Biocontrol of Cereal Pathogens

Shivaditya Gautam

DISSERTATION.COM

Boca Raton

Biocontrol of Cereal Pathogens Copyright © 2009 Shivaditya Gautam All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission from the publisher. Dissertation.com Boca Raton, Florida USA • 2010 ISBN-10: 1-59942-347-2 ISBN-13: 978-1-59942-347-0

Biocontrol Of Cereal Pathogens

ABSTRACT

Septoria leaf blotch has been the major disease of wheat in Britain and much of the rest of Europe. It was reported that the disease cause serious yield losses to range from 31 to 53%. Mycosphaerella graminicola (anamorph: Septoria tritici) is the pathogen which causes Septoria leaf blotch. The disease can be controlled by various methods such as cultural practices, chemical control, using resistant varieties and biological control. In plant pathology, the term biological control leads to the introduction of microbial antagonists or host specific pathogens to suppress diseases and populations of one or more plant pathogens. This study investigated the microbial community on and within wheat leaves which can suppress Septoria leaf blotch by reducing the inoculum level of the causative pathogen M. graminicola. Plate count and DGGE analysis techniques were used to assess the microbial community. The changes in the microbial populations of healthy, senescent and M. graminicola infected i.e. diseased wheat leaves were investigated in vitro on agar plates using plate colony count method. It was found that method was successful in assessing both bacterial and fungal population sizes showing distinct colonies. DGGE is a rapid method which can analyze large number of samples simultaneously. The bands appearing in DGGE profile represent different species present in the microbial population. The DGGE technique was successfully used in this study to assess bacterial community. Bioinformatics tools have also played a vital role in this study for identifying bacterial and fungal species. Total five bacterial species and five fungal species were identified by bioinformatics tools. The antagonistic abilities of identified bacterial and fungal species were tested against a Septoria isolate in vitro on dual culture PDA plates. Four microorganisms (Fusarium sp., Verticillium sp., Penicillium sp., Sporobolomyces sp. and Microbotryum sp.) in the dual cultures vary in their colony length, width, ratio of length and width and distance bwtween them. But after satatistical analysis all the values are insignificant observations of dual culture plates indicated that the microorganisms might inhibit Septoria by competing

Biocontrol Of Cereal Pathogens

for space. These microorganisms after testing might be good candidates for further in vivo testing of Septoria inhibition. A thorough knowledge of wheat leaf microbial communities is required for further use of biocontrol in the future.

Biocontrol Of Cereal Pathogens

TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….…4 LIST OF TABLES…………………………………………………………………...7 CHAPTER 1 INTRODUCTION & LITERATURE REVIEW…………….…….8 1.1. Septoria leaf blotch……………………………………………………………....8 1.1.1. Pathogen………………………………………………………………………8 1.1.2. Hosts………….…………………………………………………….…………9 1.1.3. Symptoms…………………….……………………………………...……......9 1.1.4. Life cycle………………………………………………………………..…...11 1.2. Today’s challenge, Septoria leaf blotch…………………………………….....12 1.3. Septoria tritici epidemiology…………………………………………..………12 1.4. Control……………………………………………………………………….…13 1.4.1. Resistant Varieties……………………………………………….………….13 1.4.2. Cultural Practices…………………………………………………….……...13 1.4.3. Chemical Control……………………………………………………………14 1.5. Biological control of plant diseases…………………………...……………….14 1.5.1. Mutualism…………………………………………………….…………….15 1.5.2. Protocooperation…………………………………………………….......….15 1.5.3. Commensalism……………………………………………………………...16 1.5.4. Neutralism…………………………………………………………………..16 1.5.5. Competition………………………………………………………………....16 1.5.6. Parasitism …………………………………………………………………..17 1.5.7. Predation………………………………………...………………………….17 1.6. Biocontrol of wheat pathogens…………………...……………………………18 1.6.1. Fungal organisms as biocontrol agent against wheat pathogens…….………18 1.6.2. Yeast species as biocontrol agent against wheat pathogens……...…….....…19 1.6.3. Bacterial organisms as biocontrol agent against wheat pathogens…………..19 1.7. Focus of the current project…………..………………………………………..20 1.7.1. Assessing microbial communities……………………..……………………..20 -1-

