Molecular diversity among viroids infecting chrysanthemum in India.

Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.

Characterization of phytotoxin producing Alternaria species isolatedfrom sesame leavesand their toxicity.

Sesame (Sesamum indicum L.), is an important oilseed crop in the tropics and subtropics, referred as "Queen of Oilseeds" owing to its high cooking quality and medicinal value. Sesame production, particularly in India, has been declining since last decade and 'Leaf blight' caused by Alternaria spp. is reported to cause yield loss up to 30-40%. Here, we investigated the fungal toxin produced by Alternaria and its pathogenicity. A total of 164 Alternaria strainswere isolated on potato dextrose agar media from the infected sesame leaves showing circular concentric rings with dark brown spots symptoms. All the isolates were screened for cultural and morphological characters. Colour of the fungus was grey to dark brown, formed smooth, raised, fluffy, and regular to irregular margins. Among 164 isolates, 43 isolates were moderately growing and 121 were fast in growth. The DNA of the isolate was amplified with ITS primers and sequence of BLAST results confirmed seven different species of Alternaria of NCBI database. Further, toxigenic potentiality of the isolates was tested with dilutions of culture filtrate (1:1 to 1:5) on sesame leaves. Among 164 isolates, 23 showed toxigenicity, varied from highly toxigenic to least toxigenic. Pathogenicity of the isolates showed that they were highly virulent to less virulent when tested by the detached leaf method. Based on the toxigenicity, the toxin was partially purified and brown coloured paste was recovered. Chemistry of the toxin was confirmed based on the IR, UV, NMR and mass spectra analyses, and it resembled the structure of alternariol mono methyl ether and altenuene which are mycotoxins in nature. Further, bioassay of toxin was carried out at different concentrations (50 to 2000 ppm) on seeds and seedlings of sesame. Maximum inhibition of seed germination of 81.1% was observed at 2000 ppm and the least was 6.67% at 50 ppm. With the increase in the concentration of toxin, the manifestation of the symptom was conspicuous and quick such as marginal, veinal necrosis, drooping and yellowing with lesion formation. From the present study, it is found that the species of Alternaria are responsible for the cause of blight disease symptoms and the toxicity of toxin produced by the pathogen was very high. The Alternaria toxin could inhibit the growth of the plant as well as seed germination rate.

PGP potential, abiotic stress tolerance and antifungal activity of Azotobacter strains isolated from paddy soils.

Azotobacter strains were isolated by serial dilution method and colonies were viscous, smooth, glistening, and brown to black colour on Jenson's N-free agar. Morphological and biochemical tests showed characteristic features of Azotobacter. Further, molecular analyses revealed the presence of different Azotobacter species viz., A. armeniacus, A. chroococcum, A. salinestris, A. tropicalis and A. vinelandii. The isolates were tested for their ability of nitrogen fixation, indole acetic acid (IAA), gibberllic acid production and phosphate solubilization. Four isolates (GVT-1, GVT-2 KOP-11 and SND-4) were efficient in fixation of highest amount of N2 (29.21 µg NmL(-1) day(-1)), produced IAA (25.50 µg mL(-1)), gibberllic acid (17.25 µg 25 mL(-1)) and formed larger P solubilizing zone (13.4 mm). Some of the Azotobacter strains were produced siderophores, hydrogen cyanide and were positive for ammonia production with respect to antifungal activity of Azotobacter was tested with dual culture method and A. tropicalis inhibited the growth of Fusarium, Aspergillus and Alternaria species. Azotobacter isolates were tested against salt (0-10%), temperature (4-55 degrees C), pH (5.0-10) and insecticide chloropyrifos (0-3%) tolerance study. Among them, A. chroococcum was found tolerant to a maximum of 6% NaCl with a temperature of 35-45 degrees C and to a pH up to 8. All the 4 strains showed effective growth against 3% chloropyrifos concentration. The studies revealed that the Azotobacter strains not only produced plant growth promoting substances but are also tolerant to abiotic stresses such as temperature, pH and insecticides.

Diversity of toxic and phytopathogenic Fusarium species occurring on cereals grown in Karnataka state, India.

