PCR-based characterisation of entomopathogenic fungi for ecological studies

The implementation of PCR-based tools for characterisation of organisms has greatly advanced our understanding of the phylogenies and species boundaries in entomopathogenic fungi, especially the widespread taxa Beauveria bassiana and Metarhizium anisopliae. These fungi have received a lot of interest due to their potential as biocontrol agents of pests. However, there is still a lack of knowledge about the fundamental ecology of these fungi in both managed and natural ecosystems, but such information is necessary both for risk assessments prior to release of biocontrol agents in the environment as well as if we wish to understand the distribution of the fungi and their impact on host populations. This latter focus is essential if the fungi are to be included in pest management strategies based on conservation biological control. A series of unspecific PCR methods have been used to characterise isolates of B. bassiana and M. anisopliae, and many studies have concluded that the fungi contain a lot of genetic diversity. The problem with these methods is that they provide little phylogenetic information of the fungi and the characters can not be used to explicitly compare data between studies. Sequences from specific targets in the DNA provide tools for explicit comparison between isolates across studies. There is now a range of primer sets published along with reference sequences in GenBank to make these comparisons possible. Multi-gene phylogenies have revealed that both B. bassiana and M. anisopliae are complexes of species that contain cryptic taxa or clades. Using merely morphological characteristics will not result in the same degree of taxa identification as will the use of DNA sequence data. Similarly, when using microsatellite markers for population genetic studies of fungal communities, it is necessary to first separate the populations in clades as allele sizes can convert across populations. In the near future, USDA-ARS in Beltsville will launch a web-based platform (MBID, Metarhizium-Beauveria ID) including lists of primers, databases of reference sequences and type isolates of new described species for the reliable identification of taxa for the scientific community. This tool should provide a common framework and nomenclature for scientists and create basis for making comparisons among studies. There should be great potential to learn more about the ecology of the entomopathogenic fungi with the application of these molecular markers. However, designing an appropriate sampling scheme in the environment is absolutely necessary to conduct these ecological studies. The use of isolates from culture collections should be avoided

Methods for isolation of entomopathogenic fungi from the soil environment - laboratory manual

Descriptions of methods and recommendation of laboratory procedures for the isolation of soil borne entomopathogenic fungi (specifically Beauveria spp. and M. anisopliae) are presented. For screening of occurrences of indigenous populations of entomopathogenic fungi the insect bait method is recommended. Further recommendations are: 1) Collect sufficient number of soil samples to cover the area of investigation; 2) if the bait method is used, apply sufficient individuals of bait insects to each sample to increase the likelihood of isolating the fungi present. Descriptions of isolation methods, statistical analyses of the data and preparation of media and bait insects are given

Ecology of entomopathogenic fungi in agroecosystems

Fungi that infect insects have received considerable attention by scientists for their potential for biological control of pests. Many research projects have focussed on the selection of virulent strains for target pests and their development as biological control agents. In contrast, surprisingly little is known about the fundamental ecology of most of these fungi in nature. This knowledge is essential in order to receive the most ecosystem services provided by entomopathogenic fungi in agricultural production. Knowledge of the basic ecology of the fungi is also necessary to include them in conservation biological control. In this biological control strategy, agricultural practices and/or habitat manipulations are applied to the farming system to favour living conditions for specific natural enemies of pests (Eilenberg et al., 2001)

Distribution, Abundance and Diversity of Fungal Entomopathogens: Foundations for Conservation Biological Control

Conservation biological control (CBC) is a pest management strategy in which farming management practices are adopted to enhance the living conditions for specific natural enemies of pests with the specific objective to suppress pest populations. Research in CBC strategies have mostly focused on provisioning of good living conditions for arthropod predators and parasitoids, usually ignoring entomopathogens as regulators of pest populations. Fungal entomopathogens are widespread in agro-ecosystems and infect a range of arthropod pests, and these fungi should therefore also be included in CBC. Knowledge of the ecology of indigenous populations of fungal entomopathogens is a prerequisite for the evaluation of their contributions to pest control and for predicting consequences of agricultural practices. The anamorphic entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales) are among the most abundant fungi infecting insects and arachnids in temperate regions. Much focus has been on the development of B. bassiana and M. anisopliae as biological control agents (for inundation and inoculation biological control) to be applied in agriculture and forestry. However, few studies have focused on the fundamental ecology of these fungi in terrestrial ecosystems, including agro-ecosystems. I will present recent results providing several new insights into the abundance, distribution and diversity of B. bassiana in agricultural fields and their surrounding habitats and discuss these in relation to CBC. Systematic uses of traditional isolation techniques show complex distributions of B. bassiana in both soil and aboveground environments. Applications of molecular markers demonstrate remarkable diverse structures of Beauveria communities as well as novel insights into the interpretation of host range. Numerous interactions among fungal entomopathogens and their hosts (including both pests and beneficials) potentially affect the impact of the fungi on pest regulation. Novel data on spatio-temporal distribution of B. bassiana and M. anisopliae in above- and belowground environments will also be presented. These results have impact on interpretation of fungal life cycles and which taxa we should expect to infect which hosts and where

