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Objective 2

To provide new information on the persistence in the environment of wild and inundatively-released genotypes of entomopathogenic microorganisms applied as microbial bio-insecticides.  The study will use soil-dwelling entomopathogenic fungi as a model system.


There is a sense of urgency to the development of bio-insecticides, but a responsible approach is also required which considers their use in relation to environmental sustainability (Bidochka et al., 1996).  The high level of host specificity of many bio-insecticides means that unwanted direct effects on nontarget organisms are likely to be rare.  However, inundative releases of a host specific genotype  could have unintended indirect effects, for example on natural entomopathogen biodiversity and effectiveness, on the abundance of arthropod natural enemies through competitive effects, or on infra specific groupings of the target host species that are associated with non-crop plants (Lockwood, 1993; Roy & Pell, 2000; Pearson and Callaway, 2003; Miller et al., 2003).  This aspect of the project will study potential indirect effects of bio-insecticide applications, focusing on the persistence of an inundatively applied genotype and its interaction with indigenous entomopathogenic fungi.  Studies of other unwanted effects are outside the scope of this project but could be included at a later stage.  Indigenous genotypes used for biological control are already in the target environment and theoretically a limited number of applications should cause only relatively short-term perturbation of the ecosystem (Goettel, 1995; Goettel et al. 2001).  However, more pronounced effects can be hypothesised for repeated applications of a genotype, and / or if the genotype is non-indigenous.  The question of indigenous vs non indigenous genotypes is very relevant in this project.  Many species of entomopathogenic fungi have global distributions, and so the opportunities for introducing non-indigenous species are limited.  However the distributions of infraspecific entities are often restricted by geography, and hence introductions of non-indigenous genotypes may become commonplace if fungal bio-insecticides are used widely.  Repeated, inundative applications of indigenous or non indigenous bio-insecticides have a number of potential outcomes, including (a) persistence of the bio-insecticide genotype with long lasting impact on the ecosystem, including displacement of native entomopathogens; (b) persistence with little consequence; (c) non establishment. The ecological factors determining these outcome include competitive exclusion (Chandler et al., 1993), community resistance to perturbation (Lockwood, 1993), and pathogen local adaptation (Dybdahl & Storfer, 2003).  The last factor is particularly important both for biological control and for generating biodiversity in host – pathogen communities (Dybdahl & Storfer, 2003).  Bidochka et al. (2001; 2002) have produced recent evidence from Canada that the infraspecific entities of two soil dwelling entomopathogenic fungi (Metarhizium anisopliae and Beauveria bassiana) are adapted to environmental conditions, which challenges a paradigm in insect pathology that the host insect is the predominant influence on population genetics.  Local adaptation would have profound implications for the ability of natural pathogen communities to compete with bio-insecticide genotypes, and hence determine both the efficacy and the sustainability of bio-insecticide applications.

Key questions for this aspect of the project are whether genotypes of entomopathogenic fungi exhibit local adaptation, and how this influences the interactions between natural populations of fungi and inundatively applied genotypes used as a bio-insecticides? Do interactions between natural entomopathogen populations and inundatively applied genotypes affect the environmental sustainabilty of microbial pest control?

This Objective will provide new information on the persistence in the environment of wild and inundatively-released genotypes of entomopathogenic fungi, using the leafy salad crop model system. Leafy salad crops grown in the UK are infested by four species of aphid (Parker et al., 2002) and chemical insecticides are used frequently.  However, aphids are also susceptible to entomopathogenic fungi and some species have been investigated as microbial bio-insecticides. In recent years we have developed experience with Metarhizium flavoviride (= M. anisopliae) var pemphigum (Foster, 1975; Driver et al., 2000) for control of the lettuce root aphid Pemphigus bursarius (Chandler, 1997) a pest of outdoor lettuce crops. When incorporated in the soil, the fungus protects plants from infestation by P. bursarius in the root zone (Parker et al., 2002).  Pemphigus bursarius colonises members of the Compositae, and recent studies of its infra-specific diversity using microsatellites have identified considerable variation within the species, associated with a preference for different host plants (Miller et al, 2003; Miller & Tatchell, unpublished (Fig. 1)). Metarhizium flavoviride var pemphigum originates from the closely related aphid Pemphigus trehernei which is associated with sea aster on UK coastal salt marshes (Foster, 1975).  Our experience with the fungus indicates strongly that it is specific to root aphids (Pemphigidae), and while it is indigenous to the UK, to the best of our knowledge it is not found naturally in agricultural habitats.  The fungus can be distinguished from other clades of Metarhizium on the basis of rDNA ITS sequence information (Driver et al., 2000).  It therefore represents an excellent opportunity to study the behaviour of a bio-insecticide that is non-indigenous to the target agroecosystem but which is native to the UK. 

This aspect of the project will:

• Investigate the effect of habitat type on the biodiversity of entomopathogenic fungi in the agro-ecosystem and look for evidence of local adaptation of indigenous populations.

• Determine the prevailing life history strategy of M. flavoviride var pemphigum and other fungal genotypes through investigation of econutritional behaviour.

• Characterise the fate of M. flavoviride var pemphigum when applied as an inundative bio-insecticide against diverse genotypes of P. bursarius.

The relationship between habitat type and biodiversity at the sub-species level will be researched for naturally occurring populations of entomopathogenic fungi, and evidence that fungal genotypes show adaptation to habitat as well as to the host organism will be investigated. The occurrence of entomopathogenic fungi will be quantified in soils collected from annual crops, field margins, Countryside Stewardship Areas, and woodland at HRI using semi selective isolation methods (Bedding & Akhurst, 1975), and the relationship between frequency of occurrence, species diversity, habitat type, and soil type will be analysed using contingency tables (Chandler et al., 1997).  Molecular methods (e.g. AFLPs) will be used to characterise fungal infraspecific diversity. Frequency distributions of genotype differences will be analysed to look for associations between alleles and habitat types. Laboratory microcosms will be used to investigate the econutritional behaviour of naturally occurring fungal genotypes and M. flavoviride var pemphigum, taking into account fungistatic mechanisms induced by other components of the soil microbiota (Lockwood & Filonow, 1981). The decay kinetics of fungal conidia populations of M. flavoviride var pemphigum and other fungal genotypes from the study site will also be described mathematically when applied in elevated concentrations in the presence and absence of microbial competition (Storey et al., 1989; Studdert et al., 1990).  Finally, the persistence of M. flavoviride var pemphigum as a bio-insecticide will be studied in a field experiment at HRI designed to maximize the biodiversity of P. bursarius and in the presence of other genotypes of entomopathogenic fungi, using a genotype specific detection method (e.g. SCAR markers, Castrillo et al. 2002).  The infra specific diversity and temporal spatial dynamics of populations of indigenous entomopathogenic fungi and P. bursarius will be determined before and after application of the bio-insecticide, along with investigation of the temporal spatial dynamics of M. flavoviride var pemphigum itself.