontrol programs to restrain noxious weeds. Allelopathy thus plays an important role
in an agroecosystem and a better understanding of this phenomenon would help in crop
improvement through sustainable agriculture (Farooq et al. 2011).
Herbicides derived from naturally occurring materials are gaining fame as these
are environmentally safe. Numerous allelopathic plants have been exploited for their use
as bioherbicides as these plants contain natural growth inhibitors (Xuan et al. 2005). For
example, sunflower (Helianthus annuus L.) cv. Suncross-42 extracts exhibited herbicidal
activity against R. dentatus and C. album (Anjum and Bajwa 2007a,b). A bioactive
annuionone H was isolated from leaves of sunflower. The isolated compound showed
pronounced herbicidal activity against P. minor, C. album, R. dentatus, C. didymus and
M. polymorpha (Anjum and Bajwa 2005). Céspedes (2006) studied some phytochemicals
of plant origin and found that some of them possessed potent herbicidal activities.
Similarly, rice allelopathy has been exploited against world’s noxious weed of rice,
barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] (Mennan et al. 2011). Ergosterol
peroxide and 7-oxo-stigmasterol proved to be the most active herbicidal compounds
isolated from rice extracts. In case of ergosterol peroxide the herbicidal activity was
higher than the commercial chemical herbicide Logran (Trisulfuron) (Macías et al. 2006).
15
Likewise, catmint [Anisomeles indica (L.) Kuntze] have shown potential herbicidal
activity like that of chemical herbicide against P. minor and other weeds of the wheat
under natural field conditions (Batish et al. 2007). Salamci et al. (2007) found that
essential oils isolated from Turkish Tanacetum aucheranum and Tanacetum
chiliophyllum var. Chiliophyllum exhibited herbicidal effects. Herbicidal effects of the
oils were evaluated on seed germination and seedling growth of test weed species viz. C.
album, curly dock (Rumex crispus L.) and A. retroflexus. Similarly, natural plant product,
benzoxazolin-2(3H)-one has been investigated to be having herbicidal activity against
lettuce (Lactuca sativa L.) (Sánchez-Moreiras et al. 2008). Likewise, essential oil of
letswaart [Origanum acutidens (Hand.-Mazz.)] has shown phytotoxicity against C.
album, R. crispus and A. retroflexus. The oil, thymol and carvacrol completely arrested
the seed germination and seedling growth of all the test plant species (Kordali et al.
2008). Glucosinolates form a group of allelochemicals produced by many species of
plants like Brassica, Sinapis, Lepidium, Nasturtium and Limnanthes spp. Glucosinolate
degradation products have shown herbicidal activity on B. tectorum coleoptile emergence
(Stevens et al. 2009). Similarly, herbicidal potential of sorghum (Sorghum bicolor L.)
water extract alone and in combination with water extracts of other allelopathic plants:
sesame, sunflower, tobacco, eucalyptus and brassica, against two problematic weeds of
wheat, P. minor and A. fatua have been demonstrated. In this study application of
sorghum and sunflower extracts each were found more effective than rest of the test plant
species (Jamil et al. 2009). In another study, wollemi pine (Wollemia nobilis Jones, Hill
& Allen) plant material exhibited herbicidal effects against L. rigidum and wild radish
(Raphanus raphanistrum L.) (Seal et al. 2010). In the same way, Aswagandha [Withania
somnifera (L.) Dunal] has been exploited as natural herbicide against Parthenium weed
(Parthenium hysterophorus L.) and P. minor under laboratory conditions, foliar spray
bioassays and soil amendment bioassay (Javaid et al. 2010a, 2011a). Similar herbicidal
activity of extracts of Datura (Datura metel L.) has also been reported against P. minor
and P. hysterophorus (Javaid et al. 2008, 2010b).
16
1.3.5.2. Herbicides from Fungi
The literature abounds with examples of herbicidal compounds (phytotoxins)
isolated from fungal world. Phytotoxins are largely low molecular weight secondary
metabolites capable of deranging the vital activity of plant cells viz. enzyme inhibition,
interference with the properties of membranes and interference with defense responses.
Phytopathogenic fungi are best known as phytotoxin producers. Phytotoxins are usually
isolated from in vitro cultures of the pathogen grown either on solid or liquid media and
have the ability to damage plants. Tal et al. (1985) isolated novel compounds, named
radianthin and radicinin having phytotoxic activity from liquid culture broth of Alternaria
helianthi (Hansf.) Tubaki & Nishih. Similarly, AAL-toxin TA isolated from Alternaria
alternata (Fr.) Keissl. and Fumonisin B1 from Fusarium moniliforme J. Sheld. proved
potent phytotoxins against duckweed (Lemna pausicostata L.) (Abbas et al. 1998).
Ascaulitoxin characterized as N2-(2,4,7-triamino-5-hydroxy)-octanedioyl-β-D-gluco-
pyranoside was isolated from the culture filtrate of Ascochyta caulina (P. Karst.) Aa &
Kesteren found a promising natural herbicide for the control of many noxious weeds.
Ascaulitoxin, when assayed in the leaf-puncture assay on weeds including C. album,
common sowthistle (Sonchus oleraceus L.), noogoora burr (Xanthium occidentale
Bertol.), annual fleabane [Erigeron annuus (L.) Pers.] and Tree of Heaven (Ailanthus
glandulosa Desf.), ascaulitoxin caused the appearance of necrotic spots (Evidente et al.
1998). Latter on, a phytotoxic metabolite trans-4 aminoproline was isolated from culture
filtrate of the same fungus and also found very effective in controlling C. album
(Evidente et al. 2000). In addition, Vurro et al. (2001) also identified another herbicidal
constituent aglycone of ascaulitoxin from culture filtrates of A. caulina that was found
very effective in controlling C. album. Latter on Vurro et al. (2012) showed that
metabolites with herbicidal properties from A. caulina can be produced on pre-industrial
level. Fukushima et al. (1998) reported that culture filtrate of Nigrospora sacchari
(Speg.) E.W. Mason showed strong herbicidal activity against weeds such as E. crus-
galli, S. viridis, A. theophrasti and slender amaranth (Amaranthus viridis L.) They
isolated three lactones from the culture broth of test fungus. The major component was
identified as (+)-phomalactone, 6-(1-propenyl)-5,6-dihydro-5-hydroxy-2H-pyran-2-one.
17
Others were 5-[1-(1-hydroxybut-2-enyl)]-furan-2-one and 5-[1-(1-hydroxybut-2-enyl)]-
dihydrofuran-2-one. The herbicidal activity of these fungal metabolites was caused by
cellular disruption when applied at concentrations higher than 50 ppm. Similarly, two
new phytotoxic nonenolides viz. herbarumins I [(7S,8S,9R)-7,8-dihydroxy-9-propyl-5-
nonen-9-olide] and herbarumins II [(2R,7S,8S,9R)-2,7,8-trihydroxy-9-propyl-5-nonen-9-
olide], were identified from Phoma herbarum Westend. Their phytotoxicity was
evaluated as pre-emergent herbicides against amaranth (Amaranthus hypochondriacus
L.). Herbarumins I appeared to be more potent herbicide than the positive control 2,4-D,
while herbarumins II exhibited a herbicidal potency similar to that of 2,4-D (Fausto
Rivero-Cruz et al. 2000). Macías et al. (2001) reported two phytotoxic naphthopyranone
derivatives by investigating fermentation broth and mycelium of the coprophilous fungus
Guanomyces polythrix M.C. González, Hanlin & Ulloa. They named these isolated
compounds as (2S, 3R)-5- hydroxy-6,8-dimethoxy-2,3-dimethyl-2,3-dihydro-4H-
naphtho[2,3-b]-pyran-4-one and (2S, 3R)-5-hydroxy-6,8,10-trimethoxy-2,3- dimethyl-
2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one. The isolated compounds significantly
arrested radicle growth of two weed seedlings, E. crus-galli and A. hypochondriacus.
Likewise, Macrocyclic trichothecene toxins produced by phytopathogen Myrothecium
verrucaria (Alb. & Schwein.) Ditmar and the non-trichothecene toxin atranone B from
Stachybotrys atra Corda have shown their phytotoxicity against kudzu (Pueraria lobata
L.) and duckweed plantlets (Abbas et al. 2002).
Destruxins are secondary metabolites isolated from entomopathogenic fungus,
Oospora destructor (Metschn.) Delacr. Destruxins exhibited a wide variety of biological
activities, but are well known for their phytotoxic activities. Phytotoxic activity of
destruxins has been demonstrated against many herbs including oat (Avena sativa L.) and
quinoa (Chenopodium quinoa Willd.) (Pedras et al. 2002). Phytotoxicity tests of the
metabolite solutions or crude toxins of A. alternata on aquatic weeds especially E.
crassipes, developed phytotoxic symptoms (Babu et al. 2003a). Moreover, an herbicidal
glycoprotein, produced by Phoma eupyrena Sacc. brought about blighting and necrosis of
leaf tissues when 1-5 µg was introduced into the mesophyll tissue of water lettuce [Pistia
stratiotes (L.) Fam.] (Babu et al. 2003b). Phytotoxic fungal metabolites namely
leptosphaerodione, elsinochrome A and cercosporin have been isolated from different
18
isolates of phytopathogenic fungal species Stagonospora convolvuli Dearn. & House.
These metabolites have been shown to be toxic to field bindweed (Convolvulus arvensis
L.) and hedge bindweed [Calystegia sepium (L.) R. Br.] (Ahonsi et al. 2005). Luis et al.
(2005) isolated two phytotoxic compounds namely 1-hydroxy-2-oxoeremophil-
1(10),7(11),8(9)-trien-12(8)-olide and penicillic acid from fungus Malbranchea
aurantiaca Sigler & Carmichael. These metabolites caused significant inhibition of
radicle growth of A. hypochondriacus. Similarly, two compounds, macrocidin A and B
have been isolated from the liquid culture filtrate of fungus Phoma macrostoma Mont.
They were the first representatives of a new family of cyclic tetramic acids. These
phytotoxic metabolites caused bleaching and chlorosis to several broadleaf weed species
(Graupner et al. 2006). Zonno et al. (2008) envisaged the use of Phyllostictine A
produced by a pathogen Phyllosticta cirsii Desm. as a natural herbicide against
Californian thistle [Cirsium arvense (L.) Scop.]. Phyllostictine A was proved to be a
promising natural herbicide against host and non host plant species. In another
investigation on the same fungus, four oxazatricycloalkenones, named phyllostictines A-
D, have been isolated, characterized and tested for herbicidal activity. Phyllostictine A
was proved highly phytotoxic against the weed C. arvense (Evidente et al. 2008).
Some plant pathogens have been found virulent enough to control weed species
and to compete commercially available synthetic chemical herbicides but most pathogens
are not sufficiently virulent to control weeds. However, this hindrance can be overcome.
As an example, there are certain amino acids that exhibit inhibitory effects on the growth
and development of certain plants. Pathogens or their mutants that can overproduce such
inhibitory amino acids can be selected. Such augmentation of biocontrol efficiency in
three separate pathogen-host systems, one with Pseudomonas and two with Fusarium has
already been reported (Sands and Pilgeram 2009). They outlined a stepwise approach that
can be followed to obtain enhanced weed control agents that would be capable of
producing inhibitory levels of selected amino acids in situ. Zhang et al. (2010) isolated
and identified the structure of herbicidal component, dimethyl o-phthalate from
phytopathogenic fungus Pythium aphanidermatum (Edson) Fitzp. When assayed on
weeds including hairy crabgrass [Digitaria sanguinalis (L.) Scop.] and A. retroflexus, the
ethyl acetate extract exhibited strongest herbicidal activity in terms of inhibition of seed
19
germination as well as seedling growth. Many species of Trichoderma are also known to
exhibit herbicidal activity. For example, Trichoderma harzianum Rifai, Trichoderma
pseudokoningii Rifai, Trichoderma reesei Simmons and Trichoderma viride Pers. have
been described to have herbicidal activity against P. minor and R. dentatus (Javaid and
Ali 2011). In order to harness maximum benefits of natural resources, total synthesis of a
number of phytotoxins have been accomplished (Nanda 2005; Leyva et al. 2008; Tanaka
et al. 2009; Selvam et al. 2009; Kamal et al. 2009).
1.4. Genus Drechslera
This Genus Drechslera is known mainly because it has a number of plant
pathogenic species and has done serious damages to crops in past. e.g. the Bengal
epiphytotic of 1942 was the most devastating plant disease in plant pathological history
(Padamadhan 1973). This epidemic was caused by Drechslera oryzae (Breda de Haan)
Subram. & B.L. Jain (Yun et al. 1988). Also in 1970, the corn crops of the Canada and
United States were severely destroyed by a corn-blight epidemic caused by the fungus
Drechslera maydis (Y. Nisik. & C. Miyake) Subram. & B.L. Jain. This epidemic incurred
the greatest crop loss in the shortest time span of any plant disease ever reported.
The genus Drechslera is well known for the production of secondary herbicidal
metabolites. Extensive research regarding isolation and purification of a number of novel
compounds having phytotoxic properties on host and non host species have been done in
past so far. A phytotoxic metabolite (─)-Dihydropyrenophorin was isolated from
Drechslera avenae (Eidam) Shoem. This toxin was found active against a number of
weed species including . A. fatua, Johnsongrass, [Sorghum halepense (L.) Pers] bermuda
grass (Cynodon dactylon Pers.) goosegrass (Eleucine indica Gaertn.), yellow foxtail
[Setaria glauca (L.), Beauv. ], and S. viridis (Sugawara and Strobel 1986). Similarly,
macrodiolide pyrenophorol (5,13-dihydroxy-8,16-dimethyl-1,9-dioxa-cyclohexadeca-
3,11-diene-2,10-dione), a metabolite isolated from D. avenae was found toxic to sterile
oat (Avena sterilis L.) and A. fatua when used at a concentration of 320 mM. Although
seed germination of A. sterilis was not affected but seedling cuttings which were partially
immersed in pyrenophorol solution showed leaf necrosis (Kastanias and Chrysayi-
Tokousbalides 2000). A similar herbicidal compound macrodiolide (8R,16R)-(-)-
20
pyrenophorin (8,16-dimethyl-1,9-dioxa-cyclohexadeca-3,11-diene2,5,10,13-tetraone) was
isolated by Kastanias and Chrysayi-Tokousbalides (2005) from culture of D. avenae. The
isolated compound inhibited seed germination of A. sativa, A. fatua and A. sterilis at a
concentration of 60 µM. The metabolite caused abnormal chlorophyll retention in leaf
sections of all the plant species tested. Also a phytotoxin named Tryptophol has been
identified from culture medium of Drechslera nodulosum Berk and curt. When tested,
this compound produced necrotic spots on leaves of goosegrass at a concentration of 6.2
x l0-4
M (Sugawara and Strobel 1987). Sugawara et al. (1987) isolated a series of
phytotoxic sesterterpenoids belonging to the ophiobolin family from culture filtrates of D.
maydis and Drechslera sorghicola (Lefebvre & Sherwin) M.J. Richardson & E.M.
