Endophytic Bacterial Diversity Research Articles

Soil Inoculation with Bacillus spp. Modifies Root Endophytic Bacterial Diversity, Evenness, and Community Composition in a Context-Specific Manner

The use of microbial inoculants containing plant growth-promoting rhizobacteria as a promoter of plant fitness and health is becoming increasingly popular in agriculture. However, whether and how these bacteria affect indigenous bacterial communities in field conditions is sparsely explored. We studied the effects of seed inoculation and field soil application of ubiquitous soil bacteria, B. cereus, B. subtilis, and B. amyloliquefaciens, on the diversity, evenness, and richness of endophytic bacterial communities in sprouting broccoli roots using high-throughput metagenome sequencing. The multiple operational taxonomic units (OTUs) assigned to different bacterial taxa clearly showed changes in ecological measures and relative abundances of certain taxa between control and treatment groups. The Bacillus inocula, themselves, failed to flourish as endophytes; however, the effects they extended on the endophytic bacterial community were both generic as well as species specific. In each case, Pseudomonadales, Rhizobiales, Xanthomonadales, and Burkholderiales were the most abundant orders in the endosphere. B. amyloliquefaciens drastically reduced the most abundant genus, Pseudomonas, while increasing the relative abundance of a range of minor taxa. The Shannon-Weiner diversity and Buzas and Gibson’s evenness indices showed that the diversity and evenness were increased in both B. amyloliquefaciens and mixed treated plants. The UniFrac measurement of beta diversity showed that all treatments affected the specific composition of the endophytic bacterial community, with an apparent interspecies competition in the mixed treatment. Taken together, Bacillus species influenced the diversity, evenness, and composition of the endophytic bacterial community. However, these effects varied between different Bacillus spp. in a context-specific manner.

Bacteriome from Pinus arizonica and P. durangensis: Diversity, Comparison of Assemblages, and Overlapping Degree with the Gut Bacterial Community of a Bark Beetle That Kills Pines.

Symbioses between plants and microorganims have been fundamental in the evolution of both groups. The endophytic bacteria associated with conifers have been poorly studied in terms of diversity, ecology, and function. Coniferous trees of the genera Larix, Pseudotsugae, Picea and mainly Pinus, are hosts of many insects, including bark beetles and especially the Dendroctonus species. These insects colonize and kill these trees during their life cycle. Several bacteria detected in the gut and cuticle of these insects have been identified as endophytes in conifers. In this study, we characterized and compared the endophytic bacterial diversity in roots, phloem and bark of non-attacked saplings of Pinus arizonica and P. durangensis using 16S rRNA gene pyrosequencing. In addition, we evaluated the degree of taxonomic relatedness, and the association of metabolic function profiles of communities of endophytic bacteria and previously reported gut bacterial communities of D. rhizophagus; a specialized bark beetle that colonizes and kills saplings of these pine species. Our results showed that both pine species share a similar endophytic community. A total of seven bacterial phyla, 14 classes, 26 orders, 43 families, and 51 genera were identified. Enterobacteriaceae was the most abundant family across all samples, followed by Acetobacteraceae and Acidobacteriaceae, which agree with previous studies performed in other pines and conifers. Endophytic communities and that of the insect gut were significantly different, however, the taxonomic relatedness of certain bacterial genera of pines and insect assemblages suggested that some bacteria from pine tissues might be the same as those in the insect gut. Lastly, the metabolic profile using PICRUSt showed there to be a positive association between communities of both pines and insect gut. This study represents the baseline into the knowledge of the endophytic bacterial communities of two of the major hosts affected by D. rhizophagus.

