Re-Thinking the Roles of Pathogens and Mutualists: Exploring the Continuum of Symbiosis in the Context of Microbial Ecology and Evolution

Systems of classification are important for guiding research activities and providing a common platform for discussion and investigation. One such system is assigning microbial taxa to the roles of mutualists and pathogens. Yet, there are often challenges and even inconsistencies in reports of research findings when microbial taxa display behaviors outside of these two static conditions (e.g. commensal). Over the last two decades, there has been some effort to highlight a continuum of symbiosis, wherein certain microbial taxa may exhibit mutualistic or pathogenic traits depending on environmental contexts, life stages, and plant host associations. However, gaps remain in understanding how to apply the continuum approach to host-microbe pairs across a range of environmental and ecological factors. This commentary presents an alternative framework for evaluating the continuum of symbiosis using dominant archetypes that define symbiotic ranges. We focus particularly on fungi and bacteria, though we recognize that archaea and other microeukaryotes play important roles in host-microbe interactions that may be described by this approach. This framework is centered in eco-evolutionary theory and aims to enhance communication among researchers, as well as prioritize holistic consideration of the factors shaping microbial life strategies. We discuss the influence of plant-mediated factors, habitat constraints, co-evolutionary forces, and the genetic contributions which shape different microbial lifestyles. Looking to the future, using a continuum of symbiosis paradigm will enable greater flexibility in defining the roles of target microbes and facilitate a more holistic view of the complex and dynamic relationship between microbes and plants.

N-cycling microbiome recruitment differences between modern and wild Zea mays

Rewilding modern agricultural cultivars by reintroducing beneficial ancestral traits is a proposed approach to improve sustainability of modern agricultural systems. In this study, we compared recruitment of the rhizosphere microbiome among modern inbred maize and wild teosinte to assess whether potentially beneficial plant microbiome traits have been lost through maize domestication and modern breeding. To do this, we surveyed the bacterial and fungal communities along with nitrogen cycling functional groups in the rhizosphere of 6 modern domesticated maize genotypes and ancestral wild teosinte genotypes, while controlling for environmental conditions and starting soil inoculum. Using a combination of high-throughput sequencing and quantitative PCR, we found that the rhizosphere microbiomes of modern inbred and wild teosinte differed substantially in taxonomic composition, species richness, and abundance of N-cycling functional genes. Furthermore, the modern vs wild designation explained 27% of the variation in the prokaryotic microbiome, 62% of the variation in N-cycling gene richness, and 66% of N-cycling gene abundance. Surprisingly, we found that modern inbred genotypes hosted microbial communities with higher taxonomic and functional gene diversity within their microbiomes compared to ancestral genotypes. These results imply that modern maize and wild maize differ in their interaction with N-cycling microorganisms in the rhizosphere and that genetic variation exists within Zea to potentially ‘rewild’ microbiome-associated traits (i.e., exudation, root phenotypes, etc.).

Vertical stratification of microbial communities in woody plants

Bacterial and fungal endophytes form diverse communities and contribute to the performance and health of their host plants. Recent evidence suggests that both host related factors and environmental conditions determine the community structure of plant endophytes. Yet, we know little about their distribution patterns, and underlying community assembly mechanisms across plant compartments. Here we analysed the structure of bacterial and fungal communities associated with tree compartments as well as their underlying soils across 12 tree individuals in boreal forests. We found that the structure of bacterial and fungal communities depends more strongly on the vertical location of tree compartments rather than the locality, species, and individuals of host trees. Microbial communities showed much stronger host specificity in aboveground than belowground compartments. While having lower compartment community variability compared to fungi, bacterial communities were markedly more distinct between below- and aboveground components but not between hosts, reflecting the greater importance of environmental filtering rather than dispersal limitation and host identity in their community assembly. Our data suggest that spatial distance from soil as a major microbiome source contributes to the formation of microbiomes in plants, and that bacterial and fungal communities may follow contrasting assembly processes.