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1.7.2. Screening microorganisms from microbial communities for disease control…...21 1.8. Aims of this study………………………………………………………….……….22 CHAPTER 2 MATERIAL & METHODS…………………………………………....23 2.1. Sampling of wheat plants………………………………………………………….23 2.2 Colony counts…………………………………………………………………...…..24 2.2.1.Recovery of leaf-associated microorganisms………………………………….....24 2.3. DNA Extraction……………………………………………………………..……...25 2.3.1. CTAB Method (Using Liquid Nitrogen)………………………………………...25 2.3.2. CTAB method for Soil samples (Using Glass Beads)………….………………..26 2.3.3. Sub cultured plates…………………..…………………………………………....27 2.3.4. Whole Leaf Extracts………….……….………………………………………….27 2.4. PCR Amplification………………………………………………………………...28 2.4.1. Bacterial 16S rDNA………..…………………………………………………...28 2.4.2. Fungal 18S rDNA…………...…………………………………………………..28 2.5. Analysis of PCR products………………………………………………………...29 2.6. Sequencing…………………………………………………………………………30 2.7. Analyzing bacterial community………………………………………………….30 2.7.1. DGGE Analysis………………………………………………………………...30 2.7.2. Sequencing of DGGE gene fragments………………………………………….31 2.8. Identification of microorganism using Bioinformatics tools…………………...31 2.8.1. 16S Bacterial samples………………………………………………………….31 2.8.2. 18S Fungal samples……………………………………………………………32 2.9. Antagonism Tests………………………………………………………………...32 2.9.1. Septoria growth inhibition trial………………………………………………..32 2.9.2. Statistical Analysis…………………………………………………………….33 CHAPTER 3 RESULTS……………………………………………………………...34 3.1 Colony counts……………………………………………………………………..34 3.2. DNA Extraction…………………………………………………………………..37 3.2.1. CTAB Method………………………………………………………………...37 -2-

Biocontrol Of Cereal Pathogens

3.2.2. Sub cultured plates………………………………………………………………37 3.2.3. Whole Leaf Extracts……………………………………………………………..43 3.3. PCR Amplification…………………………………………………………………44 3.3.1. Bacterial 16S rDNA…….……………………………………………………….44 3.3.2. Fungal 18S r DNA……….……………………………………………………...46 3.4. Analyzing bacterial community………………………………………………..…47 3.4.1. DGGE Analysis…………………………………………………………………47 3.4.2. Sequencing of DGGE gene fragments…………………………………………..49 3.5. Identification of microorganism using Bioinformatics tools…………………….51 3.5.1. 16S Bacterial samples…………………………………………………………...51 3.5.2. 18S Fungal sample………………………………………………………………55 3.6. Antagonism Tests………………………………………………………………….57 3.6.1. Septoria growth inhibition trial………………...…………………………...…..57 3.6.2. Statistical Analysis……………………………………………………………...63 CHAPTER 4 DISCUSSION…………………………………………………………..64 4.1. DNA Extraction…………………………………………………………………...64 4.2. Identification of microorganisms………………………………………………...65 4.3. DGGE analysis…………………………………………………………………….68 4.4. Antagonism test…………………………………………………………………...69 CHAPTER 5 CONCLUSION……………………………………………...…………71 ACKNOWLEDGEMENTS…………………………………………………………...72 REFERENCES………………………………………………………………………...73 APPENDICES…………………………………………………………………………85 Appendix 1. Reagent and gel preparation……………………………………………85 Appendix 2. TSA Bacterial Plates…………………………………………………….88 Appendix 3. PDA Fungal Plates………………………………………………………89 Appendix 4. Mycosphaerella graminicola isolate plate.……..…………………..…..91 Appendix 5. Analysis of variance (ANOVA) tables from statistical analysis of antagonist plates……………………………………………………………………….92 -3-