Endophytic bacterial diversity decrease in amaranth mutant lines after radiation mutagenesis

Background and objectivesA metagenomic approach was applied to analyze endophytic bacterial diversity changes in Amaranthus cruentus L. subjected to mutagenesis‐based breeding.FindingsHigh‐throughput sequencing approach of 16S rRNA variability analysis was used for monitoring of the changes of endophytic bacterial diversity in selected M generations and varieties Ficha and Pribina of Amaranthus cruentus L. A total of 15,683,430 clusters were generated in the sequencing and 15,085,381 of them passed filter. Average cluster density was 646 clusters per mm2. A 95%, 97% of sequences were Q >30. All sequences were classified into the 44 orders that were identified from all the analyzed genotypes of Amaranthus. Metagenomic libraries of Ficha and Pribina showed the similar profiles of identified bacterial orders and that are different from the profiles of M1 and M7 generations after gamma radiance treatment.ConclusionsEndophytic bacteria diversity was higher in the starting variety and final new variety of Amaranthus when comparing to the M generations. Based on the laboratory conditions of the growth of analyzed plant material, it supposes that endophytic bacterial abundance differences are a result of temporary changes in the amount of bacterial endophytes and the metagenomic limitation of their identification.Significance and noveltyMetagenomic analysis provides a sensitive approach that facilitates the study of the endophytic bacteria changes in the leaves of plant after gamma radiation mutagenesis.

Characterization of endophytic bacteria with plant growth promotion and biological control potential isolated from walnut trees

SummaryIn this study, a total of 68 endophytic bacteria were isolated from different tissues of walnut trees. About 55% and 22% of the strains had the ability to produce indole acetic acid and gibberellic acid, respectively. The capability of isolates to solubilize phosphate, growth on N‐free medium, siderophore, protease and lipase production was varied. Based on phenotypic grouping and plant growth promotion properties, twelve isolates were selected and 16S rRNA gene‐based phylogenetic analysis revealed that strains showed 99%–100% similarity to Pseudomonas, Bacillus, Arthrobacter, Roseomonas and Streptomyces genera. Amongst the selected strains, PS12, KS54, JS66 and KS74 showed root and shoot growth enhancement of poplar cutting. NS70, KS54 and PL36 strains showed antagonistic activity against Pseudomonas syringae pv. syringae;RR47, KS74 and NR69 strains had inhibition effects against Brenneria nigrifluens; and JS66 and RR26 strains had antagonistic activity against both phytopathogens under in vitro conditions. This is the first reported study to elucidate the endophytic bacterial diversity associated with walnut trees with beneficial attributes.

Illumina-based analysis of endophytic bacterial diversity of tree peony (Paeonia Sect. Moutan) roots and leaves.

Diverse communities of bacteria inhabit plant tissues and those bacteria play a crucial role for plant health and growth. Tree peony (Paeonia Sect. Moutan) is known for its excellent ornamental and medicinal values as Chinese traditional plant, but little is known about its associated bacterial community under natural conditions. To examine how endophytic bacteria in tree peony vary across tissues and cultivars, PCR-based Illumina was applied to reveal the diversity of endophytic bacteria in tree peony. A total of 149,842 sequences and 21,463 operational taxonomic units (OTUs) were obtained. The OTU abundance of roots was higher than leaves across other three cultivars except for ‘Kinkaku’ and ‘Luoyanghong’. The community was composed of five dominant groups (Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria and Actinobacteria) in all samples. Endophytic bacteria community structures had changed in leaves and roots. Sequences of Pseudomonas and Enterobacteriaceae were prevalent in root samples, whereas Succinivibrio and Acinetobacter were the dominant genus in leaf samples. Otherwise, the distribution of each dominant genus among the 5 cultivars was either varied. These findings suggested that both plant genotype and tissues contribute to the shaping of the bacterial communities associated with tree peony.

Diversity of Endophytic Bacteria in a Fern Species Dryopteris uniformis (Makino) Makino and Evaluation of Their Antibacterial Potential Against Five Foodborne Pathogenic Bacteria.

