Site and land-use associations of soil bacteria and fungi define core and indicative taxa.

Soil microbial diversity has major influences on ecosystem functions and services. However, due to its complexity and uneven distribution of abundant and rare taxa, quantification of soil microbial diversity remains challenging and thereby impeding its integration into long-term monitoring programs. Using metabarcoding, we analyzed soil bacterial and fungal communities at 30 long-term soil monitoring sites from the three land-use types arable land, permanent grassland, and forest with a yearly sampling between snowmelt and first fertilization over five years. Unlike soil microbial biomass and alpha-diversity, microbial community compositions and structures were site- and land-use-specific with CAP reclassification success rates of 100%. The temporally stable site core communities included 38.5% of bacterial and 33.1% of fungal OTUs covering 95.9% and 93.2% of relative abundances. We characterized bacterial and fungal core communities and their land-use associations at the family-level. In general, fungal families revealed stronger land-use associations as compared to bacteria. This is likely due to a stronger vegetation effect on fungal core taxa, while bacterial core taxa were stronger related to soil properties. The assessment of core communities can be used to form cultivation-independent reference lists of microbial taxa, which may facilitate the development of microbial indicators for soil quality and the use of soil microbiota for long-term soil biomonitoring.

Indicative bacterial communities and taxa of disease-suppressing and growth-promoting composts and their associations to the rhizoplane.

Compost applications vary in their plant growth promotion and plant disease suppression, likely due to differences in physico-chemical and biological parameters. Our hypothesis was that bacteria are important for plant growth promotion and disease suppression of composts and, therefore, composts having these traits would contain similar sets of indicative bacterial taxa. Seventeen composts prepared from five different commercial providers and different starting materials were classified accordingly with bioassays using cress plants and the pathogen Pythium ultimum. Using a metabarcoding approach, bacterial communities were assessed in bulk composts and cress rhizoplanes. Six and nine composts showed significant disease suppression or growth promotion, respectively, but these traits did not correlate. Growth promotion correlated positively with nitrate content of composts, whereas disease suppression correlated negatively with factors representing compost age. Growth promotion and disease suppression explained significant portions of variation in bacterial community structures, i.e. 11.5% and 14.7%, respectively. Among the sequence variants (SVs) associated with growth promotion, Microvirga, Acinetobacter, Streptomyces, Bradyrhizobium and Bacillus were highly promising, while in suppressive composts, Ureibacillus,Thermogutta and Sphingopyxis were most promising. Associated SVs represent the basis for developing prediction tools for growth promotion and disease suppression, a highly desired goal for targeted compost production and application.

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.

Environmental and Anthropogenic Factors Shape Major Bacterial Community Types Across the Complex Mountain Landscape of Switzerland.

Mountain areas harbor large climatic and geographic gradients and form numerous habitats that promote high overall biodiversity. Compared to macroorganisms, knowledge about drivers of biodiversity and distribution of soil bacteria in mountain regions is still scarce but a prerequisite for conservation of bacterial functions in soils. An important question is, whether soil bacterial communities with similar structures share environmental preferences. Using metabarcoding of the 16S rRNA gene marker, we assessed soil bacterial communities at 255 sites of a regular grid covering the mountainous landscape of Switzerland, which is characterized by close location of biogeographic regions that harbor different land-use types. Distribution of bacterial communities was mainly shaped by environmental selection, as revealed by 47.9% variance explained by environmental factors, with pH (29%) being most important. Little additional variance was explained by biogeographic regions (2.8%) and land-use types (3.3%). Cluster analysis of bacterial community structures revealed six bacterial community types (BCTs), which were associated to several biogeographic regions and land-use types but overall differed mainly in their preference for soil pH. BCT I and II occurred at neutral pH, showed distinct preferences for biogeographic regions mainly differing in elevation and nutrient availability. BCT III and IV differed only in their preferred soil pH. BCT VI occurred in most acidic soils (pH 3.6) and almost exclusively at forest sites. BCT V occurred in soils with a mean pH of 4 and differed from BCT VI in preference for lower values of organic C, total nitrogen and their ratio. Indicator species and bipartite network analyses revealed 3,998 OTUs associating to different levels of environmental factors and BCTs. Taxonomic classification revealed opposing associations of taxa deriving from the same phyla. The results revealed that pH, land-use type, biogeographic region, and nutrient availability were the main factors shaping bacterial communities across Switzerland. Indicator species and bipartite network analyses revealed environmental preferences of bacterial taxa. Combining information of environmental factors and BCTs yielded increased resolution of the factors shaping soil bacterial communities and provided an improved biodiversity framework. OTUs exclusively associated to BCTs provide a novel resource to identify unassessed environmental drivers.

Small bias of one highly dominant taxon on community analyses using amplicon sequencing.

Amplicon sequencing allows for simultaneous assessment of inoculation success and responses of indigenous communities after microbial inoculation. However, the presence of a highly dominant taxon may bias correct assessment of communities due to dilution effects, chimera formation, or preferential amplification. Here, we present a simple test to assess these biases.

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.

A species-specific multiplexed PCR amplicon assay for distinguishing between Metarhizium anisopliae, M. brunneum, M. pingshaense and M. robertsii.

The fungal species Metarhizium pingshaense, M. anisopliae, M. robertsii, and M. brunneum, a monophyletic group informally referred to as the PARB species complex, are well known facultative entomopathogens, including many commercialized strains used for biological pest control. Accurate and expedient species identification of Metarhizium isolates represents an important first step when addressing ecological as well as application-related questions involving these fungi. To this end, a species-specific multiplexed polymerase chain reaction (PCR) assay was developed for identification and discrimination among Metarhizium PARB complex species, based on unique sequence signature differences within the nuclear ribosomal intergenic spacer (rIGS) and nuclear intergenic spacer regions MzFG546 and MzIGS2. Species-specificities of the four primer pairs were assessed following a three-step approach including: (1) in silico verification of sequence signatures by BLASTN searches against publically available genome and amplicon sequence data, (2) corroboration of assay specificity and robustness by performing test PCR amplifications against a taxonomically curated reference strain collection of 68 Metarhizium strains representing 12 species, and (3) testing against a field collection of 19 unknown Metarhizium isolates from soil of a Swiss meadow. The specificity of these four primer pairs provide an efficient means to detect and discriminate PARB species in studies targeting ecological aspects of indigenous isolates, as well as efficacy, persistence and potential non-target effects of applied biocontrol strains.

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.

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.