Fraser. These ophiobolins were named as Ophiobolin I, Ophiobolin A, Ophiobolin C, 25-
Hydroxyophiobolin 1, 6-Epianhydroophiobolin A, 6-Epiophiobolin A. These ophiobolins
produced characteristic lesions on host plants. Culture filtrate and mycelia of D. maydis is
also known to produce phytotoxins named drechslerol-A [(cis) hentetracont-10-ene-12-
hydroxymethyl-4-ol], drechslerol-B [3-hydroxy-eicos-11(Z)-enyl eicos-4(Z)-enoate] and
Drechslerol-C [3-hydroxy-eicos-11(Z)-enylheptacos-11(Z)-enoate]. Drechslerol-A caused
necrotic lesion on the leaves of Wild ginger [Costus speciosus (Koenig) Smith] at 1.6 ×
10−4
M concentration. Drechslerol-C when applied with concentrations from 2.85 × 10−5
to 2.28 × 10−4
M produced characteristic necrotic and chlorotic lesions on the leaves of C.
speciosus (Shukla et al. 1987; 1989; 1990). Phytotoxic ophiobolins 6-Epiophiobolin A
and 3-anhydro-6-epiophiobolin A have also been isolated from D. maydis race T
(Canales and Gary 1988). A number of other phytotoxins from D. oryzae have also been
isolated and characterized as 6-epiophiobolin I, ophiobolin J and 8-deoxyophiobolin J
with the help of spectroscopic analyses and comparisons with already identified
ophiobolin I (Sugawara et al. 1988). Culture of Drechslera siccans (Drechsler)
Shoemaker is also reported to yield a phytotoxin named as 6,8-dihydroxy-3-(2’-
hydroxypropyl) isocoumarin (de-o-methyldiaporthin). Phyto-toxicity of this compound
has been estimated in terms of necrotic spot area when tested on A. sativa, Smooth
crabgrass [Digitaria ischaemum (Schreb.) Schreb. ex Muhl.], E. crus-galli, spiny
amaranth (Amaranthus spinosus L.), maize and soybean (Hallock et al. 1988). Triticone
A is reported to be synthesized by several fungi including Drechslera tritici- repentis
21
(Died.) Shoemaker. By undergoing racemization it forms triticone B and when tested, the
enantiomeric mixture evoke chlorosis and necrosis on a variety of plants including wild
oat (Kenfield et al. 1988). Same Drechslera species also synthesizes Triticones B, C, D,
E, and F along with Triticone A Amongst these Triticones, mixture of Triticone A and
Triticone B was found the most phytotoxic of the Triticones. Its biological activity was
assessed by observing yellowish-brown lesions by the leaf puncture method on weeds
including C. album and A. retroflexus (Hallock et al. 1993). Phytotoxic compounds
curvulin and O-methylcurvulinic acid were isolated from Drechslera indica (J.N. Rai,
Wadhwani & J.P. Tewari) Mouch. These toxins caused necroses on purslane (Portulaca
oleracea L.) and spiny amaranth (Kenfield et al. 1989). Bunkers and Strobel (1991)
proposed the mode of action of numerous phytotoxins belonging to the eremophilane
family from D. gigantea. They concluded that green island formation by these
eremophilanes proceeds through the inhibition of protein synthesis in detached oat leaves.
Although metabolites of a number of Drechslera species have been tested against
some problematic weed species, however, studies regarding the herbicidal activity of
Drechslera spp. from Pakistan are scarce. The present study was therefore, carried out to
investigate the herbicidal potential of culture filtrates of four Drechslera species from
Pakistan, namely, D. hawaiiensis, D. holmii, D. biseptata and D. australiensis against
some problematic weeds of wheat.
22
Objectives
The present research work was undertaken to seek nature friendly alternatives to
synthetic herbicides from culture filtrates of Drechslera spp. for the management of some
noxious weeds of wheat. To achieve this goal the present study was aimed:
To evaluate the in vitro and in vivo herbicidal activity of culture filtrates of four
Drechslera spp. against four noxious weeds of wheat and different wheat
varieties.
To investigate the herbicidal activity of culture filtrates Drechslera spp. against
the most susceptible weed species under field conditions.
To isolate the herbicidal compounds from the most active Drechslera species
through bioactivity guided bioassays using various chromatographic techniques.
To elucidate the structures of active herbicidal compounds through various
spectroscopic techniques.
23
Chapter 2
Materials and Methods
2.1. Selection of Test Fungal Isolates
Four species of Drechslera viz. D. australiensis, D. biseptata, D. hawaiiensis and
D. holmii, were procured from Fungal Culture Bank of Pakistan, Institute of Agricultural
Sciences, University of the Punjab, Lahore, Pakistan.
2.2. Single Spore Isolation
For the purpose of single spore isolation of the test fungi, 1 mL of autoclaved
water was poured into a vial containing the fungal culture. The vial containing fungal
culture was shaken vigorously for 1 minute and water containing the spores was poured
into 9 mL of autoclaved water to give 10-1
dilution. This procedure was repeated twice
get a 10-3
dilution. One milliliter suspension was taken from this dilution and poured on
autoclaved malt extract agar medium in 9-cm diameter Petri dish. There were three
replicates of this treatment. Sterilized glass spreader was used to spread the spores evenly
on the surface of medium in Petri plate. These plates were incubated for three days at
28±2 °C in an incubator to allow the spores to germinate until the colonies were visible.
Each colony free from contamination was removed with the help of sterilized fine needle
and transferred to another Petri plate containing malt extract agar medium. These plates
incubated at 28±2 °C for 15 days in an incubator for an appreciable conidial and mycelial
formation. After this, pure fungal cultures were confirmed and stored at 4 C.
2.3. Selection of Weeds of Wheat
Four frequently occurring and problematic weeds of wheat viz. Avena fatua L.,
Phalaris minor Retz. (Monocot.), Chenopodium album L., Rumex dentatus L. (Dicot.)
were chosen for this study. Seeds of the test weed species were collected from wheat
24
fields of University of the Punjab Lahore, Pakistan, at the end of growing season of
wheat in May 2009. These seeds were sun dried, cleaned and stored at room temperature.
2.4. Selection of Test Wheat Varieties
Three commonly cultivated wheat varieties in Punjab, Pakistan namely Inqlab 91,
Sehar 2006 and Uqab 2000 were selected to evaluate their germination and growth
response to culture filtrates of the test Drechslera species. Certified seeds of these
varieties were procured from Federal Seed Certification Department, Lahore, Pakistan.
2.5. Preparation of Culture Filtrates of the Test Fungi
Minimal medium (M-1-D) was prepared in distilled water as described by
Evidente et al. (2006b). This medium consisted of 1.2 mM Ca(NO3)2, 0.79 mM KNO3,
0.87 mM KCI, 3.0 mM MgSO4, 0.14 mM NaH2PO4, 87.6 mM sucrose, 27.1 mM
ammonium tartrate, 7.4 µM FeC13, 30 µM MnSO4, 8.7 µM ZnSO4, 22 µM H3BO3 and
4.5 µM KI. The pH was adjusted to 5.5 with 0.1 M HCl. Medium was poured into 500
mL conical flasks at 200 mL medium in each flask. Flasks were autoclaved at 121°C and
103425 P cm-2
pressure for 20 minutes and cooled to room temperature. Flasks were
individually inoculated with 5 mm agar discs of each of the four test fungal species from
the margins of actively growing fungal colonies. Inoculated flasks were incubated at
252 ºC in an incubator for 28 days. Cultures were filtered through four layers of muslin
cloth, centrifuged at 4000 rpm for ten minutes followed by filtration through sterilized
Whatman filter paper No. 1. These filtrates were stored at 4 ºC in a refrigerator. Sterilized
distilled water was added to the original filtrates (100%) to prepare dilution of 50%
(Javaid and Adrees 2009). Filtrates were generally used within a week to avoid any
contamination or chemical alteration.
2.6. Laboratory Screening Bioassays
The effect of original and diluted culture filtrates of the four selected fungal
species was evaluated on germination and early seedling growth of the test weed species
as well as against three selected wheat varieties. Seeds of weeds and wheat varieties were
25
surface sterilized with 1% sodium hypochlorite for 10 minutes. Twenty seeds of each of
the test weed species and three wheat variety were placed at equal distance in sterilized 9
cm diameter Petri dishes lined with sterilized filter papers, moistened with 3 mL of the
two concentrations of fungal culture filtrates. Treatments in a similar manner with M-1-D
medium (Original and 50% diluted) served as positive control, whereas treatment with
distilled water served as negative control. All tests were performed in quadruplicate. Petri
dishes were arranged in a completely randomized design (CRD) in a growth room
maintained at 16 C with 10 h light period daily. Data regarding germination of seeds
were recorded after 15 days. Plants were thinned and 10 uniform seedlings were selected
for measurement of different root and shoot growth parameters (Fig. 1). Materials were
dried at 60 °C in an electrical oven till constant weight (Javaid and Ali, 2011).
2.7. Foliar Spray Bioassays
Pot experiments were conducted during November-December 2009 in University
of the Punjab, Lahore, Pakistan, located on latitude 31.57 N and longitude 74.31 E.
Plastic pots of 8-cm diameter and 12-cm deep were filled with 450 g sandy loam soil
having organic matter 0.69%, pH 7.8, available phosphorus and potassium 6.3 mg kg-1
and 100 mg kg-1
respectively, with nitrogen content 350 mg kg-1
. The micronutrient Zn,
Mn, Cu, B and Fe were 1.3, 22.8, 1.9, 1.06 and 10.8 mg kg-1
respectively. NPK fertilizers
were used in each pot. Ten seeds of each weed species as well as each wheat variety were
sown in each pot. After germination, pots were arranged in two sets to perform the foliar
spray on 1-week and 2-week old seedlings. Each treatment was replicated four times. All
the pots were arranged in a completely randomized design in open under natural
environmental conditions.
Original culture filtrates of the four selected Drechslera species were sprayed on
1-week and 2-week old test weeds and wheat seedlings. Both of the sets were sprayed 4
times with an interval of four days. Treatment in a similar manner with distilled water
spray served as negative control whereas M-1-D medium without fungal inoculation was
used as positive control. All the sprays were carried out during evening hours. Plants
were harvested after 50-days growth. Plants were carefully uprooted and washed
thoroughly under tap water to remove soil. Moisture from plant surface was evaporated
26
under fan at room temperature. Parameters regarding length and fresh and dry biomass of
root and shoot were recorded (Javaid et al. 2011b).
2.8. Field Trials
2.8.1. Field Preparation
In laboratory bioassays and pot trials, R. dentatus was found the most susceptible
to various fungal culture filtrates. To evaluate the effect of culture filtrates of the four
Drechslera spp. against R. dentatus under field conditions, field trial was carried out
during the wheat growing season of 2009–2010 at Dhing Shah District Qasur, (30° 56' N
and 74° 13' E), 80 kilometers from Lahore, Pakistan.
All the recommended agronomic practices right from preparation of field till
harvesting were employed. A composite soil sample of the experimental field was taken
before launching of the experiment. Soil was got analyzed from soil and water testing
laboratory for research, Lahore, Pakistan. Soil was sandy loam in texture having available
potassium (43 mg kg-1
), ECmScm-1
(2.4), pH (7.7), organic matter (0.84%) and available
phosphorous (4.5 mg kg-1
). Experiment was laid out in randomized complete block
design (RCBD) with three replications. Each plot measured 1.54×1.54 m2. Fertilizers
were applied as recommended by the Punjab Agriculture Department, Pakistan, for
wheat. Nitrogen (N) was applied at 160 kg ha-1
as urea, P2O5 at 110 kg ha-1
as single
super phosphate and K2O at 60 kg ha-1
as sulphate of potash. Full doses of P2O5 and K2O,
and a half-dose of N were applied as basal, while half the N was top-dressed at flowering
stage.
2.8.2. Sowing of Seeds
Since all the three wheat varieties showed resistance to various fungal culture
filtrates, therefore, only one wheat variety Sehar 2006 was selected for field trials. Three
wheat seeds per hill were sown at 22 cm inter and intra row spacing accommodating
seven rows in each plot with 7 plants per row. After germination, thinning was carried
out at the stage of full emergence of first leaf to maintain only one wheat seedling at one
place. First irrigation was carried out twenty days after sowing and subsequent irrigations
27
were carried out according to the requirement of the crop. Over all six irrigations with
tube well water were carried out as dry spell was observed through out the course of
present study. Seeds of R. dentatus were sown in the field at the time first water. Weed
species belonging to different plant genera along with R. dentatus emerged in the field
after first irrigation. All weed species except R. dentatus were removed manually and 1:1
ratio of R. dentatus and wheat plants was maintained.
2.8.3. Treatments and Experimental Layout
The following twelve treatments were tried in the field trial:
T1 Weed free control.
T2 Weedy check.
T3 Application of culture filtrate of D. hawaiiensis.
T4 Application of culture filtrate of D. holmii.
