16S rRNA Gene RFLP Research Articles

Diversity of endophytic bacterial community inhabiting in tropical aerobic rice under aerobic and flooded condition.

The role and activity of bacterial endophytes remains largely unexplored and detail insight into Indian rice agro ecosystem is still little explored. In this study, we examined the diversity of endophytic bacteria in aerobic rice (variety ARB6) under aerobic and flooded field conditions. Based on 16S rRNA gene RFLP cloning sequencing, 900 clones with 144 representatives (72 aerobic and 72 flooded) revealed majority of clones affiliated toGammaproteobacteria (64.58%),Betaproteobacteria (9.72%),Alphaproteobacteria (17.36), Firmicutes (6.26%) and Bacteroidetes (2.08). The study suggests that the aerobic rice variety harbours plant growth promoting (PGP) genera (viz. Pantoea, Enterobacter, Paenibacillus, etc). Investigations on aerobic rice under aerobic and flooded conditions revealed high richness and diversity of endophytic bacteria under aerobic condition inferring that the endophytic bacteria are beneficial for rice growth and productivity, and hence, would be helpful in designing better strategies for rice cultivation under drought or water scarce conditions.

The Diversity, Growth Promoting Abilities and Anti-microbial Activities of Bacteria Isolated from the Fruiting Body of Agaricus bisporus.

Agaricus bisporus plays an important role in ecological processes and is one of the most widely cultivated mushrooms worldwide. Mushroom growth-promoting bacteria have been isolated from casing soil and compost, but microorganisms in the fruiting body have received only a little attention. To get an overview of phylogenetic diversity of microorganisms in the fruiting body of A. bisporus, as well as to screen antimicrobial and mushroom growth-promoting strains, and eventually intensify mushroom production, we isolated and characterized microorganisms from the fruiting body of A. bisporus. In total, 55 bacterial strains were isolated, among which nine isolates represented Actinomycetes. All the isolates were analyzed by 16S rRNA gene RFLP and sixteen representative strains by 16S rRNA gene sequencing. According to the phylogenetic analysis, eleven isolates represented the Gram-positive Bacillus, Lysinibacillus, Paenibacillus, Pandorea and Streptomyces genera, and five isolates belonged to the Gram-negative Alcaligenes and Pseudomonas genera. The bacteria isolated from the fruiting body of A. bisporus had broad-spectrum antimicrobial activities and potential mushroom growth-promoting abilities.

Criteria for phytoplasma 16Sr group/subgroup delineation and the need of a platform for proper registration of new groups and subgroups

As phytoplasmas are discovered at an ever-increasing pace in emerging and re-emerging plant diseases worldwide, the scheme for classification of phytoplasmas into 16S rRNA gene RFLP (16Sr) groups and subgroups is experiencing an ongoing rapid expansion. Improper delineation or designation of new groups and subgroups can open potential conflicts in classifying newly identified phytoplasma strains. To maintain the integrity of the classification scheme, criteria for the delineation of new groups and subgroups must be followed, and proper registration should be required to track established groups and subgroups.

Mesorhizobium acaciae sp. nov., isolated from root nodules of Acacia melanoxylon R. Br.

Three novel strains, RITF741T, RITF1220 and RITF909, isolated from root nodules of Acacia melanoxylon in Guangdong Province of China, have been previously identified as members of the genus Mesorhizobium, displaying the same 16S rRNA gene RFLP pattern. Phylogenetic analysis of 16S rRNA gene sequences indicated that the three strains belong to the genus Mesorhizobium and had highest similarity (100.0 %) to Mesorhizobium plurifarium LMG 11892T. Phylogenetic analyses of housekeeping genes recA, atpD and glnII revealed that these strains represented a distinct evolutionary lineage within the genus Mesorhizobium. Strain RITF741T showed >73 % DNA–DNA relatedness with strains RITF1220 and RITF909, but < 60 % DNA–DNA relatedness with the closest type strains of recognized species of the genus Mesorhizobium. They differed from each other and from their closest phylogenetic neighbours by presence/absence of several fatty acids, or by large differences in the relative amounts of particular fatty acids. While showing distinctive features, they were generally able to utilize a wide range of substrates as sole carbon sources based on API 50CH and API 20NE tests. The three strains were able to form nodules with the original host Acacia melanoxylon and other woody legumes such as Acacia aneura, Albizia falcataria and Leucaena leucocephala. In conclusion, these strains represent a novel species belonging to the genus Mesorhizobium based on the data obtained in the present and previous studies, for which the name Mesorhizobium acaciae sp. nov. is proposed. The type strain is RITF741T ( = CCBAU 101090T = JCM 30534T), the DNA G+C content of which is 64.1 mol% (T m).

