蕨类根内生菌和叶内生菌测序

三叶青(Tetrastigma hemsleyanum)作为新"浙八味"中药材之一,因其较强的抗肿瘤、抗病毒、免疫调节等药理活性而市场需求量不断增加。然而,栽培技术不成熟导致产量与品质同步提升难题,严重制约其产业化进程。植物内生菌作为宿主共生微生物,可通过多种途径调控药用植物生长发育,为解决三叶青产业瓶颈提供了新途径。本文简述了三叶青产业化发展现状与难题,阐明了药用植物内生菌促进宿主生长及品质的作用、三叶青内生菌相关研究进展以及多组学技术在"植物-内生菌"互作中的应用价值,最后展望未来发展方向,为三叶青高产优质栽培及产业化升级提供理论参考。

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Cacao cultivation is one of the main agricultural products of Ecuador, known internationally for its quality and aroma. However, it is affected by fungal diseases including Moniliophthora roreri, Moniliophthora perniciosa, Phytophthora spp., and Colletotrichum spp. The genus Colletotrichum spp. is known for its characteristics that complicate traditional taxonomic identification. In cacao cultivation, it is one of the most frequently found species as an endophyte of leaves and fruits, yet it is also reported to cause the disease known as anthracnose on leaves and fruits. The objective of this work was to identify at the species level 16 Colletotrichum isolates, 13 from healthy leaf endophytes and 3 from pods with symptoms, through multilocus analysis of the ITS1, 5.8S, and ITS2 region, and partial sequences of the TUB2 and GAPDH genes. Subsequently, their pathogenicity was evaluated by inoculating healthy cacao pods and measuring the damage caused. The 16 isolates were identified as follows: from the gloeosporioides complex, C. siamense 6, C. chrysophilum 6, C. theobromicola 2 and from the boninense complex, C. karstii 2. The most frequently found species were those that caused symptoms, especially C. siamense, to which the strains were isolated from symptomatic pods belonged. This work provides relevant and accurate information about the diversity of Colletotrichum species that colonize cocoa plantations and identifies which species might cause the disease known as anthracnose. Additionally, it allows for a more precise diagnosis and consequently better treatment.

Despite their genetic adaptation to local conditions, plants often achieve ecological success through symbiotic associations with fungal endophytes. However, the habitat-specific functionality of these interactions and their potential to drive plant adaptation to new environments remain uncertain. In this study, we tested this using the vascular flora of the Antarctic tundra (Colobanthus quitensis and Deschampsia antarctica), an extreme environment where fungal endophytes are known for playing important ecological roles. After characterizing the root-associated fungal endophyte communities of both species in two distinct Antarctic terrestrial habitats-hill and coast-we experimentally assessed the contribution of fungal endophytes to plant adaptation in each habitat. The field reciprocal transplant experiment involved removing endophytes from a set of plants and crossing symbiotic status (with and without endophytes) with habitat for both species, aiming to assess plant performance and fitness. The diversity of root fungal endophytes was similar between habitats and mainly explained by plant species, although habitat-specific endophyte community structures were identified in D. antarctica. Endophytes significantly influenced C. quitensis homeostatic regulation, including oxidative stress and osmotic control, as well as plant fitness in both environments. By contrast, the effect of endophytes on D. antarctica was particularly evident in coastal sites, suggesting an endophyte-mediated improvement in local adaptation. Altogether, our results suggest that the two Antarctic vascular plant species follow different strategies in recruiting and developing functional symbiosis with root-associated fungal communities. While C. quitensis is more generalist, D. antarctica establishes specific interactions with habitat-specific microbial symbionts, predominantly in the most stressful environmental context.

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Over the past decades, crop yields have risen in parallel with increasing use of fossil-fuel derived nitrogen (N) fertilizers, but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic and endophytic plant growth promoting bacteria (PGPB) are known to stimulate plant growth. However, compared to the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix+) or a fix- mutant strain (SmR54), compared to uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in situ laser ablation electrospray ionization mass spectrometry (LAESI-MS) to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix- strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix- strain, perhaps reflecting a plant defense response.

This study aimed to disentangle the structure, composition, and co-occurrence relationships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root endosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phenological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7).