T5 Application of culture filtrate of D. biseptata.
T6 Application of culture filtrate of D. australiensis.
T7 Bromoxynil + MCPA (recommended dose).
T8 Bromoxynil + MCPA (half dose).
T9 Culture filtrate of D. hawaiiensis + half dose of Bromoxynil + MCPA.
T10 Culture filtrate of D. holmii + half dose of Bromoxynil + MCPA.
T11 Culture filtrate of D. biseptata + half dose of Bromoxynil + MCPA.
T12 Culture filtrate of D. australiensis + half dose of Bromoxynil + MCPA.
2.8.4. Schedule of Foliar Sprays
A total of three sprays were carried out with fungal culture filtrates. First spray was
carried out when R. dentatus was at three to four leaves stage. Two successive sprays
with fungal culture filtrates were carried out with intervals of 7 days. Culture filtrates
were sprayed at 100 L ha-1
. Only one spray of synthetic herbicide Bromoxynil + MCPA
200/200EC, either alone or mixed with fungal culture filtrates was carried out with
Knapsack hand sprayer with 4T-jet nozzle. Both the recommended and half dose of the
28
herbicide was used where it was used alone. However, in combination with fungal culture
filtrates, only half dose of the herbicide was used.
2.8.5. Harvesting and Data Collection
The crop was harvested at maturity i.e. 150 days after sowing. Data for plant
height, weight, number of fertile tillers per plant, total seed weight, hundred seeds weight,
and biomass of weed plants were recorded. Wheat and weed biomass were measured
after placing plant materials in an electric oven at 60 °C to constant weight.
2.9. Statistical Analysis
All the data from laboratory screening and foliar spray bioassays as well as field
trials were subjected to analysis of variance (ANOVA) followed by Duncan’s Multiple
Range Test to delineate the treatment means using computer software COSTAT.
2.10. Organic Solvent Extraction
In the previous laboratory, pot and field trials, culture filtrate of D. australiensis
were found to be the most efficient in controlling growth of R. dentatus. This species was
thus selected for isolation and identification of active herbicidal constituents from its
culture filtrate. This fungus was grown in M-1-D medium in 500 mL conical flasks as
described in section 2.5. A total of 4 L of crude fungal culture filtrate of this were
collected and evaporated to yield 1.5 L concentrated filtrate. Four organic solvents viz. n-
hexane (C6H14), chloroform (CHCl3), ethyl acetate (C4H8O2) and n-butanol (C4H10O)
were successively used for extraction. These organic solvents were used in order of their
increasing polarity. First a volume of 300 mL of n-hexane was added to 300 mL crude
concentrated fungal culture filtrate in a separating funnel, shaken well and kept stationery
until the two phases got separated. The upper n-hexane layer was separated and the
process was repeated until all n-hexane compounds were separated from the aqueous
filtrates. Similarly, the rest of 1200 mL culture filtrates were treated with n-hexane. This
n-hexane phase was concentrated under vacuum in a rotary evaporator to yield crude n-
29
hexane fraction. The aqueous phase was then extracted similarly with chloroform, ethyl
acetate and n-butanol, yielding crude fractions.
2.11. Leaf Discs Bioassays with Crude Organic Fractions
Bioassays with crude organic fractions were carried out following procedure
described by Mahoney et al. (2003), with some modifications. R. dentatus seeds were
grown in plastic pots under natural environmental conditions. Young leaves from 30 days
old plants were detached and discs of 1-cm diameter were cut with the help of a cork
borer. The leaf discs were placed on glass slide and punctured with the help of a fine
needle. The glass slides were placed on a filter paper wetted with 2 mL of sterilized
distilled water in a Petri plate. Four milligrams of each crude fraction viz. n-hexane,
chloroform, ethyl acetate and n-butanol were dissolved in 100 µL of dimethylsulfoxide
(DMSO). Final volume of each fraction was raised to 1.0 mL with distilled water to
prepare a stock solution of 4 mg mL-1
concentration. The stock solution was serially
double diluted by adding distilled water to prepare lower concentrations of 2, 1, …,
0.0625 mg mL-1
. Droplets of 15 µL of each of the seven concentrations were applied on
the punctured leaf surface. Ten leaf discs of the test weed species were used for each
concentration. Positive control received DMSO at 100 µL mL-1
of distilled water at
highest concentration and subsequent lower concentrations were made by double diluting
it with distilled water. Treatment with distilled water alone served as negative control.
Treatments in a similar manner but with un-punctured leaf surface were also made. These
Petri plates were incubated at 25 °C under continuous fluorescent light in growth room.
Symptoms regarding appearance of necrotic spot and discolouration of leaf discs were
observed after 72 hours. Colour scale 0-3 and necrotic spot scale 4-10 was used for
comparisons (Plate 1).
30
Plate 1: Scheme for leaf discs bioassays using crude organic fractions.
water
Concentration of
Crude fraction
(mg mL-1
) Concentration of
DMSO (µl mL-1
)
0.0625
0.1250
0.2500
0.5000
4.0000
2.0000
1.0000
3.125
6.25
12
25
50
100
1.562
0.781
31
2.12. Isolation of Compounds through Chromatographic Techniques
Crude chloroform and ethyl acetate fractions of culture filtrate of D. australiensis
showing pronounced herbicidal activity were selected for Thin Layer chromatography
(TLC) analysis followed by separation through Preparative Thin Layer Chromatography
(PTLC) and Reversed Phase High Performance Liquid Chromatography (RPHPLC).
2.12.1. Thin Layer Chromatography
A thin strip of aluminum foil backed TLC (6.5 cm long and 1.5 cm wide) was cut
with a scissor. A base line was drawn near the bottom of the plate with lead pencil to
show the original position of the compound. Five milligrams of crude chloroform fraction
was taken in an eppendorf tube and was dissolved in 1 mL of methanol. A small drop of
solution was placed on the center of baseline with the help of capillary jet and allowed to
dry for few minutes. Solvent system or mobile phase was prepared in a glass jar. Ten
milliliter of solvent (chloroform, ethyl acetate and n-hexane) in 10:6:84 ratio was poured
into a glass jar to a depth of 0.9 cm. TLC strip was placed in the solvent so that its bottom
touched the solvent and solvent level remained below the baseline with the spot on it. The
container was closed with a lid and was left for a few minutes to let the solvent elute the
mixture of compounds spotted on chromatogram strip. When the solvent front moved to
about 1 cm below the upper end of the strip, the plate was removed and dried. Spot was
located under UV transilluminator, both at short and long wavelength as well as
visualized by spraying ceric sulphate solution accompanied by heating with heat gun. The
Retention factor (Rf) value for each spot was calculated using the formula:
Distance traveled by component
Rf =
Distance traveled by the solvent
Six fractions namely A (Rf 0.096), B (Rf 0.130), C (Rf 0.170), D (Rf 0.269), E (Rf
0.480) and F (Rf 0.576) were isolated from chloroform fraction of culture filtrate of D.
australiensis. Similarly, three fractions namely G (Rf 0.054), H (Rf 0.345) and I (Rf
0.618) were separated from ethyl acetate fraction of culture filtrate of the same fungal
32
species using solvent system n-hexane and ethyl acetate in a ratio of 3:7. The isolated
fractions were further purified by Preparative Thin Layer Chromatography (PTLC).
2.12.2. Preparative Thin Layer Chromatography
For preparative thick layer chromatography pre-coated silica gel GF-254
preparative plates (20 × 20 cm, 0.5 mm thick, E-Merck) were used. Solvent system was
same as in TLC. When developed with the solvent, the compounds separated in
horizontal bands. These bands were scraped from the plates and eluted with methanol.
Soluble compounds were carefully collected separately in another vial through filtration,
and were evaporated at 40 C to dryness and weighed.
2.12.3. Reversed Phase High Performance Liquid Chromatography
Compounds separated through Preparative Thin Layer Chromatography were
further subjected to Reversed Phase High Performance Liquid Chromatography. For
elution Acetonitrile (HPLC Grade) with 0.1% Formic acid added and Deionized Water
(DI water) with 0.1% Formic acid was used as solvent system. Gradient elution was
employed with initial ratio of Acetonitrile and Deionized (DI) water as 10:90 with an
increasing ratio of Acetonitrile to water as 100:0. Fractions containing purified
compounds were collected in glass vials and solvent was evaporated under continuous
currents of clean air at room temperature. The work was done at Department of
Chemistry & Biochemistry, University of California, Santa Cruz, USA.
2.13. Spectroscopic Analyses
Structural elucidation by 1D & 2D NMR techniques of only the most active
compounds was carried out. Less bioactive compounds were not subjected to mass
spectroscopy because their bioactivity was far low as compared to synthetic herbicide in
use.
Proton nuclear magnetic resonance (1H-NMR) spectra were recorded in CD3OD
using TMS as internal standard at 600 MHz and 500 MHz on Bruker AM-300, AM-600
33
and AM-500 nuclear magnetic resonance spectrometers. The 13
C-NMR spectra were
recorded in CD3OD at or 125 MHz on the same instruments.
EIMS spectra were recorded on a Finnigan MAT 311 with MASSPEC data
system.
2.14. Leaf Discs Bioassays with Purified Chromatographic Fractions
Bioassays with purified chromatographic fractions were generally carried out
using the same method as was adopted in case of crude organic fractions section 2.11,
except that these bioassays were only performed with punctured leaf disks. Stock
solutions of 2 mg mL-1
of various purified organic constituents were prepared by
dissolving 2 mg of the compound in 50 µL of DMSO and raised the volume to 1.0 mL by
adding distilled water. Lower concentrations of 1, 0.5, …, 0.03125 mg mL-1
were
prepared by serially double diluting the stock solutions. In total seven concentrations
were made with distilled water viz. 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 mg mL-1
.
For positive control 50 µL DMSO was dissolved in distilled water to make the final
volume 1.0 mL. Lower concentrations were made by double diluting this mixture with
distilled water to prepare corresponding positive control treatments of DMSO for various
concentrations of the purified compounds. A positive control using 2,4-D (2, 4-
dichlorophenoxyacetic acid) was also included in these bioassays to compare the efficacy
of isolated compounds. Treatment with distilled water alone served as negative control.
Symptoms regarding appearance of necrotic spot and discoloration of leaf discs were
observed after 72 hours.
34
Chapter 3
Results
3.1. Laboratory Bioassays
3.1.1. Effect of Fungal Culture Filtrates on Germination and Growth of C. album
3.1.1.1. Effect on Germination
Original (100%) growth medium significantly reduced germination by 12% over
control. However, the effect of diluted (50%) growth medium was nonsignificant. The
effect of various fungal culture filtrates on germination was variable. In general, all the
culture filtrates significantly suppressed the germination by 28–50% over control. The
effect of filtrates of D. australiensis and D. hawaiiensis was more pronounced as
compared to filtrates of other two fungal species (Table 1, Plate 2).
3.1.1.2. Effect on Shoot Growth
Highest length, fresh weight and dry weight of shoot was recorded in control.
Original and diluted growth medium significantly reduced shoot length by 17% and 13%,
shoot fresh weight by 31% and 25%, and shoot dry weight by 21% and 16%, over
control, respectively. In general, culture filtrates of all the four Drechslera spp.
significantly suppressed plant growth. However, a marked variation in herbicidal activity
of culture filtrates of different fungal species was evident. Culture filtrates of D.
hawaiiensis were found the most effective followed by those of D. australiensis in
reducing shoot length and biomass of C. album. There was 91% and 84% reduction in
shoot length, and 84% and 68% reduction in shoot dry weight due to original culture
filtrates of D. hawaiiensis and D. australiensis, respectively, as compared to control.
Culture filtrates of the other two fungal species exhibited comparatively less herbicidal
activity against shoot growth of the test weed species. There was 55% and 54%
reduction in shoot length and 58% decline in shoot dry weight due to original culture
filtrates of D. biseptata and D. holmii as compared to control (Table 1, Plate 2).
35
3.1.1.3. Effect on Root Growth
Growth medium exhibited variable effect on length and weight of root. Root
length was significantly reduced by 16% and 12% due to original and diluted culture
medium. Similarly, the adverse effect of original filtrates was significant on root fresh
weight. By contrast, none of the two concentrations of growth medium had significant
effect on root dry weight. Root growth exhibited high susceptibility to the application of
culture filtrates of the four Drechslera species. Root length and dry biomass were
significantly reduced by 66–88% and 56–65% due to original culture filtrates of different
fungal species as compared to control. Original filtrates of D. hawaiiensis and D.
australiensis were found the most effective in reducing length and biomass of C. album
roots (Table 1, Plate 2).
3.1.2. Effect of Fungal Culture Filtrates on Germination and Growth of R. dentatus
3.1.2.1. Effect on Germination
The effect of M-1-D broth was nonsignificant on germination of test weed
species. Culture filtrates of all the four significantly reduced germination to variable
extents. The highest herbicidal activity was shown by filtrate of D. biseptata (up to 56%
reduction) followed by those of D. australiensis, D. hawaiiensis and D. holmii,
respectively. The original culture filtrates of other Drechslera spp. significantly reduced
germination by 12–49% (Table 2, Plate 3).
3.1.2.2. Effect on Shoot Growth
The effect of original growth medium was found significant on shoot growth
resulting in 15% in each of shoot length and shoot dry weight, respectively. The effect of
all the culture filtrate treatments except 50% D. holmii was significant as compared to
control. Original filtrates of D. australiensis were found to be the most effective in
suppressing shoot length and shoot dry biomass of R. dentatus by 85% and 88%,
respectively. Similarly, D. biseptata resulted in 81% and 83% decline in shoot length
and dry biomass of R. dentatus, Generally, culture filtrates of D. hawaiiensis and D.
36
holmii proved less toxic to R. dentatus growth as these two species caused 67% and 68%,
and 73% and 72% reduction in shoot length and shoot dry weight over control,
respectively (Table 2, Plate 3).
3.1.2.3. Effect on Root Growth
In general, root growth exhibited slightly more susceptibility to the application of
culture filtrates of various Drechslera species as root length and biomass were
significantly reduced by 69–94% and 63–88%, respectively. Filtrates of D. australiensis
were found the most effective in inhibiting various root growth parameters of R. dentatus.
