Genetic structure and diversity of Coscinium fenestratum: a critically endangered liana of Western Ghats,India

Coscinium fenestratum is a critically endangered medicinal plant, well-known for its bioactive isoquinoline alkaloid berberine. The species has been over harvested from its natural habitats to meet the huge requirement of raw drug market and industrial consumption. This has lead to a rapid decline in the population size and has also led to local population extinction at few locations in the Western Ghats, India. In this study, inter-simple sequence repeat markers were used to investigate the genetic variation and population structure of seven extant populations of C. fenestratum from the central Western Ghats, India. Eight primer combination produced a total of 57 unambiguous bands, of which (47.1 %) were polymorphic. The species exhibited a moderate to low level of intra population genetic diversity (H _s = 0.347 ± 0.008; H _t = 0.378 ± 0.006 (POPGENE) and H _s = 0.262 ± 0.0028; H _t = 0.204 ± 0.020 (HICKORY)). The populations were low to moderately differentiated from one another (G _ST = 0.221) and geographical distance was not significantly correlated with genetic distance, suggesting that these long-lived, geographically distant remnant populations were once connected through gene flow. There was a significant amount of genetic variation among populations (19.85 %). The Bayesian software STRUCTURE and HICKORY were used to further reveal the genetic structure of C. fenestratum. The results revealed weak population structure (K = 2) with one single widespread gene pool, and indicated that gene flow and inbreeding are likely to be the major driving force in shaping current population genetic structure of C. fenestratum. Thus, an understanding of the genetic diversity and population structure of C. fenestratum can provide insight into the conservation and management of this species.     

Purification and characterization of polygalacturonase produced by thermophilic <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Pectin Esterification Degree in the Bioavailability of Non-heme Iron in Women

Pectins are a type of soluble fiber present in natural and processed foods. Evidence regarding the effect of esterification degree of pectins on iron absorption in humans is scarce. In the present study, the effect of pectins with different degrees of esterification on non-heme iron absorption in women was evaluated. A controlled experimental study was conducted with block design, involving 13 apparently healthy, adult women. Each subject received 5 mg Fe (FeSO₄) without pectin (control) or accompanied by 5 g citrus pectin, two with a low degree of esterification (27 and 36%), and one with a high degree of esterification (67 to 73%), each on different days. Each day, the 5 mg Fe doses were marked with radioactive ⁵⁹Fe or ⁵⁵Fe. Radioactivity incorporated into erythrocytes was determined in blood samples 14 days after the marked Fe doses were consumed. On days 18 and 36 of study, 30 and 20 mL blood samples were obtained, respectively, and blood sample radioactivity incorporated into erythrocytes was determined. Body iron status was determined from blood taken on day 18. Whole body blood volume was estimated for calculate iron bioavailability; it was assumed that 80% of absorbed radioactivity was incorporated into the Hb. All women participants signed an informed consent of participation at baseline. Iron bioavailability (mean geometric ±1 SD) alone (control) was 18.2% (12.3–27.1%), iron + pectin27 was 17.2% (10.2–29.2%), iron + pectin36 was 15.3% (9.5–24.6%), and iron + pectin67 was 19.5% (10.0–38.0%). No statistically significant differences between iron bioavailability (repeated measures ANOVA, p = 0.22) were observed. Pectin esterification degree does not influence the bioavailability of non-heme iron in women.     

Isolation and Characterization of a Marine Cyclohexylacetate-Degrading Bacterium Lutimaribacter litoralis sp. nov., and Reclassification of Oceanicola pacificus as Lutimaribacter pacificus comb. nov.