ABSTRACT Although endophytic bacteria impact the health, and ultimately the fitness, of their hosts, our understanding of the diversity of endophytic species remains limited. Here we report on the endophytic microbiota inhabiting the roots, healthy leaves and leaves attacked by a gall-inducing insect of Baccharis dracunculifolia, a species of major economic relevance in South America, using 16S rRNA gene new generation sequencing. Rhodoplanes and Nitrospira were well represented in the communities of roots and leaves; known to be important for nitrogen cycling. The difference in bacterial diversity between healthy and galled leaves was not pronounced. The leaves seem to harbor specialized bacteria with high tolerances to abiotic stresses such as wide variation in temperature, low humidity, shallow and nutrient-poor soils and high solar irradiation. These findings suggest taxon-specific ecological niches in the leaves and roots, which may be the result of different physicochemical characteristics between these structures. This study provides a basis for further investigations and adds significant new information to the current knowledge of the endophytic bacterial composition in B. dracunculifolia.

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Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

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Endophytic fungi represent large, yet unexplored components of biodiversity. This work evaluated the richness and the distribution of endophytes in the leaves of Bauhinia brevipes (Fabaceae). A total of 1110 colonies were recovered from the samples and grouped by their morphological traits into 126 taxa. The total number of taxa according to leaf development was: 102 in mature leaves, 93 in recently expanded leaves and 79 for unfolded leaves. The major endophyte genera were Phomopsis, followed by Dothiorella, Pestalotiopsis and Acremonium. The richness and the isolate numbers of endophytes were not statistically affected by leaf region. However, some taxa were leaf-age specific; six were isolated only from unfolded leaves, nine from recently expanded leaves and 17 were exclusively found in mature leaves. The composition of endophytes varied with leaf region; the similarities (Jaccard's Index) among the leaf regions of different leaf ages ranged from 0.36 to 0.46, indicating a high spatial variation in the community of endophytic fungi inside the leaves. The high richness of endophytes in this host plant highlights a significant contribution of fungi to tropical biodiversity and the need for further research in this area.

Biodiversity and biogeography of leaf-inhabiting endophytic fungi have not been resolved yet. This is because host specificity, life cycles and species concepts, in this heterogeneous ecological guild of plant-associated microfungi, are far from being understood. Even though it is known that culture-based collection techniques are often biased, this has been the method of choice for studying fungal endophytes. Isolation of fungal endophytes only through culture-based methods could potentially mask slow growing species as well as species with low prevalence, preventing the capture of the communities’ real diversity and composition. This bias can be partially resolved by the use of cultivation-independent approaches such as direct sequencing of plant tissue by next generation techniques. Irrespective of the chosen sampling method, an efficient analysis of community ecology is urgently needed in order to evaluate the driving forces acting on fungal endophytic communities. In the present study, endophytic ascomyceteous fungi from three different plant genera (Vasconcellea microcarpa, Tillandsia spp., and Heveabrasiliensis) distributed in Peru, were isolated through culture-based sampling techniques and sequenced for their ITS rDNA region. These data sets were used to assess host preferences and biogeographic patterns of endophytic assemblages. This study showed that the effect of the host’s genetic background (identity) has a significant effect on the composition of the fungal endophytic community. In other words, the composition of the fungal endophytic community was significantly related to their host’s taxonomic identity. However, this was not true for all endophytic groups, since we found some endophytic groups (e.g. Xylariales and Pleosporales) occurring in more than one host genus. Findings from this study promote the formulation of hypotheses related to the effect of altitudinal changes on the endophytic communities along the Eastern Andean slopes. These hypotheses and perspectives for fungal biodiversity research and conservation in Peru are addressed and discussed.

Abstract Foliar endophytic fungi are present in almost all vascular plants. The composition of endophyte communities varies among plant individuals. Likely, but understudied, sources of this variation are the species composition of the plant community and initial attacks by insect herbivores. We addressed these issues by characterizing fungal endophyte communities on leaves of chestnut (Castanea sativa) grown in pure vs. mixed stands. We used ITS metabarcoding methods to identify endophytic fungi associated with galls caused by the invasive gall wasp, Dryocosmus kuriphilus, and with surrounding chestnut leaf tissues. We found 1378 different OTUs. The richness, diversity and composition of endophyte communities differed between galls and surrounding leaf tissues but were independent of forest stand composition. Fungal endophyte richness was lower in galls than in surrounding leaf tissues. Most differences in the composition of fungal endophyte communities between galls and foliar tissues were due to OTU turnover. These results suggest that insect-induced galls provide a particular habitat condition for endophytic microorganisms, regardless of forest species composition. A better understanding of endophyte biology is important to improve their use as biocontrol agents of galling insects.

