Plant Microbiome
Endophytic microbial assemblage in grapevine
The plant vascular system has remained an underexplored niche despite its potential for hosting beneficial microbes. The aim of this work was to determine the origin of the microbial endophytes inhabiting grapevine. We focused on a single commercial vineyard in California over a two-year period and used an amplicon metagenomics approach to profile the bacterial (16S–V4) and fungal (ITS) communities of the microbiome across a continuum of six grapevine compartments: bulk soil, rhizosphere, root, cordon, cane and sap. Our data supported that roots are a bottleneck to microbial richness and that they are mostly colonized with soilborne microbes, including plant growth-promoting bacteria recruited by the host, but also saprophytic and pathogenic fungal invaders. A core group of taxa was identified throughout the vine; however, there was clear partitioning of the microbiome with niche adaptation of distinct taxonomic groups. Above- and belowground plant tissues displayed distinct microbial fingerprints and were intermixed in a limited capacity mostly by way of the plant sap. We discuss how cultural practices and human contact may shape the endosphere microbiome and identify potential channels for transmission of its residents.
Disease-Induced Microbial Shifts in Citrus Indicate Microbiome-Derived Responses to Huanglongbing Across the Disease Severity Spectrum
Plant microbiomes are critical components to plant health and can influence disease outcomes. We provide empirical data describing disease-induced shifts within the citrus microbiome at different levels of huanglongbing (HLB) disease severity. HLB is associated with an invasive phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’, that is introduced into the aerial portions of the tree by an insect vector. Disease manifests as aboveground foliar and fruit symptoms and significant root decline belowground. During the early phase of disease, there were depletions of putative keystone taxa in leaves and roots, followed by enrichments of putative beneficial taxa, suggesting a microbially derived immune response involved in plant protection that is ancillary to immune components encoded in the plant’s genome. In the late phase of disease, we observed enrichments of parasitic and saprophytic microorganisms, particularly in the roots. The community shifts within the root compartment are emblematic of a disease-induced dysbiosis where pathogens other than ‘Ca. L. asiaticus’ begin to dominate the community. Furthermore, we define key taxa enriched in trees with a slower rate of disease development, referred to as survivor trees, that are hallmarks of those found in trees in the early phase of disease that may be drivers of the survivor tree phenotype. We propose a disease ecology model that illustrates the relationship between the pathogen, the microbiome, and the host plant that highlights microorganisms that may serve as disease facilitators or antagonists.
An in vitro Pipeline to Screen and Select Citrus-Associated Microbiota with Potential anti-Candidatus Liberibacter asiaticus Properties
Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable “Candidatus Liberibacter asiaticus” bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium. Purified bioactive natural products with anti-“Ca. Liberibacter asiaticus” activity were identified from the fungus C. cladosporioides. Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens. This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-“Ca. Liberibacter asiaticus” bioinoculants that thrive in the citrus holosystem.
Temporal Dynamics of the Sap Microbiome of Grapevine Under High Pierce’s Disease Pressure
Grapevine is a pillar of the California state economy and agricultural identity. This study provides a comprehensive culture-independent microbiome analysis from the sap of grapevine overtime and in a context of a vascular disease. The vascular system plays a key role by transporting nutrient, water, and signals throughout the plant. The negative pressure in the xylem conduits, and low oxygen and nutrient content of its sap make it a unique and under-explored microbial environment. We hypothesized that grapevine hosts in its sap, microbes that have a beneficial impact on plant health by protecting against pathogen attack and supporting key biological processes. To address this hypothesis, we chose a vineyard under high Pierce’s disease (PD). PD is caused by the xylem-dwelling pathogenic bacterium Xylella fastidiosa. We selected ten grapevines within this vineyard with a range of disease phenotypes, and monitored them over 2 growing seasons. We sampled each vines at key phenological stages (bloom, veraison, and post-harvest) and used an amplicon metagenomics approach to profile the bacterial (16S -V4) and fungal (ITS) communities of the sap. We identified a core microbiome of the sap composed of seven bacterial (Streptococcus, Micrococcus, Pseudomonas, Bacteroides, Massilia, Acinetobacter and Bacillus) and five fungal (Cladosporium, Mycosphaerella, Alternaria, Aureobasidium, and Filobasidium) taxa that were present throughout the growing season. Overall, the sap microbial makeup collected from canes was more similar to the root microbial profile. Alpha diversity metrics indicated a microbial enrichment at bloom and in vines with moderate PD severity suggesting a host-driven microbial response to environmental cues. Beta diversity metrics demonstrated that disease condition and plant phenology impacted microbial community profiles. Our study identified several potential taxonomic targets with antimicrobial and plant growth promoting capabilities that inhabit the grapevine sap and that should be further tested as potential biological control or biofertilizer agents.
