Endophytic Bacterial Community Structure Research Articles

High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of upland rice

Upland rice is an ecotype crop resulting from the long-term domestication and evolution of rice in dry land without a water layer. Generally, the stems and leaves are thick and luxuriant, while the leaves also typically broad and light. The root system is developed with abundant root hair, and the osmotic pressure of the root and cell juice concentration in the leaves is high, while this plant is drought-resistant, heat-resistant, and water absorbent. This study aims to reveal the "core flora" of the endophytes in upland rice seeds by examining their diversity and community structures. It further intends to reveal the impact of the soil environment on the formation of endophyte community structures in upland rice seeds by comparing the environmental soil microorganisms in upland rice habitats. In this study, high-throughput sequencing technology based on the Illumina Hiseq 2500 platform was used to investigate the structure and diversity of endophytic bacterial communities using upland rice varieties collected from different locations and soil samples from unified planting sites as materials. Here, 42 endophytic OTUs were found to coexist in the 14 samples. At the phylum level, the first dominant phyla in all the samples were Proteobacteria (93.81-99.99%). At the genus level, Pantoea (8.77-87.77%), Pseudomonas (1.15-61.58%), Methylobacterium (0.40-4.64%), Sphingomonas (0.26-3.85%), Microbacterium (0.01-4.67%) and Aurantimonas (0.04-4.34%), which represent the core microflora in upland rice seeds, served as the dominant genera that coexisted in all the upland rice seeds tested. This study significant for the isolation, screening, functional evaluation, and re-action of various functional microorganisms in upland rice to improve its agronomic traits. It also provides a specific reference for the interaction between microorganisms and plants.

Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year

BackgroundThe dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites.ResultsIn this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1–3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1–3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium.ConclusionsThis study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.

Analysis of the Endophytic Bacteria Community Structure and Function of Panax notoginseng Based on High-Throughput Sequencing.

Panax notoginseng has long been used as a Chinese herb with high medicinal value. The endophytic bacteria in this medicinal plant have multiple biological functions. High-throughput sequencing is a rapidly evolving technique that helps profile the endophytic bacterial community structure of medicinal plants. However, few studies on the endophytic bacteria in P. notoginseng, particularly in dry P. notoginseng roots as a raw medicinal material, have been conducted. In this study, fresh P. notoginseng and dry P. notoginseng were analysed using high-throughput sequencing on an Illumina MiSeq platform to explore the diversity and functions of the endophytic bacteria in different parts of P. notoginseng. The results showed that a total of 201 operational taxonomic units were obtained from fresh P. notoginseng and dry P. notoginseng. The dominant phyla in the fresh and dry P. notoginseng were Proteobacteria (85.9%) and Firmicutes (99.9%), respectively, whereas the dominant genera in these samples were Enterobacter (84.4%) and Bacillus (99.6%), respectively. Fresh P. notoginseng exhibited a higher degree of endophytic bacterial diversity than dry P. notoginseng, but functional prediction of metabolism indicated that the relative abundance of the metabolic function of terpenoids and polyketides synthesis in the dry sample was higher than that in the fresh sample. Our study indicates significant differences in the diversity and metabolic function of the endophytic bacteria between fresh and dry P. notoginseng, providing useful information for the exploitation and utilization of endophytic bacteria resources from P. notoginseng.

Tissue-Specific Dynamics in the Endophytic Bacterial Communities in Arctic Pioneer Plant Oxyria digyna.

The rapid developments in the next-generation sequencing methods in the recent years have provided a wealth of information on the community structures and functions of endophytic bacteria. However, the assembly processes of these communities in different plant tissues are still currently poorly understood, especially in wild plants in natural settings. The aim of this study was to compare the composition of endophytic bacterial communities in leaves and roots of arcto-alpine pioneer plant Oxyria digyna, and investigate, how plant tissue (leaf or root) or plant origin affect the community assembly. To address this, we planted micropropagated O. digyna plants with low bacterial load (bait plants) in experimental site with native O. digyna population, in the Low Arctic. The endophytic bacterial community structures in the leaves and roots of the bait plants were analyzed after one growing season and one year in the field, and compared to those of the wild plants growing at the same site. 16S rRNA gene targeted sequencing revealed that endophytic communities in the roots were more diverse than in the leaves, and the diversity in the bait plants increased in the field, and was highest in the wild plants. Both tissue type and plant group had strong impact on the endophytic bacterial community structures. Firmicutes were highly abundant in the leaf communities of both plant types. Proteobacteria and Bacteroidetes were more abundant in the roots, albeit with different relative abundances in different plant groups. The community structures in the bait plants changed in the field over time, and increasingly resembled the wild plant endophytic communities. This was due to the changes in the relative abundances of several bacterial taxa, as well as species acquisition in the field, but with no species turnover. Several OTUs that were acquired by the bait plants in the field and represent phosphate solubilizing and diazotrophic bacterial taxa, suggesting major role in nutrient acquisition of these bacteria for this nonmycorrhizal plant, thriving in the nutrient poor arctic soils.