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Introduction The fruit of Rubus chingii Hu is a prized traditional medicine and functional food, with its quality predominantly determined by its secondary metabolites. While the metabolic dynamics during fruit ripening are documented, the role of the endophytic microbiome, a key regulator of plant physiology, remains entirely unexplored. Methods An integrated approach, combining 16S/ITS amplicon sequencing with spectroscopic and chromatographic analyses, were employed to investigate the correlation between the endophytic microbiome and the metabolome across four distinct ripening stages of R. chingii fruit. Results Significant stage-dependent shifts in the community structure of both bacterial and fungal endophytes were revealed in this study. Notably, Spearman correlation analysis identified specific microbial taxa, including the bacterial genera Geodermatophilus and Brevundimonas, and the fungal yeasts Metschnikowia and Starmerella, that were significantly positively correlated with the accumulation of key secondary metabolites (ellagic acid, flavonoids, and phenolic acids). Concurrently, the content of these beneficial metabolites and the fruit’s antioxidant capacity decreased markedly as ripening progressed. Discussion This study provides the first evidence of a structured succession in the endophytic microbiome of R. chingii fruit and its close association with the dynamics of medically relevant metabolites. The findings propose that the ripening process is a tripartite interplay between host development, microbial succession, and metabolic reprogramming. The identified keystone taxa represent promising targets for future microbiome-based strategies to manipulate fruit quality, offering novel insights into the role of the microbiome in medicinal plant biology and its potential application in sustainable agriculture.

Premise: Endophytic plant-microbe interactions range from mutualistic relationships that confer important ecological and agricultural traits to neutral or quasi-parasitic relationships. In contrast to root-associated endophytes, the role of environmental and host-related factors for acquiring leaf endophyte communities remains relatively unexplored. Here we assess leaf endophyte diversity to test the hypothesis that membership of these microbial communities is driven primarily by abiotic environment and host phylogeny. Methods: We used a broad geographic coverage of North America in the genus, Heuchera (Saxifragaceae). Bacterial and fungal communities were characterized with 16S and ITS amplicon sequencing, using QIIME2 to call operational taxonomic units and calculate species richness, Shannon diversity, and phylogenetic diversity. We assembled environmental predictors for microbial diversity at collection sites including latitude, elevation, temperature, precipitation, and soil parameters. Results: We find differing assembly patterns for bacterial and fungal endophytes; we found that only host phylogeny is significantly associated with bacteria, while geographic distance alone was the best predictor of fungal community composition. Species richness and phylogenetic diversity are very similar across sites and species, with only fungi showing a response to aridity and precipitation for some metrics. Unlike what has been observed with root-associated microbial communities, in this system microbes show no relationship with pH or other soil factors. Conclusions: Host phylogeny and geographic distance independently influence different microbial communities, while aridity and precipitation determine fungal diversity within leaves of Heuchera. Our results indicate the importance of detailed clade-based investigation of microbiomes and the complexity of microbiome assembly within specific plant organs.

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Background The difference of metabolites in medicinal plants has always been concerned to be influenced by external environmental factors. However, the relationship between endophytes and host metabolites remains unclear. Methods In this study, we used 16S and ITS amplicon sequencing to compare endophyte diversity among different tissue types and ages of Gentiana officinalis. Endophyte diversity and abundance was also analyzed in relation to the abundance of four secondary metabolites (Gentiopicroside, Loganic acid, Swertiamarine and Sweroside). Results The diversity and richness of G. officinalis endophyte differed as a function of tissue types and ages. Four metabolites of G. officinalis were significantly correlated with the abundance of dominant endophyte genera. The predictive function analysis showed that metabolism was main function of endophytic bacteria in different tissue and year root samples, while saprotroph was dominant trophic modes of endophytic fungi in the different year root samples. The dominant trophic modes of endophytic fungi was saprotroph and pathotroph, and relative abundances differed in the different tissue samples. The results of this study will help to elucidate the plant-microbial interactions and provide key information on the role of endophytes in the production of G.officinalis and its important metabolites.