Microbial Landscape of the Grapevine Endosphere in the Context of Pierce’s Disease
Microbial community structure and composition in the plant vascular endosphere has not been studied extensively especially in the context of vascular diseases. Pierce’s disease (PD) of grapevine is caused by Xylella fastidiosa, a xylem-limited bacterium. In PD impacted vineyards, there are observations of vines that remain asymptomatic despite being under high disease pressure. Because these vines are clonal, we hypothesized that the microbial community inhabiting the grapevine vascular endosphere is a major contributor to the disease escape phenotype. We used a next-generation sequencing Illumina MiSeq based platform to characterize the bacterial and fungal microbiome residing in the cane endosphere of
grapevine that displayed severely symptomatic, to mildly symptomatic or
asymptomatic disease phenotypes. Our results provide
evidence that the endophytic grapevine microbial community is composed primarily of Proteobacteria and Ascomycota with Pseudomonodales and Pleosporales as the main bacterial and fungal orders, respectively. Endophytic Pseudomonas fluorescens and Achromobacter xylosoxidans showed significant negative correlations with X. fastidiosa titer. Our data suggest that the clustering of bacterial communities appeared to be driven by the abundance of both P. fluorescens and X. fastidiosa. P. fluorescens emerged as a potential driver of the disease-escape phenotype and a promising biological control agent of PD.
Bacterial and Fungal Next Generation Sequencing Datasets and Metadata from Citrus Infected with ‘Candidatus Liberibacter asiaticus’
Citrus production throughout the world is being severely threatened by Huanglongbing (HLB), which is a disease associated with the bacteria ‘Candidatus Liberibacter asiaticus’ (CLas), africanus, and americanus. This Resource Announcement provides amplicon-based next generation sequencing (NGS) datasets of the bacterial and fungal rRNA internal transcribed spacer (ITS) region from CLas-infected citrus budwood, leaves, and roots from five orchards located in different geographical regions in Florida (USA). To our knowledge, this is the first amplicon-based NGS study (i) that describes the fungal taxa associated with citrus and (ii) that provides comparative analyses of the bacterial and fungal taxa associated with budwood, leaves, and roots from the same citrus trees. This report also provides the sample metadata linked to these sequence datasets including HLB severity rating, tissue type, citrus rootstock, citrus scion, geographical region, and year trees were planted. When analyzed with other similar datasets, we anticipate that researchers will be able to obtain a greater understanding of the factors that shape the citrus microbiome as well as identify individual microorganisms or consortia of microorganisms that play a role in HLB suppression or exacerbation.
A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds
Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the β-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across β-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.
Microbiome diversity, composition and assembly in a California citrus orchard
The citrus root and rhizosphere microbiomes have been relatively well described in the literature, especially in the context of Huanglonbing disease. Yet questions addressing the assembly of root microbial endophytes have remained unanswered. In the above ground tree tissues, leaves and stems have been the research focus point, while flush and flower microbiomes, two important tissues in the vegetative and reproductive cycles of the tree, are not well described. In this study, the fungal and bacterial taxa in five biocompartments (bulk soil, rhizosphere, root endosphere, flower and flush) of citrus trees grown in a single California orchard were profiled using an amplicon-based metagenomic Illumina sequencing approach. Trees with no observable signs of abiotic or biotic stresses were sampled for two consecutive years during the floral development phase. The rhizosphere was the most biodiverse compartment compared to bulk soil, root endosphere, flower and flush microbiomes. In addition, the belowground bacteriome was more diverse than the mycobiome. Microbial richness decreased significantly from the root exosphere to the endosphere and was overall low in the above ground tissues. Root endophytic microbial community composition shared strong similarities to the rhizosphere but also contained few taxa from above ground tissues. Our data indicated compartmentalization of the microbiome with distinct profiles between above and below ground microbial communities. However, several taxa were present across all compartments suggesting the existence of a core citrus microbiota. These findings highlight key microbial taxa that could be engineered as biopesticides and biofertilizers for citriculture.
