Preventive application of an entomopathogenic fungus in cover crops for wireworm control

Efficacy of the Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) strain ART2825 for control of wireworms (Agriotes obscurus (L.), Coleoptera: Elateridae) was examined in a semi-field pot experiment. Pots were treated in late summer during sowing of spring oat as a cover crop. Survival of wireworms was assessed four weeks after their release in October 2013, and 30 weeks after release in April 2014. Viability and persistence of the fungus was determined by counting colony forming units from substrate samples and microsatellite analyses of recovered Metarhizium isolates. The number of colonies detected in the substrate in October 2013 increased with increasing concentrations of applied conidia, and no significant reduction was observed at the second evaluation date in April 2014. Increasing conidia application rates significantly increased mycosis and reduced wireworm survival, to a level comparable to that of treatment using insecticide-coated oat seeds. The preventive application of M. brunneum conidia to reduce wireworm populations in cover crops, preceding a damage-sensitive crop like potatoes, may be a promising biocontrol strategy

Identifizierung und Charakterisierung der krankheitsunterdrückenden Mikroorganismen beim Komposteinsatz

Einleitung • Bodenbürtige Krankheiten verursachen grosse Schäden und sind schwierig zu kontrollieren. • Kompost-Zugabe kann Druck durch bodenbürtige Krankheitserreger reduzieren • ABER: Nicht jeder Komposteinsatz ist erfolgreich. • Möglicher Grund ist die sich während des Kompost-Reifeprozesses laufend ändernde mikrobielle Zusammensetzung. • Es ist weitgehend noch unbekannt, welche mikrobiellen Konsortien wichtig für die suppressive Wirkung von Komposten sind

Harnessing the Microbiomes of Suppressive Composts for Plant Protection: From Metagenomes to Beneficial Microorganisms and Reliable Diagnostics

Soil-borne diseases cause significant yield losses worldwide, are difficult to treat and often only limited options for disease management are available. It has long been known that compost amendments, which are routinely applied in organic and integrated farming as a part of good agricultural practice to close nutrient cycles, can convey a protective effect. Yet, the targeted use of composts against soil-borne diseases is hampered by the unpredictability of the efficacy. Several studies have identified and/or isolated beneficial microorganisms (i.e., bacteria, oomycetes, and fungi) from disease suppressive composts capable of suppressing pathogens (e.g., Pythium and Fusarium) in various crops (e.g., tomato, lettuce, and cucumber), and some of them have been developed into commercial products. Yet, there is growing evidence that synthetic or complex microbial consortia can be more effective in controlling diseases than single strains, but the underlying molecular mechanisms are poorly understood. Currently, a major bottleneck concerns the lack of functional assays to identify the most potent beneficial microorganisms and/or key microbial consortia from complex soil and compost microbiomes, which can harbor tens of thousands of species. This focused review describes microorganisms, which have been isolated from, amended to or found to be abundant in disease-suppressive composts and for which a beneficial effect has been documented. We point out opportunities to increasingly harness compost microbiomes for plant protection through an integrated systems approach that combines the power of functional assays to isolate biocontrol and plant growth promoting strains and further prioritize them, with functional genomics approaches that have been successfully applied in other fields of microbiome research. These include detailed metagenomics studies (i.e., amplicon and shotgun sequencing) to achieve a better understanding of the complex system compost and to identify members of taxa enriched in suppressive composts. Whole-genome sequencing and complete assembly of key isolates and their subsequent functional profiling can elucidate the mechanisms of action of biocontrol strains. Integrating the benefits of these approaches will bring the long-term goals of employing microorganisms for a sustainable control of plant pathogens and developing reliable diagnostic assays to assess the suppressiveness of composts within reach

Stability of soil microbial communities to applications of the fungal biological control agent Metarhizium brunneum

