Morels (Morchella spp.) are iconic edible mushrooms with a long history of human consumption. Some microbial taxa are hypothesized to be important in triggering the formation of morel primordia and development of fruiting bodies, thus, there is interest in the microbial ecology of these fungi. To identify and compare fungal and prokaryotic communities in soils where Morchella sextelata is cultivated in outdoor greenhouses, ITS and 16S rDNA high throughput amplicon sequencing and microbiome analyses were performed. Pedobacter, Pseudomonas, Stenotrophomonas, and Flavobacterium were found to comprise the core microbiome of M. sextelata ascocarps. These bacterial taxa were also abundant in the soil beneath growing fruiting bodies. A total of 29 bacterial taxa were found to be statistically associated to Morchella fruiting bodies. Bacterial community network analysis revealed high modularity with some 16S rDNA operational taxonomic unit clusters living in specialized fungal niches (e.g., pileus, stipe). Other fungi dominating the soil mycobiome beneath morels included Morchella, Phialophora, and Mortierella. This research informs understanding of microbial indicators and potential facilitators of Morchella ecology and fruiting body production.

Soybean (Glycine max) is an important leguminous crop that is grown throughout the United States and around the world. In 2016, soybean was valued at $41 billion USD in the United States alone. Increasingly, soybean farmers are adopting alternative management strategies to improve the sustainability and profitability of their crop. Various benefits have been demonstrated for alternative management systems, but their effects on soybean-associated microbial communities are not well-understood. In order to better understand the impact of crop management systems on the soybean-associated microbiome, we employed DNA amplicon sequencing of the Internal Transcribed Spacer (ITS) region and 16S rRNA genes to analyze fungal and prokaryotic communities associated with soil, roots, stems, and leaves. Soybean plants were sampled from replicated fields under long-term conventional, no-till, and organic management systems at three time points throughout the growing season. Results indicated that sample origin was the main driver of beta diversity in soybean-associated microbial communities, but management regime and plant growth stage were also significant factors. Similarly, differences in alpha diversity are driven by compartment and sample origin. Overall, the organic management system had lower fungal and bacterial Shannon diversity. In prokaryotic communities, aboveground tissues were dominated by Sphingomonas and Methylobacterium while belowground samples were dominated by Bradyrhizobium and Sphingomonas. Aboveground fungal communities were dominated by Davidiella across all management systems, while belowground samples were dominated by Fusarium and Mortierella. Specific taxa including potential plant beneficials such as Mortierella were indicator species of the conventional and organic management systems. No-till management increased the abundance of groups known to contain plant beneficial organisms such as Bradyrhizobium and Glomeromycotina. Network analyses show different highly connected hub taxa were present in each management system. Overall, this research demonstrates how specific long-term cropping management systems alter microbial communities and how those communities change throughout the growth of soybean.

Morel mushrooms (Morchella, Pezizales) are highly prized edible fungi. Approaches to cultivate morels indoors in pasteurized composted substrates have been successful for Morchella rufobrunnea. We used DNA amplicon sequencing of the Internal Transcribed Spacer (ITS) ribosomal DNA and 16S rRNA gene to follow bacterial and fungal communities in substrates during indoor morel cultivation. Our goal was to determine changes in microbial communities at key stages of morel cultivation, which included primordia development, fundament initiation, differentiation and maturation. Additionally, we compared microbial communities between trays that successfully fruited to those that produced conidia and primordia but aborted before ascocarp formation (non-fruiting). The prokaryotic community was dominated by Firmicutes belonging to Bacillus and Paenibacillus with a lower abundance of Flavobacteria. At earlier stages, the fungal community was dominated by Pezizomycetes including Morchella and other species, whereas, later in the cropping cycle Sordariomycetes dominated. Additionally, differences were observed between trays with successful fruiting, which were dominated by Gilmaniella; compared to trays that did not fruit, which were dominated by Cephalotrichum. Our findings inform understanding of microbial community dynamics during morel cultivation, and show that fungal genera, such as Gilmaniella, and prokaryotic genera, such as Bacillus, are abundant in substrates that support M. rufobrunnea fruiting.