Symbiotic nitrogen fixation and endophytic bacterial community structure in Bt-transgenic chickpea (Cicer arietinum L)

Symbiotic nitrogen fixation (SNF) of transgenic grain legumes might be influenced either by the site of transgene integration into the host genome or due to constitutive expression of transgenes and antibiotic-resistant marker genes. The present investigation confirmed proper nodulation of five tested Bt-chickpea events (IPCa2, IPCa4, IPCT3, IPCT10, and IPCT13) by native Mesorhizobium under field environment. Quantitative variations for nodulation traits among Bt-chickpea were determined and IPCT3 was found superior for nodule number and nodule biomass. Diversity, as well as richness indices, confirmed the changes in bacterial community structure of root and root-nodules from Bt-chickpea events IPCa2 and IPCT10. Especially, Gram-positive bacteria belonging to Firmicutes and Actinobacteria were selectively eliminated from root colonization of IPCa2. Richness indices (CHAO1 and ACE) of the root-associated bacterial community of IPCa2 was 13–14 times lesser than that of parent cv DCP92-3. Root nodule associated bacterial community of IPCT10 was unique with high diversity and richness, similar to the roots of non-Bt and Bt-chickpea. It indicated that the root nodules of IPCT10 might have lost their peculiar characteristics and recorded poor colonization of Mesorhizobium with a low relative abundance of 0.27. The impact of Bt-transgene on bacterial community structure and nodulation traits should be analyzed across the years and locations to understand and stabilize symbiotic efficiency for ecosystem sustainability.

Host identity is more important in structuring bacterial epiphytes than endophytes in a tropical mangrove forest.

Interactions between plants and microbes are involved in biodiversity maintenance, community stability and ecosystem functioning. However, differences in the community and network structures between phyllosphere epiphytic and endophytic bacteria have rarely been investigated. Here, we examined phyllosphere epiphytic and endophytic bacterial communities of six mangrove species using Illumina MiSeq sequencing of the 16S rRNA gene. The results revealed that the community structure of epiphytic and endophytic bacteria was different. Plant identity significantly affected the diversity and community structure of both epiphytic and endophytic bacteria, with a greater effect on the community structure of the former than the latter. Network analysis showed that both plant-epiphytic and plant-endophytic bacterial network structures were characterized by significantly highly specialized and modular but lowly connected and anti-nested properties. Furthermore, the epiphytic bacterial network was more highly specialized and modular but less connected and more strongly anti-nested than the endophytic bacterial network. This study reveals that the phyllosphere epiphytic and endophytic bacterial community structures differ and plant identity has a greater effect on the epiphytic than on the endophytic bacteria, which may provide a comprehensive insight into the role of plant identity in driving the phyllosphere epiphytic and endophytic microbial community structures in mangrove ecosystems.

Investigating the endophytic bacterial diversity and community structures in seeds of genetically related maize (Zea mays L.) genotypes.

This research aimed to investigate the composition and diversity of endophytic bacterial community in seeds of four hybrid maize and their parental lines, which was used to reveal the potential relationship and association of endophytic bacteria between maize genotypes and their genetic relevance. High-throughput sequencing (HTS) technology showed that a total of 1419 OTUs (46.6%) were parental lines unique and 1052 OTUs (34.5%) were hybrid varieties unique, with only 575 core OTUs revealed in all the samples. Most OTUs belonged to Proteobacteria. Enterobacter (23.2%), Shigella (21.2%), Pseudomonas (15.8%) and Achromobacter (10.1%) were the major genera; the bacterial community composition and diversity of endophytic bacteria were inconsistent among different seed genotypes. Based on principal component analysis (PCA), the results referred that the endophytic composition of hybrid sample showed obvious correlation with their female parental lines, and in 'Jingke968' and 'MC738' with the same female line the endophytic community was more similar than other hybrid samples. This was the first ever use of HTS technology for investigating the endophytic bacterial diversity and community structures in seeds of genetically related maize genotypes, it was shown that, there were core microbes shared among all genotypes of seed samples, and the female parental line was more significant to impact on the composition of their hybrid seeds than male parental line. This study would provide scientific clues for the future research on the vertical transmission of endophytes among maize generations.