‘Candidatus Phytoplasma pruni’, a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Candidatus Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1(R) as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Ca. Phytoplasma pruni' lineage. We propose that the term 'Ca. Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and--within the tolerance of a single base difference in one unique sequence--peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' taxa.

P3-S7.16 Lactobacillus crispatus colonisation reduces risk of Bacterial Vaginosis (BV) acquisition

BackgroundLongitudinal studies have found that vaginal colonisation by hydrogen peroxide-producing strains of lactobacilli is associated with a reduced risk of BV acquisition. Several Lactobacillus spp. including L crispatus, L jensenii,...

Bradyrhizobium lablabi sp. nov., isolated from effective nodules of Lablab purpureus and Arachis hypogaea

Five strains isolated from root nodules of Lablab purpureus and Arachis hypogaea grown in the Anhui and Sichuan provinces of China were classified as members of the genus Bradyrhizobium. These strains had identical 16S rRNA gene sequences which shared 99.48 %, 99.48 % and 99.22 % similarity with the most closely related strains of Bradyrhizobium jicamae PAC68(T), Bradyrhizobium pachyrhizi PAC48(T) and Bradyrhizobium elkanii USDA 76(T), respectively. A study using a polyphasic approach, including 16S rRNA gene RFLP, IGS-RFLP, BOX-PCR, comparative sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) and the recA, atpD and glnII genes, DNA-DNA hybridization and phenotypic tests, showed that the five strains clustered into a coherent group that differentiated them from all recognized species of the genus Bradyrhizobium. Sequencing of nifH and nodC genes and cross-nodulation tests showed that the representative strains CCBAU 23086(T), CCBAU 23160 and CCBAU 61434, isolated from different plants, had identical nifH and nodC gene sequences and were all able to nodulate Lablab purpureus, Arachis hypogaea and Vigna unguiculata. Based upon these results, the name Bradyrhizobium lablabi sp. nov. is proposed for this novel species and strain CCBAU 23086(T) ( = LMG 25572(T) = HAMBI 3052(T)) is designated as the type strain. The DNA G+C mol% is 60.14 (T(m)).

Arcobacter mytili sp. nov., an indoxyl acetate-hydrolysis-negative bacterium isolated from mussels

Three Arcobacter isolates, recovered from mussels (genus Mytilus), and one isolate from brackish water in Catalonia (north-east Spain) showed a novel pattern using a recently described identification method for members of the genus Arcobacter, 16S rRNA gene RFLP. Enterobacterial repetitive intergenic consensus PCR fingerprinting demonstrated that the three isolates from mussels belonged to two genotypes and that the fourth isolate from water belonged to a third genotype. Analysis of the 16S rRNA and rpoB gene sequences showed that the new isolates formed a separate lineage within the genus Arcobacter. This was also confirmed by the low DNA-DNA relatedness values (16-30 %) of the isolates with the type strains of recognized Arcobacter species. Hydrolysis of indoxyl acetate, a characteristic trait for all species of the genus Arcobacter, was negative for the novel isolates. The susceptibility of the novel isolates to cefoperazone, together with the lack of urease production and nitrate reduction, further enabled them to be differentiated from recognized Arcobacter species based on physiological characteristics. Genotypic and phenotypic characteristics indicated that the new isolates represent a novel species of the genus Arcobacter, for which the name Arcobacter mytili sp. nov. is proposed, with the type strain F2075(T) (=CECT 7386(T) =LMG 24559(T)). The DNA G+C content of strain F2075(T) was 26.9 mol%.

Bradyrhizobium spp. and Sinorhizobium fredii are predominant in root nodules of Vigna angularis, a native legume crop in the subtropical region of China

Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters.