Endophytes asymptomatically infect virtually all plant species, yet little is known about endophyte community assembly and diversity within the woody tissues of forest trees. We utilised phylogenetic null models of alpha (ses.MNTDab and ses.MPDab) and beta diversity (ses.βMNTDab and ses.βMPDab) to infer the role of deterministic and stochastic ecological processes in structuring bacterial and fungal endophyte communities in the woody tissues of Populus deltoides and the naturally occurring P. × jackii hybrid complex (P. deltoides × P. balsamifera). Microbial communities were characterised through Illumina amplicon sequencing (MiSeq) of the ITS and 16S rRNA gene. We detected 227 fungal ASVs, which were mainly classified as Ascomycota (92.4%). Among the 667 bacterial ASVs detected, the majority were classified as phylum Actinobacteriota (47.6%) and Proteobacteria (44.9%). We predicted that hybridisation could lead to a host environment that applies weaker selective effects on microbial taxa due to variability in host chemical and morphological phenotypes. Although bacterial communities did not support our prediction, fungal assemblages of the hybrid host (P. × jackii) were more phylogenetically random within (ses.MNTDab) and between assemblages (ses.βMNTDab and ses.βMPDab) then the non-hybrid (P. deltoides)—consistent with an increased role of stochastic community assembly processes and less selective host environment. Host identity had a large influence on fungal community composition (weighted UniFrac R2 = 34%), which may result from the differences in fungal selection we detected between hosts. Conversely, host identity was a weaker predictor of bacterial composition (weighted UniFrac R2 = 13%), which may reflect the more dominant role of stochasticity we detected in bacterial assembly. Our findings provide evidence that host hybridisation may alter fungal assembly processes and diversity within the woody endosphere, leading to more phylogenetically diverse associations both within and between the fungal assemblages of hybrid trees. More broadly, our results highlight how genetically diverse host populations may promote microbial biodiversity within forests and hybrid transition zones.

Endophytes play an important role in helping plants resist heavy metal stress. However, little is known about the effects of different heavy metals on the diversity and composition of endophyte communities. In this study, we used 16S and ITS amplicon sequencing to reveal the structure and function of endophytes in Symphytum officinale under different heavy metal stressors. The results showed that the endophytic fungal diversity decreased compared with the control under the different heavy metals stressors, while the diversity of endophytic bacteria showed an increasing trend. The biomarker analysis indicated that Zn and Pb stress led to obvious branches. Specific OTUs analysis showed that there were 1224, 597, and 1004 OTUs specific under Zn, Pb, and Cd stress in the bacterial community and 135, 81, and 110 OTUs specific under Zn, Pb, and Cd stress in the fungal community. The co-occurrence network showed changes in microbial interactions under heavy metal contamination conditions, suggesting that endophytic bacteria play an important role in the resistance of host plants. The Spearman analysis showed that the correlation between endophytic bacteria and endophytic fungi in relation to heavy metal transport exhibited variations. Our results expand the knowledge of the relationships of plant–microbe interactions and offer pivotal information to reveal the role of endophytes under different heavy metal stress conditions.

The plant microbiome plays a crucial role in facilitating plant growth through enhancing nutrient cycling, acquisition and transport, as well as alleviating stresses induced by nutrient limitations. Despite its significance, the relative importance of common agronomic practices, such as nitrogenous fertilizer, in shaping the plant microbiome across different cultivars remains unclear. This study investigated the dynamics of bacterial and fungal communities in leaf, root, rhizosphere, and bulk soil in response to nitrogenous fertilizer across ten sorghum varieties, using 16S rRNA and ITS gene amplicon sequencing, respectively. Our results revealed that nitrogen addition had a greater impact on sorghum-associated microbial communities compared to cultivar. Nitrogen addition significantly reduced bacterial diversity in all compartments except for the root endophytes. However, N addition significantly increased fungal diversity in both rhizosphere and bulk soils, while significantly reducing fungal diversity in the root endophytes. Furthermore, N addition significantly altered the community composition of bacteria and fungi in all four compartments, while cultivars only affected the community composition of root endosphere bacteria and fungi. Network analysis revealed that fertilization significantly reduced microbial network complexity and increased fungal-related network complexity. Collectively, this study provides empirical evidence that sorghum-associated microbiomes are predominantly shaped by nitrogenous fertilizer rather than by cultivars, suggesting that consistent application of nitrogenous fertilizer will ultimately alter plant-associated microbiomes regardless of cultivar selection.