Fungi associated with the potato taste defect in coffee beans from Rwanda
Potato taste defect (PTD) of coffee is characterized by a raw potato like smell that leads to a lower quality taste in the brewed coffee, and harms the commercial value of some East African coffees. Although several causes for PTD have been proposed, none of them have been confirmed. Recently, high throughput sequencing techniques and bioinformatic analysis have shown great potential for identifying putative causal agents of plant diseases. Toward the goal of determining the cause of PTD, we examined raw coffee beans from Rwanda exhibiting varying PTD scores using an Illumina-based sequence analysis of the fungal rRNA ITS region.
Geographic location, management strategy, and Huanglongbing disease affect arbuscular mycorrhizal fungal communities across U.S. citrus orchards
The benefits of arbuscular mycorrhizal fungi (AMF) to agroecosystems have been well recognized. Citrus is a globally grown fruit tree commonly found in association with AMF. Global citrus production is currently under the threat of the pandemic huanglongbing (HLB) disease. Since its introduction in the United States, the disease has devastated the Florida citrus industry and is now at the doorsteps of commercial orchards in California. Here, we tested how the two distinct climatic zones within the continental United States where citrus is mostly grown (California and Florida) influenced AMF community diversity and composition. We also assessed in what capacity low-input organic farming and HLB disease affected the AMF communities colonizing the citrus roots. Root samples were collected from 88 trees across 10 orchards. Orchards were selected based on conventional or organic practices in California and based on HLB symptom severity in Florida. AMF communities were characterized using high-throughput sequencing of the small-subunit ribosomal RNA gene. Taxa names were assigned based on a phylogenetic analysis that comprised a backbone of AMF reference sequences from Mycobank and virtual taxa from the MaarjAM database. AMF were detected in 78% of citrus root samples, with taxa belonging to six genera (Dominikia, Funneliformis, Glomus, Rhizophagus, Sclerocystis, and Septoglomus) and unknown Glomeraceae genera. Geographical location, management practice, and disease affected AMF community composition. We provide evidence that perennial agroecosystems are composed of generalist and specialist AMF taxa comparable with other ecosystems and identified ubiquitous taxa that could potentially be exploited for agricultural purposes.
Microbial turnover and dispersal events occur in synchrony with plant phenology in the perennial evergreen tree crop Citrus sinensis
Emerging research indicates that plant-associated microbes can alter plant developmental timing. However, it is unclear if host phenology affects microbial community assembly. Microbiome studies in annual or deciduous perennial plants face challenges in separating effects of tissue age from phenological driven effects on the microbiome. In contrast, evergreen perennial trees, like Citrus sinensis, retain leaves for years, allowing for uniform sampling of similarly aged leaves from the same developmental cohort. This aids in separating phenological effects on the microbiome from impacts due to annual leaf maturation/senescence. Here, we used this system to test the hypothesis that host phenology acts as a driver of microbiome composition. Citrus sinensis leaves and roots were sampled during seven phenological stages. Using amplicon-based sequencing, followed by diversity, phylogenetic, differential abundance, and network analyses, we examined changes in bacterial and fungal communities. Host phenological stage is the main determinant of microbiome composition, particularly within the foliar bacteriome. Microbial enrichment/depletion patterns suggest that microbial turnover and dispersal were driving these shifts. Moreover, a subset of community shifts were phylogenetically conserved across bacterial clades, suggesting that inherited traits contribute to microbe-microbe and/or plant-microbe interactions during specific phenophases. Plant phenology influences microbial community composition. These findings enhance understanding of microbiome assembly and identify microbes that potentially influence plant development and reproduction.