Winter rye cover cropping changes squash (Cucurbita pepo) phyllosphere microbiota and reduces Pseudomonas syringae symptoms

Cover cropping is a soil conservation practice that may reduce the impacts of the economically important pathogen Pseudomonas syringae on crops including squash (Cucurbita pepo). To date, no studies have directly quantified the effect of rye cover crops on P. syringae populations, nor on the bacterial community of squash leaves. In this work, we tested the hypothesis that the protective effects of cover cropping on squash may be mediated by cover cropping effects on the plant’s microbiota that in turn protects against P. syringae. Using combined 16S sequencing and culture-based approaches, we showed that rye cover cropping protects squash against P. syringae, by decreasing pathogen population size on squash leaves and increasing fruit health and marketability at harvest. We also found evidence of a strong effect of rye cover crops on bacterial communities of the squash phyllosphere. Those findings were more striking early in the growing season. Finally, we identified numerous phyllosphere bacteria belonging to the genera Sphingomonas, Methylobacterium and Pseudomonas that were promoted by rye cover crops. Overall, our findings suggest cover cropping is effective for the sustainable management of P. syringae on squash and may provide a reservoir of potential microbial biocontrol agents colonizing the phyllosphere.

Composition of the microbiomes from spinach seeds infested or non-infested with Peronospora effusa or Verticillium dahliae

The worldwide distribution of plant seeds can disseminate beneficial and plant pathogenic microorganisms. This phenomenon is of particular concern where seed production is geographically isolated from crop production, as is the case with spinach in the United States. We aimed to characterize the structure and function of spinach seed microbiomes in commercial spinach seed lots originating from Europe and New Zealand. The seed lots we analyzed were infested with Peronospora effusa and Verticillium dahliae, only infested with V. dahliae, or not infested with either of these pathogens. The microbial taxonomic composition and gene function (assessed by Gene Ontology (GO) terms) of spinach seeds were highly influenced by geographic origin and the status of pathogen infestation. Through taxonomic profiling, we found that potentially plant beneficial bacterial genera such as Pseudomonas and Pantoea were the most abundant taxa both in infested and non-infested seeds, and Stenotrophomonas was observed in seed lots infested with P. effusa and V. dahliae. Many potential plant pathogens that are not known to be associated with spinach seed were also discovered by metagenomic analysis, including Sclerotinia sclerotiorum, Botrytis cinerea, Bipolaris sorokiniana, Fusarium pseudograminearum, Alternaria brassicae, Parastagonospora nodorum, and Pyrenophora teres f. teres. Our analysis of the function of prokaryotic genes in de novo assembled metagenomes revealed distinct GO terms associated with the geographic origin of seed lots. This work provides an important first step toward identifying spinach seed-borne microorganisms that could be utilized to improve plant health and plant pathogens that could be inadvertently carried to new locations.

Frontiers and opportunities in bioenergy crop microbiome research networks

Researchers from across the four U.S. Department of Energy Bioenergy Research Centers engaged in a microbiome workshop that focused on identifying challenges and collaboration opportunities to better understand bioenergy-relevant plant–microbe interactions. The virtual workshop included hands-on educational sessions and a keynote address on current best practices in microbiome science and community microbiome standards, as well as breakout sessions aimed at identifying microbiome-related data and measurements that should be prioritized, opportunities for and barriers to integrating plant metabolites to microbiome research, and strategies for more effectively integrating microbiome data and processes into existing models. Based on participant discussion, key findings of the workshop were the need to prioritize scaling data sharing across BRCs and the broader research community and securing collaborative infrastructure in the areas of microbiome-ecosystem modeling and molecular plant-microbe interactions. This workshop review highlights additional main findings from this event, to encourage cross-site and more holistic meta-analyses while promoting wide scientific community engagement across plant microbiome sciences.

Exploring the role of fungal endophytes in the sudden death syndrome of the invasive shrub Chrysanthemoides monilifera subsp. rotundata in Australia

Pathogens that attack invasive plants can positively affect the integrity and functioning of ecosystems. Stem-tip dieback and extensive wilting followed by sudden death have been observed in Chrysanthemoides monilifera subsp. rotundata (bitou bush), one of Australia’s worst invasive shrubs. Metabarcoding and culturing methods were used to investigate if fungi are implicated in this syndrome. Metabarcoding results revealed significantly different endophytic fungal communities within healthy and diseased bitou bush, and co-located native plants. There was no difference in fungal communities between soil sampled in the root zone of healthy and diseased bitou bush at the same site. Two Diaporthe sp. operational taxonomic units (OTUs), dominant at sites with extensive wilting, explained 30% of the similarity between diseased bitou bush across all sites. Two other OTUs, Austropleospora osteospermi and Coprinellus sp., explained 20 and 40% of the similarity between diseased plants, respectively, and were only dominant at sites with dead or stunted, partially defoliated but not wilted bitou bush. A Penicillium sp. OTU explained 90% of the similarity between healthy bitou bush. Various Diaporthe spp. dominated isolations from diseased bitou bush. Manipulative experiments confirmed Diaporthe spp. pathogenicity on bitou bush excised and in-situ stems. In another experiment, Diaporthe masirevicii infected flowers and from there colonized stems endophytically, but wilting and sudden death of bitou bush did not occur within the experimental timeframe. Our study provides circumstantial evidence that bitou bush sudden death syndrome is the result of a shift in the composition of its endophytic fungal community, from mutualist to pathogenic species.