Biocontrol Of Cereal Pathogens

LIST OF FIGURES Main Document Figure 1. Images of Septoria leaf blotch on wheat leaves…………...……..…..…..10 Figure 2. Life cycle of Septoria tritici……..…………………………...…..……….11 Figure 3. Wheat planting plan……………………………………………..………..23 Figure 4. Dual culture antagonism test…………….………………….………..…...33 Figure 5. TSA plates with subcultured bacteria..….………………...…..…………..38 Figure 6. Agarose gel analysis of bacterial DNA……..….……………………….…39 Figure 7. PDA plates with subcultured fungi……………………………………..…41 Figure 8. Agarose gel analysis of fungal DNA…………………………………..….42 Figure 9. Agarose gel analysis of whole leaf extract samples…..………….....……..43 Figure 10. Agarose gel analysis of bacterial 16S rDNA gene fragments obtained after first amplification…………………………………...………………………………..44 Figure 11. Agarose gel analysis of bacterial 16S rDNA gene fragments obtained after second amplification using GC clamp VFC/VR primers…….………….……..45 Figure 12. Agarose gel analysis of fungal 18S rDNA gene fragments obtained after amplification…………...…………………………………………...….……………..46 Figure 13. DGGE analysis of bacterial community from wheat leaves……….….....48 Figure 14. Part of Band D1 (from DGGE gel, figure 13, section 3.4.1) DNA sequence viewed in FinchTV...…………………………………………..………………..……49 Figure 15. Part of Band D2 (from DGGE gel, figure 13, section 3.4.1) DNA sequence viewed in FinchTV…...………………………………………………………..……..49 Figure 16. Part of Band D3 (from DGGE gel, figure 13, section 3.4.1) DNA sequence viewed in FinchTV……...……………………………………………………..……..50 Figure 17. Part of Band D4 (from DGGE gel, figure 13, section 3.4.1) DNA sequence viewed in FinchTV……...……………………………….……………………..…….50 Figure 18. Part of Band D5 (from DGGE gel, figure 13, section 3.4.1) DNA sequence viewed in FinchTV...………………………………………….………………..…….50 Figure 19. Part of Band D6 (from DGGE gel, figure 13, section 3.4.1) DNA sequence -4-

Biocontrol Of Cereal Pathogens

viewed in FinchTV………………….………………………………………………..51 Figure 20. The bootstrap neighbour joining tree constructed from forward primer sequences of B1, B2, B3, B4 and B5 bacterial 16S rDNA samples using ClustalX...……………………………………………………………….….………..53 Figure 21. The bootstrap neighbour joining tree constructed from reverse complement primer sequences of B1, B2, B3, B4 and B5 bacterial 16S rDNA samples using ClustalX…………...……………………………………………….…..……....54 Figure 22. The bootstrap neighbour joining tree constructed from consensus sequences of F1, F2, F3, F4, F6 and F7 fungal 18S rDNA samples using ClustalX...56 Figure 23. Length and width of different antagonist colony………………………...57 Figure 24. Length and width of different Septoria colony..........................................58 Figure 25. Ratio of length and width of fungal antagonist colony……………….….59 Figure 26. Ratio of length and width of fungal Septoria colony………………...…..60 Figure 27. Distance between antagonist and Septoria colony………………….....…61 Figure 28. Septoria (SP1, SP2, SP3, SP4, SP6 and SP7) growth inhibition trial……62

Appendices Figure 1. R1, gd, 10-3 ……………………………………………………………….88 Figure 2. R1, gh, 10-1……………………………………………….……………….88 Figure 3. R1, s, 10-3 ………………………………………………...……………….88 Figure 4. R2, gd, 10-3…………………………………………….………………….88 Figure 5. R2, gh, 10-3………………………………………….…………………….88 Figure 6. R2, s, 10-3…………………………………………………………………88 Figure 7. R1, gd, 10-3, 17/06/09…………….……………………………………….89 Figure 8. R1, gh, 10-3, 17/06/09…………….……………………………………….89 Figure 9. R1, s, 10-3, 17/06/09……………….……………………………...……….89 Figure 10. R2, gd, 10-3, 18/06/09……………………………………………………89 Figure 11. R2, gh, 10-3, 18/06/09………………………………………...………….89 Figure 12. R2, s, 10-3, 18/06/09……………………………………………………...89 -5-

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Figure 13. R1, gd, 10-3, 19/06/09……………………………………………………90 Figure 14. R1, gh, 10-3, 19/06/09…………………………………………………....90 Figure 15. R1, s, 10-3, 19/06/09…………………………………………………..….90 Figure 16. R2, gd, 10-3, 19/06/09…...……………………………………………….90 Figure 17. R2, gh, 10-3, 19/06/09………………………………………………...….90 Figure 18. R2, s, 10-3, 19/06/09……………………………………………………...90 Figure 19. Control plate of Septoria isolate (CBS 100332)…………………………91

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LIST OF TABLES

Main Document Table 1. Leaf sampling categories………….………...……………...……………….24 Table 2. PCR primer sequences and PCR conditions for the amplification of 16S bacterial and 18S fungal rDNA..………………………………….……………....….29 Table 3. Colony Count on TSA Bacterial Plates……….……………………………35 Table 4. Colony Count on PDA Fungal Plates………….…………………………...36 Table 5. Bacterial isolates used for DNA extraction………………………..………37 Table 6. Fungal isolates used for DNA extraction……….....………………………40 Table 7. Bacterial species identified from BLAST search at http://bioinfo.unice.fr/blast/.…..............................................………………………..53 Table 8. One-way analysis of variance of the colony growth of Septoria tritici isolates……………………………………..…………………………………..…….63