This species incurred 94% and 88% reduction in root length and root dry weight
respectively (Table 2, Plate 3).
3.1.3. Effect of Fungal Culture Filtrates on Germination and Growth of P. minor
3.1.3.1. Effect on Germination
The effect of both original as well as diluted growth medium was nonsignificant
on seed germination. Both original and diluted culture filtrates of of all the four test
fungal species significantly reduced germination by 35–93%. The adverse effect of
original culture filtrates on germination was more pronounced as compared to diluted
ones. Original culture filtrates of D. australiensis incurred drastic effect inhibiting
germination of P. minor seeds by 93%. Culture filtrates of other three fungal species
proved less toxic to seed germination as compared to filtrates of D. australiensis (Table
3, Plate 4).
3.1.3.2. Effect on Shoot Growth
The effect of the original growth medium was significant on length as well as
biomass of shoot of P. minor seedlings as there was 19% and 23% decline in shoot length
and shoot dry weight, respectively. However, the adverse effect of the fungal culture
filtrates was much higher than the effect of growth medium. All the culture filtrates
either used in original or diluted form significantly reduced various shoot growth
37
parameters as compared to control as well as growth medium treatments. Filtrates of D.
hawaiiensis and D. australiensis incurred 65% and 60% decline in shoot length, and 64%
and 45% reduction in shoot dry weight of P. minor seedlings over control, respectively
(Table 3, Plate 4).
3.1.3.3. Effect on Root Growth
The effect of the original growth medium was significant on length as well as
biomass of root. There was 36% and 32% reduction in root length and root dry weight
due to original M-1-D broth. Root length as well as root weight were significantly
suppressed by culture filtrates of all the four Drechslera species. There was 81–90%
reduction in root length and 59–81% reduction in root dry weight of P. minor due to
different concentrations of the various culture filtrates as compared to control. Culture
filtrate of D. australiensis exhibited the highest phytotoxic activity inhibiting root length
and root dry weight of P. minor up to 90% and 81% respectively. The difference in root
fresh and dry weight was nonsignificant among the original concentrations of various
fungal culture filtrate treatments (Table 3, Plate 4).
3.1.4. Effect of Fungal Culture Filtrates on Germination and Growth of A. fatua
3.1.4.1. Effect on Germination
The effect of growth medium on germination was significant. There was 14% and
12% reduction in germination due to 100% and 50% M-1-D broth. Different culture
filtrate treatments significantly reduced the germination of A. fatua seeds by 28–54%.
Inhibitory effect of original culture filtrates of D. hawaiiensis and D. australiensis was
highest and comparable to each other (Table 4, Plate 5).
3.1.4.2. Effect on Shoot Growth
The effect of growth medium on shoot length of A. fatua was found significant as
reduction of 16% was observed in shoot length. However the effect of growth medium
was nonsignificant on shoot dry weight of A. fatua. Although all the culture filtrate
38
treatments except 50% D. biseptata significantly reduced shoot growth of A. fatua in
terms of length and biomass but adverse effects were more pronounced in case of D.
holmii where culture filtrate of this fungus consistently reduced shoot length as well as
shoot fresh and shoot dry weight by 67%, 59% and 57%, respectively. There was 27–
67% and 27–57% reduction in shoot length and shoot dry biomass due to various culture
filtrate treatments (Table 4, Plate 5).
3.1.4.3. Effect on Root Growth
The effect of growth medium on root length and biomass of A. fatua was found
significant. There was 18% reduction in length and dry biomass each due to original
growth medium. Culture filtrates of all the test fungi were found effective in arresting
various root growth parameters at both 100% as well as 50% concentration. There was
55–86% and 47–77% reduction in root length and root biomass due to various culture
filtrate treatments, respectively. The most noticeable effect was due to filtrate of D.
hawaiiensis causing 86%, 82% and 77% inhibition in length, fresh and root dry weight of
root, respectively. Second most important Drechslera species was D. holmii causing
81%, 82% and 74% inhibition in root length, root fresh weight and root dry weight,
respectively (Table 4, Plate 5).
3.1.5. Effect of Fungal Culture Filtrates on Germination and Growth of Wheat
3.1.5.1. Wheat var. Inqlab 91
3.1.5.1.1. Effect on Germination
The effect of original as well as diluted growth medium was nonsignificant on
germination of wheat var. Inqlab 91. Original culture filtrates of various Drechslera spp.
significantly reduced germination by 7-12%, whereas the effect of 50% concentration
was nonsignificant (Table 5, Plate 6).
39
3.1.5.1.2. Effect on Shoot Growth
The original growth medium significantly reduced shoot length by 17% whereas
its effect on shoot dry weight was nonsignificant. All the culture filtrates significantly
reduced various shoot growth parameters. There was 26–38%, 14–39% and 28–46%
reduction in shoot length, and fresh and dry weight over control, respectively. The
adverse effect of D. holmii was more pronounced than the effect of rest of the fungal
species (Table 5, Plate 6).
3.1.5.1.3. Effect on Root Growth
The effect of original growth medium was significant on length and biomass of
root. There was 14% and 17% reduction in length and dry biomass of root over control,
respectively. Root length and dry biomass were significantly reduced by 26–70% and 34–
56%, respectively, due to different fungal culture filtrate treatments. The highest adverse
effect on various root growth parameters was recorded due to culture filtrate of D.
hawaiiensis while filtrate of D. biseptata was found least toxic (Table 5, Plate 6).
3.1.5.2. Wheat var. Sehar 2006
3.1.5.2.1. Effect on Germination
The effect of original as well as diluted growth medium on germination was
nonsignificant as compared to negative control. Original culture filtrates of all the four
fungal species significantly reduced germination by 11–18%. The effect of culture
filtrates of D. hawaiiensis and D. holmii was more pronounced than the culture filtrates of
D. biseptata and D. australiensis (Table 6, Plate 7).
3.1.5.2.2. Effect on Shoot Growth
Growth medium exhibited variable effects on length and biomass of shoot. Shoot
length was significantly reduced by 14% and 10% due to original and diluted growth
medium, respectively, while there was a nonsignificant effect of growth medium on shoot
dry weight. D. hawaiiensis and D. holmii exhibited greatest reduction in shoot dry
40
biomass resulting in up to 24% and 22% reduction, respectively. Diluted culture filtrates
of D. australiensis, D. biseptata and D. hawaiiensis failed to induce any noteworthy
effect on shoot length, shoot fresh weight as well as shoot dry weight (Table 6, Plate 7).
3.1.5.2.3. Effect on Root Growth
The effect of original growth medium on root length as well as root dry biomass
was significant that resulted in 18% and 11% decline, respectively. Root growth was
found to be more susceptible to treatments of growth medium as well as culture filtrates
than shoot growth. The effect of all the culture filtrate treatments on root dry weight was
significant. D. holmii, D. biseptata and D. australiensis caused greatest suppression in
root dry biomass resulting in 33% reduction each, while D. hawaiensis was proved to be
the least effective one causing 30% reduction in root dry biomass (Table 6, Plate 7).
3.1.5.3. Wheat var. Uqab 2000
3.1.5.3.1. Effect on Germination
The effect of growth medium on the germination of test wheat variety was
nonsignificant when compared with control. The effect of 100% culture filtrates of D.
hawaiiensis and D. holmii was much pronounced on germination resulting in 17% and
15% reduction in germination, respectively. The effect of 50% culture filtrates of all the
test fungal species was comparable to that of original growth medium (Table 7, Plate 8).
3.1.5.3.2. Effect on Shoot Growth
Growth medium showed significant effect on shoot length at both original and
diluted concentrations causing 18% and 14% reduction respectively. All the fungal
culture treatments affected shoot length at both the concentration. The most effective
Drechslera species in this regard was D. holmii that caused 43% reduction in shoot
length when 100% culture filtrate was used. The least effective Drechslera spp. remained
D. biseptata that caused 31% decline in shoot length. In case of shoot biomass, all the
fungal culture filtrates caused deleterious effects both at 100 as well as 50%
41
concentration except 50% concentration of D. australiensis where effect on root dry
weight was similar to original growth medium (Table 7, Plate 8).
3.1.5.3.3. Effect on Root Growth
Growth medium showed significant effect on various root growth parameters at
original concentration causing 17% and 16% reduction in root length and root dry weight,
respectively. Root growth exhibited susceptibility to all the treatments of fungal culture
filtrates, D. hawaiiensis being prominent resulting in 78%, 61% and 58% reduction
followed by D. australiensis resulting in 67%, 47% and 37% reduction in root length,
root fresh weight and root dry biomass, respectively. The least effective fungal species
was D. biseptata causing 38% inhibition in root length and 33% reduction in root dry
weight (Table 7, Plate 8).
42
Table 1: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Chenopodium album in laboratory
bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 16.5 a 1.40 a 0.19 a 10.40 a 0.19 a 0.087 a
Growth medium 50 93 ab 14.4 b 1.05 b 0.16 b 9.15 b 0.18 a 0.090 a
100 88 bc 13.7 c 0.97 bc 0.15 b 8.75 b 0.15 b 0.080 a
D. hawaiiensis 50 66 e 1.9 h 0.37ef 0.04 gh 1.40 fg 0.09 de 0.040 c
100 50 f 1.5 h 0.29 f 0.03 h 1.23 g 0.08 e 0.030 c
D. holmii 50 81 cd 12.4 d 0.83 cd 0.10 cd 7.90 c 0.12 c 0.054 b
100 72 de 7.6 f 0.72 d 0.08 de 3.57 d 0.10 d 0.038 c
D. biseptata 50 82 bc 10.3 e 0.77 d 0.10 c 7.70 c 0.12 c 0.063 b
100 72 de 7.5 f 0.71 d 0.08 c-e 2.35 e 0.10 d 0.037 c
D. australiensis 50 56 f 3.3 g 0.54 e 0.07 ef 2.42 e 0.10 d 0.036 c
100 50 f 2.7 g 0.44 ef 0.06 fg 1.71 f 0.09 de 0.033 c
Table 2: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Rumex dentatus in laboratory bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 19.0 a 5.4 a 1.20 a 18.8 a 0.87 a 0.155 a
Growth medium 50 97 a 17.7 a 4.9 ab 1.15 ab 18.3 a 0.78 a 0.140 b
100 95 a 16.2 b 4.5 b 1.02 b 16.1 b 0.77 a 0.145 ab
D. hawaiiensis 50 59 c 8.4 c 2.1 c 0.60 c 8.5 c 0.38 b 0.081 c
100 51 d 6.2 d-f 1.5 cd 0.38 de 5.9 d 0.29 bc 0.057 d
D. holmii 50 95 a 7.1 cd 1.7 cd 0.47 d 5.6 d 0.25 b-d 0.050 de
100 88 b 5.2 ef 1.2 de 0.34 d-f 3.4 e 0.36 b 0.035 ef
D. biseptata 50 48 de 4.9 f 1.1 de 0.27 e-g 2.7 ef 0.13 cd 0.034 ef
100 44 e 3.6 g 0.7 e 0.21 fg 1.8 fg 0.08 d 0.019 g
D. australiensis 50 59 c 6.5 de 1.1 de 0.28 e-g 3.1 e 0.15 cd 0.032 fg
100 60 c 2.9 g 0.7 e 0.14 g 1.2 g 0.08 d 0.019 g
In a column, values with different letters show significant difference (P≤0.05) as
determined by Duncan’s Multiple Range Test.
43
Plate 2: Effect of culture filtrates of four Drechslera species on germination and
growth of C. album in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
D. hawaiiensis
1
2 3
4 5
1
2 3
4 5
D. holmii
D. biseptata
1
5
3
1
5
2 3
4 5
D. australiensis
A
C D
B
2 3
4
44
Plate 3: Effect of culture filtrates of four Drechslera species on germination and
growth of R. dentatus in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
D. hawaiiensis
1
2 3
4 5
D. holmii
1
2 3
4 5
D. biseptata
1
2 3
4 5
D. australiensis
1
2 3
4 5
A B
C D
45
Table 3: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Phalaris minor in laboratory bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 52 a 5.17 a 0.53 a 58 a 5.31 a 0.78 a
Growth medium 50 96 a 44 b 4.70 ab 0.45 b 44 b 4.31 b 0.63 b
100 96 a 42 b 4.35 b 0.41 bc 37 c 3.72 c 0.53 c
D. hawaiiensis 50 81 c 26 e 2.65 d 0.27 d 25 d 2.27 d 0.35 d
100 56 e 18 h 1.90 e 0.19 e 10 f 0.88 g 0.18 f
D. holmii 50 88 b 29 d 3.45 c 0.39 c 19 e 1.90 de 0.35 d
100 56 e 25 ef 2.62 d 0.29 d 11 f 0.96 g 0.19 f
D. biseptata 50 77 c 35 c 3.05 cd 0.38 c 12 f 1.40 f 0.28 e
100 65 d 23 fg 2.62 d 0.29 d 11 f 0.86 g 0.17 f
D. australiensis 50 13 f 21 g 3.62 c 0.37 c 16 e 1.50 ef 0.34 d
100 6 g 21 g 2.75 d 0.29 d 6 g 0.65 g 0.15 f
Table 4: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of Avena fatua in laboratory bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 100 a 59 a 4.7 a 140 a 45 a 6.8 a
Growth medium 50 88 b 87 b 53 b 4.5 a 120 b 41 b 6.0 b
100 84 b 84 b 49 c 4.3 ab 115 c 40 b 5.6 c
D. hawaiiensis 50 70 cd 51 g 34 f 3.1 d 54 g 19 e 3.4 f
100 46 e 41 h 29 g 2.5 ef 20 j 8 f 1.6 g
D. holmii 50 76 c 59 f 37 e 3.2 d 50 g 17 e 3.3 f
100 71 c 33 i 24 h 2.0 f 27 i 8 f 1.8 g
D. biseptata 50 71 c 80 c 41 d 3.9 bc 86 d 29 c 4.1 d
100 64 d 73 d 38 e 3.4 cd 62 f 21 e 3.5 f
D. australiensis 50 70 cd 67 e 37 e 3.5 cd 67 e 26 cd 4.0 de
100 52 e 48 g 31 g 2.6 e 35 h 23 d 3.6 ef
In a column, values with different letters show significant difference (P≤0.05) as
determined by Duncan’s Multiple Range Test.
