Subrata Satpathy

Endophytic colonization of white jute (Corchorus capsularis) plants by different Beauveria bassiana strains for managing stem weevil (Apion corchori)

Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales), an entomopathogenic fungus, was introduced through seed inoculation with spore suspension as an endophyte in white jute (Corchorus capsularis L.), a bast fiber crop. Out of nine B. bassiana strains, seven, viz., ITCC 6063, ITCC 4512, ITCC 4563, ITCC 5562, ITCC 4796, ITCC 5408 and ITCC 4705, became established as endophytes. The endophytic colonization was initially detected by cultivation on selective medium. Colonization was confirmed by polymerase chain reaction (PCR) using sequence characterized amplified region (SCAR) marker. Endophytic B. bassiana strains colonized in leaves of all the plants grown from treated seeds. However, the colonization frequency varied among the strains. The highest colonization frequency (70.09%) was recorded in ITCC 6063 followed by ITCC 5562 (67.67%) and ITCC 5408 (64.17%); ITCC 4512 exhibited the lowest (42.54%) colonization. ITCC 4925 and ITCC 4644 did not show any colonization. Endophytic colonization of B. bassiana reduced stem weevil infestation under pot culture. ITCC 5408 and ITCC 6063 were found most efficient, with only 10.44% and 14.06% infested plants, respectively.

First Report of Potato leaf roll virus (PLRV) Naturally Occurring on Jute (Corchorus olitorius) in India

Jute (Corchorus olitorius L.) is an important bast fiber crop that is mainly grown in the Southeast Asian countries like India, Bangladesh, Nepal, China, Indonesia, Thailand, Myanmar, and a few South American countries. In June 2013, symptoms suggestive of a viral disease were noticed on jute (cv. JRO524) in an experimental field of the CRIJAF research farm, Barrackpore, India, and the incidence of the disease was less than 2%. The infected plants showed stunted growth and short height. Mostly the upper leaves elongated with curling and coiling of lamina. Puckering and shoe string effect were also noticed. Petioles and stipules of the affected leaves were exceptionally longer. Although initially the incidence was low, it may spread to larger areas in subsequent years. Because the jute fiber is extracted from the stem, stunted growth and short height would badly affect the fiber yield and quality. Ten symptomatic and ten asymptomatic healthy looking samples were collected from the field. Corchorus golden mosaic begomovirus is common in jute; therefore, all the samples were tested by PCR using JMFL-AF/JMFL-AR, DNA-A component specific primer pair and JMFL-BF/JMFL-BR, DNA-B component specific primer pairs (1). However, there was no amplification. Because the aphid Aphis gossypii was often noticed in the jute field, all the samples were tested by double-antibody sandwich (DAS)-ELISA for common aphid transmitted viruses, e.g., Cucumber mosaic virus, Bean common mosaic virus, Cowpea mosaic virus, Papaya ring spot virus, Potato leaf roll virus (PLRV), Potato virus Y, and Watermelon mosaic virus using commercial diagnostic kits (Agdia). The symptomatic samples showed positive reaction only for PLRV. Five ELISA-positive samples and five asymptomatic healthy samples were used for RNA extraction. Total RNA was extracted by using QIAGEN RNeasy mini kit. RT-PCR was carried out with PLRV CP gene specific primer pair (3) which generated a cDNA amplicon of 627 bp in all ELISA-positive symptomatic samples. PLRV was not detected in symptomless samples. The five purified cDNA products were cloned in a pGEM-T Easy vector (Promega) and were sequenced. One of the five identical sequences was deposited in GenBank (Accession No. KF233880). The consensus sequence was analyzed by NCBI BLAST and found to share 99% similarity with the coat protein sequence of PLRV reference strain (S77421). Nucleotide span and ORF finder (NCBI) analysis indicated the 627-bp PCR amplicon coded part of a coat protein gene that had 100% identity with translated gene product (Protein ID AAB33483). PLRV is a small isometric RNA virus with worldwide distribution belonging to the family Luteoviridae whose natural host range is mainly restricted to solanaceous plants and few plants of other families (2,4). To the best of our knowledge, this is the first report of PLRV naturally occurring on jute (C. olitorius). References: (1) R. Ghosh et al. J. Virol. Methods 159:34, 2009. (2) S. Guyader and D. G. Ducray. J. Gen. Virol. 83:1799, 2002. (3) M. A. Mayo et al. J. Gen. Virol. 70:1037, 1989. (4) K. Mukherjee et al. Virus Genes 26:247, 2003.