Appendices Table 1. Phosphate buffer reagents…...………………………….……………….….85 Table 2. CTAB buffer and 25% PEG reagents…………...…………..…………...…86 Table 3. Reagents for a 10% (wt/v) polyacrylamide gel with a 30-60% denaturing gradients…..…………………..……………………………………………………...87

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Biocontrol Of Cereal Pathogens

CHAPTER-1 INTRODUCTION & LITERATURE REVIEW

1.1. Septoria leaf blotch

Septoria leaf blotch, also known as speckled leaf blotch, is caused by the fungus Septoria tritici (Wolf, 2008). The disease is widely spread in all wheat-growing areas of the world and is also a major problem in many regions (Van Ginkel et al. 1999). Septoria leaf blotch has been the major disease of wheat in Britain and much of the rest of Europe for nearly two decades (Pillinger et al. 2004). Septoria leaf blotch has the potential to cause serious losses if the environmental conditions are favourable for its spread during late May and June (Lipps et al. 2002). It was reported that the disease cause serious yield losses to range from 31 to 53 % (Eyal, 1981; Babadoost and Herbert, 1984; Polley and Thomas, 1991). This disease is common on wheat in the tillering stages but after ear emergance the disease becomes quite severe on the upper leaves (Jenkins et al.1969).

1.1.1. Pathogen Mycosphaerella graminicola (anamorph: Septoria tritici) is the pathogen which causes Septoria leaf blotch (Eyal, 1987; Eyal, 1999). The pathogen is only found on foliar parts of infected plants. It is a haploid, hemibiotrophic ascomycete having both filamentous and yeast-like growth phases i.e. yeast-like growth at 15°C and mycelial growth at 25°C (Joint Genome Institute 2008). M. graminicola can be easily cultured on both liquid and solid media. Kema et al. (1996) showed that the crosses on wheat leaves were successfully achieved in their study and found the bipolar heterothallic mating system in M. graminicola. The population genetics of M. graminicola is better understood than any other fungus due to natural occurrence of sexual recombination several times per year (Joint Genome Institute, 2008).

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Biocontrol Of Cereal Pathogens

Classification: (Source: Index of Fungorum)

Kingdom:

Fungi

Phylum:

Ascomycota

Class:

Dothideomycetes

Subclass:

Dothideomycetidae

Order:

Capnodiales

Family:

Mycosphaerellaceae

Genus:

Mycosphaerella

Species:

Graminicola

1.1.2. Hosts Major host is wheat, but also occurs on rye, triticale and some grass species (HGCA, 2007).

1.1.3 Symptoms Symptoms of Septoria leaf blotch are commonly visible in the beginning of growing season whereas lesions of Septoria tritici usually appear on young autumn-sown wheat. These lesions are elongate ovals which runs parallel to leaf veins. By early December grey water-soaked patches are apparent on the lowest leaves which quickly turns brown and necrotic (Murray et al. 1998). These patches have visible black pycnidia which are mainly characteristic feature of M. graminicola (HGCA, 2007). The presence of pycnidia is a good diagnostic characteristic (Nyvall, 1999). Pycnidia are most frequent on dead overwintering leaves of winter wheat where they are embedded in lesions, which have ashen centres (Maloy & Inglis, 1993). On mature plants, these lesions are brown and can be restricted by veins giving rectangular appearance which may combine leading to large

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areas of necrotic brown tissue (HGCA, 2007). Severely diseased leaves become yellow and die prematurely. Sometimes the entire plant may be killed.

A

B

C

D

Figure 1. Images of Septoria leaf blotch on wheat leaves. (A). Symptoms of Septoria tritici. (B). Brown necrotic lesions on young wheat plant showing black pycnidia. (C). Lesions consisting of brown necrotic tissue. (D). Coalesced lesions giving large areas of necrotic brown tissue. (Source: Cereal Disease Encyclopedia, HGCA, 2007)

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Biocontrol Of Cereal Pathogens

1.1.4.Life cycle The life cycle of M. graminicola is favoured by optimum temperatures (15-20°C), and to cause infection it requires long periods of high humidity (HGCA, 2007). The Septoria leaf blotch has two conspicuous phases. First during the winter on the basal leaves of autumn sown crops which normally infected by long distance spread of airborne ascospores, and during the summer on upper leaves of host plants (Nyvall, 1999). Initial infections are caused from wind-borne ascospores which are released from pseudothecia and asexually produced water splash-dispersed pycnidiospores produced from pycnidia (Murray et al. 1998). Infection requires at least 6 hours or more hours of leaf wetness, for maximum infection. After occurrence of infection, the fungus develops black fruiting bodies in 21 to 28 days and produces a new generation of spores. The spores produced in these fruiting bodies are discharged in sticky masses and through rain these spores are splashed onto the upper leaves and heads (Wolf, 2008). Septoria leaf blotch can become a serious disease during environmental conditions that are conducive for disease development (Nyvall, 1999).