A novel aerobic, Gram-negative, non-motile, pleomorphic, and rod-shaped bacterium designated KU5D5^T was isolated from seawater that was obtained from the coastal region of the Goto Islands, Japan, on the basis of its ability to utilize cyclohexylacetate as the sole source of carbon and energy. Strain KU5D5^T grew at pH 6.0–8.0 and 10–35 °C in the presence of 1.0–5.0 % (w/v) NaCl. Analysis of the 16S rRNA gene sequence revealed that this strain was affiliated to the family Rhodobacteraceae in the class Alphaproteobacteria and was related most closely to Lutimaribacter saemankumensis (96.6 % similarity) and Oceanicola pacificus (96.6 %). The predominant respiratory lipoquinone was ubiquinone-10 and the major cellular fatty acids were C_18:1 ω7c (66.7 %), C_16:0 (7.7 %), C_12:1 3-OH (6.1 %), and C_17:0 (6.1 %). The DNA G+C content was 58.9 mol %. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU5D5^T is suggested to represent a novel species of the genus Lutimaribacter, for which the name Lutimaribacter litoralis sp. nov. is proposed. It is also proposed that O. pacificus should be transferred to the genus Lutimaribacter as Lutimaribacter pacificus comb. nov. The type strain of L. litoralis is KU5D5^T (=JCM 17792^T = KCTC 23660^T) and the type strain of L. pacificus is W11-2B^T (=CCTCC AB 208224^T = LMG 24619^T = MCCC 1A01034^T).     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

Pelagicola litorisediminis sp. nov., a novel alphaproteobacterium isolated from tidal flat sediment

A Gram-negative, aerobic, non-motile and rod-shaped or ovoid bacterial strain, designated D1-W8^T, was isolated from a tidal flat on the South Sea in South Korea. Strain D1-W8^T was found to grow optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2.0–3.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain D1-W8^T clustered with the type strain of Pelagicola litoralis showing 97.1 % sequence identity. 16S rRNA gene sequences of the type strains of other species exhibited lower similarity values. Strain D1-W8^T was determined to contain Q-10 as the predominant ubiquinone and C_18:1 ω7c as the predominant fatty acid. The major polar lipids of strain D1-W8^T were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G+C content of strain D1-W8^T was determined to be 57.9 mol% and its DNA–DNA relatedness value with the type strain of P. litoralis was 17 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain D1-W8^T is separate from P. litoralis. On the basis of the data presented, strain D1-W8^T is considered to represent a novel species of the genus Pelagicola, for which the name Pelagicola litorisediminis sp. nov. is proposed. The type strain is D1-W8^T (= KCTC 32327^T = CECT 8287^T).     

Genetic variation and population differentiation of the endochitinase gene family in Pinus monticola

In the present study, we characterized nucleotide variations of the Pinus monticola class IV endochitinase (PmCh4) family. Using primers targeting at conserved amino acid motifs of plant class IV endochitinases, genomic DNA was amplified. Sequence data analysis identified five novel genes in the PmCh4 family with one pseudogene. Single nucleotide polymorphisms of the PmCh4 family were surveyed in seven open-pollinated seed lots representing diverse geographical distribution. Variable levels of average pairwise nucleotide diversity (π = 0.00422–0.02079) and relatively high levels of haplotype diversity (H _d = 0.85–0.96) were revealed at PmCh4 loci. Based on nucleotide variation, P. monticola populations were clustered into two main groups by phylogenetic analysis based on Nei’s genetic distance. The Mantel test revealed no correlation between geographical and genetic distances (r = ?0.11, P = 0.59). A further SNP genetic diversity study on more P. monticola populations throughout Western North America may help the design of sampling regimes for tree breeding, genetic conservation and assisted migration under climate change.     

A large-scale inventory of liana diversity in tropical forests of South Eastern Ghats,India

