Interactions between isolates of the fungus Beauveria bassiana and Zea mays

Entomopathogenic fungi from the genus Beauveria play an important role in controlling insect populations and have been utilised widely for the biological control of insect pests. Only relatively recently has research focused more on the ecology of these fungi. Various studies have reported that Beauveria bassiana have the ability to become endophytic and may colonise a broad range of plant hosts, while still maintaining pathogenicity to insects. However, the nature of these interactions within plant tissues and the mechanism for colonisation still require elucidation. The aim of this project was to address some of the fundamental questions relating to endophytic colonisation and host interaction in planta. Three putative endophytic isolates of Beauveria were subsequently investigated for interactions with a single Zea mays (maize) cultivar Pioneer 34H31. The overall hypothesis was that isolates of B. bassiana differ in their ability to colonise a single maize cultivar, as evidenced by differential effects to the plant microbiome (in the rhizosphere roots/soil), as well as plant growth and immune response following inoculation. In order to test this hypothesis, endophytic isolates had to be first obtained. Consequently, a nested PCR protocol was developed based on the translation elongation factor 1-alpha (ef1α) gene that was designed to find and amplify isolates in planta from the genus Beauveria. The nested protocol was also designed to enable species differentiation by sequence analysis and quantification of fungal biomass in planta. A prior review of the literature pertaining to Beauveria endophyte detection methodology for PCR indicated the need to optimise plant surface sterilisation for reliable detection of Beauveria in plant tissues. However, elimination of Beauveria inocula and/or DNA from the plant surfaces proved difficult. The focus of the project therefore shifted more to the plant host response to all plant-associated Beauveria. This was achieved by (1) testing the plant growth response to three different B. bassiana putativeendophytic isolates (BG11, FRh2 and J18) versus the growth-promoting Trichoderma atroviride iii isolate LU132, all of which were introduced artificially through a wound made to the emerging maize seedlings to avoid the confounding effects of surface inoculation, (2) by assessing the impact of the three B. bassiana isolates applied topically to roots of maize on the rhizosphere soil community structure and function and (3) by investigating differences in gene expression in maize roots in the response to the topical application of two different B. bassiana isolates (BG11 and J18), relative to a no-inoculum control using an RNA microarray transcriptome analysis. Results of the growth experiment showed predominantly neutral or negative effects to plant growth in terms of biomass, although plants exhibited root architecture changes as a result of one B. bassiana isolate (FRh2), and a higher chlorophyll content for another isolate (J18) when measured with a SPAD-502 chlorophyll meter, providing initial evidence for phenotypic differences between the selected study isolates. However, variation between the three B. bassiana isolates was less evident in the ecological study of the rhizosphere of maize. Neither the microbial community structure nor function was significantly affected by the presence of the isolates. However, retention of the inocula in the rhizosphere over 30 days after inoculation (DAI) was positively affected by a simulated herbivory treatment made to the maize foliage at 23 DAI, as was the general microbial community composition. The transcriptome analysis indicated putative differential gene expression in maize roots as a result of colonisation by the two B. bassiana isolates, suggesting that they may differ in their ability to colonise and/or effect the plant host immune response. Isolate J18-treated plants upregulated genes encoding for antioxidant glutathione S-transferases (GSTs) relative to BG11-treated plants, presumably to counteract excesses of reactive oxygen species (ROS). In contrast, BG11-treated plants upregulated a larger suite of genes involved in plant defence including ethylene responsive transcription factors, auxin responsive and dehydration responsive genes. Overall, this research suggests that the relationship between Beauveria and the plant host is modulated by the plant host, but may sometimes also be isolate-dependent.