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Characterization and Confirmation of Corchorus Golden Mosaic Virus Associated with Jute in Bangladesh

Hasan MM*
Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan

Meah MB
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Ali MA
Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Okazaki K
Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan

Sano Y
Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan

Abstract/ Executive Summary:

Yellow mosaic disease is a major limiting factor for jute (Corchorus capsularis L.) cultivation in Bangladesh. It cloned and sequenced three isolates of Corchorus golden mosaic virus (CoGMV) collected from different regions in Bangladesh. DNA A sequence of CoGMV-[BD:Mym:10] (BD1] shared highest identity (94.2%) with the Vietnam isolate of CoGMV, whereas DNA B shared a lower level of sequence identity (<73%) with the CoGMV isolates reported from Vietnam and India. Complete genome sequences of CoGMV-[BD: Ran: 10] [BD2] and CoGMV-[BD:Din:10] [BD3] showed at least 97% sequence identity with Indian isolates of CoGMV. The fact that the examined DNA A components of three isolates of CoGMV lack the AV2 open reading frame, indicated that BD1-3 share genetic features of New World begomoviruses. The pathogenicity of CoGMV-[BD:Din:10] [BD3] isolate was confirmed by agroinoculation and infectious clones of DNA A and DNA B induced characteristic yellow mosaic symptoms in jute plants. This was a first experimental demonstration of Koch’s postulate for a begomovirus associated with jute yellow mosaic disease.

Keywords: Jute; CoGMV; Begomovirus; Agroinoculation

Location: Mymensingh, Rangpur and Dinajpur districts of Bangladesh.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Jute

Objectives:

Considering the importance of the jute fiber and the economic impact of the viral disease, it designed research work on molecular characterization and confirmation of the causal virus of jute yellow mosaic in Bangladesh.

Methodology:

Virus source: Naturally infected plants of C. capsularis cultivar CLV-1 showing symptoms of yellow mosaic disease and healthy plants were collected from Mymensingh, Rangpur and Dinajpur districts of Bangladesh. The plants exhibiting symptoms of typical mosaic disease and healthy plants were then subjected to collect sample leaves. Sample leaves were dried using silica gel and assayed for virus infection and seed transmission at the plant virology laboratory of the Department of Agrobiology, Graduate School of Science and Technology, Niigata University, Japan. DNA extraction: Nucleic acids were extracted from symptomatic leaves (20 mg) by a modified Cetyl Trimethylammonium Bromide (CTAB) method. The total DNA was suspended in 15 μl of sterile distilled water and used in PCR procedures. PCR amplification of DNA A and DNA B components: Extracted DNAs from four samples were used in Polymerase Chain Reaction (PCR) to amplify the complete viral DNA A and DNA B genomes of CoGMV using the 3 abutting primer pairs, CoGMVAF/CoGMVAR, CoGMVB1F/CoGMVB1R and CoGMVBF/CoGMVBR, were designed according to the determined sequences (sizes 2320, 2190, 260 and 138 bp for Co-V1/R1, Co-V1/R2, Co-V2/R1 and Co-V2/R2 primer pairs, respectively). All amplifications were performed in a reaction mixture of 20 μl using PrimeStar Max enzyme according to the manufacturer’s instructions (Takara Bio Inc., Japan). The PCR cycles were programmed as 20s for initial denaturation at 98°C, and 15s each for denaturation at 98°C, annealing at 50-58°C and chain extension at 72°C (25 cycles). To confirm the completion of amplification of all the target templates, a final extension cycle was carried out at 72°C for 7 min. Cloning and sequencing: The PCR products were cloned into T-vector pMD20 and used to transform Escherichia coli (JM109) following the manufacturer’s instructions (Takara Bio Inc., Japan). Selected clones were completely sequenced by a commercial company (SolGent Co., Ltd., Korea). The sequence data were assembled and analyzed using GENETYX Win. Software package (GENETYX, Tokyo, Japan) ver. 12. Multiple sequence alignments of viral genomic sequences and pairwise comparisons were carried out with the help of ClustalW software. Phylogenetic trees were generated with Molecular Evolutionary Genetics Analysis (MEGA) software, version 6 using the neighbor-joining method. Construction of agroinfectious clones: For construction of CoGMV-[BD:Din:10] [BD3] DNA A infectious clone, the EcoRV-to-KpnI (about 1.7 kb) fragment of DNA A clone (1.0 mer) was separated and removed by gel electrophoresis and the remaining (0.4 mer) of the clone was purified, treated with T4 polymerase and self-ligated which gives pMD20A0.4mer. The viral fragment was confirmed, and a full length DNA A component released as NheI fragment was further ligated with the NheI linearized pMD20A0.4mer to generate pMD20A1.4mer. The orientation of the constructs was confirmed by restriction digestion with A flII, which was expected to release 2.7 kb fragment. The 3.7 kb band (1.4 mer), released by digestion with HindIII and EcoRI from the recombinant pMD20 clone of DNA A was subsequently cloned at the same restriction sites of a binary vector pRI201-AN-GUS and the final clone was named pRI1.4A. For construction of CoGMV-[BD: Din: 10] [BD3] DNA B infectious clone, a similar strategy was adopted. The BglII-to-KpnI (0.5 kb) fragment of DNA B clone (1.0 mer) was separated and removed by gel electrophoresis and the remaining (0.8 mer) of the clone was purified, and treated with T₄ polymerase and self-ligated to give pMD20B0.8mer. The viral fragment was confirmed and a full length DNA B component released as a FbaI fragment was further ligated with the FbaI linearized pMD20B0.8mer to generate pMd20B1.8mer. The orientation was confirmed by restriction digestion with PvuII, which was expected to release 2.7 kb fragment of DNA B. The 4.9 kb band (1.8 mer) released by digestion with HindIII and EcoRI from the recombinant pMD20 clone of DNA B was subsequently cloned at the same restriction sites of a binary vector pRI201-AN-GUS and the final clone was named pRI1.8B. Agroinoculation: Jute (Corchorus capsularis) seedlings of the cultivar CVL-1 were grown in vermiculite inside a temperature controlled growth chamber maintained at 22±2⁰C and a 16/8 h light/dark cycle. Third and fourth leaf stage jute seedlings were used for agroinoculation. CoGMV DNA A was agroinoculated either alone or with cognate DNA B. Agrobacterium tumefaciens strain C58C1 cells containing the infectious constructs pRI1.4A and pRI1.8B were grown for 48 hours on Luria Bertani medium (pH 6.8) containing kanamycin (50 μg/ml) and rifampicin (100 μg/ml). Agrobacterium cells were harvested and resuspended in MES buffer (10 mM 2-[N-morpholino) ethanesulfonic acid (MES), 10 Mm Magnesium chloride (MgCl₂ ) and 100 μM acetosyringone). DNA A and DNA B were mixed at equal concentrations (1.0 OD) and introduced by cutting of the leaf petiole with sharp blade and agroinoculated plants were grown in an insect free growth chamber at 22 ± 2°C with 12/12 hour light/dark cycle and observations were recorded periodically.

