Heterokaryosis is an integral part of the parasexual cycle used by predominantly asexual fungi to introduce and maintain genetic variation in populations. Research into fungal heterokaryons began in 1912 and continues to the present day. Heterokaryosis may play a role in the ability of fungi to respond to their environment, including the adaptation of arbuscular mycorrhizal fungi to different plant hosts. The parasexual cycle has enabled advances in fungal genetics, including gene mapping and tests of complementation, dominance, and vegetative compatibility in predominantly asexual fungi. Knowledge of vegetative compatibility groups has facilitated population genetic studies and enabled the design of innovative methods of biocontrol. The vegetative incompatibility response has the potential to be used as a model system to study biological aspects of some human diseases, including neurodegenerative diseases and cancer. By combining distinct traits through the formation of artificial heterokaryons, fungal strains with superior properties for antibiotic and enzyme production, fermentation, biocontrol, and bioremediation have been produced. Future biotechnological applications may include site-specific biocontrol or bioremediation and the production of novel pharmaceuticals.

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The soybean cyst nematode (SCN), Heterodera glycines Ichinohe (Phylum Nematoda), is a major pathogen of soybean. It causes substantial yield losses worldwide and is difficult to control because the cyst protects the eggs which can remain viable for nearly a decade. Crop rotation with non-host crops and use of biocontrol organisms such as fungi and bacteria offer promising approaches, but remain hampered by lack of knowledge of the biology of nematode parasitic organisms. We used a high-throughput metabarcoding approach to characterize fungal communities associated with the SCN cyst, a microenvironment in soil that may harbor both nematode parasites and plant pathogens. SCN cysts were collected from a long-term crop rotation experiment in Southeastern Minnesota at three time points over two growing seasons to characterize diversity of fungi inhabiting cysts and to examine how crop rotation and seasonal variation affects fungal communities. A majority of fungi in cysts belonged to Ascomycota and Basidiomycota, but the presence of several early diverging fungal subphyla thought to be primarily plant and litter associated, including Mortierellomycotina and Glomeromycotina (e.g., arbuscular mycorrhizal fungi), suggests a possible role as nematode egg parasites. Species richness varied by both crop rotation and season and was higher in early years of crop rotation and in fall at the end of the growing season. Crop rotation and season also impacted fungal community composition and identified several classes of fungi, including Eurotiomycetes, Sordariomycetes, and Orbiliomycetes (e.g., nematode trapping fungi), with higher relative abundance in early soybean rotations. The relative abundance of several genera was correlated with increasing years of soybean. Fungal communities also varied by season and were most divergent at midseason. The percentage of OTUs assigned to Mortierellomycotina_cls_Incertae_sedis and Sordariomycetes increased at midseason, while Orbiliomycetes decreased at midseason, and Glomeromycetes increased in fall. Ecological guilds of fungi containing an animal-pathogen lifestyle, as well as potential egg-parasitic taxa previously isolated from parasitized SCN eggs, increased at midseason. The animal pathogen guilds included known (e.g., Pochonia chlamydosporia) and new candidate biocontrol organisms. This research advances knowledge of the ecology of nematophagous fungi in agroecosystems and their use as biocontrol agents of the SCN.

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the number 1 pathogen of the important economic crop soybean. Bacteria represent potential biocontrol agents of the SCN, but few studies have characterized the dynamics of bacterial communities associated with cysts under different crop rotation sequences. The bacterial communities in SCN cysts in a long-term soybean–corn crop rotation experiment were investigated over 2 years. The crop sequences included long-term soybean monoculture (Ss), years 1–5 of soybean following 5 years corn (S1–S5), years 1 and 2 of corn following 5 years soybean (C1 and C2), and soybean–corn annual rotation (Sa and Ca). The bacterial 16S rRNA V4 region was amplified from DNA isolated from SCN cysts collected in spring at planting, midseason (2 months later), and fall at harvest and sequenced on the Illumina MiSeq platform. The SCN cyst microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidetes, and Verrucomicrobia. The bacterial community composition was influenced by both crop sequence and season. Although differences by crop sequence were not significant in the spring of each year, bacterial communities in cysts from annual rotation (Sa and Ca) or crop sequences of early years of monoculture following a 5-year rotation of the alternate crop (S1 and C1) became rapidly differentiated by crop over a single growing season. In the fall, genera of cyst bacteria associated with soybean crop sequences included Rhizobacter, Leptothrix, Cytophaga, Chitinophaga, Niastella, Streptomyces, and Halangium. The discovery of diverse bacterial taxa in SCN cysts and their dynamics across crop rotation sequences provides invaluable information for future development of biological control of the SCN.