Changes of Root Endophytic Bacterial Community Along a Chronosequence of Intensively Managed Lei Bamboo (Phyllostachys praecox) Forests in Subtropical China.

Endophytic bacteria widely exist inside plant tissues and have an important role in plant growth and development and the alleviation of environmental stress. However, little is known about the response of root-associated bacterial endophytes of Lei bamboo (Phyllostachys praecox) to intensive management, which is a common management practice for high bamboo shoot production in subtropical China. In this study, we comparatively investigated the root endophytic bacterial community structures in a chronosequence of intensively managed (5a, 10a, 15a, and 20a) and extensively managed plantations (as control, Con). The results showed that endophytic Proteobacteria was the dominant bacterial phylum in the bamboo roots. Intensive management significantly increased (p < 0.05) the bacterial observed species and Chao1 (except 5a) indices associated with bamboo roots. The relative abundances of Firmicutes, Bacteroidetes, and Actinobacteria (except 15a) in the intensively managed bamboo roots significantly increased (p < 0.05) compared with those in Con, while the relative abundance of Proteobacteria significantly decreased in intensively managed bamboo roots (p < 0.05). The phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were the biomarkers in Con, 5a, 15a, and 20a, respectively. Redundancy analysis (RDA) showed that soil alkali-hydrolysable N (AN), available phosphorus (AP), available K (AK), and total organic carbon (TOC) were significantly correlated (p < 0.05) with the bacterial community compositions. Our results suggest that the root endophytic microbiome of Lei bamboo was markedly influenced by intensive management practices, and the available nutrient status could be the main driving factor for such shifts. Although heavy fertilization in the intensive management system increased the diversity indices, the rapid changes in root endophyte communities and their relevant functions might indicate a high risk for sustainable management.

Endophytic Microbiota Comparison of Dendrobium huoshanense Root and Stem in Different Growth Years.

The endophytic microbiome in medicinal plants is rich and diverse, but few studies have followed the endophytic microbiome of medicinal plants in different tissues with their growth. In this study, we examined the endophytic bacterial and fungal community structures associated with both the stem and root compartments of Dendrobium huoshanense at different growth years via high-throughput sequencing of 16S rRNA genes and nrDNA fragments of internal transcribed spacer regions. Results indicated that more diverse prokaryotic and fungal operational taxonomic units were detected in roots than in stems, and the alpha diversity of endophytic prokaryotic significantly differed among the 1-, 2-, and 3-year-old roots. The dominant bacterial phyla Proteobacteria Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteria, and fungal phyla Ascomycota, Basidiomycota, and Ascomycota were detected in the stems and roots with 3 growth years. Moreover, linear discriminant effect size analysis revealed 138 differentially abundant taxonomic clades in the bacterial level, and 197 in the fungal level in six groups. Our results provide evidence for endophytic microbiota communities depending on the tissues and growth years of D. huoshanense. The results from this study should be useful to better understand medicinal plant-microbe interactions.

Use of antibiotics to control endophytic bacterial growth migration onto culture medium in Eucalyptus cloeziana F.Muell.: a micropropagation approach

