Plant-associated Microbial Communities Research Articles

Variation in the leaf and root microbiome of sugar maple (Acer saccharum) at an elevational range limit.

BackgroundBacteria, archaea, viruses and fungi live in various plant compartments including leaves and roots. These plant-associated microbial communities have many effects on host fitness and function. Global climate change is impacting plant species distributions, a phenomenon that will affect plant-microbe interactions both directly and indirectly. In order to predict plant responses to global climate change, it will be crucial to improve our understanding of plant-microbe interactions within and at the edge of plant species natural ranges. While microbes affect their hosts, in turn the plant’s attributes and the surrounding environment drive the structure and assembly of the microbial communities themselves. However, the patterns and dynamics of these interactions and their causes are poorly understood.MethodsIn this study, we quantified the microbial communities of the leaves and roots of seedlings of the deciduous tree species sugar maple (Acer saccharum Marshall) within its natural range and at the species’ elevational range limit at Mont-Mégantic, Quebec. Using high-throughput DNA sequencing, we quantified the bacterial and fungal community structure in four plant compartments: the epiphytes and endophytes of leaves and roots. We also quantified endophytic fungal communities in roots.ResultsThe bacterial and fungal communities of A. saccharum seedlings differ across elevational range limits for all four plant compartments. Distinct microbial communities colonize each compartment, although the microbial communities inside a plant’s structure (endophytes) were found to be a subset of the communities found outside the plant’s structure (epiphytes). Plant-associated bacterial communities were dominated by the phyla Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes while the main fungal taxa present were Ascomycota.DiscussionWe demonstrate that microbial communities associated with sugar maple seedlings at the edge of the species’ elevational range differ from those within the natural range. Variation in microbial communities differed among plant components, suggesting the importance of each compartment’s exposure to changes in biotic and abiotic conditions in determining variability in community structure. These findings provide a greater understanding of the ecological processes driving the structure and diversity of plant-associated microbial communities within and at the edge of a plant species range, and suggest the potential for biotic interactions between plants and their associated microbiota to influence the dynamics of plant range edge boundaries and responses to global change.

Nutrient- and Dose-Dependent Microbiome-Mediated Protection against a Plant Pathogen

Plant-associated microbial communities can promote plant nutrient uptake, growth, and resistance to pathogens [1-3]. Host resistance to infection can increase directly through commensal-pathogen interactions orindirectly through modulation of host defenses [4-6], the mechanisms of which are best described for rhizosphere-associated bacteria. For example, Arabidopsis plants infected with the foliar pathogen, Pseudomonas syringae pathovar tomato (Pst), increase their root secretion of malate, which attracts Bacillus subtillis to the roots and leads to a stronger host response against Pst [7]. Although there are numerous examples of individual defensive symbionts (e.g., [8]), it is less clear whether this type of protection is an emergent property of whole microbial communities. In particular, relatively little is known about whether and how the presence of phyllosphere (above-ground) microbial communities can increase host resistance against pathogens. In this study, we examined the ability of augmented tomato phyllosphere microbiomes to confer resistance against the causal agent of bacterial speck, Pst. Across five independent experiments, the augmented phyllosphere microbiome was found to decrease pathogen colonization. Furthermore, the dose of commensal bacteria applied affected the degree of protection conferred, and although the effect is dependent on microbial composition, it is not clearly related to overall bacterial diversity. Finally, our results suggest that resources available to the phyllosphere microbial community may play an important role in protection, as the addition of fertilizer abolished the observed microbiome-mediated protection. Together, these results have clear relevance to microbiome-mediated protection within agricultural settings and the use of plant probiotics to increase disease resistance.