Figure 2. Life cycle of Septoria tritici (Source: Cereal Disease Encyclopedia, HGCA, 2007).

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Biocontrol Of Cereal Pathogens

1.2.Today’s challenge, Septoria Leaf Blotch

In recent years, Septoria leaf blotch disease is widely spread in winter wheat crops and it is considered as the major factor of losses in the wheat crops in many countries (Lucas, 1998). In epiphytotic years crop yield losses due to this disease may be up to 20-30% of the total. It also worsens grain quality. For wheat breeders Septoria leaf blotch has emerged as a challenging disease (Zadoks, 2004). A history of Septoria leaf blotch shows that from the last century it is a serious problem about every 25 years, once per human generation (Zadoks, 2004). Zadoks (2004) showed that before 1900 severe epidemics of Septoria tritici occurred in some parts of europe but during 1970 Septoria tritici had a world wide attack and it is still a frightening fungus. Lucas (1998) deduced that there are several factors associated with the increased severity of Septoria leaf blotch such as earlier sowing of the crop, direct drilling into crop residues which may carry inoculum, and the use of dwarf varieties which are more likely to get infection.

1.3. Septoria tritici epidemiology

The primary sources of inoculum in the epidemics of Septoria leaf blotch are airborne ascospores and rain splash distributed asexual pycnidiospores from infected plant debris and possibly seed-borne infections (Eyal, 1999). Rain-splashed pycnidiospores are transported vertically upward from the base of the crop to the upper leaves where they germinate and penetrate through stomata and causes infection (Hunter et al 1999). Ascospore discharge depends on various environmental factors such as rain duration, intensity, wind and temperature. The teleomorph stage (Mycosphaerella graminicola) is also known to play an important role in the disease cycle. It was first described in the UK by Scott et al. (1988). M. graminicola ascospores contribute to the genetic diversity of the fungal population (Hunter et al. 1999). In comparison to other wheat foliar pathogens M. graminicola has relatively long latent period i.e. ranging between 11-42 days. The period between infection and the first appearance of sporulating structures is generally known as - 12 -

Biocontrol Of Cereal Pathogens

latent period (Shearer & Zadoks, 1972). The symptoms are usually developed under strong influence of environmental conditions such as moisture, optimum temperature in the range 18-25°C and light (Lovell et al. 2004). During a growing season S. tritici is able to produce several sexual cycles and ascospore generations (Kema et al. 1996).

1.4 Control

1.4.1. Resistant Varieties Resistant varieties are the best control for Septoria tritici blotch in winter wheat but only a moderate degree of resistance is exhibited in the field and several popular varieties in the Europe have partial resistance (Murray et al. 1998). Several genes regulate resistance to the leaf blotch phases of the disease. Although by providing resistance to the foliar phase serious yield losses can be prevented (Wolf, 2008). The varieties which are very prone to this disease have to be avoided as they will build up inoculum levels and cause yield loss in that variety and also in close susceptible wheat crops (DPI, 2008).

1.4.2. Cultural Practices Cultural control of Septoria leaf blotch includes crop rotation or the disposal of contaminated wheat crop debris by burning or ploughing will reduce the number of ascospores which infect the new season's crop (Murray et al. 1998) Though burning of crop debris is illegal in U.K. but still an alternative to control disease. In order to reduce the amount of inoculum crops are rotated out of cereals and grasses for 3-4 years, wheat stubble are deeply ploughed and volunteer hosts are destroyed. Even disease risk are higher with minimum tillage practices. Septoria diseases are predominant when wheat is heavily fertilized and have dense foliage (Maloy & Inglis, 1993). By crop rotation or destruction of residues of earlier wheat crops Septoria tritici blotch cannot be fully controlled as it partially effect the severity of the disease (Wolf, 2008). Cultural practices will have more effect if practiced on a district basis. In order to prevent erosion cultural practices are not used in light soil areas where stubble is stored (DPI, 2008). - 13 -