The natural endophytic bacterial growth migration onto a culture medium is commonly associated with unnecessary microplant discards. In micropropagation procedures, some bacteria can exude from the internal tissues of plants to colonize the culture medium and compete with the plants for nutrients, which may lead to a reduction in plant development. To find an efficient antibiotic protocol to control this bacterial growth migration onto the culture medium without affecting plant development, Eucalyptus cloeziana F.Muell. microstumps were subjected to four antibiotic treatments for 30 d. They were treated with gentamicin, ciprofloxacin, rifampicin, or Timentin®, in addition to the control treatment (antibiotic free). The effects of the antibiotics were monitored weekly, and the endophytic bacterial community structures were evaluated in two periods of plant development (15 d and 30 d).The denaturing gradient gel electrophoresis (DGGE) technique was used to compare the control and post-antibiotic treatment plant microbiological composition, to determine if the antibiotic treatment played a specific role on the endophytic bacterial community structure. The gentamicin treatment was composed of a distinct community from the control treatment. Nonetheless, the plants treated with ciprofloxacin and rifampicin manifested similar endophytic community structures compared to the control. In contrast, plants treated with Timentin® showed a specific bacterial community composition and a higher plant dry mass, number of shoots, and nutritional content. These results suggested that Timentin® treatment could be applied for 30 days to control endophytic bacterial growth migration onto the culture medium, without affecting the homeostatic balance between the bacteria and plants.

芽胞杆菌浸种对水稻内生细菌群落结构的影响

芽胞杆菌浸种对水稻内生细菌群落结构的影响

Composition and diversity of endophytic bacterial community in seeds of super hybrid rice ‘Shenliangyou 5814’ (Oryza sativa L.) and its parental lines

Super hybrid rice is one of the important research achievements in the field of rice super-high yield breeding in China. Compared with general high-yield rice, the yield potential of super hybrid rice is relatively high, and the average yields per hectare can exceed 10.5 t. This study aimed to study the endophytic bacterial community among a super hybrid rice combination and its parental lines and to reveal the potential relationship and association of endophytic bacteria between rice genotypes and their genetic relevance. In this research, through high-throughput sequencing based on Illumina Hiseq 2500 platform, the seeds of super hybrid rice variety ‘Shenliangyou 5814’ (sample C) and its parental lines ‘Y58S’ (sample M) and ‘C4114’ (sample F) independently cultivated by China were used as plant materials to study their endophytic bacterial structure and diversity. The number of OTUs for sample M, F and C was 327, 288 and 283 respectively, and among them 218 endophytic OTUs coexisted in the 3 samples. Pantoea (18.6–31.1%) was the first dominant groups shared in all three samples, and other dominant shared groups belonged to Methylobacterium (4.48%~17.65%), Sphingomonas (4.0%~11.4%), Rhizobium (5.69–8.78%), Microbacterium (3.85%~9.41%) and Pseudomonas (4.13%~5.68%). Although Principal Coordinates Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS) analysis showed that there were obvious differences in endophytic bacterial community composition and structure among the 3 samples, the dominant endophytic bacterial genus sample C and its parental lines remained consistency. This study would provide scientific clues for the future research on the vertical transmission of endophytes between rice generations.

Endophytic bacterial communities of Jingbai Pear trees in north China analyzed with Illumina sequencing of 16S rDNA.

Plant endophytes play a crucial role in plant growth, health and ecological function. Jingbai pear (the best quality cultivar of Pyrus ussuriensi Maxim. ex Rupr.) has important ecological and economic value in north China. Conversation of its genetics has great meanings to pear genus (Pyrus L.). However, the bacterial community associated with the cultivar remains unknown. In this study, the structure of endophytic bacterial communities associated with different tissues and soil of Jingbai Pear trees was analyzed with Illumina Miseq sequencing of bacterial 16S rDNA. This is the first report on bacterial microbiome associated with Jingbai pear. Our results demonstrated that different tissues harbored a unique bacterial assemblage. Interestingly, Cyanobacteria was the most abundant phylum, followed by Proteobacteria and Actinobacteria. Samples from three different sites (soils) had significant differences in microbial communities structure. Redundancy analysis (RDA) showed that the bacterial community structure correlated significantly with soil properties-temperature, pH, nitrogen and carbon contents. The conclusion could facilitate to understand the interaction and ecological function of endophytic bacteria with host Jingbai pear trees, so as to benefit the pear variety genetic resource conservation and protection.

Endophytic Bacterial Community Structure and Function of Herbaceous Plants From Petroleum Hydrocarbon Contaminated and Non-contaminated Sites.