Ecotype-Dependent Response of Bacterial Communities Associated with Arabidopsis to Cold Acclimation

Low temperature is a primary factor limiting geographical distribution of plants and crop yield in large areas of the world. Plant-associated microbial communities have rarely been acknowledged as possible determinants of cold acclimation, the process leading to freezing tolerance in plants. Here we studied the impact of cold acclimation on leaf-associated bacteria by analyzing 10 different Arabidopsis ecotypes differing widely in their freezing tolerance. In climate chamber experiments, we found that leaves of all Arabidopsis accessions were colonized by highly diverse bacterial taxa (852 operational taxonomic units) mainly from Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. During cold acclimation, in all ecotypes the bacterial community composition and diversity, and especially the core species composition changed drastically. However, the response of the bacterial communities was highly ecotype-dependent; sensitive and tolerant accessions shared only eight responders. Unique responders in tolerant accessions were identified as putative indicators of freezing tolerance. Thus, leaf bacteria appear to be genotype-dependent associated with cold acclimation, which suggests an additional function of plant−microbe interaction and may open new possibilities for biotechnological applications.

Defining the Core Citrus Leaf- and Root-Associated Microbiota: Factors Associated with Community Structure and Implications for Managing Huanglongbing (Citrus Greening) Disease.

Stable associations between plants and microbes are critical to promoting host health and productivity. The objective of this work was to test the hypothesis that restructuring of the core microbiota may be associated with the progression of huanglongbing (HLB), the devastating citrus disease caused by Liberibacter asiaticus, Liberibacter americanus, and Liberibacter africanus The microbial communities of leaves (n = 94) and roots (n = 79) from citrus trees that varied by HLB symptom severity, cultivar, location, and season/time were characterized with Illumina sequencing of 16S rRNA genes. The taxonomically rich communities contained abundant core members (i.e., detected in at least 95% of the respective leaf or root samples), some overrepresented site-specific members, and a diverse community of low-abundance variable taxa. The composition and diversity of the leaf and root microbiota were strongly associated with HLB symptom severity and location; there was also an association with host cultivar. The relative abundance of Liberibacter spp. among leaf microbiota positively correlated with HLB symptom severity and negatively correlated with alpha diversity, suggesting that community diversity decreases as symptoms progress. Network analysis of the microbial community time series identified a mutually exclusive relationship between Liberibacter spp. and members of the Burkholderiaceae, Micromonosporaceae, and Xanthomonadaceae This work confirmed several previously described plant disease-associated bacteria, as well as identified new potential implications for biological control. Our findings advance the understanding of (i) plant microbiota selection across multiple variables and (ii) changes in (core) community structure that may be a precondition to disease establishment and/or may be associated with symptom progression.IMPORTANCE This study provides a comprehensive overview of the core microbial community within the microbiomes of plant hosts that vary in extent of disease symptom progression. With 16S Illumina sequencing analyses, we not only confirmed previously described bacterial associations with plant health (e.g., potentially beneficial bacteria) but also identified new associations and potential interactions between certain bacteria and an economically important phytopathogen. The importance of core taxa within broader plant-associated microbial communities is discussed.

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere.

The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.

Response of Microbial Populations on the Creeping Bentgrass Phyllosphere to Periodic Fungicide Applications

Fungicides are frequently applied on golf course putting greens to combat the myriad of diseases that can affect them; however, it is unknown how these fungicides may affect microbial populations. Plant-associated microbial communities are intimately involved with plant development and soil biological processes. This two-year study was designed to evaluate the impact of five selected fungicides on culturable actinomycetes, fungi, general bacteria, and fluorescent pseudomonads residing in the turfgrass phyllosphere. Fungicides were applied at label rates on a 14-day interval. Four samples, consisting of five individual turf plants, were taken five days post-application. Samples were homogenized in 1 ml sterile deionized water and serial diluted to 10−3 and 10−5. Each dilution was then plated onto four different media (actinomycete isolation agar, acidified potato dextrose agar, nutrient agar + 1% sucrose, and King’s B) for enumeration of microbial populations. Fungicides had both positive and negative impacts on the culturable microbes tested, although population shifts across all treatments were observed with the prevailing weather conditions. These results show that while response to repeated fungicide application appears to be organism dependent, microbes are not completely removed from the environment.

Disease Management in the Genomics Era-Summaries of Focus Issue Papers.