The decay and fungal succession of apples with bitter rot across a vegetation diversity gradient

Bitter rot is a disease of apple caused by fungi in the genus Colletotrichum. Management begins with removal of infected twigs and fruits from tree canopies to reduce overwintering inoculum. Infected apples are usually tossed to the orchard floor, which is generally managed as herbicide-treated weed-free tree rows, separated by grass drive rows. We monitored decay rates and succession of fungi of apples with bitter rot in tree canopies, and on the soil surface in tree rows, grass drive rows, and nearby diverse plant communities. We hypothesized that decay would occur most rapidly within diverse plant communities, which would provide a more diverse array of potential fungal decomposers. Apples in tree canopies became dry and mummified and had more Colletotrichum gene marker copies the following growing season than did apples on the soil surface. Of the soil surface samples, those in grass drive rows and diverse plant communities had higher moisture, faster decay rates, and sharper decreases in Colletotrichum gene marker copies than apples in tree rows. Fungal composition across all decaying apples was dominated by yeasts, with higher genus-level richness, diversity, and evenness in apples from tree canopies than those on the soil surface. In soil surface apples, we observed clear successional waves of Pichia, Kregervanrija, and [Candida] yeasts, with similar but distinctly diverging fungal composition. Our results show that orchard floor management can influence fungal succession in apples with bitter rot, but suggests that bitter rot management should primarily focus on removing infected apples from tree canopies.

From roots to leaves: the capacity of Micromonospora to colonize different legume tissues

An important number of Micromonospora strains have been reported from nitrogen fixing root nodules of legume and actinorhizal plants. However, the question of whether this bacterium can also be found in other parts of these plants remains unanswered. Over 150 strains were recovered from different Lupinus angustifolius and Pisum sativum tissues including leaves, stems, roots, and nodules. Ninety-seven percent of the isolates were identified by 16S rRNA gene sequence in the target genus and were associated with 27 different Micromonospora species. Plant-polymer degrading enzymes are suspected to play a role in the colonization of plants. To this end, bacterial enzymatic activity assays for amylases, cellulases, chitinases, pectinases and xylanases were determined. All strains produced xylanases and pectinases, while 98.6%, 98%, and 94.6% of them produced amylases, cellulases, and chitinases, respectively. The most productive strains included seven isolates from P. sativum and one from L. angustifolius. Strain Micromonospora lupini ML01-gfp was used to determine its capacity to reach and colonize different plant organs using P. sativum as the plant model. Stem and leaf samples were monitored by optical and fluorescence microscopy to locate the tagged strain. These results strongly suggest that Micromonospora is able, not only to infect nitrogen-fixing nodules, but also of reaching other parts of the host plant, especially the leaves.

Endophytic microbiome variation among single plant seeds

Like other plant compartments, the seed harbors a microbiome. Seed microbiome members are the first to colonize a germinating seedling, and they may initiate the trajectory of microbiome assembly for the next plant generation. Therefore, the members of the seed microbiome are important for the dynamics of plant microbiome assembly and the vertical transmission of potentially beneficial symbionts. However, it remains challenging to assess the microbiome at the individual seed level (and, therefore, for the future individual plants) due to low endophytic microbial biomass, seed exudates that can select for particular members, and high plant and plastid contamination of resulting reads. Here, we report a protocol for extracting microbial DNA from an individual seed (common bean, Phaseolus vulgaris L.) with minimal disruption of host tissue, which we expect to be generalizable to other medium- and large-seed plant species. We applied this protocol to determine the 16S rRNA V4 and rRNA ITS2 amplicon composition and examine the variability of individual seeds harvested from replicate common bean plants grown under standard, controlled conditions to maintain health. Using DNA extractions from individual seeds, we compared seed-to-seed, pod-to-pod, and plant-to-plant microbiomes, and found highest microbiome variability at the plant level. This suggests that several seeds from the same plant could be pooled for microbiome assessment, given experimental designs that apply treatments at the parent plant level. This study adds protocols and insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems.