Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance.

KEY MESSAGE: RNAi mediated silencing of pectin degrading enzyme of R. solani gives a high level of resistance against sheath blight disease of rice. Rice sheath blight disease caused by Rhizoctonia solani Kuhn (telemorph; Thanatephorus cucumeris) is one of the most devastating fungal diseases which cause severe loss to rice grain production. In the absence of resistant cultivars, the disease is currently managed through fungicides which add to environmental pollution. To explore the potential of utilizing RNA interference (RNAi)-mediated resistance against sheath blight disease, we identified genes encoding proteins and enzymes involved in the RNAi pathway in this fungal pathogen. The RNAi target genes were deciphered by RNAseq analysis of a highly virulent strain of the R. solani grown in pectin medium. Additionally, pectin metabolism associated genes of R. solani were analyzed through transcriptome sequencing of infected rice tissues obtained from six diverse rice cultivars. One of the key candidate gene AG1IA_04727 encoding polygalacturonase (PG), which was observed to be significantly upregulated during infection, was targeted through RNAi to develop disease resistance. Stable expression of PG-RNAi construct in rice showed efficient silencing of AG1IA_04727 and suppression of sheath blight disease. This study highlights important information about the existence of RNAi machinery and key genes of R. solani which can be targeted through RNAi to develop pathogen-derived resistance, thus opening an alternative strategy for developing sheath blight-resistant rice cultivars.

Description of Rhodobacter azollae sp. nov. and Rhodobacter lacus sp. nov.

Three strains (JA826T, JA912T and JA913), which were yellowish brown colour, rod to oval shaped, Gram-stain-negative, motile, phototrophic bacteria with a vesicular architecture of intracytoplasmic membranes, were isolated from different pond samples. The DNA G+C content of the three strains was between 64.6 and 65.5 mol%. The highest 16S rRNA gene sequence similarity of all three strains was with the type strains of the genus Rhodobacter sensu stricto in the family Rhodobacteraceae. Strain JA826T had highest sequence similarity with Rhodobacter maris JA276T (98.5 %), Rhodobacter viridis JA737T (97.5 %) and other members of the genus Rhodobacter (<97 %). Strain JA912T had highest sequence similarity with Rhodobacter viridis JA737T (99.6 %), Rhodobacter sediminis N1T (99.3 %), Rhodobacter capsulatus ATCC 11166T (98.8 %) and less than 97 % similarity with other members of the genus Rhodobacter. The 16S rRNA gene sequence similarity between strains JA826T and JA912T was 96.9 %. DNA-DNA hybridization showed that strains JA826T and JA912T (values among themselves and between the type strains of nearest members <44 %) did not belong to any of the nearest species of the genus Rhodobacter. However, strains JA912T and JA913 were closely related (DNA-DNA hybridization value >90 %). The genomic distinction was also supported by differences in phenotypic and chemotaxonomic characteristics in order to propose strains JA826T (=KCTC 15478T=LMG 28758T) and JA912T (=KCTC 15475T=LMG 28748T) as new species in the genus Rhodobacter sensu stricto with the names Rhodobacter lacus and Rhodobacter azollae, respectively.

Lacinutrix himadriensis sp. nov., a psychrophilic bacterium isolated from a marine sediment, and emended description of the genus Lacinutrix.

A novel gram-negative, rod-shaped, non-motile, psychrophilic bacterium, designated strain E4-9a(T), was isolated from a marine sediment sample collected at a depth of 276 m from Kongsfjorden, Svalbard, in the Arctic Ocean. The colony colour was golden yellow. Strain E4-9a(T) was positive for amylase activity at 5 °C. The predominant fatty acids were iso-C(15 : 1) G (21.8 %), anteiso-C(15 : 0) (19.1 %), anteiso-C(15 : 1) A (18.6 %), iso-C(15 : 0) (13.8 %) and iso-C(16 : 1) H (6.4 %). Strain E4-9a(T) contained MK-6 as the major respiratory quinone. The polar lipids consisted of phosphatidylethanolamine, three unidentified aminolipids (AL1, AL4 and AL5), an unidentified phospholipid and four unidentified lipids (L1, L4 to L6). Based on 16S rRNA gene sequence similarity, it was ascertained that the closest related species to E4-9a(T) were Lacinutrix copepodicola, L. algicola and L. mariniflava, with sequence similarity to the respective type strains of 98.5, 96.5 and 95.8 %. Phylogenetic analysis showed that strain E4-9a(T) clustered with the type strain of L. copepodicola and with those of L. algicola and L. mariniflava at distances of 1.5 and 4.8 % (98.5 and 95.2 % similarity), respectively. However, DNA-DNA hybridization with L. copepodicola DJ3(T) showed 59 % relatedness with respect to strain E4-9a(T). The DNA G+C content of strain E4-9a(T) was 29 mol%. Based on the results of DNA-DNA hybridization and phenotypic data, it appears that strain E4-9a(T) represents a novel species of the genus Lacinutrix, for which the name Lacinutrix himadriensis sp. nov. is proposed. The type strain is E4-9a(T) ( = CIP 110310(T)  = KCTC 23612(T)).

Winogradskyella psychrotolerans sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from Arctic sediment.

A novel Gram-negative, rod-coccus shaped, non-motile, strain, RS-3(T), was isolated from a sediment sample collected from the marine transect of Kongsfjorden, Ny-Ålesund, Svalbard, Arctic. Colonies and broth cultures were yellowish in colour due to the presence of carotenoids. Strain RS-3(T) was positive for oxidase, aesculinase, caseinase, gelatinase and urease activities and negative for amylase, catalase, lipase, lysine decarboxylase, ornithine decarboxylase, DNase and ß-galactosidase activities. The predominant fatty acids were iso-C15 : 0 (18.0), anteiso-C15 : 0 (16.8), iso-C15 : 1 G (14.2), anteiso-C15 : 1 A (6.0) and iso-C15 : 0 3-OH (6.8). Strain RS-3(T) contained MK-6 (72.42 %) and MK-7 (27.58 %) as the major respiratory quinones and phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids make up the polar lipid composition. The DNA G+C content of strain RS-3(T) was 34.7±1.2 mol%. The 16S rRNA gene sequence analysis indicated that Winogradskyella pacifica and Winogradskyella thalassocola are the most closely related species with sequence similarities to the type strains of these species of 98.5 and 97.7 %, respectively. However, DNA-DNA hybridization with Winogradskyella pacifica KCTC 22997(T) and Winogradskyella thalassocola DSM 15363(T) showed a relatedness of 22 and 42.5 % with respect to strain RS-3(T). Based on the DNA-DNA hybridization values, phenotypic and chemotaxonomic characteristics and phylogenetic inference, strain RS-3(T) is proposed as a novel species of the genus Winogradskyella, for which the name Winogradskyella psychrotolerans sp. nov. is proposed. The type strain of Winogradskyella psychrotolerans sp. nov. is RS-3(T) ( = CIP 110154(T) = NBRC 106169(T)). An emended description of the genus Winogradskyella is provided.

Iodobacter arcticus sp. nov., a psychrotolerant bacterium isolated from meltwater stream sediment of an Arctic glacier.

Two novel violet-pigmented, Gram-negative, rod-shaped, non-motile bacteria, designated strains M4-16(T) and M4-9, were isolated from sediment from an Arctic glacier. The predominant fatty acids of both strains were C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3), C16 : 0, C14 : 0 and C18 : 1ω7c and/or C18 : 1ω6c (summed feature 8) and both strains contained ubiquinone-8 as the respiratory quinone. The polar lipids consisted of phosphatidylethanolamine, two unidentified phospholipids and one unidentified aminolipid. 16S rRNA gene sequence analysis indicated that strains M4-16(T) and M4-9 were members of the genus Iodobacter and closely related to Iodobacter fluviatilis ATCC 33051(T) with pairwise sequence similarity of 98.9 %. The DNA-DNA relatedness between strains M4-9 and M4-16(T) was 92.5 %, while strains M4-9 and M4-16(T) had DNA-DNA relatedness values of 21.5 and 18.2 %, respectively, with Iodobacter fluviatilis JCM 9044(T). The RAPD-PCR banding patterns of strains M4-9 and M4-16(T) were similar but differed from that of Iodobacter fluviatilis JCM 9044(T). Based on data from the current polyphasic study, strains M4-16(T) and M4-9 represent a novel species of the genus Iodobacter, for which the name Iodobacter arcticus sp. nov. is proposed. The type strain of Iodobacter arcticus is M4-16(T) ( = CIP 1103011(T) = MTCC 11351(T)).

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

The molecular diversity and evolution of Rice tungro bacilliform virus from Indian perspective.

