Metarhizium strains associated with grass grub (Costelytra giveni) in New Zealand, and their potential as biocontrol agents : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Grass grub (Costelytra giveni) (Coleoptera: Scarabaeidae), an endemic pest of improved pasture in New Zealand, is a damaging root herbivore which lives in soils for most of its life cycle. The pest is resistant to most microbes, but larvae are susceptible to some strains of the fungi belonging to the genus Metarhizium. However, fungal diseases are rarely sufficient to control the pest naturally. It has been shown that other microbes can influence infection by the fungal propagules, but it is not known if this is due to specific species of microbes, and the mode of action of microbial interactions is yet to be elucidated. Plant and soil type can also influence the effectiveness of fungal entomopathogens, as they release compounds which can directly and indirectly affect soil microbial communities and potentially the infection process. Also, the abundance of Metarhizium in pasture soils and the host range of Metarhizium to New Zealand insect pests is unknown for most strains. In this project, the goal was to find Metarhizium strains with the potential to be used as a biocontrol agent of grass grub in New Zealand and to assess the effect of microbial interactions with Metarhizium on the control achieved. Metarhizium spp. were isolated from the environment, identified through molecular approaches, and bioassays conducted against second and third instar grass grub larvae to determine relative virulence. A number of Metarhizium strains were isolated from infected grass grub larvae, soil and obtained from existing collections. At one field site, Metarhizium spp. were infecting around 5% of field-collected larvae. These isolates were shown to belong to the species, M. anisopliae, M. novozealandicum, M. robertsii. M. guizhouense, M. pemphigi, M. brunneum, M. frigidum and M. pinghaense. Among these isolates M. novozealandicum (C14), M. novozealandicum (F99), M. anisopliae (M2), M. anisopliae (F672) and M. pinghaense (JK) caused high grass grub larval mortality after 35 days in bioassays (Chapter 2). M. novozealandicum (C14) caused the highest larval mortality in second and third instar grass grub in both semi-sterilised and non-sterilised soil. The soil type did not affect the pathogenicity of Metarhizium. The LT50 and LC50 values of M. novozealandicum (C14) were lower than for other Metarhizium isolates. M. novozealandicum (C14) at 107 conidia/ml (LC50 < 2.5 x 107 conidia/ 10 g soil after 28 days) was effective against both second and third instar grass grub (nearly 100% larval mortality after 35 days) (Chapter 3). Interactions between M. novozealandicum (C14) and another potential deterrent of grass grub, the grass endophyte Epichloë, were investigated. M. novozealandicum (C14) applied at a rate of 105 conidia/ml had no interaction with meadow fescue endophyte (Epichloë uncinatum) and could not be detected as an endophyte inside grass tissues. However, M. novozealandicum (C14) established as an endophyte inside meadow fescue when soil was inoculated with 107 conidia/ml. After surface sterilisation and culturing M. novozealandicum (C14) was isolated from inside colonised plants, and inhibition of other endophytic fungi was found when isolating on MSM medium. Fungal hyphae were seen inside plant tissue using fluorescent microscopy, but could not specifically be confirmed as M. novozealandicum (C14) (Chapter 4). A range of bacteria were isolated from soil and dead field collected grass grub larvae to examine the effect on M. novozealandicum (C14) infection and larval mortality, as a proxy for bacteria- fungal interactions. Most selected bacteria had a synergistic interaction with M. novozealandicum (C14) against the second instar larvae but there was no synergism against the third instar larvae. Isolate 6-1 (Yersinia enterocolitica) when combined with M. novozealandicum (C14) was more effective than the other bacterial isolates. This bacterial isolate also produced more chitinase than other bacteria tested but not proteinase. Isolate Yersinia enterocolitica 6-1 produced volatiles that inhibited the growth of M. novozealandicum (C14) when not in direct contact, but did not show direct antagonism against M. novozealandicum (C14) on PDA medium. There is potential to combine isolate Yersinia enterocolitica 6-1 with M. novozealandicum (C14) for the biocontrol of grass grub (Chapter 5). The host range of Metarhizium was assessed for some New Zealand insect pests in bioassays. M. novozealandicum (C14) had high pathogenicity against two insect pests, Helicoverpa armigera (Lepidoptera) and Tenebrio molitor (Coleoptera) while there was less effective against three insect pests, Plutella xylostella, Wiseana sp. and Myzus persicae (Chapter 6). The distribution of Metarhizium in pasture soil from three sites (Lincoln, Oxford and West Coast) was also investigated. Both M. novozealandicum and M. anisopliae were isolated, but M. novozealandicum was dominant at all three sites. At the Lincoln site there were high Metarhizium CFU counts and relatively low grass grub larval counts while the Oxford site had low CFU counts and high grass grub larval counts, but the West Coast site produced low counts of both. Although a correlation between high Metarhizium abundance and low grass grub abundance at Lincoln and the opposite at Oxford site was indicated, the number of samples was low, and this study needs to be repeated with further replicates to provide more robust data (Chapter 7). Overall, the results obtained in this thesis have provided essential biological evidence to help understand the potential of M. novozealandicum (C14) as a biocontrol agent against grass grub in New Zealand. Combining M. novozealandicum (C14) with bacteria, such as Y. enterocolitica, may achieve even better control than with M. novozealandicum (C14) alone. M. novozealandicum (C14) can also colonise meadow fescue endophytically, which may have implications for other modes of action, although no evidence of additional effects were found in this study. M. novozealandicum (C14) was also able to infect other insect pest species. A wide host range could be beneficial depending on impacts on natural enemies. The correlation between the presence of Metarhizium and decreasing populations of grass grub at two sites was shown, but this result needs to be replicated over more areas. In conclusion, M. novozealandicum (C14) has the potential to become a biocontrol agent against grass grub in New Zealand