Among the plant life-forms, lianas, the wood climbers still remain less studied than trees. The forests of Eastern Ghats of India are also relatively under studied compared with the Western Ghats biodiversity hotspot. We conducted a large-scale, landscape-level investigation of liana diversity in six hill complexes of the South Eastern Ghats, which covers 4297 km². We divided the study area into 6.25 km × 6.25 km grids and within each grid a 0.5 ha (5 m × 1000 m) transect was established and all lianas ≥1.5 cm diameter at breast height (dbh) were inventoried in 110 transects totalling a 55-ha area. Liana diversity totalled 143 species in 83 genera and 37 families in the 55 ha sampled. Of these 20 species (28.6%) were endemic to peninsular India and 7 (10%) species belonged to the rare and endangered category. Liana species richness ranged from 8–35 species and density 95–544 individuals per transect. A total of 32 033 liana individuals were enumerated in the 55 ha and the mean abundance was 291 individuals per transect. Across sites, liana abundance varied significantly, but not species richness and basal area. Asclepiadaceae (13 species, 9%) and Apocynaceae (11 species, 8%) constituted the most diverse liana families, followed by Papilionaceae, Vitaceae (10 each, 7%), Convolvulaceae, Mimosaceae, Oleaceae (8 each, 6%), Capparaceae, Rhamnaceae (7 each, 5%) and Menispermaceae (5 species, 3%). In liana stem size distribution, the lowest diameter class (1.5–3 cm dbh) accounted for greatest species richness (137 species, 96%), abundance (27 358 individuals, 85%) and basal area (13.5 m², 36%). The stem twiners were the predominant climber type in terms of species richness (61 species, 42.65%), whereas the armed scramblers were abundant due to stem density (21 571 individuals, 67.34%). The dispersal modes of lianas, assessed by fruit types, revealed zoochory as the prevalent mode (85 species, 59%) indicating the faunal dependence of lianas in the Eastern Ghats landscape. Liana diversity of the Eastern Ghats was compared with inventories made across the tropics. With these baseline data generated on lianas, the importance of biodiversity conservation of the already fragmented South Eastern Ghats region is underlined and potential areas of further research on liana ecology are suggested.     

Fine- and local- scale genetic structure of <Emphasis Type="Italic">Dysoxylum malabaricum,</Emphasis> a late-successional canopy tree species in disturbed forest patches in the Western Ghats,India

Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, which is the world’s most densely populated biodiversity hotspot. In this study, we used variation at ten nuclear simple sequence repeat loci to investigate genetic diversity and fine scale spatial genetic structure (FSGS) in seedlings and adults of D. malabaricum from four forest patches in the northern part of the Western Ghats. When genetic variation was compared between seedlings and adults across locations, significant differences were detected in allelic richness, observed heterozygosity, fixation index (F _IS), and relatedness (P < 0.05). Reduced genetic diversity and increased relatedness at the seedling stage might be due to fragmentation and disturbance. There was no FSGS at the adult stage and FSGS was limited to shorter distance classes at the seedling stage. However, there was clear spatial genetic structure at the landscape level (<50 km), regardless of age class, due to limited gene flow between forest patches. A comparison of the distributions of size classes in the four locations with published data from a more southern area, showed that large trees (diameter at breast height, DBH, >130 cm) are present in the southern sacred forests but not in the northern forest reserves. This pattern is likely due to stronger harvesting pressure in the north compared to the south, because in the north there are no cultural taboos regulating the extraction of natural resources. The implications for forest conservation in this biodiversity hotspot are discussed.     

Endemic Asian Chytrid Strain Infection in Threatened and Endemic Anurans of the Northern Western Ghats,India

The Western Ghats of India harbors a rich diversity of amphibians with more than 77% species endemic to this region. At least 42% of the endemic species are threatened due to several anthropogenic stressors. However, information on amphibian diseases and their impacts on amphibian populations in this region are scarce. We report the occurrence of Batrachochytridium dendrobatidis (Bd), an epidermal aquatic fungal pathogen that causes chytridiomycosis in amphibians, from the Western Ghats. In the current study we detected the occurrence of a native Asian Bd strain from three endemic and threatened species of anurans, Bombay Night Frog Nyctibatrachus humayuni, Leith''s Leaping Frog Indirana leithii and Bombay Bubble Nest Frog Raorchestes bombayensis, for the first time from the northern Western Ghats of India based on diagnostic nested PCR, quantitative PCR, DNA sequencing and histopathology. While, the Bd infected I. leithii and R. bombayensis did not show any external symptoms, N. humayuni showed lesions on the skin, browning of skin and sloughing. Sequencing of Bd 5.8S ribosomal RNA gene, and the ITS1 and ITS2 regions, revealed that the current Bd strain is related to a haplotype endemic to Asia. Our findings confirm the presence of Bd in northern Western Ghats and the affected amphibians may or may not show detectable clinical symptoms. We suggest that the significance of diseases as potential threat to amphibian populations of the Western Ghats needs to be highlighted from the conservation point of view.     