Naturally occurring entomopathogens, including fungi, bacteria, protozoa, nematodes and viruses, are important for natural regulation of insect populations and therefore used in biological control of insect pests. Biological control using entomopathogens is an alternative pest management strategy to chemical control, is considered to be more environmental friendly than chemical control and is often part of integrated pest management (IPM). Beneficial effects of entomopathogens as compared to chemicals may include conservation of other natural enemies, reduction of pesticide residues in soil and plants as well as safety for non-target organisms. The development of existing biological control for important soil dwelling pests in Europe by exploiting novel application strategies and synergistic effects of entomopathogenic fungi, entomopathogenic nematodes and natural substances, has been the major aim of the EU-project supporting this thesis (INBIOSOIL). Biological control using entomopathogenic fungi may include soil applications of large quantities of fungal propagules, often resulting in densities of up to 1014 propagules per ha. Such mass applications and introductions of microorganisms to soil may affect indigenous soil microbial communities and the ecosystem functions they fulfil. Assessment of potential effects of applied microorganisms on native soil microbial communities is therefore important and also required in the registration process of novel products by European and Swiss regulations. Species of the entomopathogenic fungal genus Metarhizium are widely used in biological control and potential effects of Metarhizium on native soil microbial communities have barely been investigated. As a basic principal of risk assessment of mass applications it is mandatory to verify that soil microbial communities are exposed to the applied fungal biological control agent (BCA). It is important to be able to determine both exposure (presence and abundance) as well as effects, e.g., changes in microbial community structures, of a BCA in soil as basis of a risk assessment. Therefore, the aims of this thesis were I) to improve molecular tools to assess presence and abundance of the BCA in soil in order to track exposure of soil microbial communities and II) to assess potential effects of applications of two strains of the BCA on soil microbial communities in pot and field experiments and III) to investigate potential analytical constraints of the presence of one highly abundant microbial strain on the assessment of changes in soil microbial community structures. Exposure of soil microbial communities to the BCA was assessed by isolation of Metarhizium spp. from soil on selective medium and subsequently identifying the genotype of the applied strains using simple sequence repeat (SSR) marker analyses. To optimize the typing tool the transferability of 41 existing SSR markers to different Metarhizium spp. were investigated (Chapter 2). This was particularly important since structure and taxonomy within the genus Metarhizium have been drastically revised in the recent past. Among other changes, the species M. anisopliae has been recognized as a species complex and has been divided into 10 species. Suitable markers were selected for genotyping of the applied strains and discriminating them from native Metarhizium strains present in soils. Biocontrol experiments were performed to control Agriotes obscurus larvae in a pot and a field experiment and Diabrotica virgifera virgifera larvae in a pot experiment using the BCA M. brunneum ART2825 and M. brunneum EAMa01/58-Su over a period of four months (Chapter 3 and 4). Treatments included three different formulations of the BCA, i.e. fungus colonized barley kernels (FCBK), fungal capsules (Fcap) and fungal granules (Fgran), unformulated fungal spores, combinations of the BCA and garlic extract, garlic extract alone and controls. Potential effects of M. brunneum on fungal and prokaryotic soil communities were assessed using next generation sequencing (NGS) of ribosomal marker regions, i.e., the internal transcribed spacer 2 (ITS2) for fungal soil communities and part of the small subunit of the ribosomal RNA gene (16S V3-V4) for prokaryotic communities. One of the biases occurring in NGS is preferential amplification of target sequences in the PCR due to the occurrence of one highly abundant sequence. In order to assess if the sequence of the applied strain, which was highly abundant, will affect the assessment of changes of soil microbial communities, an experiment was performed in which different concentration of a plasmid containing the target sequence of the BCA was added to a soil DNA extract (Chapter 5). Results revealed that M. brunneum ART2825 formulated as FCBK was the most efficient treatment against A. obscurus larvae in the pot experiment, which resulted in 77 % reduction of damaged potato tubers compared to the untreated control (Chapter 3). No biocontrol effect was detected in the Agriotes-field experiment as well as in the Diabrotica-pot experiment (Chapter 3 and 4). Selective plating and SSR marker analyses confirmed exposure of soil microbial communities to the applied strains in the pot and field experiments and built the basis for effect analyses (Chapter 3 and 4). In the Agriotes-pot experiment, M. brunneum ART2825 affected the fungal community structures only slightly and only if formulated. These effects were in the same range as effects caused by the carrier material only and no effects were detected if pure fungal spores were applied to soil. In contrast, prokaryotic community structures were not affected by any fungal applications. Also, fungal and prokaryotic communities did not change upon the application of the BCA in the Diabrotica-pot and the Agriotes-field experiment. Finally, assessment of effects, i.e., changes in soil microbial community structures, were not impeded by the presence of one highly abundant sequence or sequencing errors that potentially corroborate the reliability of the results (Chapter 5). In conclusion, genotyping using SSR marker analyses was a valuable tool for confirming inoculation success and exposure of soil microbial communities to the applied strains. Although, constraints of amplicon sequencing, such as PCR bias, erroneous sequences and limitations in species identification, may occur, it is currently the most suitable technique, for the sequencing depth and the large number of samples and replicates required in experiments as performed in this study. Natural fluctuations including spatial and temporal differences of soil microbial community structures were similar or greater than any treatment-effects detected by applying different formulations of the BCA or unformulated fungal spores. Therefore, it is unlikely that applications of M. brunneum ART2825 and M. brunneum EAMa01/58-Su adversely affect soil microbial communities beyond the extent induced by fluctuations occurring in natural habitats. However, the findings of this study are restricted to specific BCA strains, soils, experimental designs, and analytical procedures and may require confirmation in other systems or if new analytical tools become available

