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Thesis FINAL PRINT Preface This study was conducted at the Department of Chemistry, Biotechnology and Food Science (IKBM), Norwegian University of Life Sciences (UMB) during November 2009 to November 2010. My supervisors were Professor Dr Arne Tronsmo and PhD student Md. Hafizur Rahman, Department of IKBM, UMB. I express my sincere appreciation and gratitude to Professor Arne Tronsmo and Dr. Linda Gordon Hjeljord for their dynamic guidance throughout the period of the study, constant encouragement, constructive criticism and valuable suggestion during preparation of the thesis. I also want to thank Md. Hafizur Rahman for his guidance during my research work. Moreover I express my sincere gratitude to Grethe Kobro and Else Maria Aasen and all other staffs and workers at IKBM, UMB for their helpful co-operation to complete the research work in the laboratory. Last but not the least; I feel the heartiest indebtedness to Sabine and my family members for their patient inspirations, sacrifices and never ending encouragement. Ås, January 2011 Latifur Rahman Shovan i Abstract This thesis has been focused on methods to control diseases caused by Botrytis cinerea. B. cinerea causes grey mould disease of strawberry and chickpea, as well as many other plants. The fungal isolates used were isolated from chickpea leaf (Gazipur, Bangladesh) or obtained from the Norwegian culture collections of Bioforsk (Ås) and IKBM (UMB). Both morphological and molecular characterization helped to identify the fungal isolates as Botrytis cinerea (B. cinerea 101 and B. cinerea-BD), Trichoderma atroviride, T. asperellum Alternaria brassicicola, and Mucor piriformis. The identity of one fungal isolate, which was obtained from the culture collection of Bioforsk under the name Microdochium majus, could not be confirmed in this study. Growth rate and morphology of the Trichoderma species and B. cinerea strains were affected by temperature. The optimum temperature for B. cinerea was found to be 250C and 25-300C for T. atroviride and T. asperellum. B. cinerea strains could grow at 50C and 300C, but their growth was very slow at those temperatures. T. asperellum could grow at 370C. Both B. cinerea strains germinated well in a pH range from 3 to 8.5 and the optimum pH of B. cinerea101 was 5. Assessment of antagonistic activity and sensitivity to fungicides of T. atroviride and T. asperellum were investigated using in vitro tests and it was found that the antagonistic properties of both Trichoderma isolates were more effective in reducing radial growth of B. cinerea, as well as other test fungi, in high nutrient media (PDA) than in low nutrient media (SNA). In most cases T. atroviride showed more antagonistic activity than T. asperellum. Water-soluble inhibitory metabolites produced by both Trichoderma isolates showed fungicidal and fungistatic activity. T. atroviride was highly sensitive to the fungicides Amistar and Signum. T. asperellum was not sensitive to low concentrations of Amistar and Signum. T. atroviride and T. asperellum were moderately sensitive to Switch. Teldor stimulated conidia germination of both Trichoderma isolates. Four fungicides and unhydrolyzed chitosan were evaluated to investigate their antifungal activity against B. cinerea 101 and other test pathogens in synthetic media. Switch and Signum successfully inhibited conidia germination of B. cinerea 101. Though Teldor did not inhibit conidia germination, it was very effective in inhibiting germ tube elongation at low concentration. Amistar was less effective against B. cinerea 101. Almost all of the fungicides were effective against the other test pathogens as well. Among the tested fungicides, Signum was the most effective fungicide against all of the test pathogens. Even 1/10 of the recommended dose of Signum completely inhibited conidia germination of all test fungal ii pathogens. Unhydrolyzed chitosan showed dose response effect against B. cinerea and 0.25% (vol/vol) unhydrolyzed chitosan completely inhibited conidia germination of B. cinerea. It was also effective against A. brassicicola. Hydrolysis did not improve the antifungal activity of chitosan against B. cinerea 101 in this study. Synergistic interactions of fungicides and chitosan on inhibition of conidia germination of the test fungi were studied in synthetic media and on detached strawberry flowers, with the aim to investigate if the effective concentrations of fungicides could be reduced when combined with chitosan. Combinations of chitosan and fungicides showed synergistic effect in reducing the germination and growth of B. cinerea 101, B. cinerea-BD and M. majus in synthetic media, and also on the disease infection rate, disease severity and sporulation of B. cinerea 101 in strawberry flowers. Combination of chitosan and Teldor showed better synergism than the other treatment combinations of chitosan and other fungicides on inhibition of conidia germination of B. cinerea 101 and B. cinerea-BD in synthetic media. Combination of chitosan with Teldor also showed better inhibition of B. cinerea 101 on detached strawberry flowers than combination of chitosan with other fungicides. iii Contents 1 Introduction .......................................................................................................................................... 1 1.1 Background .................................................................................................................................... 1 1.2 Botrytis cinerea .............................................................................................................................. 1 1.2.1 Taxonomy .......................................................................................................................... 1 1.2.2 Morphology ....................................................................................................................... 2 1.2.3 Disease cycle of Botrytis cinerea ....................................................................................... 2 1.2.4 Economical importance ..................................................................................................... 4 1.3 Microdochium majus ..................................................................................................................... 6 1.3.1 Taxonomy .......................................................................................................................... 6 1.3.2 Economic important .......................................................................................................... 6 1.4 Mucor piriformis ............................................................................................................................ 7 1.4.1 Taxonomy .......................................................................................................................... 7 1.4.2 Morphology ....................................................................................................................... 7 1.4.3 Economic importance ........................................................................................................ 7 1.5 Alternaria brassicicola ................................................................................................................... 8 1.5.1 Taxonomy .......................................................................................................................... 8 1.5.2 Morphology ....................................................................................................................... 8 1.5.3 Economic importance ........................................................................................................ 8 1.6 Disease management .................................................................................................................... 8 1.6.1 Cultural control .................................................................................................................. 8 1.6.2 Chemical control ................................................................................................................ 9 1.6.3 Biological control ............................................................................................................. 10 1.6.3.1 The need for biological control agents ............................................................ 10 1.6.3.2 Trichoderma as a Biological control agent....................................................... 11 1.6.3.3 Mechanism of action ....................................................................................... 11 1.6.4 Chitin............................................................................................................................... 12 1.6.4.1 Chitosan ........................................................................................................... 13 1.6.4.2 Chitosan as antimicrobial agents ..................................................................... 13 1.6.4.3 Mechanism of action ....................................................................................... 14 2 Aims of the study ................................................................................................................................ 14 3 Materials and Methods ...................................................................................................................... 15 3.1 Morphological and molecular characterization of the fungal isolates ....................................... 15 3.1.1 Collection of fungal isolates............................................................................................
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