The fern plant Dryopteris uniformis has traditionally been used in herbal medicine and possesses many biological activities. This study was conducted to explore the endophytic bacterial diversity associated with D. uniformis and evaluate their antibacterial potential against foodborne pathogenic bacteria (FPB). Among 51 isolated endophytic bacteria (EB), 26 EB were selected based on their morphological characteristics and identified by 16S rRNA gene analysis. The distribution of EB was diverse in the leaf and the stem/root tissues. When the EB were screened for antibacterial activity against five FPB, Listeria monocytogenes, Salmonella Typhimurium, Bacillus cereus, Staphylococcus aureus, and Escherichia coli O157:H7, four EB Bacillus sp. cryopeg, Paenibacillus sp. rif200865, Staphylococcus warneri, and Bacillus psychrodurans had a broad spectrum of antibacterial activity (9.58 ± 0.66 to 21.47 ± 0.27 mm inhibition zone). The butanol solvent extract of B. sp. cryopeg and P. sp. rif200865 displayed effective antibacterial activity against the five FPB, which was evident from the scanning electron microscopy with irregular or burst cell morphology in the EB-treated bacteria compared to smooth and regular cells in case of the control bacteria. The minimum inhibitory concentration and minimum bactericidal concentration values ranged between 250-500 μg/mL and 500-100 μg/mL, respectively. The above outcomes signify the huge prospective of the selected EB in the food industry. Overall, the above results suggested that D. uniformis contains several culturable EB that possess effective antibacterial compounds, and that EB can be utilized as a source of natural antibacterial agents for their practical application in food industry to control the spread of FPB as a natural antibacterial agent.

Rhizobium oryziradicis sp. nov., isolated from rice roots.

Two Gram-stain-negative, aerobic, rod-shaped endophytic bacterial strains, N19T and N11-2, were isolated from fresh rice (Oryza sativa) roots during investigation of the rice endophytic bacterial diversity. The 16S rRNA gene sequence results indicated that the similarity between strains N19T and N11-2 was 100 %. Both of them belong to the genus Rhizobium, with close similarity to Rhizobium taibaishanense CCNWSX 0483T (97.7 %), followed by Rhizobium vitis NCPPB 3554T (97.5 %). The sequence similarities of the housekeeping genes recA, gyrB and glnA between the novel isolates and members of the established species of the genus Rhizobium were less than 87 %. The DNA-DNA hybridization rates between strains N19T and N11-2 were 87.9 % using the initial renaturation rate method. Based on draft genome sequences, strain N19T showed 18.2 % and 19.6 % DNA-DNA hybridization values to R. taibaishanense CCNWSX 0483T and R. vitis S4, which demonstrated that these new isolates represent a novel species in the genus Rhizobium. The main cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The DNA G+C content of strain N19T was 58.7 mol% (Tm). The polar lipid profile of N19T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown lipid, two unknown aminolipids and an unidentified aminophospholipid. According to physiological and biochemical characteristics and genotypic data, strains N19T and N11-2 are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium oryziradicis sp. nov. is proposed, with N19T (=ACCC 19962T=KCTC 52413T) as the type strain.

Endophytic bacterial diversity of Avicennia marina helps to confer resistance against salinity stress in Solanum lycopersicum

ABSTRACTThe current study aimed to explore the endophytic bacterial diversity of Avicennia marina and the potential roles of these endophytes in counteracting saline conditions in tomato plants. Molecular analysis revealed strains from Paenibacillus, Bacillus, Microbacterium, Citrobacter, Lysinibacillus, Halomonas, Virgibacillus, Exiguobacterium, and Vibrio. However, Bacillus pumilus AM11 and Exiguobacterium sp. AM25 showed significantly higher growth in saline media. In response to salinity stress, tomato plants treated with AM11 and AM25 showed significantly higher (∼15–23%) biomass, photosynthetic rate and pigment accumulation compared to controls. Salinity-exposed plants had significantly reduced growth and increased (three-fold) lipid peroxidation, whilst glutathione, catalase, and peroxidase activities were significantly reduced. In contrast, AM11, AM25, and methionine improved these physiochemical attributes. The study concludes that the application of bacterial endophytes from plants growing in saline conditions can offer other plants similar stress-resistance potential. Such halophytic bacterial strains can be used to improve plant growth in saline conditions.

Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria.

The interior of plants constitutes a unique environment for microorganisms with various organisms inhabiting as endophytes. Unlike subterranean plant parts, aboveground parts are relatively less explored for endophytic microbial diversity. We employed a combination of cultivation and molecular approaches to study the endophytic bacterial diversity in banana shoot-tips. Cultivable bacteria from 20 sucker shoot-tips of cv. Grand Naine included 37 strains under 16 genera and three phyla (Proteobacteria, Actinobacteria, Firmicutes). 16S rRNA gene-ribotyping approach on 799f and 1492r PCR-amplicons to avoid plant organelle sequences was ineffective showing limited bacterial diversity. 16S rRNA metagene profiling targeting the V3-V4 hypervariable region after filtering out the chloroplast (74.2%), mitochondrial (22.9%), and unknown sequences (1.1%) revealed enormous bacterial diversity. Proteobacteria formed the predominant phylum (64%) succeeded by Firmicutes (12.1%), Actinobacteria (9.5%), Bacteroidetes (6.4%), Planctomycetes, Cyanobacteria, and minor shares (<1%) of 14 phyla including several candidate phyla besides the domain Euryarchaeota (0.2%). Microbiome analysis of single shoot-tips through 16S rRNA V3 region profiling showed similar taxonomic richness and diversity and was less affected by plant sequence interferences. DNA extraction kit ominously influenced the phylogenetic diversity. The study has revealed vast diversity of normally uncultivable endophytic bacteria prevailing in banana shoot-tips (20 phyla, 46 classes) with about 2.6% of the deciphered 269 genera and 1.5% of the 656 observed species from the same source of shoot-tips attained through cultivation. The predominant genera included several agriculturally important bacteria. The study reveals an immense ecosystem of endophytic bacteria in banana shoot tissues endorsing the earlier documentation of intracellular "Cytobacts" and "Peribacts" with possible roles in plant holobiome and hologenome.

High-Throughput Sequencing Analysis of the Endophytic Bacterial Diversity and Dynamics in Roots of the Halophyte Salicornia europaea.

Endophytic bacterial communities of halophyte Salicornia europaea roots were analyzed by 16S rRNA gene pyrosequencing. A total of 20,151 partial 16S rRNA gene sequences were obtained. These sequences revealed huge amounts of operational taxonomic units (OTUs), that is, 747-1405 OTUs in a root sample, at 3 % cut-off level. Root endophytes mainly comprised four phyla, among which Proteobacteria was the most represented, followed by Bacteroidetes, Actinobacteria, and Firmicutes. Gammaproteobacteria was the most abundant class of Proteobacteria, followed by Betaproteobacteria and Alphaproteobacteria. Genera Pantoea, Halomonas, Azomonas, Serpens, and Pseudomonas were shared by all growth periods. A marked difference in endophytic bacterial communities was evident in roots from different host life-history stages. Gammaproteobacteria increased during the five periods, while Betaproteobacteria decreased. The richest endophytic bacteria diversity was detected in the seedling stage. Endophytic bacteria diversity was reduced during the flowering stage and fruiting stage. The five libraries contained 2321 different OTUs with 41 OTUs in common. As a whole, this study first surveys communities of endophytic bacteria by tracing crucial stages in the process of halophyte growth using high-throughput sequencing methods.

Endophytic bacterial diversity in the phyllosphere of Amazon Paullinia cupana associated with asymptomatic and symptomatic anthracnose.