46
Plate 4: Effect of culture filtrates of four Drechslera species on germination and
growth of P. minor in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
D. hawaiiensis D. holmii
D. biseptata D. australiensis
1
47
Plate 5: Effect of culture filtrates of four Drechslera species on germination and
growth of A. fatua in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
D. hawaiiensis
1
2 3
4 5
D. holmii
1
2 3
4 5
5
2 3
4
1
D. biseptata D. australiensis
1
2 3
4 5
A B
C D
48
Table 5: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of wheat var. Inqlab 91 in laboratory
bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 115 a 71 a 4.3 a 92.1 a 49 a 7.5 a
Growth medium 50 98 ab 104 b 70 ab 4.2 a 91.1 a 42 b 6.5 b
100 96 a-c 95 c 66 ab 3.9 ab 79.1 b 39 b 6.2 b
D. hawaiiensis 50 95 a-c 79 ef 49 ef 2.8 de 31.3 f 24 ef 3.6 fg
100 88 d 75 fg 46 f 2.6 de 28.0 f 21 f 3.3 g
D. holmii 50 94 a-d 89 cd 59 cd 3.4 bc 68.3 c 33 cd 4.7 cd
100 88 d 71 g 43 f 2.3 e 40.2 e 29 de 4.1 ef
D. biseptata 50 95 a-c 92 c 63 bc 3.8 ab 77.2 b 37 bc 5.4 c
100 92 cd 85 de 57 cd 3.1 cd 68.2 c 34 cd 4.9 cd
D. australiensis 50 94 a-d 92 c 63 bc 3.8 ab 65.0 c 32 cd 5.2 c
100 93 b-d 79 ef 54 de 3.1 cd 50.3 d 30 d 4.3 d-f
Table 6: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of wheat var. Sehar 2006 in laboratory
bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 105 a 85 a 4.9 a 98 a 63 a 9.0 a
Growth medium 50 96 ab 94 b 77 b 4.6 ab 96 a 59 a 8.2 b
100 96 ab 90 bc 71 bc 4.4 a-c 80 b 51 b 8.0 b
D. hawaiiensis 50 90 bc 85 c 66 cd 4.2 a-d 72 b 39 c 6.6 cd
100 82 d 60 e 43 f 3.7 d 38 e 43 c 6.3 d
D. holmii 50 87 cd 75 d 51 e 4.0 b-d 39 e 35 c 6.2 d
100 82 d 59 e 45 ef 3.8 cd 34 e 33 c 6.0 d
D. biseptata 50 93 bc 87 c 66 cd 4.2 b-d 50 d 46 d 7.0 c
100 89 bc 76 d 61 d 4.1 b-d 39 e 35 c 6.0 cd
D. australiensis 50 94 ab 87 c 67 cd 4.2 b-d 61 c 40 c 6.7 cd
100 87 cd 65 e 49 ef 3.9 a-d 56 c 39 c 6.0 cd
Table 7: Effect of original (100%) and diluted (50%) culture filtrates of four Drechslera
species on germination and growth of wheat var. Uqab 2000 in laboratory
bioassays.
Fungal species Conc.
(%)
Germi-
nation
(%)
Shoot
length
(mm)
Shoot
fresh wt.
(mg)
Shoot
dry wt.
(mg)
Root
length
(mm)
Root
fresh wt.
(mg)
Root dry
wt. (mg)
Control 0 100 a 131 a 90 a 5.0 a 109 a 65 a 9.6 a
Growth medium 50 97 ab 113 b 86 ab 4.8 ab 101 a 56 b 8.9 a
100 94 a-c 107 c 81 bc 4.5 bc 91 b 51 bc 8.1 b
D. hawaiiensis 50 88 c-e 91 f 58 e 3.5 fgh 33 f 27 gh 4.4 e
100 83 ef 78 gh 53 e 3.3 gh 24 g 25 h 4.0 e
D. holmii 50 90 b-d 100 d 76 c 4.0 de 81 c 42 de 6.6 cd
100 85 de 75 h 51 e 3.1 h 49 e 36 ef 6.4 cd
D. biseptata 50 92 b-d 96 de 67 d 3.9 def 76 c 43 de 6.7 cd
100 88 c-e 91 f 54 e 3.4 gh 68 d 39 ef 6.4 cd
D. australiensis 50 89 b-d 100 d 80 c 4.2 cd 82 c 48 cd 6.9 c
100 80 f 81 g 54 e 3.6 efg 35 f 34 fg 6.0 d
In a column, values with different letters show significant difference (P≤0.05) as
determined by Duncan’s Multiple Range Test.
49
Plate 6: Effect of culture filtrates of four Drechslera species on germination and
growth of Inqlab 91 in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
1
\2
4
5 1
4
2
1
D. hawaiiensis D. holmii
D. biseptata D. australiensis
1
2 3
2
4
1
2 3
4 5
1
3
5 5 4
1
3 2
4 5
A B
C D
50
Plate 7: Effect of culture filtrates of four Drechslera species on germination and
growth of Sehar 2006 in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
4
2
D. holmii
D. hawaiiensis D. holmii
D. biseptata
B A
D C 1
4
2 3
5
1
3 2
4 5
1
2
4 5
3
2 3
5
4
1
4
51
D. hawaiiensis D. holmii
D. biseptata
Plate 8: Effect of culture filtrates of four Drechslera species on germination and
growth of Uqab 2000 in laboratory bioassays.
1: Control (water) 2: 50% Medium 3: 100% Medium 4: 50% Fungal
Culture Filtrates (FCF) 5: 100% FCF
A B
D
3 2
1
4
1
2
3
4
5
1
2 3
4 5
1
2 3
4 5
5
D. australiensis
C D
B
3
5
1
5
3
1
52
3.2. Foliar Spray Bioassays
3.2.1. Effect of Fungal Culture Filtrates on Growth of C. album
3.2.1.1. Effect on Shoot Growth
Foliar spray with growth medium had negligible effect on length as well as dry
biomass of C. album shoot as compared to control where water was used for spray. In
general, the effect of various fungal culture filtrates was more pronounced on 1-week
than on 2-week old plants. Culture filtrate of D. hawaiiensis exhibited the highest
herbicidal effect resulting in significant reduction of 9% and 20% in shoot length and
shoot dry biomass, respectively, of 1-week old plants, over control. Similarly, foliar spray
of culture filtrate of D. australiensis significantly suppressed shoot dry biomass of 1-
week old plants by 14% over control. The effect of all other fungal culture filtrate
treatments including 1-week as well as 2-week old plants was nonsignificant as compared
to control (Fig.1 A & B, Plate 9).
3.2.1.2. Effect on Root Growth
All the fungal culture filtrates reduced root biomass of C. album by 6–17% over
control in 1-week old plants. The adverse effect of culture filtrate of D. hawaiiensis was
more pronounced as compared to rest of the treatments. However, the effect of foliar
application of all the four fungal culture filtrates was nonsignificant over control both in
1-week as well as 2-week old plants (Fig.1C).
3.2.2. Effect of Fungal Culture Filtrates on Growth of R. dentatus
3.2.2.1. Effect on Shoot Growth
Statistical analysis of data demonstrated that foliar spray with growth medium had
nonsignificant effect on length as well as dry biomass of R. dentatus shoot. Foliar spray
with culture filtrates of all the four Drechslera species significantly reduced shoot length
of 1-week old R. dentatus plants. Similarly, spray with culture filtrates of all the four test
fungal species except D. holmii significantly declined the shoot length of 2-week old
53
weed plants. Culture filtrate of D. australiensis was found to be the most effective
causing 42% reduction in shoot length followed by filtrate of D. hawaiiensis resulting in
41% reduction in the studied parameter of 1-week old plants. Culture filtrates of D.
holmii and D. biseptata showed comparatively less pronounced herbicidal activity against
the test weed species causing 33% and 23% reduction in shoot length of 1-week old
plants, respectively. The adverse effect of culture filtrates of various test fungal species
on shoot biomass was generally similar to that of their effect on shoot length. The highest
decline of 60% in shoot biomass of 1-week old plants was recorded due to application of
culture filtrate of D. australiensis followed by D. hawaiiensis (56%), D. biseptata (56%)
and D. holmii (54%). Adverse effect of foliar spray on shoot biomass was more
pronounced in 1-week than in 2-week old plants (Fig. 2 A & B, Plate 10).
3.2.2.2. Effect on Root Growth
The data indicate significant herbicidal potential of culture filtrates of various
Drechslera species against root growth of R. dentatus. Root biomass of the weed was
severely suppressed in 1-week old plants by 68–82% due to foliar spray of different
Drechslera species. Culture filtrates of D. australiensis and D. hawaiiensis appeared to
be the most effective inhibiting root biomass by 82% and 80%, followed by 74% and
68% reduction in root biomass due to filtrates of D. holmii and D. biseptata, respectively.
Herbicidal activity of culture filtrates of various fungal species on 2-week old plants was
comparatively less pronounced where 58-73% reduction in root biomass was recorded
(Fig. 2C).
3.2.3. Effect of Fungal Culture Filtrates on Growth of P. minor
3.2.3.1. Effect on Shoot Growth
All the fungal culture filtrates reduced shoot length and shoot biomass of P. minor
by 2–7% and 4–9% over negative control, respectively. However, the effect of foliar
spray treatments of all the four Drechslera species was nonsignificant both in 1-week and
2-week old P. minor plants (Fig. 3 A & B, Plate 11).
54
3.2.3.2. Effect on Root Growth
All the fungal culture filtrates reduced root biomass of P. minor by 1–10% over
negative control. However, the effect of foliar spray treatments of all the Drechslera
species was nonsignificant both in 1-week and 2-week old P. minor plants (Fig. 3 C).
3.2.4. Effect of Fungal Culture Filtrates on Growth of A. fatua
3.2.4.1. Effect on Shoot Growth
Growth medium showed a nonsignificant effect on shoot growth in terms of
length and biomass. None of the fungal culture filtrate treatments exhibited significant
effect against the shoot length of A. fatua. By contrast, shoot biomass of the weed showed
a variable response to foliar application of culture filtrates of different Drechslera
species. Highest and significant reduction of 37% and 42% in shoot biomass was
recorded due to culture filtrates of D. australiensis and D. biseptata in both 1-week as
well as 2-week old plants. Similarly, culture filtrate of D. holmii significantly suppressed
shoot biomass by 15% in 1-week old plants. The effect of rest of the fungal culture
treatment was nonsignificant against shoot biomass of A. fatua (Fig. 4 A and B, Plate 12).
3.2.4.2. Effect on Root Growth
Data presented in Fig. 4 C reveals that foliar spray with culture filtrate of D.
hawaiiensis significantly enhanced root biomass over control. All other foliar spray
treatment exhibited nonsignificant effect on root biomass of the target weed species.
3.2.5. Effect of Fungal Culture Filtrates on Growth of Wheat
3.2.5.1. Effect on Shoot Growth
Data presented in Fig. 5a–8a reveals a variable response of shoot length in various
wheat varieties to the four fungal culture filtrates. Foliar spray of culture filtrates of D.
holmii and D. australiensis significantly reduced shoot length of 1-week old plants of
Inqlab 91. Similarly, culture filtrates of D. biseptata and D. australiensis also
55
significantly reduced shoot length of 2-week old plants. In contrast, the effect of all the
fungal culture filtrate treatments on shoot length of Sehar 2006 and Uqab 2000 was
nonsignificant. The effect of culture filtrates of all the four Drechslera species on shoot
biomass of all the three test wheat varieties was nonsignificant (Fig. 5b–7b, Plate 13-15).
3.2.5.2. Effect on Root Growth
Effect of foliar spray treatments of all the four Drechslera species was
nonsignificant on root biomass of all the three test wheat varieties, at 1-week as well as 2-
week growth stage (Fig. 5C, 6C and 7C).