A total of 198 cereal samples (53 maize, 54 sorghum, 37 paddy and 54 wheat) were collected from 11 districts of Karnataka to understand the percent infection (PI), relative density (RD) and their frequency (Fr) caused by Fusarium spp. All samples were screened by agar plating method using MGA 2.5 agar media and incubated at 25 ± 2 °C for 3-5 days. The study revealed the association of 10 different Fusarium species known trichothecene producers such as Fusarium acuminatum, F. avenaceum, F. crookwellense, F. culmorum, F. equiseti, F. graminearum, F. nivale, F. poae, F. sambucinum and F. sporotrichioides along with non-trichothecene producers like F. anthophilum, F. oxysporaum, F. proliferatum, F. semitectum, F. solani, and F. verticillioides. All the ten isolated potential trichothecene producing Fusarium species were analyzed for their ability to produce trichothecenes by using thin layer chromatography method. The highest infection of Fusarium spp. in maize was by F. verticillioides with PI of (2.95 %), with RD of (15.16 %) and highest Fr was by F. graminearum (79.24 %) and the lowest was F. avenasium with PI of (0.13 %). For sorghum maximum PI was by F. verticillioides (3.02 %), with F. graminearum having highest RD (14.39 %) and with F. verticillioides highest Fr. (72.22 %). In paddy highest PI was by F. verticillioides (3.21 %) and the least was by F. avenaceum (0.09 %). Similarly in wheat the highest PI was by F. verticillioides (2.76 %) while lowest was by F. avenaceum (0.10 %). The highest Fr was with F. graminearum (79.62 %) while the lowest was by F. avenaceum (3.70 %) and the highest RD was by F. graminearum (22.04 %) and lowest was by F. solani (0.72 %). The manually identified Fusarium spp. were further confirmed by PCR-based detection using ITS1 and ITS4 primers followed by sequencing of the PCR amplicons. PCR studies confirmed that all the tested fungal isolates belongs to Fusarium spp. with the amplicon size of 600 bp. Sequencing and the blast data from NCBI data base confirmed the sequence similarity of 99 % to the genus Fusarium and accession numbers were obtained. Chemotyping studies showed that the isolated Fusarium species are known to produce different types of trichothecenes. The study revealed the diversity in phytopathogenic Fusarium spp. in major cereal crops growing in different agro-climatic regions of Karnataka, India.

Detection of antimicrobial traits in fluorescent pseudomonads and molecular characterization of an antibiotic pyoluteorin.

Thirty isolates of fluorescent pseudomonads were obtained from rhizosphere of different crops in Raichur, India. The fluorescent pseudomonad strains were characterized in vitro for biochemical traits, antimicrobial traits, and pyoluteorin antibiotic production. All the isolates that showed fluorescent pigment production under UV light were rod shaped, Gram negative, positive for oxidase, catalase and citrate utilization tests, and negative for indole test. Out of 30 isolates, 07 isolates were positive for HCN production, 15 isolates were positive for H2S production, and all the isolates were positive for siderophore production. Among all the isolates, RFP-22 showed the maximum percent inhibition of mycelium (46.66 %) of Rhizoctonia solani, the pathogen, and the remaining isolates showed the moderate to least inhibition of mycelium growth of R. solani. The 16S rRNA analysis confirmed that the antibiotic positive isolates belonged to genus Pseudomonas. The amplification of 779 bp region in isolates RFP- 4 and RFP-19 corresponded to pyoluteorin antibiotic-coding pltB gene. Further characterization of pyoluteorin antibiotic through TLC and TOF-MS analysis confirmed the presence of pyoluteorin at 274.26 (g/mol) peak and 2.10 min retention time. Biochemical and molecular analyses confirmed the antagonism of Pseudomonas and isolate through pyoluteorin production.

Probiotic potential of lactobacillus strains isolated from sorghum-based traditional fermented food.

Sorghum-based traditional fermented food was screened for potential probiotic lactic acid bacteria. The isolates were identified by biochemical, physiological and genetic methods. Species identification was done by 16s rRNA sequence analysis. The functional probiotic potential of the two Lactobacillus species viz., Lactobacillus plantarum (Lact. plantarum) and Lactobacillus pentosus (Lact. pentosus) was assessed by different standard parameters. The strains were tolerant to pH 2 for 1 h and resistant to methicillin, kanamycin, vancomycin and norfloxacin. Two (Lact. plantarum COORG-3 and Lact. pentosus COORG-8) out of eight isolates recorded the cell surface hydrophobicity to be 59.12 and 64.06%, respectively. All the strains showed tolerance to artificial duodenum juice (pH 2) for 3 h, positive for bile salt hydrolase test and negative for haemolytic test. The neutralized cell-free supernatant of the strains Lact. pentosus COORG-4, Lact. plantarum COORG-1, Lact. plantarum COORG-7, Lact. pentosus COORG-8 and Lact. plantarum COORG-3 showed good antibiofilm activity. Lact. pentosus COORG-8 exhibited 74% activity against Pseudomonas aeruginosa-MTCC 7903 and Lact. plantarum COORG-7 showed 68% inhibition of biofilm against Klebsiella pneumonia MTCC 7407. Three (Lact. plantarum COORG-7, Lact. pentosus COORG-5 and Lact. pentosus COORG 8) out of eight isolates exhibited a good antimicrobial activity against Listeria monocytogenes and five isolates (Lact. pentosus COORG 2, Lact. plantarum COORG 1, Lact. plantarum COORG 4, Lact. pentosus COORG 3 and Lact. plantarum COORG 6) are active against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumonia, Enterococcus faecalis. The study also evaluated the cholesterol lowering property of the Lactobacillus strains using hen egg yolk as the cholesterol source. The cholesterol in hen egg yolk was assimilated by 74.12 and 68.26% by Lact. plantarum COORG 4 and Lact. pentosus COORG 7, respectively. The results of the present study suggest that the Lactobacillus strains isolated and characterized from sorghum-based fermented product may be used as probiotic strains for therapeutic applications.

