A new approach for delivery of entomopathogenic fungi for plant protection against insect pests and plant diseases via maize seed : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Entomopathogenic fungi have been recognized mainly for their ability to kill insects which has seen as an advantage for the development of these particular fungi as biocontrol agents in sustainable agriculture programmes. Recently, it was determined that some species of entomopathogenic fungi, including Metarhizium spp., also have the capability to associate with roots and aerial parts of the plant. The potential benefits from the plant-fungal association are as broad as nutrient acquisition, plant growth promotion, protection against biotic or abiotic factors and even increase of the induced defence response in the plant through modification of the phytohormone content. However, many of these the positive effects are uncertain and we are far from understanding the complex interaction between the plant, the fungus and a biotic challenger. Additionally, the establishment of the plant-fungal association relies on the specificity of the fungus and plant interaction. The complex taxon Metarhizium anisopliae had gone through an intense molecular revision, where more than 30 new species of this genus have been described. M. anisopliae, and some other members of the genus such as M. robertsii and M. brunneum, have been described as strongly associated with plants roots, and at the same time with the ability to infect insect pests. Some strains are associated with the rhizosphere and can even colonise the plant as endophytes. One of the main constrains in the use of this type of fungi for the control of soil dwelling insects or pathogens in the agriculture are the contact to the target pest, and the survival of the biocontrol agent in the environment. The soil offers an appropriate environment for fungi, where conditions of moisture and temperature are suitable. This PhD study contributes to the goal of developing improved biological control agents from selected rhizosphere competent, entomopathogenic fungi, delivered efficiently through seed coating. Using molecular approach, isolates of several genera of entomopathogenic fungi were characterised and species assigned based on phylogenetic comparisons. Among the species described, this is the first report of the presence of recently described new species of Metarhizium spp. in New Zealand, such as Metarhizium novozealandicum, M. robertsii, M. brunneum, M. guizhouense and M. frigidum. Presence of specific genes associated with plant and insect colonisation abilities were also determined, to aid selection of potential plant associated isolates. In addition, the selection of Metarhizium isolates with the potential to survive in the plant rhizosphere was evaluated for first time by spectrophotometric determination of fungal growth in root exudates. Conidia from the fungal isolates where then used to coat maize seed, to determine the effect of each fungal isolate –plant interaction on plant growth and resistance to insect and disease damage. In presence of the grass grub Costelytra giveni (Coleoptera: Scarabaeidae) and the maize disease fungus, Fusarium graminearum (Ascomycete), some entomopathogenic fungal treatments produced improved growth performance of the maize seedlings. It was also determined the fungal infection of the grass grubs and the decrease in the Fusarium graminearum rot root due to the seed coating with entomopathogenic fungi. Particularly, the M. anisopliae and M. robertsii isolates had significantly higher colonization of the rhizosphere than the other isolates. It was also demonstrated that colonisation of the rhizosphere by M. anisopliae produced changes in the levels of salicylic acid and jasmonic acid when compared to control plants. This is the first report where the effect of M. anisopliae on the content of the phytohormones was evaluated. The seed coating was further developed by replacing fungal conidia with resistant structures known as microsclerotia (MS). Production of these resistant structures in M. guizhouense and M. novozealandicum was evaluated for first time. Maize seeds coated with MS grew better than control plants when grown in presence of F. graminearum. The association between Metarhizium spp and maize roots, after seeds were coated with MS, was possible to determine using fluorescent and laser confocal microscopy. These observations on maize have not been previously published where soil conditions were appropriate for MS germination and hyphae developed along the maize roots which associated with the ecto- and endo-rhizosphere. These studies also confirmed the suitability of using conidia of isolates of Metarhizium transformed with the fluorescent proteins mcherry and GFP to coat maize seeds for the observation of plant colonisation. This is the first report of using a blastospore-based transformation system for M. anisopliae. Overall the results suggest that seed coating with microsclerotia of Metarhizium can be used as delivery system for the control of plant pathogens and insect pests and improve the opportunity for close association with plant roots after conidia germination and hyphal growth.