Rice tungro disease is caused by a combination of two viruses: Rice tungro spherical virus and Rice tungro bacilliform virus (RTBV). This study was performed with the objective to decipher the molecular variability and evolution of RTBV isolates present in the tungro-affected states of Indian subcontinent. Phylogenetic analysis based on ORF-I, ORF-II, and ORF-IV sequences showed distinct divergence of Indian RTBV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Chinsura West Bengal, and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic analysis were further supported with the single nucleotide polymorphisms (SNPs), insertion and deletion (INDELs) and evolutionary distance analysis. In addition, sequence difference count matrix revealed a maximum of 56 (ORF-I), 13 (ORF-II) and 73 (ORF-IV) nucleotides differences among all the Indian RTBV isolates taken in this study. However, at the protein level these differences were not significant as revealed by K (a)/K (s) ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100 % (ORF-I), 96-100 % (ORF-II), 94-100 % (ORF-IV) and 86-100 % (ORF-I), 98-100 % (ORF-II) and 95-100 % (ORF-IV), respectively, among Indian isolates of RTBV. The divergence of RTBV isolates into two independent clusters of Indian and non-Indian was shown with the help of the data obtained from phylogeny, SNPs, and INDELs, evolutionary distance analysis, and conserved motifs analysis. The important role of ORF-I and ORF-IV in RTBV diversification and adaptation to different rice growing regions is also discussed.

Bacterial diversity of soil in the vicinity of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones.

Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria.

Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of kongsfjorden and Ny-alesund, Svalbard, Arctic.

Culturable bacterial diversity of seven marine sediment samples of Kongsfjorden and a sediment and a soil sample from Ny-Alesund, Svalbard, Arctic was studied. The bacterial abundance in the marine sediments of Kongsfjorden varied marginally (0.5 x 10(3)-1.3 x 10(4) cfu/g sediment) and the bacterial number in the two samples collected from the shore of Ny-Alesund also was very similar (0.6 x 10(4) and 3.4 x 10(4), respectively). From the nine samples a total of 103 bacterial isolates were obtained and these isolates could be grouped in to 47 phylotypes based on the 16S rRNA gene sequence belonging to 4 phyla namely Actinobacteria, Bacilli, Bacteroidetes and Proteobacteria. Representatives of the 47 phylotypes varied in their growth temperature range (4-37 degrees C), in their tolerance to NaCl (0.3-2 M NaCl) and growth pH range (2-11). Representatives of 26 phylotypes exhibited amylase and lipase activity either at 5 or 20 degrees C or at both the temperatures. A few of the representatives exhibited amylase and/or lipase activity only at 5 degrees C. None of the phylotypes exhibited protease activity. Most of the phylotypes (38) were pigmented. Fatty acid profile studies indicated that short chain fatty acids, unsaturated fatty acids, branched fatty acids, the cyclic and the cis fatty acids are predominant in the psychrophilic bacteria.

Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Enrofloxacin and Its Degradation Products in Tablet Dosage Forms.

The present work was the development of a simple, efficient, and reproducible stability-indicating reverse-phase high performance liquid chromatographic (RP-HPLC) method for simultaneous determination enrofloxacin (EFX) and its degradation products including ethylenediamine impurity, desfluoro impurity, ciprofloxacin impurity, chloro impurity, fluoroquinolonic acid impurity, and decarboxylated impurity in tablet dosage forms. The separation of EFX and its degradation products in tablets was carried out on Kromasil C-18 (250 × 4.6 mm, 5 µm) column using 0.1% (v/v) TEA in 10 mM KH2PO4 (pH 2.5) buffer and methanol by linear gradient program. Flow rate was 1.0 mL min(-1) with a column temperature of 35°C and detection wavelength was carried out at 278 nm and 254 nm. The forced degradation studies were performed on EFX tablets under acidic, basic, oxidation, thermal, humidity, and photolytic conditions. The degraded products were well resolved from the main active drug and also from known impurities within 65 minutes. The method was validated in terms of specificity, linearity, LOD, LOQ, accuracy, precision, and robustness as per ICH guidelines. The results obtained from the validation experiments prove that the developed method is a stability-indicating method and suitable for routine analysis.

Outbreak of waterborne hepatitis E in Hyderabad, India, 2005.

A large outbreak of hepatitis E occurred in 2005 in Hyderabad, Andhra Pradesh, India. A total of 1611 cases were reported between 1 March and 31 December 2005 (attack rate 40/100,000). The epidemic curve suggested a continuing common source outbreak. Cases were centred around open sewage drains that crossed the old city. The attack rate was significantly higher in neighbourhood blocks supplied by water supply lines that crossed open drains (203/100,000) than in blocks supplied by non-crossing water pipes with a linear trend (38/100 000, P<0.00001). Crossing water pipelines were repaired and the attack rates declined.