56
Fig. 1: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old Chenopodium album plants. Vertical bars show standard
errors of means of four replicates. Values with different letters show significant
difference as determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
3
6
9
12
15
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
) a a
ba a a
ab ab abab abab
B
0
0.02
0.04
0.06
0.08
0.1
0.12
1-week old 2-week old
Sh
oo
t b
iom
ass (
g)
cd ab
bcabd
ab
ab
a aba ab
a ab
C
0
0.009
0.018
0.027
0.036
0.045
1-week old 2-week old
Ro
ot
bio
mass (
g)
ab ab ab ab ab ab ab
b ab
ab
a
ab
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
57
Plate 9: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of 1-
week and 2-week old Chenopodium album plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
1
A
B
Spray started on 1-Week old plants
Spray started on 2-Week old plants
2 3 4 5 6
1 2 3 4 5 6
58
Fig. 2: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old Rumex dentatus plants. Vertical bars show standard errors of
means of four replicates. Values with different letters show significant difference as
determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
2
4
6
8
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
) a a
d
a a bbc
c c
a
cd
B
0
0.02
0.04
0.06
0.08
1-week old 2-week old
Sh
oo
t b
iom
ass (
g)
e
ccd
a a
e
ab
e
aa
e
b
d
C
0
0.02
0.04
0.06
0.08
1-week old 2-week old
Ro
ot
bio
mass (
g)
e
a
d cd
aa a
cd c
b
de
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
59
Plate 10: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old Rumex dentatus plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
A
B
Spray started on 1-Week old plants
Spray started on 2-Week old plants
1 2 3 4 5 6
1 2 3 4 5 6
60
Fig. 3: Effect of foliar spray of culture filtrates of Drechslera spp. on growth of 1-
week and 2-week old Phalaris minor plants. Vertical bars show standard errors of
means of four replicates. Values with different letters show significant difference as
determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
3
6
9
12
15
18
21
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
) a a a
a aa
a a
a a aa
B
0
0.03
0.06
0.09
0.12
1-week old 2-week old
Sh
oo
t b
iom
ass (
g)
aa a a a a
a a a a a a
C
0
0.03
0.06
0.09
0.12
1-week old 2-week old
Ro
ot
bio
mass (
g)
a a
a a a a a a
a a a a
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
61
Plate 11: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old Phalaris minor plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
A
B
Spray started on 1-Week old plants
Spray started on 2-Week old plants
1 2 3 4 5 6
1 2 3 4 5 6
62
Fig. 4: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old Avena fatua plants. Vertical bars show standard errors of
means of four replicates. Values with different letters show significant difference as
determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
7
14
21
28
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
) aaaaaaaaaa a a
B
0
0.05
0.1
0.15
0.2
0.25
1-week old 2-week old
Sh
oo
t b
iom
ass (
g)
a
cccc
abb ab abaa a
C
0
0.04
0.08
0.12
0.16
0.2
1-week old 2-week old
Ro
ot
bio
ma
ss
(g
)
bbab
bbbbbbb b
a
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
63
Plate 12: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old Avena fatua plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
A
B
Spray started on 1-Week old plants
Spray started on 2-Week old plants
1 2 3 4 5 6
1 2 3 4 5 6
64
Fig. 5: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old plants of wheat var. Inqlab 91. Vertical bars show standard
errors of means of four replicates. Values with different letters show significant
difference as determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
8
16
24
32
40
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
)
c c c a-c bc c a-c a-c ab a ab a
B
0
0.08
0.16
0.24
0.32
0.4
1-week old 2-week old
Sh
oo
t b
iom
ass (
g)
ab ab
a a a
a a a a a a
aa a
C
0
0.06
0.12
0.18
0.24
0.3
1-week old 2-week old
Ro
ot
bio
mass (
g)
a a a a a a a
a a a a a
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
65
Plate 13: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old wheat var. Inqlab 91 plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
A
B
Spray started on 1-Week old plants
Spray started on 2-Week old plants
1 2 3 4 5 6
1 2 3 4 5 6
66
Fig. 6: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old plants of wheat var. Sehar 2006. Vertical bars show standard
errors of means of four replicates. Values with different letters show significant
difference as determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
8
16
24
32
40
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
)
ab
a ab ab ab b
a ab
ab ab
ab ab ab
B
0
0.08
0.16
0.24
0.32
0.4
1-week old 2-week old
Sh
oo
t b
iom
ass (
g) a a a a a a a a a a a a
C
0
0.06
0.12
0.18
0.24
1-week old 2-week old
Ro
ot
bio
mass (
g) a a a a a a a a
a a a a
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
67
Plate 14: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth
of 1-week and 2-week old wheat var. Sehar 2006 plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
A
B
Spray started on 1-Week old plants
Spray started on 2-Week old plants
1 2 3 4 5 6
1 2 3 4 5 6
68
Fig. 7: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old plants of wheat var. Uqab 2000. Vertical bars show standard
errors of means of four replicates. Values with different letters show significant
difference as determined by Duncan's Multiple Range Test at P ≤ 0.05.
A
0
8
16
24
32
40
1-week old 2-week old
Sh
oo
t le
ng
th (
cm
) a a a a a a a a a a a a
B
0
0.1
0.2
0.3
0.4
1-week old 2-week old
Sh
oo
t b
iom
ass (
g)
a a a
a a a a
a a a a
a
C
0
0.06
0.12
0.18
0.24
1-week old 2-week old
Ro
ot
bio
mass (
g) a a
a a
a a a
a a a
a a
Control Growth medium D. hawaiiensis
D. biseptataD. holmii D. australiensis
69
A
Plate 15: Effect of foliar spray of culture filtrates of four Drechslera spp. on growth of
1-week and 2-week old wheat var. Uqab 2000 plants.
1: Negative control (water), 2: Positive control (M-1-D), 3: Culture filtrate (CF) of D.
hawaiiensis, 4: CF of D. holmii, 5: CF of D. biseptata, and 6: CF of D. australiensis
.
Spray started on 1-Week old plants
Spray started on 2-Week old plants
1 2 3 4 5 6
1 2 3 4 5 6
B
70
3.3. Field Experiment
3.3.1. Effect of Fungal Culture Filtrates on Weed Biomass
Both recommended and half dose of the herbicide Bromoxynil+MCPA
completely checked the growth of the target weed species. Similarly, weed growth was
also completely checked when half dose of the herbicide was used in combination with
culture filtrates of different Drechslera spp. In general, culture filtrates of all the four
Drechslera spp. significantly reduced biomass of R. dentatus as compared to weedy
check. However, variability among the herbicidal activity of the culture filtrates of
different Drechslera spp. was evident. Among the four Drechslera spp., culture filtrates
of D. australiensis and D. hawaiiensis were found to be more effective against R.
dentatus than the culture filtrates of other two Drechslera species. There was 58, 57, 31
and 39% reduction in dry biomass of R. dentatus due to foliar application of culture
filtrates of D. australiensis, D. hawaiiensis, D. biseptata and D. holmii, respectively
(Table 8).
3.3.2. Effect of Fungal Culture Filtrates on Wheat Growth and Yield
Maximum and significant reduction of 14% in height of wheat plants was
recorded due to R. dentatus in weedy check as compared to weed free control. Plants
height was also significantly reduced due to presence of the weed in treatments where
only culture filtrates of different Drechslera species was used in foliar spray. There was
6, 8, 11 and 13% reduction in plant height due to foliar spray of D. australiensis, D.
hawaiiensis, D. biseptata and D. holmii, respectively as compared to weed free control.
The effect of weed competition on wheat plants height was nonsignificant over control
where synthetic herbicide Bromoxynil+MCPA was sprayed either alone or in
combination with the culture filtrates of the four Drechslera species (Fig. 8A). The
response of number of tillers and total above ground dry biomass to the weed competition
and foliar spray applications was generally similar to that of the response of plant height.
Maximum and significant reduction of 30% and 28% in tillering and above ground dry
matter, respectively, was recorded in weedy check as compared to control. Similarly,
71
there was 9–21% and 12–23% reduction in number of tillers and above ground dry
matter, respectively, due to weed competition in various treatments where only the fungal
culture filtrates were used as foliar spray. In contrast, in all the synthetic herbicide
treatments, either alone or in combination with fungal culture filtrates, the effect of R.
dentatus interference on the two studied parameters was insignificant as compared to
control (Fig. 8 B & C).
Data regarding the effect of weed competition and various types of foliar spray
applications on grain yield and 100 grains weight is presented in Fig. 9. Highest reduction
in grain yield (43%) was recorded in weedy check over control. Similarly, significant
reduction in grain yield was also recorded due to the weed competition over control in
treatments where only fungal culture filtrates were used in the foliar spray application.
There was 21, 22, 35 and 36% reduction in grain yield over control in treatments of foliar
spray applications of culture filtrates of D. australiensis, D. hawaiiensis, D. biseptata and
D. holmii, respectively (Fig. 9A). R. dentatus interference significantly reduced the
weight of 100 grains by 30, 25 and 27% in weedy check, and in treatments where culture
filtrates of D. biseptata and D. holmii, respectively, were used in the foliar spray
application (Fig. 9B). The effect of R. dentatus on grain yield and 100 grains weight was
insignificant in Bromoxynil+MCPA treatments, either alone or in combination with
fungal culture filtrates (Fig. 9 A & B).
72
Table 8: Effect of foliar spray of herbicide bromoxynil+MCPA and culture filtrates of
four Drechslera spp. on biomass of Rumex dentatus.
Treatments Weed dry biomass
(g plot-1
)
Reduction over
weedy check
(%)
Weed free 0 ± 0 d -
Weedy check 915 ± 45 a 0
Drechslera hawaiiensis 391 ± 12 c 57
D. holmii 642 ± 30 b 30
D. biseptata 630 ± 13 b 31
D. australiensis 384 ± 22 c 58
Bromoxynil+MCPA (Full dose) 0 ± 0 d 100
Bromoxynil+MCPA (Half dose) 0 ± 0 d 100
Bromoxynil+MCPA (Half dose) + D. hawaiiensis 0 ± 0 d 100
Bromoxynil+MCPA (Half dose) + D. holmii 0 ± 0 d 100
Bromoxynil+MCPA (Half dose) + D. biseptata 0 ± 0 d 100
Bromoxynil+MCPA (Half dose) + D. australiensis 0 ± 0 d 100
±, Indicates standard errors of means of three replicates.
In a column, values with different letters show significant difference (P ≤ 0.05) as
determined by Duncan’s Multiple Range Test.
73
Fig. 8: Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA
and culture filtrates of four Drechslera spp. on different growth parameters of field
grown wheat. Vertical bars show standard errors of means of three replicates. Bars
with different letters show significant difference (P ≤ 0.05) as determined by Duncan’s
Multiple Range Test.
A
0
21
42
63
84
105
Pla
nt
heig
ht
(cm
)
aaaaaabb
cdd bc
d
a
B
0
2
4
6
8
10
12
14
No
. o
f ti
llers
/p
lan
t
aa aa a a b
c c b
d
a
C
0
200
400
600
800
1000
1200
1400
1600
1800
To
tal d
ry b
iom
as
s (
g)
aaaaaa
abbcbc
a-c
c
a
Weed free
D. holmii
Bromoxynil+MCPA (FD)
HD + D. holmii
Weedy check
D. biseptata
Bromoxynil+MCPA (HD)
HD + D. biseptata
D. hawaiiensis
D. australiensis
HD + D. hawaiiensis
HD + D. australiensis
74
Fig. 9: Effect of foliar spray of full (FD) and half dose (HD) of Bromoxynil+MCPA
and culture filtrates of four Drechslera spp. on grain yield and 100 grains weight of
field grown wheat. Vertical bars show standard errors of means of three replicates.
Bars with different letters show significant difference (P ≤ 0.05) as determined by
Duncan’s Multiple Range Test.
A
0
200
400
600
800
Gra
in y
ield
/plo
t (g
)
dede
e
cd b-d
a-cab
ab a-c a-c aba
B
0
0.5
1
1.5
2
2.53
3.5
4
4.5
5
5.5
10
0 g
rain
s w
eig
ht
(g)
a
bbb
aaaa
aaaa
Weed free
D. holmii
Bromoxynil+MCPA (FD)
HD + D. holmii
Weedy check
D. biseptata
Bromoxynil+MCPA (HD)
HD + D. biseptata
D. hawaiiensis
D. australiensis
HD + D. hawaiiensis
HD + D. australiensis
75
T3 D. hawaiiensis
T5 D. biseptata T6 D. australiensis T4 D. holmii
T1 Weed free T2 Weedy check
T7 Herbicide (RD) T8 Herbicide (HD) T9 HD + D. hawaiiensis
T10 HD +D. holmii T11 HD +D. biseptata
Plate 16: Effect of foliar spray of recommended (RD) and half dose (HD) of
Bromoxynil+MCPA and culture filtrates of four Drechslera spp. on field grown weed
and wheat.
T12 HD +D. australiensis
76
3.4. Leaf Discs Bioassays Using Crude Organic Fractions
Positive reaction showing necrotic spots was observed on punctured R. dentatus
leaf surface, while no necrotic spot was observed in case of unpunctured leaf discs. In
punctured leaf surface, chloroform fraction was found to be highly effective in producing
necrotic spot followed by ethyl acetate fraction. n-hexane and n-butanol fractions did not
produce any necrotic spot. Crude chloroform fraction produced necrotic spot at minimum
concentration of 1.0 mg mL-1
, while ethyl acetate fraction produced necrotic spots at
minimum concentration of 2.0 mg mL-1
. Severe discoloration was also observed in case
of bioassays performed with punctured leaves. In case of chloroform fraction, severe
discoloration was observed up to concentration of 0.25 mg mL-1
followed by ethyl acetate
fraction, severe discoloration was observed only at highest concentration of 4.0 mg mL-1
.
In case of n-hexane and n-butanol fractions, discoloration was not much pronounced.
Leaf sections treated with DMSO as positive control exhibited only light discoloration,
while distilled water used as negative control had no effect on treated leaf sections. In
case of unpunctured leaf surface, although chloroform and ethyl acetate fractions did
produce moderate discoloration, but this effect was less pronounced when compared with
similar treatments using punctured leaf sections (Table 9 & 10, Plate17).
3.5. Leaf Discs Bioassays Using Purified Chromatographic Fractions
In these bioassays out of six purified chromatographic fractions from crude
chloroform fraction of culture filtrate of D. australiensis, four viz. A, C, D and F were
found effective in producing necrotic spot on punctured leaf discs of R. dentatus leaves.
Among these, A and F were found most bioactive in producing necrotic spots on
punctured leaf discs surfaces at minimum concentration of 0.5 mg mL-1
. Fraction C was
found active at minimum concentration of 1.0 mg mL-1
, while fraction D was found
active at highest concentration of 2.0 mg mL-1
. Chromatographic fractions B and E did
not produce any necrotic spot. In case of bioassays performed with 2,4-D, as positive
control, maximum bioactivity was observed at minimum concentration of 0.25 mg mL-1
.
Discolouration of cut leaf sections was also observed to variable extent in all the
treatments. Fraction C and F induced severe discolouration at concentration of 2.0 mg
77
mL-1
. Interestingly, only light discolouration was observed in case of 2,4-D, even at
highest concentration of 2.0 mg mL-1
(Table 11, Plate18).
In case of bioassays conducted with purified chromatographic fractions from
crude ethyl acetate fraction, only fraction H was found active in producing necrotic spot
on cut R. dentatus leaves. This fraction induced necrosis as well as severe discolouration
only at highest concentration of 2.0 mg mL-1
(Table 12, Plate18).
78
Table 9: Leaf discs bioassays using crude organic fractions on punctured leaf surface.
DMSO Conc.
(µL 1000µL-1
)
DMSO effect
Organic
fraction
Conc.