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PREMISE Endophytic plant-microbe interactions range from mutualistic relationships that confer important ecological and agricultural traits to neutral or quasi-parasitic relationships. In contrast to root-associated endophytes, the role of environmental and host-related factors in the acquisition of leaf endophyte communities at broad spatial and phylogenetic scales remains sparsely studied. We assessed endofoliar diversity to test the hypothesis that membership in these microbial communities is driven primarily by abiotic environment and host phylogeny. METHODS We used a broad geographic coverage of North America in the genus Heuchera L. (Saxifragaceae), representing 32 species and varieties across 161 populations. Bacterial and fungal communities were characterized using 16S and ITS amplicon sequencing, respectively, and standard diversity metrics were calculated. We assembled environmental predictors for microbial diversity at collection sites, including latitude, elevation, temperature, precipitation, and soil parameters. RESULTS Assembly patterns differed between bacterial and fungal endophytes. Host phylogeny was significantly associated with bacteria, while geographic distance was the best predictor of fungal community composition. Species richness and phylogenetic diversity were consistent across sites and species, with only fungi showing a response to aridity and precipitation for some metrics. Unlike what has been observed with root-associated microbial communities, in this system microbes show no relationship with pH or other soil factors. CONCLUSIONS Overall, this work improves our understanding of the large-scale patterns of diversity and community composition in leaf endophytes and highlights the relative significance of environmental and host-related factors in driving different microbial communities within the leaf microbiome.

Next generation sequencing (NGS) enables rapid analysis of the composition and diversity of microbial communities in several habitats. We applied the high throughput techniques of NGS to the metagenomics study of endophytic bacteria in Aloe vera plant, by assessing its PCR amplicon of 16S rDNA sequences (V3–V4 regions) with the Illumina metagenomics technique used to generate a total of 5,199,102 reads from the samples. The analyses revealed Proteobacteria, Firmicutes, Actinobacteria and Bacteriodetes as the predominant genera. The roots have the largest composition with 23% not present in other tissues. The stems have more of the genus—Pseudomonas and the unclassified Pseudomonadaceae. The α-diversity analysis indicated the richness and inverse Simpson diversity index of the bacterial endophyte communities for the leaf, root and stem tissues to be 2.221, 6.603 and 1.491 respectively. In a similar study on culturable endophytic bacteria in the same A. vera plants (unpublished work), the dominance of Pseudomonas and Bacillus genera was similar, with equal proportion of four species each in root, stem and leaf tissues. It is evident that NGS technology captured effectively the metagenomics of microbiota in plant tissues and this can improve our understanding of the microbial–plant host interactions.

Falcataria falcata is an exotic tree species that was imported to southern China around 1940 and has been widely planted in the Guangdong province of China. Using the 16S rRNA amplicon sequencing approach, we investigated the composition of the bacterial endophytes in the root nodules of naturally grown F. falcata and elucidated the core bacterial endophyte group. Across all samples, there were 575 bacterial genera and 29 bacterial phyla. Proteobacteria accounted for 42–90% relative abundance in all regions. Notably, Bradyrhizobium, Paucibacter, Rhizobium, and Mesorhizobium were consistently detected in all regions studied. Among these, Bradyrhizobium (13–37%) and Paucibacter (1–34%) were the dominant genera. Despite the differences in endophytic amplicon sequence variants (ASVs) across all regions, our results demonstrate that some ASVs, which have been termed herein as commonly shared core ASVs (c-ASVs), still inhabit F. falcata root nodules across multiple regions simultaneously. More importantly, some c-ASVs dominated in F. falcata root nodules across multiple regions. This study demonstrated the consistency of the bacterial endophyte communities of F. falcata root nodules.