First Report of a 16SrV-C Phytoplasma Causing Little Leaf and Bunchy Top of Tossa Jute (Corchorus olitorius) in India

Jute is the most important phloem fiber crop of the world, and is mainly grown in the South East Asian countries of India, Bangladesh, Nepal, China, Indonesia, Thailand, and Myanmar, and few South American countries. The fiber is used in making sacks, ropes, bags, carpets, shoes, geo-textiles, and home decorations. There are two kinds of jute: tossa jute (Corchorus olitorius L.) and white jute (C. capsularis). In June 2012, symptoms suggestive of phytoplasma infection (little leaf and bunchy top) were noticed on tossa jute in different experimental fields of the CRIJAF research farm, Barrackpore, India, and the incidence of the disease varied from 5 to 20%. The infected plants showed profuse lateral branching with a bushy appearance. In many plants, branching at the apical portion developed a bunchy top symptom with tufts of smaller leaves. Leafy stem was also common in many plants with main stems covered with numerous little leaves. Total DNA was extracted from leaf midveins of 15 symptomatic and 5 asymptomatic plants by using an improved salt concentration and simple sodium acetate CTAB method (1). PCR was carried out with universal P1/P7 primer set followed by nested primer pair R16F2n/R16R2 (3), resulting in DNA amplicons that were 1.8 kb and 1.2 kb, respectively, in all symptomatic samples tested. Phytoplasma was not detected in symptomless samples. The five purified nested products were cloned in a pGEM-T Easy vector (Promega) and sequenced. One of the sequences that proved to be identical was deposited in GenBank (Accession No. KF501045). The consensus sequence was analyzed by NCBI BLAST and found to share 99% similarity with the 16Sr DNA sequence of the alder yellows phytoplasma reference strain (GenBank Accession No. AY028789), which belongs to the 16SrV group. The phylogenetic tree based on the 16SrDNA sequence of phytoplasmas belonging to group 16SrV and other distinct phytoplasma groups also showed that the phytoplasma clustered with members of subgroup 16SrV (4). Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TruI restriction site used for 16SrV subgroups A, B, C, D, and E indicated that the 16SrV Corchorus strain belonged to subgroup C. RFLP patterns from all symptomatic C. olitorius samples were identical to the 16SrV-C pattern (2). The vector species transmitting the concerned phytoplasma in C. olitorius still needs to be identified. The leaf hopper, Amrasca biguttula biguttula, may be a potential vector as it is often noticed in jute fields. To the best of our knowledge, this is the first report of 16SrV-C phytoplasma associated with tossa jute (C. olitorius) in India. Initiative has to be taken to manage this disease; otherwise, branching of the main stems would badly affect the fiber quality as well as yield. References: (1) C. Biswas et al. Lett. Appl. Microbiol. 56:105, 2012. (2) B. Duduk et al. J. Phytopathology 152:575, 2004. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) N. Saitou and M. Nei. Mol. Biol. Evol. 4:406, 1987.