First Report of Grapevine yellow speckle viroid-1 and Hop stunt viroid Infecting Grapevines (Vitis vinifera) in India.

During March through July 2012, 10 to 15% of the Vitis vinifera cultivars Thompson Seedless and Anab-e-Shahi exhibited yellow leaf spots and flecks, shortened internodes, and tiny yellow leaves in vineyards of the Bijapur, Doddaballapur, and Kolar districts of Karnataka State, India. These are the major grapevine cultivation regions in India. Samples were collected from four different plants from each district (12 samples in total) and RNA was extracted using 2X CTAB buffer (1). Presence of Grapevine yellow speckle viroid1 (GYSVd-1, genus Apscaviroid) was tested by reverse transcription (RT)-PCR with primer pair PBCVd100C/194H (4) for the amplification of a 220-bp region of the genome. In agarose gel electrophoresis, five samples showed amplicons of the expected size. These amplicons were cloned and sequenced. BLAST analysis confirmed the presence of GYSVd-1. Based on this data, the full-length genome of GYSVd-1 was amplified by RT-PCR using primer pair 341M (5'-CACTCGCGGGGCGCGTTGGA-3') and 342P (5'-CAATCCCCGGAACCCCCGCT-3') and the amplicons were cloned and sequenced. Sequence analysis revealed two sequence variants namely Kar-1 (GenBank Accession No. AB742222) and Kar-2 (AB742223) with 98% and 99% identity to GYSVd-1 variants IXc (X87913) and II (X87906), respectively. GYSVd-1 variants Kar-1 and Kar-2 clustered in two distinct phylogenetic sub-clades. All 12 samples also tested positive for Hop stunt viroid (HpSVd, genus Hostuviroid) in two separate sets of RT-PCR using HSV-78P (5'-AACCCGGGGCAACTCTTCTC-3') and HSV-83M (5'-AACCCGGGGCTCCTTTCTCA-3'); and HSV-7P (5'-AATTCTCGAGTTGCCGC-3') and HSV-220M (5'-CGAACCGAGAGGTGATGCCA-3'), with the expected size of 303 and 213 bp, respectively (3). Sequence analysis of the amplicons confirmed the presence of HpSVd. Alignment of HpSVd nucleotide sequences obtained from the 12 samples showed the presence of a single type of sequence variant, namely Ind-2 (AB742225). BLAST analysis showed 99% sequence identity of Ind-2 with a HpSVd variant isolated from a 100-year-old grapevine in China. All 12 grapevine samples were also tested for the presence of Australian grapevine viroid (AGVd, genus Apscaviroid), Grapevine yellow speckle viroid 2 (GYSVd 2, genus Apscaviroid), and Citrus exocortis viroid (CEVd, genus Pospiviroid) by RT-PCR as described previously (2). None of the samples showed any positives. Northern blot assay using appropriate digoxigenin-labeled riboprobes performed as described previously (2) further confirmed RT-PCR results. Positive controls for RT-PCR and Northern blot were obtained from viroid-infected grapevines maintained in the greenhouse. GYSVd-1 and HpSVd were detected in symptomatic and symptomless plants. Hence, the symptoms observed in the vineyard cannot be attributed to viroid infection. More work is needed to identify the causal agent(s) of the decline of Thompson Seedless and Anab-e-Shadi cultivars. References: (1) C. R. Adkar-Purushothama et al. Plant Dis. 97:149, 2013. (2) D. Jiang et al. Virus Res, 169:237, 2012. (3) Y. Kawaguchi-Ito et al. PLoS One 4:e8386, 2009. (4) L. I. Ward et al. Plant Dis. 95:617, 2011.