Assessment of microbial diversity in the rhizosphere of Pinus roxburghii (Sarg.) and bio-inoculant potential of selected pine bacterial isolates for wheat varieties based on cultureindependent and culture-dependent techniques.

Evidence is lacking regarding compatibility of pine bacteria as bio-inoculants for crops. The diversity and abundance of rhizosphere bacteria of Pinus roxburghii has never been investigated with simultaneous application of culture-dependent and culture-independent techniques. The present study was aimed to isolate, characterise, check the bio-inoculant potential of pine bacteria and assess rhizosphere bacterial diversity using culture-independent advanced approaches. Forty bacteria isolated from the rhizoplane of P. roxburghii growing in a cold climate at high altitude in Murree, were morphologically characterised; nine were identified by 16S rRNA sequence analyses and used in experiments. Diversity and abundance of the 16S rRNA gene and nifH gene in the rhizosphere was assessed by cloning, RFLP analysis, 454-amplicon pyrosequencing and qPCR. The bacterial isolates significantly improved dry weight of shoot, root, root area, IAA and GA3 content, number of grains plant-1 , weight of grains plant-1 in wheat varieties Chakwal-50 and Fareed-06 under axenic and field conditions. The number of 16S rRNA sequences (2979) identified by pyrosequencing shared similarity with 13 phyla of bacteria and archaea. The results confirm the existence of diverse bacteria of agricultural and industrial importance in the rhizosphere and compatibility of rhizoplane bacteria as bio-inoculants for wheat varieties.

Obesity, Visceral Fat, and NAFLD: Querying the Role of Adipokines in the Progression of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of clinicopathologic conditions ranging from steatosis alone to nonalcoholic steatohepatitis (NASH), with varying risks for progression to cirrhosis and hepatocellular carcinoma. There is mounting evidence that NAFLD not only complicates obesity, but also perpetuates its metabolic consequences. Critical event that leads to progressive liver injury in NAFLD is unknown. Obesity reflects a generalized proinflammatory state with its increased inflammatory markers like C reactive protein, IL-6, IL-8, IL-10, PAI-1, TNF-α, and hepatocyte growth factor. The elevated production of these adipokines is increasingly considered to be important in the development of diseases linked to obesity and the metabolic syndrome. Disordered cytokine production is likely to play a role in the pathogenesis of NAFLD. There is no effective treatment for NAFLD, though weight loss may halt disease progression and revert histological changes, the underlying mechanism remaining elusive. All stages of the disease pathway from prevention, early identification/diagnosis, and treatment require an understanding of the pathogenesis of liver injury in NAFLD.

Isolation and 16S rRNA sequence analysis of the beneficial bacteria from the rhizosphere of rice.

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 10(5)-10(6) counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using 15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.

Experimentally determined microcracking around a circular hole in a flat plate of bone: comparison with predicted stresses.

We examined the microcracking (damage) in the vicinity of a circular hole in bovine femoral bone specimens. The stresses near the hole were derived by a finite element analysis model using the bone's elastic constants and yield stresses, which were determined from a series of mechanical tests specifically for the type of bone under examination. The spatial occurrence and distribution of microcracking was compared to the patterns of the predicted maximum principal stress, the von Mises stress, and the strain energy density function (all implicated by various workers as stimuli for bone remodelling) and to the predictions derived by the use of two engineering criteria for anisotropic yield under mixed mode of stress. The predictions for stresses and the strain energy density were all very similar, making it impossible to claim that any of them is superior to the others. However, empirical examination of the results of the Hencky-von Mises and Tsai-Wu anisotropic yield criteria showed that the Tsai-Wu criterion approximated reasonably the pattern of microcracking around the hole. We suggest that, in the light of the considerable damage observed in the vicinity of stress concentrators, similar damage in irregular material interfaces (i.e. near orthopaedic implants) would require the re-examination of the theories concerning bone remodelling so as to account for the possibility of occurrence of damage and the quantification of its magnitude and likely effect. The presence of considerable microdamage in bone long before it fails suggests that damage-based criteria are more likely to be successful predictors of bone remodelling behaviour than would stress or strain-based criteria.

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.

Phylogenetic studies on uncultured Frankia populations in nodules of Datisca cannabina.

Part of the 16S rRNA gene was amplified directly from uncultured endophyte populations within the root nodules of Datisca cannabina and three strains isolated from nodules of Alnus glutinosa (AgKG'84/4), Coriaria nepalensis (Cn3), and D. cannabina (Dc2). Sequence comparison based on 930 nucleotides indicated that the endophyte of D. cannabina nodules belongs to the genus Frankia and is highly related to the endophyte of C. nepalensis root nodules. The relatedness of the endophytes of C. nepalensis and D. cannabina nodules was also reflected by closely related nifH sequences amplified from the nodules. 16S rRNA sequence analysis of the noninfective strains obtained from both D. cannabina (Dc2) and C. nepalensis (Cn3) nodules also revealed the close relationship of these strains to the genus Frankia.

Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter michiganensis subsp. sepedonicus.

In this article we report on the polymerase chain reaction amplification of a partial 16S rRNA gene from the plant pathogenic bacterium Clavibacter michiganensis subsp. sepedonicus. A partial sequence (about 400 base pairs) of the gene was determined that covered two variable regions important for oligonucleotide probe development. A specific 24mer oligonucleotide probe targeted against the V6 region of 16S rRNA was designed. Specificity of the probe was determined using dot blot hybridization. Under stringent conditions (60 degrees C), the probe hybridized with all 16 Cl. michiganensis subsp. sepedonicus strains tested. Hybridization did not occur with 32 plant pathogenic and saprophytic bacteria used as controls under the same conditions. Under less stringent conditions (55 degrees C) the related Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. nebraskensis, and Clavibacter michiganensis subsp. tesselarius also showed hybridization. At even lower stringency (40 degrees C), all Cl. michiganensis subspecies tested including Clavibacter michiganensis subsp. michiganensis showed hybridization signal, suggesting that under these conditions the probe may be used as a species-specific probe for Cl. michiganensis.