Background Biological soil crusts (biocrusts) are a key component of arid land ecosystems, where they render critical services such as soil surface stabilization and nutrient fertilization. The bundle-forming, filamentous, non-nitrogen-fixing cyanobacterium Microcoleus vaginatus is a pioneer primary producer, often the dominant member of the biocrust microbiome, and the main source of leaked organic carbon. We hypothesized that, by analogy to the rhizosphere of plant roots, M. vaginatus may shape the microbial populations of heterotrophs around it, forming a specialized cyanosphere. Results By physically isolating bundles of M. vaginatus from biocrusts, we were able to study the composition of the microbial populations attached to it, in comparison to the bulk soil crust microbiome by means of high-throughput 16S rRNA sequencing. We did this in two M. vaginatus -dominated biocrust from distinct desert biomes. We found that a small, selected subset of OTUs was significantly enriched in close proximity to M. vaginatus . Furthermore, we also found that a majority of bacteria (corresponding to some two thirds of the reads) were significantly more abundant away from this cyanobacterium. Phylogenetic placements suggest that all typical members of the cyanosphere were copiotrophs and that many were diazotrophs (Additional file 1 : Tables S2 and S3). Nitrogen fixation genes were in fact orders of magnitude more abundant in this cyanosphere than in the bulk biocrust soil as assessed by qPCR. By contrary, competition for light, CO 2, and low organic carbon concentrations defined at least a part of the OTUs segregating from the cyanobacterium. Conclusions We showed that M . vaginatus acts as a significant spatial organizer of the biocrust microbiome. On the one hand, it possesses a compositionally differentiated cyanosphere that concentrates the nitrogen-fixing function. We propose that a mutualism based on C for N exchange between M . vaginatus and copiotrophic diazotrophs helps sustains this cyanosphere and that this consortium constitutes the true pioneer community enabling the colonization of nitrogen-poor soils. On the other hand, a large number of biocrust community members segregate away from the vicinity of M . vaginatus , potentially through competition for light or CO 2 , or because of a preference for oligotrophy. Electronic supplementary material The online version of this article (10.1186/s40168-019-0661-2) contains supplementary material, which is available to authorized users.

Biological soil crusts (biocrusts) are slow-growing, phototroph-based microbial assemblages that develop on the topsoils of drylands. Biocrusts help maintain soil fertility and reduce erosion. Because their loss through human activities has negative ecological and environmental health consequences, biocrust restoration is of interest. Active soil inoculation with biocrust microorganisms can be an important tool in this endeavor. We present a culture-independent, two-step process to grow multispecies biocrusts in open greenhouse nursery facilities, based on the inoculation of local soils with local biocrust remnants and incubation under seminatural conditions that maintain the essence of the habitat but lessen its harshness. In each of four U.S. Southwest sites, we tested and deployed combinations of factors that maximized growth (gauged as chlorophyll a content) while minimizing microbial community shifts (assessed by 16S rRNA sequencing and bioinformatics), particularly for crust-forming cyanobacteria. Generally, doubling the frequency of natural wetting events, a 60% reduction in sunlight, and inoculation by slurry were optimal. Nutrient addition effects were site specific. In 4 months, our approach yielded crusts of high inoculum quality reared on local soil exposed to locally matched climates, acclimated to desiccation, and containing communities minimally shifted in composition from local ones. Our inoculum contained abundant crust-forming cyanobacteria and no significant numbers of allochthonous phototrophs, and it was sufficient to treat ca. 6,000 m 2 of degraded dryland soils at 1 to 5% of the typical crust biomass concentration, having started from a natural crust remnant as small as 6 to 30 cm 2 .IMPORTANCE Soil surface crusts can protect dryland soils from erosion, but they are often negatively impacted by human activities. Their degradation causes a loss of fertility, increased production of fugitive dust and intensity of dust storms with associated traffic problems, and provokes general public health hazards. Our results constitute an advance in the quest to actively restore biological soil covers by providing a means to obtain high-quality inoculum within a reasonable time (a few months), thereby allowing land managers to recover essential, but damaged, ecosystem services in a sustainable, self-perpetuating way as provided by biocrust communities.