Endophytes colonize an ecological niche similar to that of phytopathogens, which make them candidate for disease suppression. Anthracnose is a disease caused by Colletotrichum spp., a phytopathogen that can infect guarana (Paullinia cupana), an important commercial crop in the Brazilian Amazon. We investigated the diversity of endophytic bacteria inhabiting the phyllosphere of asymptomatic and symptomatic anthracnose guarana plants. The PCR-denaturation gradient gel electrophoresis (PCR-DGGE) fingerprints revealed differences in the structure of the evaluated communities. Detailed analysis of endophytic bacteria composition using culture-dependent and 16S rRNA clone libraries revealed the presence of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria phyla. Firmicutes comprised the majority of isolates in asymptomatic plants (2.40E−4). However, cloning and sequencing of 16S rRNA revealed differences at the genus level for Neisseria (1.4E−4), Haemophilus (2.1E−3) and Arsenophonus (3.6E−5) in asymptomatic plants, Aquicella (3.5E−3) in symptomatic anthracnose plants, and Pseudomonas (1.1E−3), which was mainly identified in asymptomatic plants. In cross-comparisons of the endophytic bacterial communities as a whole, symptomatic anthracnose plants contained higher diversity, as reflected in the Shannon–Weaver and Simpson indices estimation (P < 0.05). Similarly, comparisons using LIBSHUFF and heatmap analysis for the relative abundance of operational taxonomic units (OTUs) showed differences between endophytic bacterial communities. These data are in agreement with the NMSD and ANOSIM analysis of DGGE profiles. Our results suggest that anthracnose can restructure endophytic bacterial communities by selecting certain strains in the phyllosphere of P. cupana. The understanding of these interactions is important for the development of strategies of biocontrol for Colletotrichum.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1037-0) contains supplementary material, which is available to authorized users.

Diversity and functional characterization of bacterial endophytes dwelling in various rice (Oryza sativa L.) tissues, and their seed-borne dissemination into rhizosphere under gnotobiotic P-stress

Endophytic bacterial diversity in four rice cultivars grown in two soil-types, their plant-probiotic features and rhizospheric deployment under P-stress were investigated. Oryza sativa cvs. TCN1, TCS10, TK8, and TN71 were cultivated in greenhouse using non-sterile acidic and near-neutral paddy soils for 60 days. Root, stem and leaf tissues were screened for culturable bacterial endophytes using nutrient agar. Endophytes were identified and profiled for plant-probiotic features. The richness, Shannon-Weiner diversity, evenness and Venn’s distribution in terms of endophytic strains were evaluated. Seed-borne endophytes were characterized through DGGE. The deployment of endophytes into the rhizosphere in TCN1 and TK8 under gnotobiotic P-stress was assessed. A total of 52 distinct endophytic bacterial strains affiliated to 5 classes and 20 discrete genera exhibiting differential plant-probiotic features were isolated from various tissues of four different rice cultivars. The diversity and distribution of endophytes fluctuated with soil-type, tissue-type and rice genotype. Gnotobiotic insoluble P treatment revealed significantly enhanced deployment of P-solubilizing rhizobacteria in TCN1 as compared to soluble P and P-lacking control. Rice endophytic bacteria are diverse, and their distribution within the plant and deployment as rhizobacteria were found to be influenced by host genotype, edaphic factors and nutrient stress.

Shifts in Symbiotic Endophyte Communities of a Foundational Salt Marsh Grass following Oil Exposure from the Deepwater Horizon Oil Spill.

Symbiotic associations can be disrupted by disturbance or by changing environmental conditions. Endophytes are fungal and bacterial symbionts of plants that can affect performance. As in more widely known symbioses, acute or chronic stressor exposure might trigger disassociation of endophytes from host plants. We tested this hypothesis by examining the effects of oil exposure following the Deepwater Horizon (DWH) oil spill on endophyte diversity and abundance in Spartina alterniflora – the foundational plant in northern Gulf coast salt marshes affected by the spill. We compared bacterial and fungal endophytes isolated from plants in reference areas to isolates from plants collected in areas with residual oil that has persisted for more than three years after the DWH spill. DNA sequence-based estimates showed that oil exposure shifted endophyte diversity and community structure. Plants from oiled areas exhibited near total loss of leaf fungal endophytes. Root fungal endophytes exhibited a more modest decline and little change was observed in endophytic bacterial diversity or abundance, though a shift towards hydrocarbon metabolizers was found in plants from oiled sites. These results show that plant-endophyte symbioses can be disrupted by stressor exposure, and indicate that symbiont community disassembly in marsh plants is an enduring outcome of the DWH spill.

Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects.

This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8–9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen.

Endophytic bacterial diversity in Korean kimchi made of Chinese cabbage leaves and their antimicrobial activity against pathogens

The diversity of bacterial endophytes associated with Chinese cabbage (CC) leaves from two cultivation areas in Korea, namely, Seosang-gun (SS) and Haenam-gun (HN), and from a laboratory transgenic plant (TP) was investigated. A total of 5.76 log CFU/g isolates were identified from the interior leaves of the three cultivars of CC, which were grouped into 5.08, 5.36, and 5.34 log CFU/g from SS, HN, and TP samples, respectively. Phylogenetic analysis based on the 16S rRNA sequences indicated that the isolates belonged to four major phylogenetic groups: high-G + C Gram-positive bacteria (HGCGPB), low-G + C Gram-positive bacteria (LGCGPB), Proteobacteria, and Bacteroidetes. The most predominant group of species in the leaves of the SS, HN and TP CC cultivars were LGCGPB (50%), LGCGPB (65.2%), and HGCGPB (43%), respectively. A total of 23 bacterial genera were identified from the three cultivars of Chinese cabbage. Most extracellular hydrolytic enzyme-producing colonies among the isolates belong to the genus Bacillus those were predominant in the HN cultivars. In addition, some isolates especially Bacillus sp. (TPL08), Bacillus subtilis (SSL16, HNL10) exhibited potential activity as biocontrol agents against food-borne pathogenic bacteria and phytopathogenic fungi tested in this study. This study first revealed the endophytic bacterial communities in leaves of Chinese cabbage (main ingredients of kimchi) grown in Korea.

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.

Screening and Production of Biodegradable Polyester Poly(3-hydroxybutyrate) by Bacteria Endophytic to Brassica nigra L.

Aims: Poly(3-hydroxybutyrate) [P(3HB)], the microbially produced biodegradable thermoplastics has find wide range of applications in recent years. Development of low cost production strategies utilizing novel organisms is a crucial challenge. Present study is aimed to isolate and screen bacterial endophytes of Brassica nigra L. for the production of P(3HB). Place and Duration of Study: The experiments were performed in the Microbiology Laboratory, Department of Botany, University of Calcutta, Kolkata during 2012-2014. Methodology: Culturable bacterial endophytes were isolated from surface sterilized healthy tissues of B. nigra L. and screened for P(3HB) production in mineral salts medium. The chloroform extracted dried polymer was treated with H2SO4 and quantified spectrophotometrically at 235 nm. Original Research Article Das et al.; BBJ, 7(3): 134-146, 2015; Article no.BBJ.2015.054 135 Results: About 78% of the bacterial endophytes recovered from surface sterilized B. nigra L. tissues showed different degrees of P(3HB) accumulation. Isolates (9) showing P(3HB) accumulation exceeding 10% of the cell dry weight (CDW) were characterized and tentatively identified as members of Bacillus, Pseudomonas, Xanthomonas, Alcaligenes and Acetobacter. The most potent isolate, BNL 06 identified as Bacillus pumilus BNL 06 (GenBank Accession No. KP202723), accumulated P(3HB) accounting 18% of CDW with an yield of 0.55 g/l. Finally the nature of the polymer was further confirmed by FTIR analysis. Conclusion: Exploration of the endophytic bacterial diversity of B. nigra L. have clearly revealed the potential of Bacillus pumilus BNL 06 for P(3HB) production as an alternative source of thermoplastics.