(mg mL-1
)
Effect of crude organic fractions
n-hexane Chloroform Ethyl acetate n-butanol
Colour Necrotic
Spot Colour
Necrotic
Spot Colour
Necrotic
Spot Colour
Necrotic
Spot Colour
Necrotic
Spot
Water 0 4 Water 0 4 0 4 0 4 0 4
1.560 0 4 0.0625 0-1 4 1-2 4 0 4 0 4
3.125 1 4 0.1250 1 4 2 4 1 4 0 4
6.250 0-1 4 0.2500 1 4 2-3 4 1 4 1 4
12.500 0-1 4 0.5000 1-2 4 3 4 1-2 4 1 4
25.000 0-1 4 1.0000 2 4 3 5 2 4 1-2 4
50.000 0-1 4 2.0000 2 4 3 7 2 5 2 4
100.000 0-1 4 4.0000 2 4 3 10 3 7 2 4
Colour scale
0 = no change
1 = Light discoloration
2 = Moderate discoloration
3 = Severe discoloration
Necrotic spot scale
4 = No necrotic spot
5 = Necrotic spot ≤ 1mm
6 = Necrotic spot ≤ 2 > 1mm
7 = Necrotic spot ≤ 3 > 2mm
8 = Necrotic spot ≤ 4 > 3mm
9 = Necrotic spot ≤ 5 > 4mm
10 = Necrotic spot ≤ 6 > 5mm
79
Table 10: Leaf discs bioassays using crude organic fractions on unpunctured leaf surface.
DMSO Conc.
(µL 1000 µL-1
)
DMSO effect
Organic
fraction
Conc.
(mg mL-1
)
Effect of crude organic fractions
n-hexane Chloroform Ethyl acetate n-butanol
Colour Necrotic
Spot Colour
Necrotic
Spot Colour
Necrotic
Spot Colour
Necrotic
Spot Colour
Necrotic
Spot
Water 0 4 Water 0 4 0 4 0 4 0 4
1.560 0 4 0.0625 0 4 0 4 0 4 0 4
3.125 0 4 0.1250 0 4 0-1 4 1 4 0 4
6.250 0-1 4 0.2500 0-1 4 1 4 2 4 1 4
12.500 0-1 4 0.5000 0-1 4 2 4 2 4 1 4
25.000 0-1 4 1.0000 0-1 4 2 4 2 4 1 4
50.000 0-1 4 2.0000 0-1 4 2 4 2 4 1 4
100.000 0-1 4 4.0000 0-1 4 2 4 2 4 1 4
Colour scale
0 = no change
1 = Light discoloration
2 = Moderate discoloration
3 = Severe discoloration
Necrotic spot scale
4 = No necrotic spot
5 = Necrotic spot ≤ 1mm
6 = Necrotic spot ≤ 2 > 1mm
7 = Necrotic spot ≤ 3 > 2mm
8 = Necrotic spot ≤ 4 > 3mm
9 = Necrotic spot ≤ 5 > 4mm
10 = Necrotic spot ≤ 6 > 5mm
80
Plate 17: Effect of crude chloroform (A) and ethyl acetate (B) fraction
of culture filtrate of Drechslera australiensis on punctured leaf discs of
Rumex dentatus.
Necrotic spot
Necrotic spot
Concentration of
crude chloroform
fraction (mg mL-1
) A
B
4
2
1
2
4
Concentration of
crude ethyl acetate
fraction (mg mL-1
)
81
Table 11: Leaf discs bioassays using purified chromatographic fractions from chloroform fraction of D. australiensis on punctured leaf surface.
DMSO Conc.
(µL 1000 µL-1)
DMSO effect
2,4-D/
Compound
Conc.
(mg mL-1)
2,4-D effect
Effect of purified chromatographic fractions
A B C D E F
Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S
Water 0 4 Water 0 4 0
4 0 4 0 4 0 4 0 4 0 4
0.780 0 4 0.0312 0-1 4 0
4 0 4 0 4 0 4 0 4 0 4
1.560 0 4 0.0625 0-1 4 0-1
4 0-1 4 0-1 4 0-1 4 0-1 4 0-1 4
3.125 0 4 0.1250 1 4 0-1
4 0-1 4 1 4 1 4 0-1 4 0-1 4
6.250 0 4 0.2500 1 6 1
4 0-1 4 1 4 1 4 0-1 4 0-1 4
12.500 0-1 4 0.5000 1 7 1
6 1 4 2 4 1 4 1 4 1 6
25.000 0-1 4 1.0000 1 10 1-2
7 1 4 2 7 2 4 1 4 2 7
50.000 0-1 4 2.0000 1 10 2
9 1 4 3 8 2 7 1 4 3 9
Colour scale
0 = no change
1 = Light discoloration
2 = Moderate discoloration
3 = Severe discoloration
Necrotic spot scale
4 = No necrotic spot
5 = Necrotic spot ≤ 1mm
6 = Necrotic spot ≤ 2 > 1mm
7 = Necrotic spot ≤ 3 > 2mm
8 = Necrotic spot ≤ 4 > 3mm
9 = Necrotic spot ≤ 5 > 4mm
10 = Necrotic spot ≤ 6 > 5mm
N.S = Necrotic spot
82
Table 12: Leaf discs bioassays using purified chromatographic fractions from ethyl acetate fraction of D. australiensis on punctured leaf
surface.
DMSO Conc.
(µL 1000 µL-1
)
DMSO effect
2,4-D/
Compound
Conc.
(mg mL-1
)
2,4-D effect Effect of purified chromatographic fractions
G H I
Colour N.S Colour N.S Colour N.S Colour N.S Colour N.S
Water 0 4 Water 0 4 0 4 0 4 0 4
0.780 0 4 0.0312 0-1 4 0 4 0 4 0 4
1.560 0 4 0.0625 0-1 4 0-1 4 0-1 4 0-1 4
3.125 0 4 0.1250 1 4 1 4 1 4 1 4
6.250 0 4 0.2500 1 6 1-2 4 2 4 1 4
12.500 0-1 4 0.5000 1 7 2 4 2 4 1-2 4
25.000 0-1 4 1.0000 1 10 2 4 2 4 2 4
50.000 1 4 2.0000 1 10 2 4 3 7 2 4
Necrotic spot scale
4 = No necrotic spot
5 = Necrotic spot ≤ 1mm
6 = Necrotic spot ≤ 2 > 1mm
7 = Necrotic spot ≤ 3 > 2mm
8 = Necrotic spot ≤ 4 > 3mm
9 = Necrotic spot ≤ 5 > 4mm
10 = Necrotic spot ≤ 6 > 5mm
Colour scale
0 = no change
1 = Light discoloration
2 = Moderate discoloration
3 = Severe discoloration
N.S = Necrotic spot
83
Plate 18: Effect of 2,4-D and chromatographic fractions (A), (C), (D), (F)
and (H) of culture filtrate of Drechslera australiensis on punctured leaf
discs of Rumex dentatus.
2,4-D
0.25
0.5
1
2
A
1
2
0.5
C
1
2
D
2
H
2
F
1
2
0.5
Necrotic spot
Necrotic spot
Concentration of
pure fraction
(mg mL-1
)
Concentration of
pure fraction
(mg mL-1
)
Concentration of
pure fraction
(mg mL-1
) Concentration of
pure fraction
(mg mL-1
)
Necrotic spot
Concentration of
pure fraction
(mg mL-1
)
Concentration of
pure fraction
(mg mL-1
)
84
3.6. Spectroscopic Data of Isolated Compounds
Chromatographic fractions A and F exhibited the highest herbicidal activity,
therefore spectroscopic analyses of only these two compounds were carried out.
Chromatographic fraction A was identified as compound 1 and that of F as compound 2.
3.6.1. Compound 1
Holadysenterine
Colorless amorphous powder.
[α]D28
14.6° (c = 1.0, MeOH).
M.p. 219-220.5 °C.
HREIMS m/z: [M]+
390.3030 (Calcd. for C23H38N2O3 390.3083).
EIMS m/z: [M]+ 390, 317, 354, 307, 289, 278, 154, 136, 115, 107, 85.
1H-NMR (CD3OD, 600 MHz) δH : 1.19 (1H, m, H-1), 1.33 (1H, m, H-1), 1.35 (1H, m, H-
2), 1.44 (1H, m, H-2), 2.99 (1H, brm, H-3), 5.44 (brs 1H, m, H-6), 1.84 (2H, m, H-12),
1.33 (1H, m, H-17), 4.01 (1H, d, J = 10.9, H-18), 3.70 (1H, d, J = 10.9 ), 1.29 (s, 3H, H-
19), 3.45 (1H, m, H-20), 1.38 (1H, d, J = 7.1 H-21), 2.01 (s, 3H, CO-Me).
H2N
H
HH3C
N
H H
H
OH
OCH3
HO
1
2
3
4
5
6
7
8
9
10
11
12
13
1415
16
17
18
19
20
21
Fig. 10. Chemical structure of holadysenterine
85
3.6.2. Compound 2
(Z)- docos-5-en-1-oic acid
Viscous oil.
HREIMS m/z: [M]+
338.5702 (calcd. 338.5727 for C22H42O2).
EIMS (rel int) m/z, [M]+ 338 (26), 324 (30), 294 (10), 293 (60), 279 (40), 265 (35), 225
(28), 117 (100), 113 (75), 87 (32).
1H-NMR (CH3OD, 500 MHz) δ: 0.88 ( 3H, t, J = 6.8 Hz, H-22), 2.89 ( 2H, t, J = 7.2 Hz,
H-2), 5.25 (2H, dt, J = 11.6 Hz, H-5 and H-6), 2.21 (4H, m, H-4 and H-7), 1.32-1.84 (28
H, br s, H-3, H-8-H-21).
13C-NMR (CH3OD, 125 MHz) δ: 180.1 (C-1), 130.1 (C-5), 127.5 (C-6), 14.1 (C-22),
22.1-38.8 (C-2-C-4, C-7-C-21).
HOOC1
2
3
4
5
6
7
8
9
10
11
1214
1315
16
17
18
19
20
21
22
Fig. 11. Chemical structure of (Z)- docos-5-en-1-oic acid
86
Chapter 4
Discussion Discovery of natural herbicidal compounds from plants and microbes is an
extraordinary challenge and is an area of intense research. In the past years, a number of
highly successful herbicidal compounds based upon fungal metabolites have been
discovered (Berestetskiy 2008). The genus Drechslera is well known as bioherbicides for
its use in weed control programs (Peng and Boyetchko 2006; Hirase et al. 2006; Casella
et al. 2010; Rabbani et al. 2011). Besides this, a number of herbicidal compounds have
also been isolated from culture filtrates of different species of Drechslera (Evidente et al.
2006c). However, studies regarding the herbicidal activity of metabolites of Drechslera
species from Pakistan are lacking. The present study was, therefore, carried out to
investigate the herbicidal potential of metabolites of various Drechslera spp. from
Pakistan against some problematic weeds of wheat. In general, metabolites of all the four
test Drechslera spp. namely D. australiensis, D. biseptata, D. hawaiiensis and D. holmii,
exhibited herbicidal activity to variable extent against various target weeds of wheat.
In laboratory bioassays, generally original concentration of the M-1-D growth
medium significantly reduced germination, length as well as fresh and dry biomass of the
seedlings. However, this effect was far less pronounced as compared to the effect of
fungal culture filtrates. Original growth medium reduced germination, shoot length, shoot
dry biomass, root length and root biomass by 4–16%, 15–19%, 9–23%, 14–36% and 6–
32%, respectively. On the other hand, original culture filtrates of various Drechslera
species suppressed germination, shoot length, shoot dry biomass, root length and root
biomass by 12–94%, 27–91%, 17–88%, 56–94% and 47–88%, respectively. Although
contents of the original growth medium exhibited adverse effect on germination and
seedlings growth to some extent, however, it is very likely that most of the medium
contents were used during the 28 days growth period of the test fungal species, and the
effect of these medium components was probably negligible in the fungal culture filtrate
treatments.
In laboratory bioassays, culture filtrates of all the test Drechslera spp. reduced
seed germination of the four target weed species by 12–94%. Earlier, Idrees and Javaid
87
(2008) have reported 23% reduction in germination of parthenium (Parthenium
hysterophorus L.) seeds due to culture filtrates of D. hawaiiensis. In a similar study,
Javaid and Adress (2009) reported 20%, 30% and 93% reduction in germination of
parthenium seeds due to culture filtrates of Drechslera biseptata, D. australiensis and D.
rostrata, respectively. Herbicidal activity of fungal metabolites against germination of
weed seeds is not restricted to the culture filtrates of Drechslera species only. There are
also reports of herbicidal activity of culture filtrates of Trichoderma spp., Fusarium spp.,
Cladosporium spp. and Alternaria alternata (Idrees and Javaid 2008; Javaid and Adrees
2009; Javaid and Ali 2011). Akbar and Javaid (2010) studied the effect culture filtrates of
presently tested Drechslera species using malt extract as growth medium. The results of
the two studies reveals that M-1-D is comparatively better growth medium than malt
extract for the preparation of fungal culture filtrates for management of weeds of wheat.
Recently, Javaid et al. (2012) have also reported similar differential effects of culture
filtrates of Trichoderma spp. prepared in M-1-D and malt extract growth media and
tested against parthenium weed. The variable herbicidal potential of the fungal
metabolites prepared in different growth media could be due to the formation of different
quantities of culture filtrates in different growth media (Zonno et al. 2008). In the present
study, seedling growth of various weed species was also adversely affected by culture
filtrates of the test Drechslera species. Similar inhibition in seedling growth of other
weed species such as parthenium has also been reported due to culture filtrates of
Drechslera and other fungal species (Javaid and Adrees 2009; Javaid et al. 2011b).