The genomics revolution has contributed enormously to research and disease management applications in plant pathology. This development has rapidly increased our understanding of the molecular mechanisms underpinning pathogenesis and resistance, contributed novel markers for rapid pathogen detection and diagnosis, and offered further insights into the genetics of pathogen populations on a larger scale. The availability of whole genome resources coupled with next-generation sequencing (NGS) technologies has helped fuel genomics-based approaches to improve disease resistance in crops. NGS technologies have accelerated the pace at which whole plant and pathogen genomes have become available, and made possible the metagenomic analysis of plant-associated microbial communities. Furthermore, NGS technologies can now be applied routinely and cost effectively to rapidly generate plant and/or pathogen genome or transcriptome marker sequences associated with virulence phenotypes in the pathogen or resistance phenotypes in the plant, potentially leading to improvements in plant disease management. In some systems, investments in plant and pathogen genomics have led to immediate, tangible benefits. This focus issue covers some of the systems. The articles in this focus issue range from overall perspective articles to research articles describing specific genomics applications for detection and control of diseases caused by nematode, viral, bacterial, fungal, and oomycete pathogens. The following are representative short summaries of the articles that appear in this Focus Issue .

Bacterial Abilities and Adaptation Toward the Rhizosphere Colonization.

The rhizosphere harbors one of the most complex, diverse, and active plant-associated microbial communities. This community can be recruited by the plant host to either supply it with nutrients or to help in the survival under stressful conditions. Although selection for the rhizosphere community is evident, the specific bacterial traits that make them able to colonize this environment are still poorly understood. Thus, here we used a combination of community level physiological profile (CLPP) analysis and 16S rRNA gene quantification and sequencing (coupled with in silico analysis and metagenome prediction), to get insights on bacterial features and processes involved in rhizosphere colonization of sugarcane. CLPP revealed a higher metabolic activity in the rhizosphere compared to bulk soil, and suggested that D-galacturonic acid plays a role in bacterial selection by the plant roots (supported by results of metagenome prediction). Quantification of the 16S rRNA gene confirmed the higher abundance of bacteria in the rhizosphere. Sequence analysis showed that of the 252 classified families sampled, 24 were significantly more abundant in the bulk soil and 29 were more abundant in the rhizosphere. Furthermore, metagenomes predicted from the 16S rRNA gene sequences revealed a significant higher abundance of predicted genes associated with biofilm formation and with horizontal gene transfer (HGT) processes. In sum, this study identified major bacterial groups and their potential abilities to occupy the sugarcane rhizosphere, and indicated that polygalacturonase activity and HGT events may be important features for rhizosphere colonization.

Nutrient supplementation of pinewood biochar for use as a bacterial inoculum carrier

Biochar can function as an effective low-cost inoculum carrier for plant growth-promoting bacteria. Here, we examined the shelf life and inoculum potential of Pseudomonas putida UW4 (UW4) and the potential for further optimization by adding nutrient supplements to a pinewood biochar (PBC) that was used as an inoculum carrier. PBC-600 was produced by pyrolysis at 600 °C and modified with Luria–Bertani broth, a worm casting extract, or mixed with earthworm castings. All treatments were compared with peat as a standard reference material. Subsequent experiments examined survival and root colonization potential of UW4. Results showed that the addition of LB broth to PBC-600 increased the growth of UW4 in storage, but that both supplemented and non-supplemented biochar were similarly effective for establishing a high population density of UW4 in the rhizosphere of inoculated plants. The population density represented by16S ribosomal RNA (rRNA) gene copy numbers for UW4 was correlated with above ground plant biomass (r 2 = 0.6725), which had a positive effect on alleviating salinity stress for the host plant. Analysis of community compositions in the rhizosphere by high throughput sequencing and Kohonen self-organizing maps indicated that high population abundances of UW4 were positively correlated with the abundance of Arthrobacter, Sphingomonas, Rubellimicrobium, and Gematimonas, but were negatively correlated with other dominant taxa. The results suggest that organic nutrient supplementation of the carrier does not promote increased shelf life or inoculum efficacy, but suggests the need for further studies on how biochar and inoculants can be used to shape plant-associated microbial communities.