Lipase production by Serratia marcescens strain SN5gR isolated from the scat of lion-tailed macaque (Macaca silenus) in Silent Valley National Park, a biodiversity hotspot in India

An extracellular lipase-producing bacterium was isolated from a fecal sample of lion-tailed macaque (Macaca silenus), an endangered Old World monkey that is endemic to the Western Ghats of South India. Morphological, biochemical and molecular analyses identified the bacterium as Serratia marcescens. Production of lipase was investigated in shake-flask culture. Optimum tributyrin concentration of 1.5 % was found to be the most suitable triglyceride to increase lipase production (13.3 U ml^?1). The next best lipid source observed was olive oil (11.94 U ml^?1), followed by castor oil, coconut oil and palm oil. Analyzing the effect of different carbon sources on lipase production revealed that 2 % glucose yielded higher lipase production than the other tested carbon sources. Investigations on suitable nitrogen source for lipase production revealed that 2 % meat extract yielded higher lipase production. The most suitable trace element for maximum lipase production was zinc sulfate, followed by magnesium sulfate and copper sulfate. Partial characterization of the crude lipase revealed that pH 7.0 and a temperature of 40 °C gave optimal lipase activity. Enzymatic activity of the crude sample was retained over a wide temperature range (20–75 °C), and 70 % of enzyme activity was retained at 60 °C. Testing the effect of various organic solvents on lipase activity revealed that hexadecane increased lipase activity by 85 % over the control.     

Distribution of detritivores in tropical forest streams of peninsular Malaysia: role of temperature,canopy cover and altitude variability

The diversity and abundance of macroinvertebrate shredders were investigated in 52 forested streams (local scale) from nine catchments (regional scale) covering a large area of peninsular Malaysia. A total of 10,642 individuals of aquatic macroinvertebrates were collected, of which 18.22 % were shredders. Biodiversity of shredders was described by alpha (α_average ), beta (β) and gamma diversity (γ) measures. We found high diversity and abundance of shredders in all catchments, represented by 1,939 individuals (range 6–115 and average per site of 37.29?±?3.48 SE) from 31 taxa with 2–13 taxa per site (α_average?=?6.98?±?0.33 SE) and 10–15 taxa per catchment (γ?=?13.33?±?0.55 SE). At the local scale, water temperature, stream width, depth and altitude were correlated significantly with diversity (Adj-R ²?=?0.205). Meanwhile, dissolved oxygen, stream velocity, water temperature, stream width and altitude were correlated to shredder abundance (Adj-R ²?=?0.242). At regional scale, however, water temperature was correlated negatively with β and γ diversity (r ²?=?0.161 and 0.237, respectively) as well as abundance of shredders (r ²?=?0.235). Canopy cover was correlated positively with β diversity (r ²?=?0.378) and abundance (r ²?=?0.266), meanwhile altitude was correlated positively with β (quadratic: r ²?=?0.175), γ diversity (quadratic: r ²?=?0.848) as well as abundance (quadratic: r ²?=?0.299). The present study is considered as the first report describing the biodiversity and abundance of shredders in forested headwater streams across a large spatial scale in peninsular Malaysia. We concluded that water temperature has a negative effect while altitude showed a positive relationship with diversity and abundance of shredders. However, it was difficult to detect an influence of canopy cover on shredder diversity.     