Biocontrol Of Cereal Pathogens

1.4.3. Chemical Control Chemical control of the disease is widely practiced in Europe. The application of azole based fungicides such as triazoles, usually to the flag leaf of wheat, can be highly effective in reducing Septoria tritici blotch (Murray et al. 1998). Fungicidal treatment on seed can also reduce disease severity (Maloy & Inglis, 1993). Before spraying fungicide it is important to correctly identify Septoria tritici blotch as nutritional disorders such as aluminium toxicity can be confused with Septoria tritici blotch (DPI, 2008). Moreover before application of foliar fungicide, the important thing to keep in mind is to protect the last two leaves because these leaves supply most of the energy which is required in production of grain. In order to protect of these leaves the best way is to apply the fungicide between emergence of the flag leaf and the beginning of flower (Wolf, 2008). There is a widespread resistance to the benzimidazole (MBC) group of fungicides in UK populations of Septoria tritici (Murray et al. 1998). There are various foliar fungicides which are effective against Septoria leaf blotch such as Bumper, Headline, Proline, Propimax, Quilt, Quadris, Stratego, and Tilt (DPI, 2008). These fungicides belong to strobilurins and triazoles chemical group. Early applications of foliar fungicides and fungicide seed treatments can only provide partial protection from Septoria leaf blotch during early season, as these applications fails to protect the upper canopy during grain fill when the plants are more prone to disease (Wolf, 2008).

1.5. Biological control of plant diseases

Any living organism that can be manipulated by man to suppress the pest or pathogen is known as the biological control agent or BCA (Baker & Cook, 1974). In plant pathology, the term biological control leads to the introduction of microbial antagonists or host specific pathogens to suppress diseases and populations of one or more plant pathogens (Pal & Gardener, 2006). There are various factors implied in biological control such as host, pathogen or parasite, physical environment and antagonists. Biological control also involves the use of microbial inoculants in suppression of single type of plant disease. - 14 -

Biocontrol Of Cereal Pathogens

Although diseases can be suppressed by managing soils in order to raise the combined activities of native soil and plant associated organisms (Pal & Gardener, 2006). Moreover to understand the mechanisms of biological control, it is very useful to know the different ways through which organisms interact. All plants and pathogens throughout their life cycle interact with a wide variety of organisms by direct or indirect contact. These interactions have drastic affect on plant health (Pal & Gardener, 2006). The interactions of two populations are defined by the outcomes for each (Odum et al. 1953). There are various types of interactions such as mutualism, protocooperation, commensalism, neutralism, competition, parasitism, and predation.

1.5.1. Mutualism Mutualism is a biological interaction between two organisms that benefits both individuals, such as increased survival. Sometimes, it also becomes a lifelong interaction, which involves close physical and biochemical contact for example mutuality between plants and mycorrhizal fungi. (Pal & Gardener, 2006). Mycorrhizas are symbiotic relationship between rhizosphere fungi and the roots of plants. Many studies have shown that exchange of mineral and other organic resources between plant and fungal symbionts has benefited both individuals (Johnson et al. 1997). However, they are generally facultative and opportunistic. These types of mutualistic association led to biological control, by strengthening the plant with improved nutrition or by stimulating host defenses.

1.5.2. Protocooperation Protocooperation is a form of mutualism in which the organisms involved is not fully dependant on each other for survival. Examples are protocooperation between soil bacteria or fungi, and the plants that occur growing in the soil. Soil bacteria and fungi interrelate with each other, producing nutrients essential to the plant’s survival. Plants benefit by getting essential mineral nutrients and carbon dioxide from root nodules and decomposing organic substances. Fowler et al. (1989) showed protocooperation between - 15 -

Biocontrol Of Cereal Pathogens

soil inhabiting mole crickets and nematophagous fungi in South America. This protocooperation depends on the parasitic nematode of mole crickets, which has proven to be a promising biological control agent against pest insects. A lot of isolated microorganisms which are classified as biological control agents can be considered as mutualists involved in protocooperation, because their survival does not depends on any specific host and disease suppression also varies with the prevailing environmental conditions (Pal & Gardener, 2006).

1.5.3. Commensalism It is a symbiotic interaction between two living organisms, where one individual benefits and the other is unaffected. Mostly microorganisms which are plant-associated are supposed to be commensals in relation to the host plant (Pal & Gardener, 2006). Peterson et al. (2006) showed that peptidoglycan from Bacillus cereus mediates commensalism with rhizosphere bacteria from the Cytophaga-Flavobacterium group and also enhances Cytophaga-Flavobacterium rhizosphere isolates growth in root exudate medium.