Sustainable agriculture by the regulation of plant-associated microbial community

Sustainable agriculture by the regulation of plant-associated microbial community

Taxonomy, Physiology, and Natural Products of Actinobacteria.

Actinobacteria are Gram-positive bacteria with high G+C DNA content that constitute one of the largest bacterial phyla, and they are ubiquitously distributed in both aquatic and terrestrial ecosystems. Many Actinobacteria have a mycelial lifestyle and undergo complex morphological differentiation. They also have an extensive secondary metabolism and produce about two-thirds of all naturally derived antibiotics in current clinical use, as well as many anticancer, anthelmintic, and antifungal compounds. Consequently, these bacteria are of major importance for biotechnology, medicine, and agriculture. Actinobacteria play diverse roles in their associations with various higher organisms, since their members have adopted different lifestyles, and the phylum includes pathogens (notably, species of Corynebacterium, Mycobacterium, Nocardia, Propionibacterium, and Tropheryma), soil inhabitants (e.g., Micromonospora and Streptomyces species), plant commensals (e.g., Frankia spp.), and gastrointestinal commensals (Bifidobacterium spp.). Actinobacteria also play an important role as symbionts and as pathogens in plant-associated microbial communities. This review presents an update on the biology of this important bacterial phylum.

Biological control in the microbiome era: Challenges and opportunities

Biocontrol research has long been focused on the study of single strains of biocontrol agents (BCAs) and on their interaction with pathogens and host plants. Further focus on plant-associated microbial communities was suggested several years ago, but significant advances only occurred recently. The advent of high-throughput sequencing (or next-generation sequencing – NGS) technologies is now driving a paradigm change that allows researchers to integrate microbial community studies into the traditional biocontrol approach. This integration could answer old scientific questions, and will raise new biocontrol hypotheses. Microbial communities could impact disease control through their interaction with host plants, pathogens, and BCAs. A better understanding of these interactions will provide unexpected opportunities to develop innovative biocontrol methods against plant pathogens. For example, formulation or timing of BCA application can be improved, “helper” microbial strains can be selected, or molecules driving the microbiota to a pathogen-resistant composition (“prebiotic” approach) can be developed. The five main challenges of microbiome implementation in biocontrol research are also described, i.e. (i) the management of technical errors and biases, (ii) the growing importance of bioinformatics, (iii) the adaptation of experimental schemes, (iv) the appropriate interplay between NGS and other technologies, and (v) the need to complete current genome databases.

Microbial genome-enabled insights into plant-microorganism interactions.

Advances in genome-based studies on plant-associated microorganisms have transformed our understanding of many plant pathogens and are beginning to greatly widen our knowledge of plant interactions with mutualistic and commensal microorganisms. Pathogenomics has revealed how pathogenic microorganisms adapt to particular hosts, subvert innate immune responses and change host range, as well as how new pathogen species emerge. Similarly, culture-independent community profiling methods, coupled with metagenomic and metatranscriptomic studies, have provided the first insights into the emerging field of research on plant-associated microbial communities. Together, these approaches have the potential to bridge the gap between plant microbial ecology and plant pathology, which have traditionally been two distinct research fields.

Rôle des micro-organismes bénéfiques pour aider les plantes à acquérir une tolérance aux stresses environnementaux