Fungicides reduce fungal pathogen populations and are essential to food security. Understanding the impacts of fungicides on crop microbiomes is vital to minimizing unintended consequences while maintaining their use for plant protection. However, fungicide disturbance of plant microbiomes has received limited attention, and has not been examined in different agricultural management systems. We used amplicon sequencing of fungi and prokaryotes in maize and soybean microbiomes before and after foliar fungicide application in leaves and roots from plots under long-term no-till and conventional tillage management. We examined fungicide disturbance and resilience, which revealed consistent non-target effects and greater resiliency under no-till management. Fungicides lowered pathogen abundance in maize and soybean and decreased the abundance of Tremellomycetes yeasts, especially Bulleribasidiaceae, including core microbiome members. Fungicide application reduced network complexity in the soybean phyllosphere, which revealed altered co-occurrence patterns between yeast species of Bulleribasidiaceae, and Sphingomonas and Hymenobacter in fungicide treated plots. Results indicate that foliar fungicides lower pathogen and non-target fungal abundance and may impact prokaryotes indirectly. Treatment effects were confined to the phyllosphere and did not impact belowground microbial communities. Overall, these results demonstrate the resilience of no-till management to fungicide disturbance, a potential novel ecosystem service provided by no-till agriculture.

Tar spot is a fungal disease complex of corn that has been destructive and yield limiting in Central and South America for nearly 50 years. Phyllachora maydis, the causal agent of tar spot, is an emerging corn pathogen in the United States, first reported in 2015 from major corn producing regions of the country. The tar spot disease complex putatively includes Monographella maydis (syn. Microdochium maydis), which increases disease damage through the development of necrotic halos surrounding tar spot lesions. These necrotic halos, termed “fish-eye” symptoms, have been identified in the United States, though Monographella maydis has not yet been confirmed. A recent surge in disease severity and loss of yield attributed to tar spot in the United States has led to increased attention and expanded efforts to understand the disease complex and how to manage it. In this study, next-generation sequencing of the internal transcribed spacer-1 (ITS1) ribosomal DNA was used to identify fungal taxa that distinguish tar spot infections with or without fish-eye symptoms. Fungal communities within tar spot only lesions were significantly different from communities having fish-eye symptoms. Two low abundance OTUs were identified as Microdochium sp., however, neither were associated with fish-eye symptom development. Interestingly, a single OTU was found to be significantly more abundant in fish-eye lesions compared to tar spot lesions and had a 91% ITS1 identity to Neottiosporina paspali. In addition, the occurrence of this OTU was positively associated with Phyllachora maydis fish-eye symptom networks, but not in tar spot symptom networks. Neottiosporina paspali has been reported to cause necrotic lesions on various monocot grasses. Whether the related fungus we detected is part of the tar-spot complex of corn and responsible for fish-eye lesions remains to be tested. Alternatively, many OTUs identified as Phyllachora maydis, suggesting that different isolate genotypes may be capable of causing both tar spot and fish-eye symptoms, independent of other fungi. We conclude that Monographella maydis is not required for fish-eye symptoms in tar spot of corn.

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

Highlights d A novel nitrogen-fixing heterocyst has evolved multiple times in Fischerella d The envelopes of these heterocysts are less permeable than those of ancestral cells d This is adaptive at high temperature but results in ecological specialization d Adaptation involved loss of phenotypic plasticity and has a simple genetic basis

Fungal communities are known to contribute to the functioning of living plant microbiomes as well as to the decay of dead plant material and affect vital ecosystem services, such as pathogen resistance and nutrient cycling. Yet, factors that drive structure and function of phyllosphere mycobiomes and their fate in leaf litter are often ignored. We sought to determine the factors contributing to the composition of communities in temperate forest substrates, with culture-independent amplicon sequencing of fungal communities of pre-senescent leaf surfaces, internal tissues, leaf litter, underlying humus soil of co-occurring red maple (Acer rubrum) and shagbark hickory (Carya ovata). Paired samples were taken at five sites within a temperate forest in southern Michigan, USA. Fungal communities were differentiable based on substrate, host species, and site, as well as all two-way and three-way interactions of these variables. PERMANOVA analyses and co-occurrence of taxa indicate that soil communities are unique from both phyllosphere and leaf litter communities. Correspondence of endophyte, epiphyte, and litter communities suggests dispersal plays an important role in structuring fungal communities. Future work will be needed to assess how this dispersal changes microbial community functioning in these niches.