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China.

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S-23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.

Polyphasic characterization of mung bean ( Vigna radiata L.) rhizobia from different geographical regions of China

Polyphasic characterization of 54 indigenous mung bean ( Vigna radiata L.) rhizobia from different geographic regions of China was determined by analyzing the variability of 16S rRNA gene RFLP, 16S–23S rRNA gene Intergenetic Spacer (IGS) RFLP, G-C rich RAPD and phenotype assays. Based on these characteristics, mung bean rhizobia were clustered into four groups. Group I comprised 16 slow-growing isolates from a variety of geographic regions. This group was genetically distinct from Bradyrhizobium japonicum and Bradyrhizobium liaoningense, and may represent a new species. Group II was composed of 18 isolates, which could be sub-divided into two sub-groups that were respectively related to B. japonicum and B. liaoningense. Group III comprised 12 isolates from South China and clustered together with Bradyrhizobium elkanii. Group IV formed a miscellany of 8 fast-growing isolates variously related to the genera Sinorhizobium, Rhizobium and Mesorhizobium.

Isolation and characterization of a diverse group of phenylacetic acid degrading microorganisms from pristine soil

A diverse range of microorganisms capable of growth on phenylacetic acid as the sole source of carbon and energy were isolated from soil. Sixty six different isolates were identified and grouped according to 16S rRNA gene RFLP analysis. Subsequent sequencing of 16S rDNA from selected strains allowed further characterization of the phenylacetic acid degrading population isolated from soil. Nearly half (30) of the isolates are Bacillus species while the rest of the isolates are strains from a variety of genera namely, Arthrobacter, Pseudomonas, Rhodococcus, Acinetobacter, Enterobacter, Flavobacterium, and Paenibacillus. Sixty-one of the sixty-six strains reproducibly grew in defined minimal liquid culture medium (E2). All strains isolated grew when at least one hydroxylated derivative of phenylacetic acid was supplied as the carbon source, while 59 out of the 61 strains tested, accumulated ortho-hydroxyphenylacetic acid in the assay buffer; when pulsed with phenylacetic acid. Oxygen consumption experiments failed to indicate a clear link between phenylacetic acid and hydroxy-substituted phenylacetic acid in isolates from a broad range of genera.

Characterization of bacteria isolated from wild legumes in the north-western regions of China.

Nodule isolates from 11 species of wild legumes in north-western China were characterized by numerical taxonomy, PCR-based 16S rRNA gene RFLP and sequence analyses, DNA-DNA hybridization, restriction patterns of nodDAB and nifH genes, and symbiotic properties. Based on the results of numerical taxonomy, most of the 35 new isolates were grouped into five clusters (clusters 7, 9, 12, 14 and 15). Clusters 7 and 12 were identified as Mesorhizobium amorphae and Agrobacterium tumefaciens, respectively, based on their high DNA homologies with the reference strains for these species, their 16S rRNA gene analysis and their phenotypic features. Results of 16S rDNA PCR-RFLP analysis showed that cluster 9 belonged to Rhizobium. Clusters 14 and 15 were identified as Mesorhizobium based on their moderately slow-growing, acid-producing characters and the high similarity of their 16S rDNA PCR-RFLP patterns to those of Mesorhizobium species. These two clusters were genomic species distinct from all described species based on analysis of DNA relatedness within this genus. The isolates in cluster 12 (Agrobacterium tumefaciens) failed to nodulate their original host and other selected hosts and they did not hybridize to nif or nod gene probes. The possibility of opportunistic nodulation of these isolates is discussed. Identical restriction patterns were obtained in the nif or nod gene hybridization studies from the three isolates within cluster 15, which were isolated from the same host species. The isolates from different host plants in each of clusters 9 and 14 produced different nodDAB RFLP patterns, but similar nifH RFLP patterns appeared (one band for each isolate). Different patterns were observed among different clusters from both the nod and nif gene hybridization studies. Crossnodulation was recorded among the isolates and the host plants in the same cluster and promiscuous properties were found among some of the hosts tested.

DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates.

To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified by PCR and the PCR products were sequenced. Three isolates had identical 16S rRNA sequences and two isolates had sequences that differed from the others by only one nucleotide.