Due to their sessile nature, plants have always been confronted to various abiotic and biotic stresses in their immediate environment. As a consequence, the survival of plants depended on their ability to adjust rapidly their physiology, development and growth to escape or mitigate the impacts of stress. All plants are known to perceive and respond to stress signals such as drought, heat, salinity, attacks by herbivores and pathogens. Some biochemical processes are common to all plant stress responses including the production of certain stress proteins and metabolites, as well as the modification of the reactive oxygen species (ROS) metabolism. Although there has been extensive research in the plant stress response field, it is not yet known which factors are responsible for conferring to some plant species the capacity to colonize extreme habitats. Although considerable progress has been made in our understanding of plant stress physiology, the contribution of the plant-associated microbial community in the soil, commonly called the rhizosphere, has only recently received enhanced attention. Recent studies showed that some plant species in natural habitats require microbial associations for stress tolerance and survival. Since plants have colonized land, they have evolved mechanisms to respond to changing environmental conditions and settle in extreme habitats. Although many plants lack the adaptive capability to adapt to stress conditions, the ability of a variety of plants to adapt to stress conditions appears to depend on the association with microbes, raising a number of questions: can all plants improve stress tolerance when associated with their appropriate microbial partners? Did we miss identifying the right partners for a given plant species or variety? What distinguishes the microbes and plants that are adapted to extreme environmental conditions from those living in temperate zones? Answers to these questions are likely to revolutionize plant biology and could lead to new methods for a sustainable agriculture.

Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO 2

Rising atmospheric CO(2) levels are predicted to have major consequences on carbon cycling and the functioning of terrestrial ecosystems. Increased photosynthetic activity is expected, especially for C-3 plants, thereby influencing vegetation dynamics; however, little is known about the path of fixed carbon into soil-borne communities and resulting feedbacks on ecosystem function. Here, we examine how arbuscular mycorrhizal fungi (AMF) act as a major conduit in the transfer of carbon between plants and soil and how elevated atmospheric CO(2) modulates the belowground translocation pathway of plant-fixed carbon. Shifts in active AMF species under elevated atmospheric CO(2) conditions are coupled to changes within active rhizosphere bacterial and fungal communities. Thus, as opposed to simply increasing the activity of soil-borne microbes through enhanced rhizodeposition, elevated atmospheric CO(2) clearly evokes the emergence of distinct opportunistic plant-associated microbial communities. Analyses involving RNA-based stable isotope probing, neutral/phosphate lipid fatty acids stable isotope probing, community fingerprinting, and real-time PCR allowed us to trace plant-fixed carbon to the affected soil-borne microorganisms. Based on our data, we present a conceptual model in which plant-assimilated carbon is rapidly transferred to AMF, followed by a slower release from AMF to the bacterial and fungal populations well-adapted to the prevailing (myco-)rhizosphere conditions. This model provides a general framework for reappraising carbon-flow paths in soils, facilitating predictions of future interactions between rising atmospheric CO(2) concentrations and terrestrial ecosystems.

Next-Generation Communication

In the past decade, there have been major advances in our understanding of the molecular interplay between plants and various classes of microbial pathogens (bacteria, fungi, oomycetes, viruses, and nematodes) and microbial symbionts (rhizobia or mycorrhizal fungi). These fundamental insights, reviewed in a special section in this issue (p. 741), provide a conceptual framework for rational human intervention through the breeding or engineering of crops for durable and broad-spectrum disease resistance. However, such engineered plants will eventually be deployed into soils of varying geochemistry that harbor a staggering diversity of microbes. Many of these microbes can associate intimately with crops or other plants in natural habitats. A deeper understanding of plant-associated microbial communities is likely to offer exciting opportunities for controlling plant growth and pathogen burden in sustainable agricultural settings over the next two decades.

Friends and foes: streptomycetes as modulators of plant disease and symbiosis

The ecological role of soil streptomycetes within the plant root environment is currently gaining increased attention. This review describes our recent advances in elucidating the complex interactions between streptomycetes, plants, pathogenic and symbiotic microorganisms. Streptomycetes play diverse roles in plant-associated microbial communities. Some act as biocontrol agents, inhibiting plant interactions with pathogenic organisms. Owing to the antagonistic properties of streptomycetes, they exert a selective pressure on soil microbes, which may not always be for plant benefit. Others promote the formation of symbioses between plant roots and microbes, and this is in part due to their direct positive influence on the symbiotic partner, expressed as, e.g., promotion of hyphal elongation of symbiotic fungi. Recently, streptomycetes have been identified as modulators of plant defence. By repressing plant responses to pathogens they facilitate root colonisation with pathogenic fungi. In contrast, other strains induce local and systemic resistance against pathogens or enhance plant growth. In conclusion, while streptomycetes have a clear potential of acting as biocontrol agents, care has to be taken to avoid strains that select for virulent pathogens or enhance disease development. We argue towards the use of an integrated screening approach in the search for efficient biocontrol agents, including assays on in vitro antagonism, plant growth, and disease suppression.