Biological soil crusts (biocrust) are microbial communities that develop at the soil surface of drylands and play an important role in erosion control and fertility. Soil surface disturbance from a broad range of natural and human processes (e.g. fire, livestock grazing, off-road traffic) cause significant losses in biocrust cover and associated ecosystems services. Hence, biocrust restoration is emerging as an important intervention strategy to rehabilitate degraded dryland soils. In a multistep process, we designed protocols for the establishment of "microbial nurseries" to produce photosynthetic cyanobacterial inoculum for biocrust seeding at scale. We first report on the strategy for isolation, directly from the target site, of a large culture collection of cyanobacteria that included multiple representatives of the five most common biocrust taxa. After genetic pedigreeing of these isolates, we could select those that best matched field populations genetically for scale-up cultivation. We then developed protocols for effective cyanobacterial biomass production to obtain sufficient inoculum. This was followed by conditioning treatments (hardening off) to preacclimate this inoculum to the stressful conditions expected in the field. Finally, we show that the inoculum obtained was fit to thrive in its original soil under natural outdoor conditions if sufficient water was available. We repeated this process successfully for four sites, two in the hot Chihuanuan desert and two in the cooler Great Basin Desert, and on two textural types of soils in each. The cyanobacterial biocrust nursery approach represents a versatile, viable, and safe tool for the rehabilitation of dryland soils.

Microcoleus vaginatus plays a prominent role as both primary producer and pioneer in biocrust communities from dryland soils. And yet, it cannot fix dinitrogen, essential in often nitrogen-limited drylands. But a diazotroph-rich “cyanosphere” has been described in M. vaginatus, hinting that there exists a C for N exchange between the photoautotroph and heterotrophic diazotrophs. We provide evidence for this by establishing such a symbiosis in culture and by showing that it is selective and dependent on nitrogen availability. In natural populations, provision of nitrogen resulted in loss of diazotrophs from the cyanosphere of M. vaginatus compared to controls, but provision of phosphorus did not. Co-culturing of pedigreed cyanosphere diazotroph isolates with axenic M. vaginatus resulted in copious growth in C and N-free medium, but co-culture with non-cyanosphere diazotrophs or other heterotrophs did not. Unexpectedly, bundle formation in M. vaginatus, diacritical to the genus but not seen in axenic culture, was restored in vitro by imposed nitrogen limitation or, even more strongly, by co-culture with diazotrophic partners, implicating this trait in the symbiosis. Our findings provide direct evidence for a symbiotic relationship between M. vaginatus and its cyanosphere and help explain how it can be a global pioneer in spite of its genetic shortcomings.

Cyanobacteria are a widespread and important bacterial phylum, responsible for a significant portion of global carbon and nitrogen fixation. Unfortunately, reliable and accurate automated classification of cyanobacterial 16S rRNA gene sequences is muddled by conflicting systematic frameworks, inconsistent taxonomic definitions (including the phylum itself), and database errors. To address this, we introduce Cydrasil 3 (https://www.cydrasil.org), a curated 16S rRNA gene reference package, database, and web application designed to provide a full phylogenetic perspective for cyanobacterial systematics and routine identification. Cydrasil 3 contains over 1300 manually curated sequences longer than 1100 base pairs and can be used for phylogenetic placement or as a reference sequence set for de novo phylogenetic reconstructions. The web application (utilizing PaPaRA and EPA-ng) can place thousands of sequences into the reference tree and has detailed instructions on how to analyze results. While the Cydrasil web application offers no taxonomic assignments, it instead provides phylogenetic placement, as well as a searchable database with curation notes and metadata, and a mechanism for community feedback.