ABSTRACT Sugarcane mosaic virus (SCMV) causes mosaic disease in crops such as maize and sugarcane by its vector—an aphid—and is transmitted top-down into the root system. However, understanding of the effects of the aphid-borne virus on root-associated microbes after plant invasion remains limited. The current project investigated maize root-associated (rhizosphere and endosphere) bacterial communities, potential interspecies interaction, and assembly processes in response to SCMV invasion based on 16S rRNA gene amplicon sequencing. SCMV was detected in the roots 9 days after inoculation, and leaf mosaic and chlorosis appeared. The SCMV invasion markedly reduced the α-diversity of endosphere bacteria compared with uninoculated controls (Mock). The connectivity and complexity of the bacterial co-occurrence network in the root endosphere decreased after SCMV invasion, implying that the plant virus may alter root endophyte-microbial interactions. Moreover, a signature that deviates more from stochastic processes was observed in virus-infected plants. Unexpectedly, the rhizosphere bacterial communities were rarely affected by the viral invasion. This study lays the foundation for elucidating the fate of the microbial component of the plant holobiont following aphid-borne virus exposure. IMPORTANCE Biotic (e.g., soil-borne viruses) stress can alter root-associated bacterial communities, essential in maintaining host plant growth and health. However, the regulation of root-associated microorganisms by plant viruses from shoots is still largely unknown. Our results show that plant virus invasion leads to reduced and simpler inter-microbial communication in the maize endosphere. In addition, stochastic processes act on bacterial community assembly in both rhizosphere and endosphere, and bacterial communities in virus-invaded plant endosphere tend to shift toward deterministic processes. Our study highlights the negative effects of plant viruses on root endophytes from the microbial ecology perspective, which may be microbially mediated mechanisms of plant diseases. Biotic (e.g., soil-borne viruses) stress can alter root-associated bacterial communities, essential in maintaining host plant growth and health. However, the regulation of root-associated microorganisms by plant viruses from shoots is still largely unknown. Our results show that plant virus invasion leads to reduced and simpler inter-microbial communication in the maize endosphere. In addition, stochastic processes act on bacterial community assembly in both rhizosphere and endosphere, and bacterial communities in virus-invaded plant endosphere tend to shift toward deterministic processes. Our study highlights the negative effects of plant viruses on root endophytes from the microbial ecology perspective, which may be microbially mediated mechanisms of plant diseases.

The microbial structure and metabolic function of plant-associated endophytes play a key role in the ecology of various environments, including trees. Here, the structure and functional profiles of the endophytic bacterial community, associated with Paulownia elongata × fortunei, in correlation with seasonality, were evaluated using Biolog EcoPlates. Biolog EcoPlates was used to analyse the functional diversity of the microbiome. The total communities of leaf endophyte communities were investigated using 16S rRNA V5–V7 region amplicon deep sequencing via Illumina MiSeq. Community level physiological profiling (CLPP) analysis by the Biolog EcoPlate™ assay revealed that the carboxylic acids (19.67–36.18%) and amino acids (23.95–35.66%) were preferred by all by all communities, whereas amines and amides (0.38–9.46%) were least used. Seasonal differences in substrate use were also found. Based on the sequencing data, mainly phyla Proteobacteria (18.4–97.1%) and Actinobacteria (2.29–78.7%) were identified. A core microbiome could be found in leaf-associated endophytic communities in trees growing in different locations. This work demonstrates the application of Biolog EcoPlates in studies of the functional diversity of microbial communities in a niche other than soil and shows how it can be applied to the functional analyses of endomicrobiomes. This research can contribute to the popularisation of Biolog EcoPlates for the functional analysis of the endomicrobiome. This study confirms that the analysis of the structure and function of the plant endophytic microbiome plays a key role in the health control and the development of management strategies on bioenergy tree plantations.

One of the most common methods to survey bacterial communities is targeted amplification of the hypervariable regions of the 16s rRNA gene followed by sequencing. This protocol details Illumina library preparation of such amplicons from communities isolated from maize. We include both staggered PCR primers to improve Illumina base calling and peptide nucleic acids (PNAs) to reduce the presence of plant organelles. Primers are designed with Illumina adapter sequences for the addition of sample-specific indexes (barcodes). We also briefly discuss alternative primer sets, including ones that directly discriminate against plant organelles or that amplify different organisms (e.g., fungal internal transcribed spacer [ITS] sequences).