The effect of irradiance and temperature responses and the phenology of a native alga,Undaria pinnatifida (Laminariales), at the southern limit of its natural distribution in Japan

Phenology, irradiance, and temperature characteristics of an edible brown alga, Undaria pinnatifida (Laminariales), were examined from the southernmost natural population in Japan, both by culturing gametophytes and examining the photosynthetic activity of sporophytes using dissolved oxygen sensors and pulse amplitude-modulated chlorophyll fluorometer (IMAGING-PAM). Our surveys confirmed that sporophytes were present between winter and early summer, but absent by July. IMAGING-PAM experiments were used to measure maximum effective quantum yield (ΦII at 0 μmol photons m^?2 s^?1) for each of 14 temperatures (8–36 °C). Oxygen production was also determined over a coarser temperature gradient. Net photosynthesis and ΦII (at 0 μmol photons m^?2 s^?1) were observed to be temperature-dependent; the maximum ΦII was estimated to be 0.67, occurred at 21.2 °C, and was nearly identical to the optimal temperature of the net photosynthetic rate (21.7 °C). A net photosynthesis–irradiance (P–E) model revealed that saturation irradiance (E _k) was 119.5 μmol photons m^?1 s^?1, and the compensation irradiance (E _c) was 17.4 μmol photons m^?1 s^?1. Culture experiments on the gametophytes revealed that most individuals could not survive temperatures over 28 °C and that growth rates were severely inhibited. Based on our observations, temperatures greater than 20 °C are likely to influence photosynthetic activity and gametophyte survival, and therefore, it is possible that this species might become locally extinct if seawater temperatures in this region continue to rise.     

Bioprospecting of thermo- and osmo-tolerant fungi from mango pulp–peel compost for bioethanol production

The persistent edaphic stress on microbial succession due to dynamic changes during composting was explored for selection of multi-stress tolerant microbe(s) desirable for ethanol production. A total of 23 strains were isolated from mango compost using four successive enrichments in YP broth (g l^?1): glucose, 100; 150; 250 with ethanol (40) and cycloheximide (0.4) at 40 °C, pH 6.0. Based on multi-gene ribotyping, 14 yeasts (61 %) of Saccharomycetaceae, 2 filamentous fungi (8.6 %) and 7 bacteria (30.4 %) were obtained. Phenetic and phylogenetic analysis of the 14 yeasts revealed 64.3 % tolerant to 500 g l^?1 glucose, growth at 45 °C and resemblance to Candida sp. (14.3 %), Kluyveromyces marxianus (35.7 %), Pichia kudriavzevii (21.4 %) and Saccharomyces cerevisiae (28.6 %). Assessment of the 14 yeasts in glucose fermentation medium (pH 4.5 at 40 °C) showed ethanol productivity of ≥92 % by 12 yeasts with theoretical yields of 90–97 %. Fermentation of molasses (150 g l^?1 glucose equivalent) by P. kudriavzevii D1C at 40 °C resulted in 73.70 ± 0.02 g l^?1 ethanol and productivity of 4.91 ± 0.01 g l^?1 h^?1. Assessment of P. kudriavzevii D1C revealed multi-stress tolerance towards 5-hydroxymethyl furfural, ethanol (20 %, v/v), high gravity and H₂O₂ (0.3 M) indicating suitability for ethanol production using high gravity molasses and pre-treated lignocellulosic biomass fermentation.     

Altererythrobacter oceanensis sp. nov., isolated from the Western Pacific

A Gram-stain negative, ovoid-rod shaped, strictly aerobic bacterium, strain Y2^T, was isolated from a deep-sea sediment of the Western Pacific. Phylogenetic and phenotypic properties of the organism indicated that it belongs to the genus Altererythrobacter. Strain Y2^T shares highest 16S rRNA gene sequence similarity of 96.6 % with Erythrobacter jejuensis CNU001^T, followed by the type strains of recognized members of the genus Altererythrobacter (94.8–96.5 %). Strain Y2^T forms a clade with E. jejuensis CNU001^T in the cluster of species of the genus Altererythrobacter. Growth of strain Y2^T was observed at 4–40 °C (optimum, 35–37 °C), at pH 6.0–10.0 (optimum, pH 7.0–8.0) and in the presence of 0–5 % (w/v) NaCl (optimum, 2–3 %). The major cellular fatty acids were found to be C_17:1 ω6c (41.5 %), summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c; 17.2 %), C_17:1 ω8c (11.0 %) and C_15:0 2OH (8.1 %). The major respiratory quinone was determine to be ubiquinone 10 (Q-10). The polar lipid analysis indicated the presence of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, one sphingoglycolipid, three unidentified phospholipids, two unidentified glycolipids, two unidentified aminolipids and three unknown lipids. The DNA G + C content of the type strain is 60.0 mol %. On the basis of the data from the polyphasic characterization, strain Y2^T represents a novel species, for which the name Altererythrobacter oceanensis sp. nov. is proposed. The type strain is Y2^T (=CGMCC 1.12752^T =LMG 28109^T).     