Distribution patterns of fungal entomopathogens in soil habitats: Natural occurrence, diversity and dynamics

Fungal entomopathogens are seemingly ubiquitous in soils. An increasing number of surveys around the world have shown that ascomycete fungi from the Hypocreales can be isolated from soils of many habitats. Based on morphological characteristics of the fungi some patterns of distribution have emerged. However, recent developments in molecular characterization of entomopathogenic fungi have revealed significant new insights into the distribution of genetic groups of some taxa, including Beauveria spp, and possible restrictions to certain habitats. I will present and discuss some of these distribution patterns and include new findings which indicate that some fungi are restricted to underground lifestyles while others cycle between below- and aboveground habitats within an agroecosystem

Insect pathogenic fungi in biological control: status and future challenges

In Europe, insect pathogenic fungi have in decades played a significant role in biological control of insects. With respect to the different strategies of biological control and with respects to the different genera of insect pathogenic fungi, the success and potential vary, however. Classical biological control: no strong indication of potential. Inundation and inoculation biological control: success stories with the genera Metarhizium, Beauveria, Isaria/Paecilomyces and Lecanicillium (previously Verticillium). However, the genotypes employed seem to include a narrow spectrum of the many potentially useful genotypes. Conservation biological control: Pandora and Entomophthora have a strong potential, but also Beauveria has a potential to be explored further. The main bottleneck for further exploitation of insect pathogenic fungi in biological control is the limited knowledge of host pathogen interaction at the fungal genotype level

Diversitet af insekternes svampesygdomme i økologisk jordbrug - hvorledes kan den øges?

Det er et vigtigt mål i økologisk jordbrug at inddrage skadedyrenes naturlige fjender i reguleringen af skadedyrpopulationer. En vigtig gruppe af disse fjender er insekternes naturligt forekommende svampesygdomme, som blandt andet findes i jorden. Danske studier viser, at landbrugslandet rummer en stor diversitet af disse svampe, og det er en fremtidig udfordring at udnytte denne diversitet i regulering af skadedyrspopulationer

Økologisk dyrkning fremmer den naturlige regulering af skadedyr

Naturlige svampesygdomme, som hjælper med til at regulere skadedyr i grønsager, er hyppigere i økologiske dyrkningssystemer sammenlignet med konventionelle. Samtidig er der færre overvintrende kålfluepupper. Det viser de foreløbige resultater fra den første dyrkningssæson i FØJO III-projektet VegQure

Entomopathogenic fungi infecting non-pest insects: Implications for ecosystem services and relevance of behavioural ecology

Entomopathogenic fungi infect a wide array of insects from most orders and they are among the natural enemies that contribute to the regulation of insect populations. However, only a limited number of studies have focused on the impact of fungal pathogens on populations of non-pest insects. Effects of entomopathogenic fungi on non-pest host populations should receive more attention based on the increasing interest in conservation biological control. In this strategy, founded on competition theory, non-pest host populations adjacent to cropping systems will in principle affect pest populations through shared natural enemies. We present examples of selected non-pest host-fungus systems from temperate ecosystems that are relevant for the expected ecosystem service provided by entomopathogenic fungi. Predators are among the non-pest hosts that are infected by fungi. Recent advances in our understanding of the effect of pathogens on the behaviour of predators may shed light on the significance of entomopathogenic fungi for the regulation of predator populations. We discuss what we can learn about host-pathogen interactions from behavioural ecology and which life history parameters in the host that may be important for the impacts of fungal pathogens on their host populations

Pathogen avoidance by insect predators

Insects can detect cues related to the risk of attack by their natural enemies. Pathogens are among the natural enemies of insects and entomopathogenic fungi attack a wide array of host species. Evidence documents that social insects in particular have adapted behavioural mechanisms to avoid infection by fungal pathogens. These mechanisms are referred to as 'behavioural resistance'. However, there is little evidence for similar adaptations in non-social insects. We have conducted experiments to assess the potential of common insect predators to detect and avoid their entomopathogenic fungal natural enemy Beauveria bassiana. The predatory bug Anthocoris nemorum was able to detect and avoid nettle leaves that were treated with B. bassiana. Females laid fewer eggs on leaf halves contaminated with the pathogen. Similarly, females were very reluctant to contact nettle leaves contaminated with the fungus compared to uncontaminated control leaves in ‘no-choice’ experiments. Adult seven spot ladybirds, Coccinella septempunctata, overwinter in the litter layer often in groups. Adult C. septempunctata modified their overwintering behaviour in relation to the presence of B. bassiana conidia in soil and sporulating conspecifics by moving away from sources of infection. Furthermore active (non-overwintering) adult C. septempunctata were also able to detect and avoid B. bassiana conidia on different substrates; bean leaves, soil and sporulating on dead conspecifics. Our studies show that insect predators have evolved mechanisms to detect and avoid pathogens that they are susceptible to. Fungal pathogens may be significant mortality factors among populations of insect predators, especially long-lived species that must diapause before reproduction. Likewise, actively foraging species are more likely to come in contact with pathogens than predators that sit and wait for prey. These particular groups of insects will benefit from adaptations to avoid pathogens