1.5.4. Neutralism It is the biological interaction when the organisms occupy different and non-overlapping niches in the same microhabitat together without affecting each other (Campbell, 1989). Although both species are not affected by the population density of each other. In terms of biological control, an inability to associate the population dynamics of a pathogen with that of another organism shows neutralism (Pal & Gardener, 2006). In contrast, antagonism between organisms results in a negative outcome for one or both.

1.5.5. Competition Competition is defined as an interaction between organisms in which one organism is harmed by the presence of another and also reduces the activity, growth rate and fertility of the interacting organisms (Begon et al. 1996). Biological control occurs when nonpathogens compete with pathogens for nutrients in and around the host plant. The - 16 -

Biocontrol Of Cereal Pathogens

understanding of biological control is also affected by the direct interactions that benefit one population at the expense of another (Pal & Gardener, 2006).

1.5.6. Parasitism It is a type of symbiotic correlation between two unrelated organisms where one organism (the parasite) benefits or obtains its food from the host, and both organisms coexist over a prolonged period of time (Campbell, 1989). In this type of interaction parasite benefits from host for growth and reproduction. It also emphasizes that excessive harm done to a host by parasite makes host less competitive and also endangers the survival of the parasite species. The activities of various hyperparasites that parasitize plant pathogens can result in biocontrol (Pal & Gardener, 2006).

1.5.7. Predation Predation is an interaction between organisms in which biomass is captured by one organism from another for consumption and sustenance (Begon et al. 1996). Predators are generally animals that feed at higher trophic levels in the macroscopic world such as microarthropods and fungal feeding nematodes that consume pathogen biomass for sustenance (Pal & Gardener, 2006).

From all of these types of interactions, biological control results in varying degrees depending on the environment within which they occur. Biological control commonly arises from manipulating antagonisms between microorganisms and pathogens or from manipulating mutualisms between microorganisms and their plant hosts (Pal & Gardener, 2006).

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1.6. Biocontrol of wheat pathogens

1.6.1. Fungal organisms as biocontrol agents against wheat pathogens Most of the colonizers on leaves of cereals are capable of antagonism (Fokkema, 1978). Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens (Green et al. 1999). It is an antagonistic fungus that is widely recognized as a potential biocontrol agent against several soilborne plant pathogens (Jensen et al. 1995 & Papavizas, 1985). Trichoderma harzianum isolates have shown potential to reduce levels of fusarium head blight of wheat by reducing perithecial and ascospore production of Gibberella zeae on wheat straw (Inch & Gilbert, 2007). One Trichoderma isolate T-22, is registered as a biocontrol agent in the USA. In their study it was found that when T-22 strain was inoculated with G. zeae it reduced perithecial formation by 70%. Application of biocontrol agents to wheat residues has proven to be useful for controlling Septoria leaf blotch (Perelló et al. 2009). Trichoderma harzianum, T. aureovide and T. koningii isolates have potential to inhibit mycelial growth of the necrotrophic pathogen Pyrenophora tritici-ripentis, causing tan spot of wheat (Perelló et al. 2003).

Trichoderma spp. is a most successful antagonistic agent used for biocontrol of pathogens on plant surfaces of cruciferous, solanaceous and gramineous plants (Perelló et al. 2006). The earlier studies of Perelló et al. (1997, 2003) have shown biocontrol potential of Trichoderma isolates to suppress P. tritici-repentis and M. graminicola on wheat under laboratory experiments and greenhouse conditions. Most recently Perelló et al. (2006) found that on application of Trichoderma isolates in some trial pots it gave disease control comparable to that achieved with conventional fungicides. It is believed that T. harzianum acts as an effective antagonist against Septoria leaf blotch by causing a biochemical induced response in wheat plants (Cordo et al. 2007).

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Biocontrol Of Cereal Pathogens

1.6.2. Yeast species as biocontrol agent against wheat pathogens Some commonly found yeast species have also proven to be good biocontrol agents against wheat pathogens. These include Aureobasidium sp., Sporobolomyces sp., and Cryptococcus sp. which has shown antagonistic, effects towards foliar pathogens (Fokemma et al. 1976). Fokemma et al. (1976) showed that these yeast species have reduced the superficial mycelial growth of Septoria nodorum and the infection of wheat leaves by 50%. The studies have also revealed that strain J121 of the yeast Pichia anomala reduces Penicillium roqueforti growth on wheat cereal grains (Druvefors & Schnurer, 2005).