Although fungal endophytes are commonly investigated for their ability to deter microbial plant pathogens, few studies have examined the activity of fungal root endophytes against nematodes. The soybean cyst nematode (SCN; Heterodera glycines), the most severe yield-limiting pathogen of soybean (Glycine max), is commonly managed through rotation of soybean with corn (Zea mays), a nonhost of the SCN. A total of 626 fungal endophytes were isolated from surface-sterilized corn and soybean roots from experimental plots in which soybean and corn had been grown under annual rotation and under 1, 3, 5, and 35 years of continuous monoculture. Fungal isolates were grouped into 401 morphotypes, which were clustered into 108 operational taxonomic units (OTUs) based on 99% sequence similarity of the full internal transcribed spacer region. Morphotype representatives within each OTU were grown in malt extract broth and in a secondary metabolite-inducing medium buffered with ammonium tartrate, and their culture filtrates were tested for nematicidal activity against SCN juveniles. A majority of OTUs containing isolates with nematicidal culture filtrates were in the order Hypocreales, with the genus Fusarium being the most commonly isolated nematicidal genus from corn and soybean roots. Less commonly isolated taxa from soybean roots included the nematophagous fungi Hirsutella rhossiliensis, Metacordyceps chlamydosporia, and Arthrobotrys iridis. Root endophytic fungal diversity in soybean was positively correlated with SCN density, suggesting that the SCN plays a role in shaping the soybean root endophytic community.

The rhizosphere effect shapes microbial communities around and within plant roots and may lead to the enrichment of fungi involved in both positive and negative plant-soil feedbacks. We used internal transcribed spacer 1 amplicon sequencing to investigate how continuous monoculture affects the fungal communities in the corn and soybean rhizosphere, rhizoplane, and root endosphere in a long-term crop rotation experiment where soils were infested with a major soybean pathogen, the soybean cyst nematode (SCN, Heterodera glycines). Community-level statistical analyses showed evidence of selective filtering and enrichment of fungi in and around corn and soybean roots and in SCN cysts. Patterns of relatedness between fungal communities in various agroecosystem compartments suggested that SCN cysts are colonized by soybean root endophytic fungi and that colonization of roots and cysts may aid proliferation of these fungi in the bulk soil agroecosystem compartment over time. Natural antagonists of the SCN such as nematode-trapping fungi and nematode endoparasites increased in relative abundance in the rhizosphere and root endosphere, respectively, over continuous soybean monoculture. In contrast, arbuscular mycorrhizal and plant-pathogenic fungi, several of which were negatively correlated with corn yield, increased in relative abundance over continuous corn monoculture. These results suggest the possibility of positive plant-soil feedbacks involving nematophagous fungi over continuous soybean monoculture and negative plant-soil feedbacks involving plant pathogens and some nonbeneficial arbuscular mycorrhizal fungi over continuous corn monoculture.

The microbiome is known to play a role in many human diseases, but identifying key microbes and their functions generally requires large studies due to the vast number of species and genes, and the high levels of intra-individual and inter-individual variation. 16S amplicon sequencing of the rRNA gene is commonly used for large studies due to its comparatively low sequencing cost, but it has poor taxonomic and functional resolution. Deep shotgun sequencing is a more accurate and comprehensive alternative for small studies, but can be cost-prohibitive for biomarker discovery in large populations. Shallow or moderate-depth shotgun metagenomics may serve as a viable alternative to 16S sequencing for large-scale and/or dense longitudinal studies, but only if resolution and reproducibility are comparable. Here we applied both 16S and shallow shotgun stool microbiome sequencing to a cohort of 5 subjects sampled twice daily and weekly, with technical replication at the DNA extraction and the library preparation/sequencing steps, for a total of 80 16S samples and 80 shallow shotgun sequencing samples. We found that shallow shotgun sequencing produced lower technical variation and higher taxonomic resolution than 16S sequencing, at a much lower cost than deep shotgun sequencing. These findings suggest that shallow shotgun sequencing provides a more specific and more reproducible alternative to 16S sequencing for large-scale microbiome studies where costs prohibit deep shotgun sequencing and where bacterial species are expected to have good coverage in whole-genome reference databases.

Aim: Swine manure foaming is a major problem, causing damage to property, livestock, and people. Here, we identified the main chemicals and microbes that contribute to foaming. Methods and results:Foaming and non-foaming swine manure were sampled from farms in Iowa and Illinois. Targeted and untargeted metabolomics analyses identified chemical markers that differed between foaming and non-foaming manure and between manure layers. Microbial community analysis and metagenomics were performed on a subset of samples. Foam contained significantly higher levels of total bile acids and long chain fatty acids like palmitic, stearic and oleic acid than the other manure layers. Foam layers also had significantly higher levels of ubiquinone 9 and ubiquinone 10. The slurry layer of foaming samples contained more alanine, isoleucine/leucine, diacylglycerols (DG), phosphtatidylethanolamines, and vitamin K2, while ceramide was significantly increased in the slurry layer of non-foaming samples. Eubacterium coprostanoligenes and Methanoculleus were more abundant in foaming samples, and E. coprostanoligenes was significantly correlated with levels of DG. Genes involved in diacylglycerol biosynthesis and in the biosynthesis of branched-chain hydrophobic amino acids were overrepresented in foaming samples. Conclusions:A mechanism for manure foaming is hypothesized in which proliferation of Methanoculleus leads to excessive production of methane, while production of DG by E. coprostanoligenes and hydrophobic proteins by Methanosphaera stadtmanae facilitates bubble formation and stabilization. Significance and impact of study:While some chemical and biological treatments have been developed to treat swine manure foaming, its causes remain unknown. We identified key microbes and metabolites that correlate with foaming and point to possible roles of other factors like animal feed.