Saccharopolyspora cavernae sp. nov., a novel actinomycete isolated from the Swallow Cave in Yunnan, south-west China

A Gram-positive, aerobic, non-motile actinobacterial strain, designated YIM C01235^T, was isolated from a soil sample collected from the Swallow Cave, Yunnan province, south-west China. The isolate grew at 10–30 °C, pH 6.0–9.0 and 0–8 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed highest similarity to Saccharopolyspora gloriosae YIM 60513^T (96.8 %), and lower 16S rRNA gene sequence similarities (95.1–96.7 %) with the other species of the genus Saccharopolyspora. The whole-cell hydrolysates contained meso-diaminopimelic acid (meso-DAP), arabinose and galactose. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, two unknown glycolipids, two unknown phospholipids and one polar lipid. MK-9(H₄) was the predominant menaquinone. The major fatty acids were iso-C_15:0, iso-C_16:0, anteiso-C_15:0 and anteiso-C_17:0. Mycolic acids were not detected. The genomic DNA G+C content was 69.1 mol%. These chemotaxonomic data, together with its morphological properties, were consistent with the assignment of strain YIM C01235^T to the genus Saccharopolyspora. The results of physiological and biochemical tests allowed strain YIM C01235^T to be differentiated phenotypically from all the recognized Saccharopolyspora species. On the basis of evidence from this polyphasic study, the novel species Saccharopolyspora cavernae sp. nov. is proposed. The type strain is YIM C01235^T (=DSM 45825^T = CCTCC AA 2012022^T).     

Sphingobacterium pakistanensis sp. nov., a novel plant growth promoting rhizobacteria isolated from rhizosphere of Vigna mungo

The taxonomic status of a bacterium, strain NCCP-246^T, isolated from rhizosphere of Vigna mungo, was determined using a polyphasic taxonomic approach. The strain NCCP-246^T can grow at 16–37 °C (optimum 32 °C), at pH ranges of 6–8 (optimum growth occurs at pH 7) and in 0–4 % (w/v) NaCl. Phylogenetic analysis based upon on 16S rRNA gene sequence comparison revealed that strain NCCP-246^T belonged to genus Sphingobacterium. Strain NCCP-246^T showed highest similarity to the type strain of Sphingobacterium canadense CR11^T (97.67 %) and less than 97 % with other species of the genus. The DNA–DNA relatedness value of strain NCCP-246^T with S. canadense CR11^T and Sphingobacterium thalpophilum JCM 21153^T was 55 and 44.4 %, respectively. The chemotaxonomic data revealed the major menaquinone as MK-7 and dominant cellular fatty acids were summed feature 3 [C_16:1 ω7c/C_16:1 ω6c] (37.07 %), iso-C_15:0 (28.03 %), C_16:0 (11.85 %), C_17:0 cyclo (8.84 %) and C_14:0 (2.42 %). The G+C content of the strain was 39.2 mol%. On the basis of DNA–DNA hybridization, phylogenetic analyses, physiological and, biochemical data, strain NCCP-246^T can be differentiated from the validly named members of genus Sphingobacterium and thus represents as a new species, for which the name, Sphingobacterium pakistanensis sp. nov. is proposed with the type strain NCCP-246^T (= JCM18974 ^T = KCTC 23914^T).     

Molecular Characterization and Genetic Diversity of Insecticidal Crystal Protein Genes in Native Bacillus thuringiensis Isolates

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.