Phylogenomics of Plant-Associated Botryosphaeriaceae Species
The Botryosphaeriaceae is a fungal family that includes many destructive vascular pathogens of woody plants (e.g., Botryosphaeria dieback of grape, Panicle blight of pistachio). Species in the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum, and Neoscytalidium attack a range of horticultural crops, but they vary in virulence and their abilities to infect their hosts via different infection courts (flowers, green shoots, woody twigs). Isolates of seventeen species, originating from symptomatic apricot, grape, pistachio, and walnut were tested for pathogenicity on grapevine wood after 4 months of incubation in potted plants in the greenhouse. Results revealed significant variation in virulence in terms of the length of the internal wood lesions caused by these seventeen species. Phylogenomic comparisons of the seventeen species of wood-colonizing fungi revealed clade-specific expansion of gene families representing putative virulence factors involved in toxin production and mobilization, wood degradation, and nutrient uptake. Statistical analyses of the evolution of the size of gene families revealed expansions of secondary metabolism and transporter gene families in Lasiodiplodia and of secreted cell wall degrading enzymes (CAZymes) in Botryosphaeria and Neofusicoccum genomes. In contrast, Diplodia, Dothiorella, and Neoscytalidium generally showed a contraction in the number of members of these gene families. Overall, species with expansions of gene families, such as secreted CAZymes, secondary metabolism, and transporters, were the most virulent (i.e., were associated with the largest lesions), based on our pathogenicity tests and published reports. This study represents the first comparative phylogenomic investigation into the evolution of possible virulence factors from diverse, cosmopolitan members of the Botryosphaeriaceae.
Two Food Waste By-Products Selectively Stimulate Beneficial Resident Citrus Host-Associated Microbes in a Zero-Runoff Indoor Plant Production System
The global production of food waste is a far-reaching problem with sizable financial, ethical, social, and environmental costs. Over 66 million tons of food waste is produced annually in the United States alone. This waste can be converted into valuable digestate by-products that promote a circular economy within agri-food systems. The present work investigated the use of two liquid digestates of microaerobic fermentation from mixed food waste and beer mash, respectively, as biostimulants for non-bearing citrus plants (nursery stock) grown in a zero-runoff greenhouse system with recirculating irrigation. The digestates' impact on the structure and diversity of the microbiota was determined on the irrigation water, soil, leaves, roots, and rhizosphere of citrus plants. A combination of culture-dependent (selective media) and culture-independent approaches (Next-Generation Sequencing) was used to assess the composition of the microbial communities and to single out the presence of foodborne pathogens. Our results suggest that the use of digestates is safe (i.e., no human or plant pathogens were present in the digestates or enriched in the plant production system following amendments). Digestates application to the irrigation water reduced the bacterial diversity within 24–48 h and selectively and significantly stimulated beneficial resident host-associated microorganisms (Pseudomonas putida) by two to three orders of magnitude. Carbon dynamics were analyzed in the nutrient solutions by measuring dissolved organic carbon and characterizing carbon species through gas chromatography-electron ionization-mass spectrometry. Our results indicate that dissolved organic carbon in the recirculating irrigation water spikes after each digestate amendment and it is quickly metabolized by bacteria, plateauing 24 h after application. Soil carbon, nitrogen, and nutrient dynamics were also analyzed, and results suggest that digestates increased the concentration of some plant nutrients in soils without causing a surge of potentially toxic elements. This study represents a proof-of-concept for the safe re-use of organic wastes, from farming and consumers, in agriculture. Implementing this type of integrated plant production system could reduce the environmental impact of food waste and benefit the public by improving soil health, reducing agricultural footprint, and increasing crop fitness by deploying a method based on a circular economy and sustainable food production approaches.
Profiling grapevine trunk pathogens in planta: a case for community-targeted DNA metabarcoding
DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology.
Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk disease complex
Grapevines, like other perennial crops, are affected by so-called ‘trunk diseases’, which damage the trunk and other woody tissues. Mature grapevines typically contract more than one trunk disease and often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development, especially under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e. metatranscriptomics) approach that could monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most relevant co-infecting GTPs in diseased grapevine wood provided the unprecedented opportunity to generate a multi-species reference for the mapping and quantification of DNA and RNA sequencing reads. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Initial testing on grapevine wood experimentally inoculated with individual GTPs confirmed the validity of the method. Using naturally infected field samples expressing a variety of trunk disease symptoms, we show that our approach provides quantitative assessments of species composition, as well as genome-wide transcriptional profiling of potential virulence factors, namely cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting GTPs.
The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.