Findings of the present study reveals that culture filtrates of different test Drechslera spp.
showed variable herbicidal activity against the germination and seedling growth of target
weed species. Culture filtrates of D. australiensis and D. hawaiiensis were found more
effective in suppressing germination of the test weed species than the culture filtrates of
other two fungal species. Variation in herbicidal activity of different Drechslera spp. has
also been reported against germination of parthenium seeds (Javaid and Adrees 2009;
Javaid et al. 2011b). Variation in herbicidal activity of culture filtrates of different
Drechslera spp. could be attributed to the variation in chemical constituents of different
fungal species (Evidente et al. 2005; Zhou et al. 2008; Eneyskaya et al. 2009; Yang et al.
2009). In laboratory bioassays, generally root growth was more susceptible to various
88
employed culture filtrates of the four Drechslera species. As herbicidal compounds are
first absorbed by roots from the surroundings, resulting in reduced growth (Noor and
Khan, 1994).
In pot trials, the effect of M-1-D medium as foliar spray treatments on the growth
of weeds as well as wheat plants was generally nonsignificant. The effect of various
culture fultrates on the growth of the test weed species was highly variable with respect
to the target weed species. In laboratory bioassays, generally all the test plant species
showed pronounced susceptibility to various fungal culture filtrates. In contrast, in pot
trials, generally culture filtrates of all the four Drechslera species exhibited marked
herbicidal activity against R. dentatus while their effect on growth of rest of the test weed
species and wheat varieties was nonsignificant. Such differential herbicidal effects of
fungal culture filtrates in laboratory and pot bioassays has also been reported in other
similar studies (Javaid and Adrees 2009; Javaid et al. 2011b). The differential effect of
fungal culture filtrates in laboratory and pot trials may be attributed to the two factors.
First, in laboratory bioassays seeds were directly exposed to various culture filtrate
treatments. Consequently, the very delicate germinating seedling’s growth was severely
affected by the applied culture filtrates. In contrast, in pot trials spray was done on 1-
week and 2-week old seedlings which were comparatively more tolerant. Secondly, in
laboratory bioassays, seedlings were exposed to various fungal culture filtrate treatments
through out the experimental period while in pot trials, spray was done at some regular
intervals. Although culture filtrates of all the Drechslera species exhibited pronounced
herbicidal activity against R. dentatus, however, D. australiensis and D. hawaiiensis were
found to be the most effective fungal species causing 42% and 41% reduction in shoot
length of 1-week old plants. Adverse effects of foliar spray on shoot biomass of R.
dentatus was more pronounced in 1-week old than in 2-week old plants. Although roots
were not directly exposed to foliar spray application, however, root biomass in R.
dentatus was also severely suppressed in 1-week old plants by 68–82% due to foliar
spray of different Drechslera species. Earlier, Javaid and Ali (2011) studied the effect of
culture filtrates of various Trichoderma species on pot grown plants of some problematic
weeds of wheat and found that R. dentatus was the most susceptible weed species. The
differential response of the four weed species to the same or different culture filtrates
89
could be attributed to the different morphological and physiological characteristics of the
test plant species involved. Toxicity is assumed to be associated with the presence of
strong electrophilic or nucleophilic systems. Action by such systems on specific positions
of proteins or enzymes would alter their configuration and affect their activity (Macías et
al. 1992). Previously, various studies conducted regarding the effect of foliar spray of
culture filtrates of different pathogenic fungi including species of Fusarium, Alternaria
and Drechslera against parthenium weed support the findings of the present study and
suggested that fungal culture filtrates can be exploited as herbicides (Idrees and Javaid
2008; Javaid and Adrees 2010; Javaid et al. 2011b).
In both laboratory bioassays and pot trials, R. dentatus found to be the most
susceptible weed species to the application of culture filtrates of Drechslera spp. so this
species was selected for field trials. On the other hand, all the test wheat varieties
exhibited almost similar behaviour to the application of fungal culture filtrates thus only
one wheat variety Sehar 2006 was selected for field experiment. In the field study,
herbicidal activity of culture filtrates of four Drechslera spp. was compared with a
commercial herbicide Bromoxynil+MCPA. The chemical herbicide was used either alone
in recommended dosage or its half dose was applied in combination with culture filtrates
of various Drechslera species. R. dentatus reduced wheat grain yield by 43% over weed
free control. Application of recommended dose of chemical herbicide completely killed
the weed plants. Although none of the fungal culture filtrates treatments completely
eliminated the weed, however, these markedly reduced the weed biomass by 30–58%
over weedy check. Wheat grain yield losses in treatments where foliar spray of culture
filtrates of D. australiensis, D. hawaiiensis, D. biseptata and D. holmii was carried out
were 21, 22, 35 and 36%, respectively, as compared to 43% losses in weedy check. In the
present study, original culture filtrates were applied as foliar spray. It is highly likely that
efficacy of these filtrates can be enhanced markedly if these are applied in a concentrated
form because generally, quantity of active herbicidal constituents in fungal culture
filtrates is very low (Evidente et al. 2006bc). In the field trials, half dose of synthetic
herbicide also completely killed the R. dentatus plants. Consequently, the effects of
combined application of fungal culture filtrates and half dose of herbicide could not be
assessed. Further studies are required in this regards using lower concentrations of the
90
herbicide. To best of our knowledge, the present study is the first report of using fungal
toxins as herbicidal agents in true field conditions. Generally, earlier experiments were
carried out in trays or pots (Vurro et al. 2001; Javaid et al. 2011b).
In both laboratory and pot trials, the effect of culture filtrates of various test
Drechslera species was more severe on germination and growth of weeds than that of
wheat. In these bioassays, culture filtrates of the four Drechslera species reduced the
shoot length, shoot biomass and root biomass of various target weed species by 22–91%,
17–88% and 47–48%, respectively. By contrast, shoot length, shoot biomass and root
biomass of wheat varieties Inqlab 91, Sehar 2006 and Uqab 2000 was reduced by 0–
4.9%, 0–13% and 0–28%, respectively. In foliar spray bioassays culture filtrate
applications had variable effects on growth of the test weeds. Shoot and root growth of 1-
week old R. dentatus plants was significantly reduced by culture filtrates of all the fungal
species. Conversely, the effect of foliar application of culture filtrates of all the fungal
species was nonsignificant on growth of all the three test wheat varieties. Likewise, in
field trials culture filtrates of D. australiensis, D. biseptata, D. hawaiiensis and D. holmii
reduced the biomass of R. dentatus by 58%, 31% and 57% and 30%, respectively, while
had no adverse effect on growth of wheat. The differential response of the weeds
especially R. dentatus and wheat to fungal metabolites can be best exploited in the
management of R. dentatus and possibly other weeds by the culture filtrates of
Drechslera spp. under field conditions.
In the present study, culture filtrates of D. australiensis exhibited the best
herbicidal activity in laboratory bioassays, foliar spray pot experiments as well as under
field conditions. Therefore, this fungal species was selected for the isolation of active
herbicidal compounds. The crude culture filtrates of D. australiensis were fractionated
using four organic solvent viz. n-hexane, chloroform, ethyl acetate and n-butanol. Leaf
discs bioassays were carried out using different concentrations of these crude organic
fractions of culture filtrates of D. australiensis. Chloroform fraction exhibited the highest
herbicidal activity followed by ethyl acetate fraction in terms of necrotic spot formation
and causing leaf disc discolouration. Isolation and purification of compounds from
chloroform and ethyl acetate fractions using various chromatographic techniques
revealed the presence of six compounds from chloroform fraction and three compounds
91
from ethyl acetate fraction. Leaf discs bioassays using these purified compounds revealed
that the most active herbicidal compounds were present in chloroform fraction From
chloroform fraction, chromatographic fractions A and F showed the highest efficacy in
producing necrotic spot at punctured leaf surface at minimum concentration of 0.5 mg
mL-1
as compared to 0.25 mg mL-1
of reference compound 2,4-D. However, when
compared the efficacy of chromatographic fractions A and F with that of 2,4-D in terms
of discoloration of leaf discs, 2,4-D was found less bioactive than the two isolated natural
compounds as only light discolouration was observed in case of 2,4-D, even at highest
concentration of 2.0 mg mL-1
.
Structural elucidation of the most active chromatographic fractions A and F were
carried out using various spectroscopic techniques and these fractions were identified as
holadysenterine as (compound 1) and (Z)- docos-5-en-1-oic acid as (compound 2),
respectively. In many earlier studies, several herbicidal constituents has been isolated
from other species of Drechslera. Culture of Drechslera siccans (Drechsler) Shoemaker
is also reported to yield a phytotoxin named as 6,8-dihydroxy-3-(2’-hydroxypropyl)
isocoumarin (de-o-methyldiaporthin). Phyto-toxicity of this compound has been
estimated in terms of necrotic spot area when tested on Avena sativa, Echinochloa crus-
galli and Amaranthus spinosus (Hallock et al. 1988). Capio et al. (2004) isolated two
phytotoxic compounds namely cytochalasin B and dihydrocytochalasins from extracts of
dried mycelia and liquid culture filtrates of Drechslera wirreganensis Wallwork, Lichon
& Sivan. and D. campanulata (Lév.) B. Sutton. Similarly, another metabolite namely
drazepinone with broad spectrum herbicidal activity has been isolated from Drechslera
siccans. This compound was characterized as 3,5,12a-trimethyl-2,5,5a,12a-tetrahydro-1H
naphtha [20,30:4,5]furo[2,3-b]azepin-2-one (Evidente et al. 2005).
Compound 1 (holadysenterine) was isolated as amorphous solid from the
chloroform extraction. Molecular formula was obtained by HREIMS, showing peak at
m/z 390.3030 for C23H38N2O3 showing six degrees of unsaturation in the compound. Four
unsaturations were accounted for by a tetra-cyclic pregnane type skeleton, two were due
to the endocyclic double bond and was due to carboxyl function. The UV spectrum was
inconclusive. Inspection of 1H-NMR spectrum of compound 1 showed olefinic proton at
δ 5.44 (1H, br singlet). The spectra showed two doublets at δ 3.70 (1H, d, J = 10.9 Hz)
92
assignable to the hydroxyl methylene protons. At δ 2.99 and 3.45 two broad multiplets
were observed and were assigned to the H-3α and 20β, respectively. Two methyl singlets
were observed at δ 1.29 and δ 2.0 while at δ 1.38 a characteristic methyl doublet was
observed having J = 7.1 Hz. The downfield shift of methyl group at δ 2.0 indicated the
presence of acetamide functionality in the molecule. A solvent exchangeable proton
singlet due to N-hydroxy was observed at δ 4.98 (Bhutani, 1990), indicated its attachment
at amine side chain functionality. EIMS spectrum gave peaks at m/z 317 [M+H-
CH3CONOH]+ (-C-N-bond cleavage), 289 [M+H-C4H8NO2]+ (C17-C20 bond cleavage)
and 278 [M+H-C5H8NO2]+ (C13-C17 and C16-C17 bands cleavage). This proves the
attachment of hydroxyl methyl group at amine centre. On the basis of these evidences
and comparison with the literature data, compound 1 was assigned as (20S)- 20
acetylhydroxylamine, 3β- amino, 13β hydroxymethylenepregn-5-ene and named
previously as holadysenterine (Kumar et al. 2007). Due to lack of amount, 13
C spectrum
was unpredictable, however some 2D NMR spectra (HMBC, 1H-
1H COSY) were
showing some signals of 13
C and from which final structure of compound 1 was
concluded.
Compound 2, (Z)- docos-5-en-1-oic acid was isolated as viscous oil. Molecular
formula was calculated from HREIMS, which gave [M+] peak at m/z 338.5702 (calcd.
338.5727) corresponding to the molecular formula C22H42O2. The peaks in EIMS
spectrum differ each by 14 mass indicating the aliphatic hydrocarbon nature of the
molecule. The 1H-NMR spectrum indicate the characteristic peaks at δ 5.25 as a doublet
of triplet integrated for 2H having J value 11.6 showing olefinic moiety in the molecule.
Terminal methyl group was appeared at δ 0.88 as a triplet with J = 6.8 Hz. A
characteristic triplet integrated for two protons appeared at δ 2.89 with J = 7.2 Hz
indicating the attachment of methylene with carboxylic moiety. An envelop of 34 protons
was appeared as broad singlet inferred for 17 methylene units at δ 1.32-1.84. Another
prominent 4 proton integrated signal was appeared at δ 2.21 indicated the attachment of 2
methylene with the olefinic band which was further confirmed in HMBC correlation. The
13C-NMR spectrum displayed signal at 180.1 while the olefinic bands were appeared at δ
130.1 and 127.5. The terminal methyl group was appeared at 14.1 while the remaining 19
methylene units appeared in between δ 22.1-38.8. The exact location of the double bond
93
in the molecule was confirmed by the loss of peaks in EIMS spectrum at m/z 251 and
265, 225 and 211 due to α and β fission of the double bonds, which confirmed its location
at C-5. The stereochemistry of the molecule was obtained by the calculated coupling
constant value, which indicates small J value, 11.6 Hz. On the basis of these evidences
and comparison with the literature data the molecule was identified as (Z)- docos-5-en-1-
oic acid (Misra and Wagner 2006).
94
Conclusions
In laboratory bioassays, culture filtrates of all the four test Drechslera species
showed variable herbicidal activity against weeds and wheat. Weeds were
generally more susceptible than wheat varieties.
In foliar spray bioassays, fungal culture filtrates significantly reduced the
growth of R. dentatus while the effect on other weed species and wheat varieties
was generally nonsignificant.
Under field conditions, culture filtrates of D. australiensis and D. hawaiiensis
reduced biomass of R. dentatus by 58% and 57%, consequently increased the
wheat grains yield by 22% and 21%, respectively.
Culture filtrates of D. australiensis exhibited the highest herbicidal activity.
Two herbicidal compounds were isolated from chloroform fraction of culture
filtrate of D. australiensis and identified as holadysenterine and (Z)- docos-5-
en-1-oic acid.
Future Prospects
The two identified compounds can be used as structural lead for the total
synthesis of natural product based eco-friendly analogues to be used as
commercial herbicides.
The shelf life of the identified compounds should be investigated.
Studies are required to investigate the genes responsible for the production of
effective herbicidal compounds; this may help to the boost up the production of
these compounds.
95
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