In silico analysis of the 16S rRNA gene of endophytic bacteria, isolated from the aerial parts and seeds of important agricultural crops.

Because of human population growth, increased food production and alternatives to conventional methods of biocontrol and development of plants such as the use of endophytic bacteria and fungi are required. One of the methods used to study microorganism diversity is sequencing of the 16S rRNA gene, which has several advantages, including universality, size, and availability of databases for comparison. The objective of this study was to analyze endophytic bacterial diversity in agricultural crops using published papers, sequence databases, and phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S rRNA gene was used to identify endophytic bacteria, in important agricultural crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes, located in different geographical regions (America, Europe, and Asia). The corresponding 16S rRNA gene sequences were selected from the NCBI database, aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The most common orders present in the analyzed cultures were Bacillales, Enterobacteriales, and Actinomycetales and the most frequently observed genera were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that only approximately 1.56% of the total sequences were not properly grouped, demonstrating reliability in the identification of microorganisms. This study identified the main genera found in endophytic bacterial cultures from plants, providing data for future studies on improving plant agriculture, biotechnology, endophytic bacterium prospecting, and to help understand relationships between endophytic bacteria and their interactions with plants.

Effect of different treatment on endophytic bacterial communities in continuous cropping of Chrysanthemum morifoliu

To reveal the effect of rotation cropping and bacterial manure on the growth of Chrysanthemum morifolium and screen the beneficial endophytic, the diversity of endophytic and dominant genera of different treatment groups were analyzed. Four different treatments were continuous cropping, rotation, self-made organic fertilizer and commercially available fertilizer, respectively. Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results showed that enzyme Hae III was more appropriate than enzyme Hinfl because the number of TRFs digested by enzyme Hae III was more than that of enzyme Hinfl. In comparison of diversity, the endophytic bacterial communities' diversity index in group of cropping rotation and fertilizer was higher than that of continuous cropping which indicated that the addition of exogenous microorganism in soil could increase the diversity of plant endophyte. 18 dominant species were selected, including 3 kinds of Firmicutes, 4 kinds of Actinomycetes and 11 kinds of Proteobacteria. The results of dominant species comparison showed that the number of dominant species in continuous cropping of Ch. morifolium was significantly less than that of the rotation group. Some dominant bacteria in rotation group and fertilizer group such as Arthrobacter, Streptomyces, Streptomyces, Flavobacterium and Mycobacterium were not found in the continuous cropping of Ch. mortfolium group. Dominant species of fertilizer treatment group was similar with the rotation group, and the continuous cropping group's dominant species was more abundant. It indicates that these bacteria may be able to mitigate hindrance in continuous cropping, especially the Flavobacterium which can decompose the pathogenic fungi is worthy of further attention. Compared with leaves, there are more dominant species in roots and stems. The diversity of edophytic bacterial communities in continuous cropping of Ch. morifolium stays below than that in the rotation of Ch. morifolium, and fertilizer treatment can increase the diversity of continuous cropping so that it could mitigate hindrance in continuous cropping.

Analysis of endophytic bacterial community composition by 16S rDNA clone library in Achnatherum inebrians

The endophytic bacterial diversity of drunken horse grass (DHG) (Achnatherum inebrians) growing in native habitat station in Xinjiang Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of DHG, a pair of bacterial PCR primers was selected to specifically amplify bacterial 16S rDNA sequences directly from DHG root tissues. Among 249 positive clones in the 16S rDNA library of endophytes, 57 OTUs were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Flavobacteria, Actinobacteria, Sphingobacteria, and uncultured bacteria. The dominant group was Proteobacteria, whie the most numerous genus was Bacillus. More than 8.00% of the total clones showed a high similarity to uncultured bacteria, suggesting that nonculturable bacteria make a significant part of DHG endophytic bacterial community. To our knowledge, this is the first report that endophytic bacteria associated with DHG are revealed by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in DHG.