Aspects of the Ecology and Population Dynamics of the Fungus Beauveria Bassiana Strain F418 in Soil

Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Aspects of the ecology and population dynamics of the fungus Beauveria bassiana strain F418 in soil A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy At Lincoln University By Céline Blond Lincoln University 2012 Abstract Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. Abstract Aspects of the ecology and population dynamics of the fungus Beauveria bassiana strain F418 in soil by Céline Blond This research aimed to improve understanding of the ecology of Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) strain F418 in soil, aided by the use of gfp transformants, to improve the use of this fungus as a biopesticide in New Zealand pastures. Prior to using B. bassiana F418 gfp transformants (F418 gfp tr1 and F418 gfp tr3), their phenotypes were comprehensively compared to the wild-type F418. Compared to F418, F418 gfp tr3 had a faster rate of germination at 15 °C for 24 h and 20 °C for 14 h and F418 gfp tr1 had a slower rate of germination at 25 °C for 14 h; however this was not apparent at longer incubation times at all temperatures. F418 gfp tr3 colony growth rate was slower than F418 at 15 and 20 °C but it was not different at 25 °C. There was no difference in conidial production and conidia size at different incubation temperatures. There were no differences in virulence against Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae and conidia production on cadavers using different fungal concentrations, T. molitor larval size or using either single or mixed fungal strains. F418 gfp tr1 phenotypical characteristics were more similar to F418 than F418 gfp tr3, however, F418 gfp tr3 fluorescence was substantially brighter than F418 gfp tr1. Therefore, both transformed strains could be used as substitutes for the wild-type in ecological studies. In an effort to explain the observed differences, an attempt was made to find the insertion point of the gfp cassette inside the transformant genomes. F418 persistence was initially assessed in microcosms of pasteurised soil under eight different abiotic, biotic and pre-application factors. F418 persistence was better with (i) low water content (14 and 22 % gravimetric water content) compared to high water content (31 % gravimetric water content), (ii) soil at 10 and 15 °C compared to 20 °C, (iii) soil containing 7.5 % peat compared to 0 and 15 % peat and (iv) superphosphate fertiliser than with nitrogen. Persistence of F418 was lower in the presence of non-host insects than in soil without insects. Generally there was an increase in F418 populations in the first few weeks in ii pasteurised soil. The results using the transformants with different moisture levels, fertilisers and the presence or absence of insects were similar to those observed for F418 in pasteurised soil but the findings were different in non-sterile soil. Moisture levels and presence of host, non-host and no insects were chosen as key factors warranting further investigation. F418 gfp tr3 was used to study the effect of these two factors more intensively in pasteurised and non-sterile soil. This study reports the development of a modified soil sandwich method to assess fungal propagule dynamics (conidia vs. mycelia). Results from the moisture experiments showed that the increase of F418 and F418 gfp tr3 observed in the first few weeks in pasteurised soil was likely due to the development of mycelia. Soil communities assessed using single strand conformation polymorphism (SSCP) showed a correlation with F418 gfp tr3 persistence in pasteurised and non-sterile soil. The lower F418 gfp tr3 populations observed in pasteurised soil with a high moisture level may have been due to antagonistic microbes. F418 gfp tr3 populations were lower in pasteurised soil in the presence of non-host Costelytra zealandica White (Coleoptera: Scarabaeidae) larvae than without insects. Further investigations showed that F418 gfp tr3 could attach, germinate and form appressoria on C. zealandica cuticle. Conidia of C. zealandica larvae were shown to survive passage through the C. zealandica gut. Differences in the soil communities, as assessed by SSCP, correlated with differences in persistence in pasteurised and non-sterile soil with host, non- host and without insects. The lower F418 gfp tr3 populations observed in pasteurised soil with C. zealandica could have been due to non-specific germination on non-host cuticle and/or presence of antagonistic microbes. F418 gfp tr3 interactions with red clover plants were studied in the final chapter to add some complexity to the simplified microcosms and be more similar to the pasture reality. F418 gfp tr3 could colonise the rhizosphere in pasteurised soil but this was not observed in non-sterile soil. F418 gfp tr3 was found as an endophyte in red clover roots and stems after 18 weeks in pasteurised and non-sterile soil. Overall, this research provides new insights on B. bassiana F418 ecology in soil and highlighted the importance of studying B. bassiana population dynamics in non-sterile soil where soil microorganisms have a strong impact. Keywords: Beauveria bassiana, entomopathogenic fungus, soil, gfp transformant, persistence, soil moisture, non-host insect, Costelytra zealandica, clover, endophyte, population dynamics, soil sandwich. iii Acknowledgements I would like to thanks my supervisors Dr Hayley Ridgway, Prof. Travis Glare, Dr Mickael Brownbridge, Assoc. Prof. R. Bruce Chapman and Prof. Leo Condron for their invaluable advices and guidance throughout this PhD. I would not have finished this doctorate without your encouragement. I would also like to thanks the following people for their help: Dave Saville for his statistical expertise, Norma Merrick for the sequencing, Richard Townsend for providing grass grubs, Dalin Brown for teaching me the SSCP method, Sean Marshall for showing me some techniques with grass grubs, Tracey Nelson with diverse methods in microbiology, Nic Cummings for his help with PCR and phylogenic studies, Neil Smith for the use of Equi-pf and the pressure plates, Aimee McKinnon for proof reading my thesis at the last minute, Sophie Mambrini and Clément Lignon for their help digging hundreds of kilograms of soil while reshaping Europe and particularly Sophie for spending so many hours helping me remove roots and worms, all of that for having used only a tenth of the soil. Enfin, j’aimerai tout particulièrement remercier mes parents qui m’ont supporté pendant toutes ces années. I would like to acknowledge the Tertiary Education Commission for their financial support. iv Table of Contents Abstract ................................................................................................................................ ii Acknowledgements ............................................................................................................ iv Table of Contents ................................................................................................................ v List of Tables....................................................................................................................... xi List of Figures ................................................................................................................... xiii Abbreviations and Symbols ............................................................................................ xvii Chapter 1 Introduction ........................................................................................................ 1 1.1 Research background ................................................................................................... 1 1.2 Biocontrol of pest arthropods with entomopathogenic fungi .......................................... 2 1.3 Mode of action of entomopathogenic fungi ................................................................... 3 1.4 Factors affecting the use of entomopathogenic fungi against soil dwelling insect pests ............................................................................................................................. 4 1.5 Beauveria bassiana ...................................................................................................... 5 1.6 Factors affecting Beauveria bassiana persistence and virulence in soil ........................ 6 1.6.1 Abiotic factors .................................................................................................... 6 1.6.2 Biotic factors .................................................................................................... 11 1.6.3 Pre-application factors ..................................................................................... 17 1.7 Techniques to isolate, quantify and identify entomopathogenic fungi in soil ................ 17 1.7.1 Isolation and quantification techniques ............................................................ 17 1.7.2 Identification

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