Bacterial endophytes (BEs) are non-pathogenic residents of healthy plant tissues that can confer benefits to plants. Many Bacterial endophytes have been shown to contribute to plant growth and health, alleviation of plant stress and to in-planta contaminant-degradation. This study examined the endophytic bacterial communities of plants growing abundantly in a heavily hydrocarbon contaminated site, and compared them to those found in the same species at a non-contaminated. We used culture- dependent and independent methods to characterize the community structure, hydrocarbon degrading capabilities, and plant growth promoting traits of cultivable endophytes isolated from Achillea millefolium, Solidago Canadensis, and Daucus carota plants from these two sites. Culture- dependent and independent analyses revealed class Gammaproteobacteria predominated in all the plants regardless of the presence of petroleum hydrocarbon, with Pantoea spp. as largely dominant. It was interesting to note a >50% taxonomic overlap (genus level) of 16s rRNA high throughput amplicon sequences with cultivable endophytes. PERMANOVA analysis of TRFLP fragments revealed significant structural differences between endophytic bacterial communities from hydrocarbon-contaminated and non-contaminated soils—however, there was no marked difference in their functional capabilities. Pantoea spp. demonstrated plant beneficial characteristics, such as P solubilization, indole-3-acetic acid production and presence of 1-aminocyclopropane-1-carboxylate deaminase. Our findings reveal that functional capabilities of bacterial isolates being examined were not influenced by the presence of contamination; and that the stem endosphere supports ubiquitous BEs that were consistent throughout plant hosts and sites.

Insights into Endophytic Bacterial Community Structures of Seeds Among Various Oryza sativa L. Rice Genotypes

This study aimed to investigate the endophytic bacterial communities among various rice seed genotypes, to define the core microbiome and to further explore the relationship between rice genotypes and their endophytic bacterial flora. Simple sequence repeats molecular marker technology was used to investigate the genetic polymorphism in five different genotypes of rice. Endophytic bacterial communities in rice seeds were investigated using the Illumina-based 16S rRNA gene. The results showed that rice genotype had little impact on the diversity and richness of endophytic bacteria in seeds. The various rice genotypes do not have significantly different communities of endophytic bacterial in seeds, but the endophyte abundance distributions are obviously different, especially the dominant endophytic genera. Some phyla, such as Acidobacteria, Fusobacteria, Chlamydiae and Gemmatimonadetes, were first detected in rice seeds using high-throughput sequencing technique. As expected, five different rice genotypes were found to have a shared microbiome. At the genus level, Pantoea (28.33–72.77%), Acinetobacter (0.16–34.23%) and Xanthomonas (3.20–13.51%), which are probably the core microflora in indica rice seeds, served as the dominant genera that coexisted in all rice seeds tested. However, the current studies cannot confirm the correlation of rice phylogeny and seed bacterial microbiome clearly, but provide some clues of technical possibility and valuable experiences for the future study.

Diversity of cultivable bacterial endophytes in Paullinia cupana and their potential for plant growth promotion and phytopathogen control

Endophytic bacteria occupy the same niche of phytopathogens and may produce metabolites that induce the host plant systemic resistance and growth. Host and environmental variables often determine the endophytic community’s structure and composition. In this study, we addressed whether the plant genotype, organ, and geographic location influence the structure, composition, and functionality of endophytic bacterial communities in Paullinia cupana. To characterize the communities and identify strains with potential application in agriculture, we analyzed two P. cupana genotypes cultivated in two cities of the State of Amazonas, Brazil. Endophytic bacteria were isolated from surface-disinfested root, leaf, and seed tissues through the fragmentation and maceration techniques. The colonization rate, number of bacteria, richness, diversity, and functional traits were determined. The plant growth-promoting ability of selected bacterial strains was assessed in Sorghum bicolor. We identified 95 bacterial species distributed in 29 genera and 3 phyla (Proteobacteria, Actinobacteria, and Firmicutes). The colonization rate, richness, diversity, and species composition varied across the plant organs; the last parameter also varied across the plant genotype and location. Some strains exhibited relevant plant growth-promoting traits and antagonistic traits against the main phytopathogens of P. cupana, but they were not separated by functional traits. The main bacterial strains with plant growth-promoting traits induced S. bicolor growth. Altogether, our findings open opportunities to study the application of isolated endophytic bacterial strains in the bioprospection of processes and products.