1.6.3. Bacterial organisms as biocontrol agent against wheat pathogens Besides fungal and yeast bacterial species have also played a vital role in suppression of wheat diseases as biocontrol agents. A Bacillus licheniformis strain when inoculated with Pyrenophora trichostoma reduces tan spot disease of wheat (Ghazanfari & Gough, 1981). Ghazanfari & Gough (1981) showed that Bacillus strains have great potential to suppress wheat foliar diseases. Other bacterial species such as Pseudomonas sp. isolated from soil environment also have ability to reduce disease levels of two foliar wheat pathogens (Levy et al. 1989). Levy et al. (1989) concluded that a fluorescent Pseudomonas sp. strain when inoculated onto wheat seedlings has reduced Septoria tritici disease by 88% and Puccinia recondita by 98%.

It is clear from the work discussed that phyllosphere microorganisms have potential for biocontrol of foliar diseases. It will also be a promising strategy in managing Septoria leaf blotch in the fields which has been the major disease of wheat in Britain and much of the rest of Europe for nearly two decades (Perelló et al. 2009).

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Biocontrol Of Cereal Pathogens

1.7. Focus of the current project

An increasing volume of literature on plant-associated microorganisms as biocontrol agents shows that these agents have great potential to control plant diseases. But due to multiple interactions between plant pathogens and biocontrol agents it is an intricate process to identify and assess biocontrol agents which can successfully control plant disease. So the focus of the current project is to study the microbial community on and within wheat leaves which could suppress Septoria leaf blotch by reducing the inoculum level of the causative pathogen M. graminicola. To study plant microbial community initial step is to assess the potential of resident microflora for biocontrol of plant diseases.

1.7.1. Assessing microbial communities Researchers have used several methods for analyzing structure and diversity of microbial communities Øvreås et al. (1997). These methods were categorized into two groups, biochemical-based techniques for example plate count and molecular-based techniques for example DGGE analysis (Kirk et al. 2004). Kirk et al. (2004) showed that these techniques used for assessing microbial communities are not very accurate and reliable due to their limitations. There are several advantages and disadvantages associated with both methods such as that the plate count method is fast and cheap method but cannot detect unculturable microorganisms (Kirk et al. 2004), on the other hand DGGE is a rapid method which can analyze large number of samples simultaneously but can only detect dominant species and each band can represent more than one species (Kirk et al. 2004). Legard et al. (1994) investigated seasonal changes in the microbial populations of spring wheat and found that both bacterial and fungal population sizes could be assessed successfully using plate colony count method whereas Øvreås et al. (1997) showed that plate count method is an incomplete method for assessing biodiversity, as less than 1% of the bacterial population was cultivable on the selected media.

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Biocontrol Of Cereal Pathogens

Moreover, in case of molecular techniques denaturing gradient gel electrophoresis (DGGE) is used for comparing rDNA gene fragments of microbial communities (Saito et al. 2007). To generate the rDNA gene fragments for DGGE PCR amplification was done with total extracted community DNA samples using universal bacterial and fungal primers (Øvreås et al. 1997; Lane 1991; Muyzer et al. 1993; Borneman & Hartin 2000). DGGE separates gene fragments of the same length but with different base-pair sequence on the basis of their melting properties which are sequence specific (Muyzer et al. 1993). Muyzer et al. (1993) showed that all the bands appearing in DGGE profile represent different species present in the microbial population. They also proved by their study that it is possible to identify a species that represents only 1% of the total population. Thus DGGE is a useful method for studying bacterial and fungal communities.

Although limitations were associated with both methods used for assessing these microbial communities, but still both the biochemical technique i.e. plate count and molecular techniques i.e. DGGE were successfully used to assess microbial communities in terms of size and diversity (Nakatsu et al. 2000). Therefore in this project also plate count and DGGE technique is used for assessing microbial community.

1.7.2. Screening microorganisms from microbial communities for disease control For screening the antagonistic activity of microorganisms towards plant pathogens, a coculture of the test pathogen and the antagonist was established in vitro on agar plates (Gachomo et al. 2008). It is a simple and inexpensive method mostly used as a prelude to more complex in vivo studies of antagonism. For assessment of antagonistic ability of microorganisms against pathogen the time needed for colony overgrowth is considered as an important parameter (Begum et al. 2008). Innocenti et al. (2003) showed that success of biocontrol measures depends on the characteristics of both pathogen and antagonist microorganisms. Agar plate dual cultures have been widely used in many studies to observe the specific effects of particular pathogen-antagonist pairings on pathogen growth and spore production (Andrews et al. 1983; Begum et al. 2008; Gachomo et al. - 21 -