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Interactions of insect and soil microbial communities with insect pathogenic fungi

Periodic Reporting for period 1 - INMIfungi (Interactions of insect and soil microbial communities with insect pathogenic fungi)

Reporting period: 2018-06-01 to 2020-05-31

Metarhizium are arthropods-pathogen fungi found particularly in soils in diverse ecosystems all over the world. They are important antagonists of arthropods and have been widely used as biological control agents (BCA) to control pest insects in agriculture for decades. Presence, abundance and community structure of these fungi in the environment may depend on many abiotic (e.g. pH, organic matter- or nitrogen content) and biotic factors (e.g. host presence, plant- and microbial communities). Previous studies have demonstrated effects of some soil factors, e.g. pH or organic matter content, on Metarhizium communities in particular habitats, however, many of them with contradictory results. Furthermore, studies addressing Metarhizium occurrence and diversity mostly have focussed on differences on a field scale and investigation of these aspects on a low spatial scale are missing.
The goal of this project was to assess to what extend abiotic factors as well as arthropod communities might explain Metarhizium community composition in particular soils or land-use types. Further exploitation of the potential of Metarhizium for pest control is important to provide alternatives to chemical pest control and to foster sustainable agriculture. A crucial base for the achievement of this goal is to enlarge and elaborate our ecological knowledge and to understand how environmental factors drive community development of these important antagonists of arthropods. This knowledge may open up approaches that can be used to regulate these fungi and make use of their function as regulators of arthropod populations particularly in an agricultural context.
The work performed in this project was focused on the effects of soil factors on Metarhizium abundance and community structure in different land-use types. In addition, it was assessed how these factors as well as arthropod communities may drive Metarhizium communities within a particular land-use type at low special scale.
The first objective of the project was to investigate abundance and community structure of Metarhizium in three land-use types (arable land, grassland and forest). Each land-use type included 10 sites, which are part of the Swiss soil-monitoring network (NABO), and are distributed across Switzerland. Soil sampling was performed in 2016 and 13 physical chemical soil parameters measured in each sample (Figl.1).
The results revealed that Metarhizium spp. was not present in all the sites. Whereas in grassland Metarhizium was detected in all the 10 site sampled, in arable land it was present in 8 and in forest only in 4 sites. The abundance was highest in grassland, followed by forest and arable land. multilocus genotypes (MLG) were detected using 14 microsatellite markers (SSR) among the 349 Metarhizium isolates collected from the 30 sites. Of these, 14 MLGs (259 isolates) were identified as M. brunneum, 7 (80 isolates) as M. robertsii and 3 (10 isolates) as M. guizhouense applying EF-1ɑ sequence analysis. The genetic diversity was highest in grassland, followed by arable land and forest. The genetic Metarhizium spp. structures were significantly different among the three land-use types and the distribution of the different species across the country varied significantly. Of the 13 soil physical and chemical parameters evaluated, only 5 (“C:N ratio”, “basal respiration”, “organic carbon”, “total carbon” and “bulk density”) significantly explained the variation of Metarhizium communities among the three land-use type. Results have confirmed that single soil factors alone do not allow prediction of Metarhizium abundance or their community composition. Results from this first part of the project were presented in four meetings and some seminars during the last 2 years. The goal of the second part of the project was to investigate how and to what extend arthropod communities in a particular soil affect Metarhizium community structure at a low spatial scale and throughout one year. Experiments were performed in three different permanent grassland plots, i.e. extensive grassland, orchard and low intensive grassland, located within a radius of 400m at Agroscope, Zürich,Switzerland. Each of the 3 10 x 10 m plots was divided into 25 subplots (2 x 2 m). Two soil core were taken per subplot in each plot, one to isolate Metarhizium spp. and measure soil factors, and the second to extract arthropods communities. Sampling was performed 4 times over the course of 1 year. Metarhizium spp. isolation as well as abundance and community structures were assessed as above. A protocol including arthropod extraction (using a MacFadyen extractor) from soil samples, DNA extraction and metabarcoding analysis based on partial mitocondrial C oxidase subunit one (COI) amplicon sequencing was established.
The protocol was used to process the 300 samples obtained from the three grassland plots. At all the four sampling time points, extensive grassland showed the highest mean abundance of the three plots followed by low intensive grassland and orchard. Highest abundances were detected in late summer in all the three plots. In total, 670 isolates were collected from the three plots at the four sampling times. Twenty-two microsatellite MLGs were detected across the 3 grassland sites and the 4 sampling time points. Seven of the 22 MLGs were present in all the 3 grassland plots at least at one of the 4 sampling time points and 3 of them were present at each sampling time point. In addition, some MLGs were plot specific and present at each or just at one of the four sampling time points. Eleven MLGs were identified as M. robertsii (429 isolates), 8 as M. brunneum (161 isolates) and 3 as M. guizhouense (80 isolates). Overall PERMANOVA revealed that the plot significantly explained the variation in Metarhizium communities among sampling plots at the different sampling time point. Correlation of Metarhizium abundance with soil physical and chemical parameter revealed significant results for 4 parameters (potassium, silt, phosphate and sand content). Metabarcoding of soil arthropod communities was performed for the 300 samples applying the developed protocol. Unfortunately, these analyses got delayed by the Covid-19 situation. Sequence data became available only after the project has terminated. Preliminary analyses showed that arthropod communities, similar to Metarhizium, differ among the three plots. However, detailed analyses are ongoing to finalize the study.
Results from the project have been presented in several international meetings, workshops and seminars. Two manuscripts for publication, which correspond to the data obtained from the first and the second objectives are in preparation, and are expected to be published in early 2021.
The developed metabarcoding approach for soil arthropods has great potential for application in different ecological studies on soil arthropod community structures The knowledge generated in this project contributes significantly to the understanding of the ecology and distribution of indigenous Metarhizium communities. It will help to improve approaches used in biological pest control and develop new approaches that may rely more on conservation biological control concepts, where naturally present fungi are regulated by modification of environmental factors. Such approaches may provide important alternatives to chemical pest control and foster sustainable agriculture and IPM strategies.
Georeferenced NABObio sampling sites of the three land-use types: arable land, grassland and forest