Bt Jute Expressing Fused δ-Endotoxin Cry1Ab/Ac for Resistance to Lepidopteran Pests

Jute (Corchorus sp.) is naturally occurring, biodegradable, lignocellulosic-long, silky, golden shiny fiber producing plant that has great demands globally. Paper and textile industries are interested in jute because of the easy availability, non-toxicity and high yield of cellulosic biomass produced per acre in cultivation. Jute is the major and most industrially used bast fiber-producing crop in the world and it needs protection from insect pest infestation that decreases its yield and quality. Single locus integration of the synthetically fused cry1Ab/Ac gene of Bacillus thuringiensis (Bt) in Corchorus capsularis (JRC 321) by Agrobacterium tumefaciens-mediated shoot tip transformation provided 5 potent Bt jute lines BT1, BT2, BT4, BT7 and BT8. These lines consistently expressed the Cry1Ab/Ac endotoxin ranging from 0.16 to 0.35 ng/mg of leaf, in the following generations (analyzed upto T4). The effect of Cry1Ab/Ac endotoxin was studied against 3 major Lepidopteran pests of jute- semilooper (Anomis sabulifera Guenee), hairy caterpillar (Spilarctia obliqua Walker) and indigo caterpillar (Spodoptera exigua Hubner) by detached leaf and whole plant insect bioassay on greenhouse-grown transgenic plants. Results confirm that larvae feeding on transgenic plants had lower food consumption, body size, body weight and dry weight of excreta compared to non-transgenic controls. Insect mortality range among transgenic feeders was 66–100% for semilooper and hairy caterpillar and 87.50% for indigo caterpillar. Apart from insect resistance, the transgenic plants were at par with control plants in terms of agronomic parameters and fiber quality. Hence, these Bt jutes in the field would survive Lepidopteran pest infestation, minimize harmful pesticide usage and yield good quality fiber.

In planta detection of Macrophomina phaseolina from jute (Corchorus olitorius) by a sodium acetate-based direct PCR method

Macrophomina phaseolina (Tassi) Goid. is the most important pathogen of jute and primarily causes seedling blight, leaf spot and stem rot. The pathogen was detected from field samples by a simple method of direct PCR (dPCR) which obviates the steps of DNA extraction. The leaf bits were treated with a lysis buffer at 65°C for 25 min, whereas the stem pieces were initially incubated at 65°C for 5 min followed by incubation at 60°C for 25 min and the lysate was used as PCR template. Based on the type of tissue, the composition and concentration of lysis buffer systems were optimized. For leaf samples the optimized buffer system composed of 20 mmol l-1 tris (hydroxymethyl aminomethane (Tris)-Cl (pH 8.0), 1.5 mmol l-1 ethylene diamine tetra acetate (EDTA) (pH 8.0), 1.4 mol l-1 sodium acetate and 200 μg/ml proteinase K. Further, 3% PVP (w/v) and β-mercaptoethanol (1% w/v) were added into the buffer. In case of stem samples, PVP was not applied and higher concentrations were used for other components. M. phaseolina could be detected from both leaf and stem samples generating amplicon of 350 bp. This is the first report of detecting M. phaseolina by a direct PCR method without DNA extraction.

First report of bacterial leaf blight of jute (Corchorus capsularis L.) caused by Xanthomonas campestris pv. capsularii in India

Xanthomonas campestris pv. capsularii causing blight on jute (Corchorus capsularis) leaves was reported for the first time in India. The symptom of the disease initially observed was appearance of small angular brown leaf spots and later as blighted areas on leaf lamina. The disease-causing pathogen was isolated and identified on the basis of its colony morphology, PCR, sequencing and subsequent BLASTn analysis.

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).

First report of bacterial leaf spot caused by Xanthomonas campestris pv. olitorii on jute grown for seed in India.