Source of Information: J Plant Pathol Microb, Volume 6, Isue 2, 1000256, ISSN: 2157-7471 JPPM, an open access journal

Article Link (Online Journal): http://www.omicsonline.org/open-access/characterization-and-confirmation-of-corchorus-golden-mosaic-virus-associated-with-jute-in-bangladesh-2157-7471-1000256.pdf

Funding Source:

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Achievement/Findings:

Yellow mosaic disease has immense economic importance, including through its association with jute plants in Bangladesh. In phylogenetic analyses, DNA A sequences of CoGMV BD1-3 isolates were clustered into a group comprising other isolates of CoGMV reported from India and Vietnam. On the basis of DNA A sequence similarity, BD1 was most closely related to Vietnam isolate (94.2%), whereas BD2 and BD3 were closely related to Indian isolates (98.2- 98.8%). The level of nucleotide identity (92.1-92.8%) found between DNA A sequences of BD1 and other Indian and BD3 isolates of CoGMV was slightly lower than the strain demarcation level of 93%. However, the DNA A of Vietnam isolate exhibited intermediate levels of nucleotide identity of 94.2% and 93.9% with BD1 and BD2, respectively, indicating that BD1, Vietnam and the other CoGMV isolates should be regarded as variants. It is also noteworthy that DNA B of BD1 had only 72.1-72.6% nucleotide identity with other CoGMV isolates. Possible recombination events could not be detected in DNA B of BD1 with other CoGMV and CoYVV isolates, using RDP ver. 3 (results not shown). DNA B component, by virtue of encoding no overlapping genes, has a greater capacity for variation.

Therefore, to satisfy the Koch’s postulates, the infectivity of CoGMV-[BD: Din: 10] [BD3] was established by inoculating the agro infectious clones in jute plant. The agro inoculation in jute plants resulted in the disease symptoms that are similar to those occurred in the virus infected jute plant in fields. Thus, it fulfilled the Koch’s postulates and showed for the first time that CoGMV-[BD: Din: 10] [BD3] is responsible for the newly emerging yellow mosaic disease of jute in Bangladesh. Empty agrobacterium cells (control) or only DNA A of CoGMV-[BD: Din: 10] [BD3] inoculated plants did not show any symptom by 48 day dpi. This showed that only DNA A of CoGMV-[BD: Din: 10] [BD3] was neither able to sustain nor produce systemic infection in the host plants. This phenomenon is observed for most of the geminiviruses with the bipartite genomes.

Knowledge Management: Journal