Biological Diabrotica Management and Monitoring of Metarhizium Diversity in Austrian Maize Fields Following Mass Application of the Entomopathogen Metarhizium brunneum

Inundative mass application of Metarhizium brunneum BIPESCO 5 (Hypocreales, Clavicipitaceae) is used for the biological control of Diabrotica v. virgifera (Coleoptera, Chrysomelidae). Long-term field trials were performed in three Austrian maize fields—with different cultivation techniques and infestation rates—in order to evaluate the efficacy of the treatment to control the pest larvae. In addition, the indigenous Metarhizium spp. population structure was assessed to compare the different field sites with BIPESCO 5 mass application. Annual application of the product Granmet-PTM (Metarhizium colonized barley kernels) significantly increased the density of Metarhizium spp. in the treated soil above the upper natural background level of 1000 colony forming units per gram dry weight soil. Although a decrease in the pest population over time was not achieved in heavily infested areas, less damage occurred in treated field sites in comparison to control sites. The Metarhizium population structure was significantly different between the treated field sites. Results showed that inundative mass application should be repeated regularly to achieve good persistence of the biological control agent, and indicated that despite intensive applications, indigenous populations of Metarhizium spp. can coexist in these habitats. To date, crop rotation remains the method of choice for pest reduction in Europe, however continuous and preventive application of M. brunneum may also present an alternative for the successful biological control of Diabrotica

Multiplexed microsatellite markers for seven Metarhizium species

Cross-species transferability of 41 previously published simple sequence repeat (SSR) markers was assessed for 11 species of the entomopathogenic fungus Metarhizium. A collection of 65 Metarhizium strains including all 54 used in a recent phylogenetic revision of the genus were characterized. Between 15 and 34 polymorphic SSR markers produced scorable PCR amplicons in seven species, including M. anisopliae, M. brunneum, M. guizhouense, M. lepidiotae, M. majus, M. pingshaense, and M. robertsii. To provide genotyping tools for concurrent analysis of these seven species fifteen markers grouped in five multiplex pools were selected based on high allelic diversity and easy scorability of SSR chromatograms.ISSN:0022-2011ISSN:1096-080

Land-Use Type Drives Soil Population Structures of the Entomopathogenic Fungal Genus Metarhizium

Species of the fungal genus Metarhizium are globally distributed pathogens of arthropods, and a number of biological control products based on these fungi have been commercialized to control a variety of pest arthropods. In this study, we investigate the abundance and population structure of Metarhizium spp. in three land-use types—arable land, grassland, and forest—to provide detailed information on habitat selection and the factors that drive the occurrence and abundance of Metarhizium spp. in soil. At 10 sites of each land-use type, which are all part of the Swiss national soil-monitoring network (NABO), Metarhizium spp. were present at 8, 10, and 4 sites, respectively. On average, Metarhizium spp. were most abundant in grassland, followed by forest and then arable land; 349 Metarhizium isolates were collected from the 30 sites, and sequence analyses of the nuclear translation elongation factor 1α gene, as well as microsatellite-based genotyping, revealed the presence of 13 Metarhizium brunneum, 6 Metarhizium robertsii, and 3 Metarhizium guizhouense multilocus genotypes (MLGs). With 259 isolates, M. brunneum was the most abundant species, and significant differences were detected in population structures between forested and unforested sites. Among 15 environmental factors assessed, C:N ratio, basal respiration, total carbon, organic carbon, and bulk density significantly explained the variation among the M. brunneum populations. The information gained in this study will support the selection of best-adapted isolates as biological control agents and will provide additional criteria for the adaptation or development of new pest control strategies

Assessing effects of the entomopathogenic fungus Metarhizium brunneum on soil microbial communities in Agriotes spp. biological pest control

The release of large quantities of microorganisms to soil for purposes such as pest control or plant growth promotion may affect the indigenous soil microbial communities. In our study, we investigated potential effects of Metarhizium brunneum ART2825 on soil fungi and prokaryota in bulk soil using high-throughput sequencing of ribosomal markers. Different formulations of this strain, and combinations of the fungus with garlic as efficacy-enhancing agent, were tested over 4 months in a pot and a field experiment carried out for biological control of Agriotes spp. in potatoes. A biocontrol effect was observed only in the pot experiment, i.e. the application of FCBK resulted in 77% efficacy. Colony counts combined with genotyping and marker sequence abundance confirmed the successful establishment of the applied strain. Only the formulated applied strain caused small shifts in fungal communities in the pot experiment. Treatment effects were in the same range as the effects caused by barley kernels, the carrier of the FCBK formulation and temporal effects. Garlic treatments and time affected prokaryotic communities. In the field experiment, only spatial differences affected fungal and prokaryotic communities. Our findings suggest that M. brunneum may not adversely affect soil microbial communities.ISSN:0168-6496ISSN:1574-694