Jute (Corchorus olitorius L.) is the second most important fiber crop after cotton in terms of global production (3). In November 2011, symptoms suggestive of bacterial infection were observed on a seed crop of jute at the CRIJAF research farm, Barrackpore, West Bengal, India. The disease appeared as small, brown, circular spots, usually less than 5 mm in diameter on the leaves and some of the spots were surrounded by a yellow halo. The lesions on the stems were elongated and in some cases were found to girdle the stem. In the later stages of disease, brown sunken spots were found on the green capsules. Disease incidence varied from about 20% to 90% of the total plants in different affected fields at the CRIJAF research farm. Bacterial leaf spot of jute with similar symptoms was reported in 1957 from Sudan (4). Five symptomatic and three asymptomatic leaf samples were collected from different jute fields. Bacterial colonies isolated on nutrient agar medium from infected young leaves were Xanthomonas-like and pale yellow cream in color. Total DNA was extracted from symptomatic as well as asymptomatic leaf samples by using an improved salt concentration and simple sodium acetate CTAB method (2). Single bacterial colonies were transferred to nutrient agar (NA) medium plates and incubated at 28°C for 48 h. Pure colonies from plates were used directly for DNA extraction using the QIAGEN DNeasy Blood and Tissue kit. PCR was carried out with Xanthomonas campestris specific primers NZ8F3/NZ85R3 (1), which generated an amplicon of 530 bp from all the symptomatic leaf samples as well as pure cultures of the isolated bacteria. No amplification was obtained from asymptomatic leaves. The amplicons from the five symptomatic samples collected from the field were sequenced and showed 100% identity with one another, and one sequence (strain JB-CO-13) was deposited in GenBank (Accession No. KC342185). The BLASTn analysis revealed that bacterial strain JB-CO-13 had 100% identity with X. campestris pv. olitorii (EU285213). Nucleotide span and ORF finder (NCBI) analysis indicated the 530-bp PCR amplicon coded part of a gyrase B gene that had 100% identity with a translated gene product (Protein ID: ABX84334). Three leaves of five 1-month-old jute plants (cv. JRO 204) in pot culture were infiltrated each with a separate bacterial strain using suspensions (1 × 105 CFU/ml) in distilled water. The negative control consisted of leaves infiltrated with sterile distilled water. The plants were kept in a greenhouse with mean maximum and minimum temperatures of 28.96 and 21.8°C, respectively. The plants were covered with plastic bags to maintain high relative humidity (>80%). Typical bacterial lesions were recorded on all the inoculated plants after 1 week. No lesions were seen on the negative control. To the best of our knowledge, this is the first report of bacterial leaf spot on C. olitorius caused by X. campestris pv. olitorii from India. References: (1) J. Adriko et al. Plant Pathol. 61:489, 2012. (2) C. Biswas, et al. Lett. Appl. Microbiol. 56:105, 2013. (3) Food and Agriculture Organization of the United Nations. Agricultural Commodities: Profiles and Relevant WTO Negotiating Issues. Online: http://www.fao.org/docrep/006/Y4343E/y4343e03.htm , 2003. (4) K. A. Sabet. Ann. Appl. Biol. 45:516, 1957.

Molecular identification of a Candidatus phytoplasma (Group16SrV-D) coding partial uvrB gene and degV gene on a new host – mesta (Hibiscus sabdariffa) – with phyllody and reddening of leaves in India

Mesta (Hibiscus sabdariffa) is an important bast fiber crop. In August 2011, there was an outbreak of a phytoplasma-like disease on H. sabdariffa in different villages of the northern coastal mesta-growing region of Andhra Pradesh, India, covering mainly two districts – Srikakulam and Vijayanagaram. The infected plants showed characteristic symptoms such as phyllody and reddening of leaves. PCR with P1/P7 universal primer pair of 16 S rDNA yielded amplicons of 1850 bp from all symptomatic mesta leaf samples similar to samples of brinjal little leaf (phytoplasma positive reference control). However, asymptomatic samples were not amplified. Multiplex nested-PCR showed simultaneous amplification of DNA fragments with phytoplasma specific primers, viz., P1/P7 universal primer pair of 16 S rDNA, nested primer pair R16F2n/R2, uvrB and DegV gene-specific uvrB-degVF/R primer generating amplicons of 1850 bp, 1200 bp and 1023bp, respectively. However, SecY-map gene specific primer SecY-mapF/R was not amplified. The 1023 bp nucleotide sequence of uvrB and DegV gene of the phytoplasma was deposited in the GenBank (NCBI) with the accession no. JX975061. NCBI BLASTn analysis of the 1023 bp products showed that the phytoplasma strain belonged to elm yellows group (16SrV-D). This is the first report that Hibiscus sabdariffa is infected by a phytoplasma and we named it mesta phyllody disease (MPD).