Shifts in Rhizoplane Communities of Aquatic Plants after Cadmium Exposure

In this study we present the comparative molecular analysis of bacterial communities of the aquatic plant Lemna minor from a contaminated site (RCP) and from a laboratory culture (EPA), as well as each of these with the addition of cadmium. Plants were identified as L. minor by analysis of the rpl16 chloroplast region. Comparative bacterial community studies were based on the analyses of 16S rRNA clone libraries, each containing about 100 clones from the root surfaces of plants. Bacterial communities were compared at three phylogenetic levels of resolution. At the level of bacterial divisions, differences in diversity index scores between treatments, with and without cadmium within the same plant type (EPA or RCP), were small, indicating that cadmium had little effect. When we compared genera within the most dominant group, the beta-proteobacteria, differences between unamended and cadmium-amended libraries were much larger. Bacterial diversity increased upon cadmium addition for both EPA and RCP libraries. Analyses of diversity at the phylotype level showed parallel shifts to more even communities upon cadmium addition; that is, percentage changes in diversity indices due to cadmium addition were the same for either plant type, indicating that contamination history might be independent of disturbance-induced diversity shifts. At finer phylogenetic levels of resolution, the effects of cadmium addition on bacterial communities were very noticeable. This study is a first step in understanding the role of aquatic plant-associated microbial communities in phytoremediation of heavy metals.

Impact of genetically modified crops on soil- and plant-associated microbial communities.

Transgenic or genetically modified plants possess novel genes that impart beneficial characteristics such as herbicide resistance. One of the least understood areas in the environmental risk assessment of genetically modified crops is their impact on soil- and plant-associated microbial communities. The potential for interaction between transgenic plants and plant residues and the soil microbial community is not well understood. The recognition that these interactions could change microbial biodiversity and affect ecosystem functioning has initiated a limited number of studies in the area. At this time, studies have shown the possibility that transgenes can be transferred to native soil microorganisms through horizontal gene transfer, although there is not evidence of this occurring in the soil. Furthermore, novel proteins have been shown to be released from transgenic plants into the soil ecosystem, and their presence can influence the biodiversity of the microbial community by selectively stimulating the growth of organisms that can use them. Microbial diversity can be altered when associated with transgenic plants; however, these effects are both variable and transient. Soil- and plant-associated microbial communities are influenced not only by plant species and transgene insertion but also by environmental factors such as field site and sampling date. Minor alterations in the diversity of the microbial community could affect soil health and ecosystem functioning, and therefore, the impact that plant variety may have on the dynamics of the rhizosphere microbial populations and in turn plant growth and health and ecosystem sustainability, requires further study.

Metabolic profiling as a means of characterizing plant-associated microbial communities

Microbial communities from leaf and root habitats associated with sugar beet ( Beta vulgaris) were characterized according to their capacity to metabolize a range of 95 sole carbon sources available in a commercial assay, GN-BIOLOG MicroPlates. Metabolic profiling was assessed as a method for evaluating perturbation of microbial communities of glasshouse-grown sugar beet inoculated with a genetically modified microorganism. This technique has allowed microbial communities, colonising the immature leaves of treated and untreated plants to be differentiated, although no differences were observed when plants inoculated with genetically modified microorganisms and unmodified inoculated plants were compared. As plants developed and differentiated, the carbon utilization patterns observed allowed communities to be grouped according to the habitat from which they were isolated, irrespective of treatment. These studies demonstrate that the genetically modified microorganism, introduced as a seed dressing colonised developing immature tissue throughout the 231-day study but did not disrupt the natural succession of microbial communities in glasshouse-grown sugar beet plants.