Endophytic bacterial communities in in vitro shoot cultures derived from embryonic tissue of hybrid walnut (Juglans × intermedia)

A detailed picture of the endophytic bacterial community structure on phylum and genus level in in vitro shoot cultures of sixteen Juglans genotypes including Juglans regia, Juglans nigra and mainly Juglans hybrids was generated by using culture-dependent and culture-independent methods. A total of eight endophytic bacterial strains with a high identity score (97–100%) were isolated from tissue culture shoot material derived from embryonic tissue of Juglans hybrids by the culture-dependent approach. By sequence comparison of a 16S rRNA fragment to the NCBI database, they were identified as Acinetobacter, Brevundimonas, Moraxella, Pseudomonas, and Roseomonas species. The culture-independent approach involved Illumina MiSeq 16S rRNA amplicon sequencing. Five different phyla (Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria) were analyzed in more detail. The phylum Proteobacteria including genera such as Brevundimonas spp., Acinetobacter spp. and Moraxella spp. showed the highest relative abundance of endophytic bacteria. Thus, the results of the sequencing approach accorded well with those obtained in the culture-dependent approach. The abundance of Moraxella spp. was correlated with a good micropropagation in the Juglans genotypes under investigation, whereas, Brevundimonas spp. and Acinetobacter spp. were only detected in clones with medium propagation rates. Other bacterial endophytes known as plant growth promoting bacteria such as Pseudomonas spp., Erwinia spp., Burkholderia spp., Pelomonas spp., or Sphingomonas spp. were also found in in vitro cultures of a wide range of Juglans genotypes.

Inoculation of a phenanthrene-degrading endophytic bacterium reduces the phenanthrene level and alters the bacterial community structure in wheat.

Colonization by polycyclic aromatic hydrocarbon (PAH)-degrading endophytic bacteria (PAHDEB) can reduce the PAH contamination risk in plant. However, little information is available on the impact of PAHDEB colonization on the endophytic bacterial community of inner plant tissues. A phenanthrene-degrading endophytic bacterium (PDEB), Massilia sp. Pn2, was inoculated onto the roots of wheat and subjected to greenhouse container experiments. The endophytic bacterial community structure in wheat was investigated using high-throughput sequencing technology. The majority of endophytic bacteria in wheat were Proteobacteria, and the dominant genus was Pseudomonas. Phenanthrene contamination clearly increased the diversity of endophytic bacteria in wheat. The cultivable endophytic bacteria counts in wheat decreased with increasing the level of phenanthrene contamination; the endophytic bacterial community structure changed correspondingly, and the bacterial richness first increased and then decreased. Inoculation of strain Pn2 reduced the phenanthrene contamination in wheat, enlarged the biomass of wheat roots, changed the bacterial community structure and enhanced the cell counts, diversity and richness of endophytic bacteria in phenanthrene-contaminated wheat in a contamination level-dependent manner. The findings of this investigation provide insight into the responses of endophytic bacterial community in plant to external PAH contamination and PAHDEB colonization.

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, “SDS-extracted” sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.

A comparative analysis of endophytic bacterial communities associated with hyperaccumulators growing in mine soils

Interactions between endophytic bacterial communities and hyperaccumulators in heavy metal-polluted sites are not fully understood. In this study, the diversity of stem-associated endophytic bacterial communities of two hyperaccumulators (Solanum nigrum L. and Phytolacca acinosa Roxb.) growing in mine soils was investigated using molecular-based methods. The denaturing gradient gel electrophoresis (DGGE) analysis showed that the endophytic bacterial community structures were affected by both the level of heavy metal pollution and the plant species. Heavy metal in contaminated soil determined, to a large extent, the composition of the different endophytic bacterial communities in S. nigrum growing across soil series (five sampling spots, and the concentration of Cd is from 0.2 to 35.5 mg/kg). Detailed analysis of endophytic bacterial populations by cloning of 16S rRNA genes amplified from the stems of the two plants at the same site revealed a different composition. A total of 51 taxa at the genus level that included α-, β-, and γ-Proteobacteria (68.8% of the two libraries clones), Bacteroidetes (9.0% of the two libraries clones), Firmicutes (2.0% of the two libraries clones), Actinobacteria (16.4% of the two libraries clones), and unclassified bacteria (3.8% of the two libraries clones) were found in the two clone libraries. The most abundant genus in S. nigrum was Sphingomonas (23.35%), while Pseudomonas prevailed in P. acinosa (21.40%). These results suggest that both heavy metal pollution and plant species contribute to the shaping of the dynamic endophytic bacterial communities associated with stems of hyperaccumulators.