Diversity of Bacillus-like bacterial community in the rhizospheric and non-rhizospheric soil of halophytes (Salsola stocksii and Atriplex amnicola), and characterization of osmoregulatory genes in halophilic Bacilli.

Salinity is one of the major abiotic stresses; a total of 3% of the world's land mass is affected by salinity. Approximately 6.3 million hectares of land in Pakistan is affected by salinity to varying degrees, and most of the areas are arid to semiarid with low annual precipitation. The aim of the present study is to identify and characterize Bacillus and Bacillus-derived bacterial genera from the rhizospheric and non-rhizospheric soil samples from the Khewra Salt Mine, Pakistan, by using culture-independent and -dependent methods. Seven Bacillus-like bacterial genera, Bacillus, Halobacillus, Virgibacillus, Brevibacillus, Paenibacillus, Tumebacillus, and Lysinibacillus, were detected by using pyrosequencing analysis, whereas only four genera, Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus, were identified by culture-dependent methods. Most of the Bacillus-like isolates identified in this study were moderately halophilic, alkaliphilic, and mesophilic bacteria and were considered a good source of hydrolytic enzymes because of their ability to degrade proteins, carbohydrates, and lipids. Eight Bacillus-like strains from the genera Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus showed positive results for the presence of ectABC gene cluster (ectoine), six strains could synthesize betaine from choline, and six strains tested positive for the synthesis of proline from either glutamate or ornithine by using proline dehydrogenase enzyme.

Comparison of Microbial Communities Associated with Halophyte (Salsola stocksii) and Non-Halophyte (Triticum aestivum) Using Culture-Independent Approaches.

Halophyte microbiome contributes significantly to plant performance and can provide information regarding complex ecological processes involved in osmoregulation of these plants. The objective of this study is to investigate the microbiomes associated with belowground (rhizosphere), internal (endosphere) and aboveground (phyllosphere) tissues of halophyte (Salsola stocksii) through metagenomics approach. Plant samples were collected from Khewra Salt Mines. The metagenomic DNA from soil, root and shoot samples was isolated with the help of FastDNA spin kit. Through PCR, the 16S rRNA gene from four different Salsola plants and wheat plants was amplified and cloned in InsTAclone PCR cloning kit. Metagenomic analyses from rhizosphere, endosphere and phyllosphere of Salsola showed that approximately 29% bacteria were uncultured and unclassified. Proteobacteria and Actinobacteria were the most abundant phyla in Salsola and wheat. However, Firmicutes, Acidobacteria, Bacteriodetes, Planctomycetes, Cyanobacteria, Thermotogae, Verrucomicrobia, Choroflexi and Euryarchaeota were predominant groups from halophyte whereas Actinobacteria, Proteobacteria, Firmicutes, Cyanobacteria, Acidobacteria, Bacteriodetes, Planctomycetes and Verrucomicrobia were predominant phyla of wheat samples. Diversity and differences of microbial flora of Salsola and wheat suggested that functional interactions between plants and microorganisms contribute to salt stress tolerance.

Marinactinospora endophytica sp. nov., isolated from a medicinal plant.

A novel endophytic actinobacterium, designated strain YIM 690053(T), was isolated from healthy stems of Salsola ferganica Drob, and was characterized using a polyphasic taxonomic approach. The strain formed well-branched substrate mycelia with no fragmentation, and abundant aerial mycelia that differentiated into long spores. The strain was found to grow at 10-55 °C, pH 6.0-10.0 and in the presence of 0-7 % (w/v) NaCl. The genomic DNA G+C content was determined to be 70.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 690053(T) belongs to the genus Marinactinospora, and it shared the highest 16S rRNA gene sequence similarity with Marinactinospora thermotolerans SCSIO 00652(T) (98.96 %). However, the DNA-DNA relatedness values between the two strains was 37.85 ± 2.77 %. Based on the differences in the molecular and biochemical characteristics from its closest relative, strain YIM 690053(T) is concluded to represent a novel species of the genus Marinactinospora, for which the name Marinactinospora endophytica sp. nov. is proposed. The type strain is YIM 690053(T)(=KCTC 29664(T) = DSM 46799(T)).

Arthrobacter endophyticus sp. nov., an endophytic actinobacterium isolated from root of Salsola affinis C. A. Mey.

A Gram-staining-positive, white-coloured, aerobic, non-motile, catalase-positive and oxidase-negative, endophytic actinobacterium, designated strain EGI 6500322(T), was isolated from the surface-sterilized root of the halophyte Salsola affinis C. A. Mey collected from Urumqi, Xinjiang province, north-west China. Growth occurred at 5-35 °C (optimum 25-30 °C), at pH 5-10 (optimum pH 7-8) and with 0-13% NaCl (w/v) (optimum 0-5%). The predominant menaquinone was MK-9 (93.1%). The major cellular fatty acids were anteiso-C15:0 (49.5%) and iso-C15:0 (15.1%). The cell-wall peptidoglycan contained lysine, alanine and glutamic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two unknown phospholipids and one unknown glycolipid. The DNA G+C content of strain EGI 6500322(T) was 62.0 mol%. Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain EGI 6500322(T) were identified as Arthrobacter ardleyensis DSM 17432(T) (98.38%) and Arthrobacter bergerei DSM 16367(T (98.37%). The DNA-DNA relatedness between strain EGI 6500322(T) and Arthrobacter ardleyensis DSM 17432(T) and Arthrobacter bergerei DSM 16367(T) was 53.4 ± 4.1% and 30.5 ± 1.7%, respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain EGI 6500322(T) should represent a novel species of the genus Arthrobacter, for which the name Arthrobacter endophyticus sp. nov. is proposed. The type strain is EGI 6500322(T) ( = CTC 29490(T) = JCM 30091(T)).

Okibacterium endophyticum sp. nov., a novel endophytic actinobacterium isolated from roots of Salsola affinis C. A. Mey.

A white bacterial strain, designated EGI 650022(T), was isolated from the roots of Salsola affinis C. A. Mey, collected from Urumqi City, Xinjiang, north-western China. The strain was found to be aerobic, Gram-stain positive, oxidase-positive and catalase-positive. Cells were non-motile and irregular rods. Growth occurred at NaCl concentrations between 0 and 7 % (w/v), temperatures between 5 and 45 °C, and pH 6.0-9.0. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain EGI 650022(T) belongs to a clade with the genera Okibacterium and Plantibacter in the family Microbacteriaceae. The novel strain EGI 650022(T) showed highest levels of 16S rRNA gene sequence similarity with members of the genera Okibacterium and Plantibacter (97.2-98.0 %). The cell-wall peptidoglycan contained glutamate, homoserine, glycine, alanine and lysine. The predominant menaquinones (MKs) were MK-11, MK-12 and MK-12 (H4). The polar lipid pattern comprised phosphatidylglycerol, diphosphatidylglycerol, two unknown glycolipids and two unknown phospholipids. The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The DNA G+C content was 66.0 mol%. The DNA-DNA relatedness values of strain EGI 650022(T) with Okibacterium fritillariae DSM 12584(T), Plantibacter flavus DSM 14012(T) and Plantibacter auratus DSM 19586(T) were 39.7, 19.7 and 22.0 %. Based on phylogenetic, phenotypic, chemotaxonomic and DNA-DNA hybridization data, strain EGI 650022(T) is considered to represent a novel species of the genus Okibacterium, for which the name Okibacterium endophyticum sp. nov. is proposed; the type strain is EGI 650022(T) (=JCM 30086(T) = KCTC 29492(T)).