Aspects of the ecology and population dynamics of the fungus Beauveria bassiana strain F418 in soil

Abstract

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 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.