A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta.

UNLABELLED: We have developed a simple method of direct PCR (dPCR) without time-consuming procedures of DNA extraction by directly using the leaf bits for rapid detection of begomoviruses in jute and mesta. The leaf bits were treated with a lysis buffer for 35 min, and the lysate was used as PCR template. Different components and their concentration in lysis buffer systems were optimized and the optimal buffer system composed of 20 mmol l(-1) tris (hydroxymethyl aminomethane (Tris)-Cl (pH 8·0), 1·5 mmol l(-1) ethylenediaminetetraacetic acid (EDTA) (pH 8·0), 1·4 mol l(-1) NaCl and 200 µg/mL Proteinase K. Further, 3% PVP (w/v) and ß-marcaptoethanol (1% v/v) were additionally added into the buffer in case of jute. Under optimized PCR conditions, both viral DNA as well as plant (jute and mesta) genomic DNA were amplified from the lysate. dPCR required fewer reagents and less incubation time reducing both time and cost of detection. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of begomoviruses by serology is not suitable due to difficulty in preparing high titre and specific antisera. Begomoviruses are routinely detected by PCR-based techniques using universal or specific primers. However, it is a prerequisite to isolate pure DNA from the samples before PCR. DNA extraction from some plants such as jute, mesta is very difficult due to the presence of mucilage and other impurities. Therefore, we have developed a method of direct PCR without DNA extraction for detection of begomoviruses from these crops. It is the first report of a direct PCR method in jute and mesta.

Molecular and biological characterization of corchorus mottle virus, a new begomovirus from Brazil.

A begomovirus infecting Orinoco jute (Corchorus hirtus) from Brazil was characterized. Molecular analysis revealed a bipartite genomic organization, which is typical of the New World begomoviruses. Sequence analysis and phylogenetic data showed that both genomic components have the closest relationship with abutilon mosaic Brazil virus, with an identity of 87.3 % for DNA-A, indicating that this virus is a member of a new begomovirus species for which the name "Corchorus mottle virus" (CoMoV) is proposed. Sida rhombifolia plants inoculated by biolistics with an infectious clone of CoMoV showed systemic vein chlorosis, mottling and leaf deformation symptoms, while Nicotiana benthamiana and tomato plants had symptomless infection. CoMoV is the first corchorus-infecting begomovirus reported in Brazil.

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.).

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V-C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA-A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl2 , Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low-cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V-C.

Molecular characterization and phylogenetic relationships of two new bipartite begomovirus infecting malvaceous plants in Yucatan, Mexico.

Sida acuta and Corchorus siliquosus plants showing yellow mosaic and yellow vein symptoms, respectively, were collected in the Yucatan Peninsula, Mexico. Total DNA was isolated from both plant species and used for the amplification, cloning, and sequencing of the Begomovirus genome. Nucleotide comparison of the complete DNA-A component isolated from S. acuta and C. siliquosus confirmed the presence of two distinct begomoviruses species. Based on phenotypic symptoms observed in infected field plants, the names Sida yellow mosaic Yucatan virus (SiYMYuV) and Corchorus yellow vein Yucatan virus (CoYVYuV) were proposed. The SiYMYuV DNA-A shared the highest nucleotide identity (86%) with the Okra yellow mosaic Mexico virus (OkYMMV). The complete DNA-B component shared the highest nucleotide identity (80%) with CoYVYuV. The CoYVYuV DNA-A shared the highest nucleotide identity (84%) with SiYMYuV. The 166-nt common region (CR) sequence for the DNA-A and DNA-B components of SiYMYuV shared a high nucleotide identity of 99%, and the 151 nt of CoYVYuV CR shared 95% of nucleotide identity. The organization and the iterated sequence of the putative AC1 binding site (located within the common region) of both isolates, were similar to that of the begomoviruses of the Western Hemisphere. Phylogenetic analyses placed the DNA-A and DNA-B of SiYMYuV and CoYVYuV in the clade containing the Abutilon mosaic virus (AbMV).

Corchorus yellow vein virus, a New World geminivirus from the Old World.

A bipartite begomovirus infecting Jute mallow (Corchorus capsularis, Tilliaceae) in Vietnam was identified using novel degenerate PCR primers. Analysis of this virus, which was named Corchorus yellow vein virus (CoYVV), showed that it was more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. Evidence is provided that CoYVV is probably indigenous to the region and may be the remnant of a previous population of New World begomoviruses in the Old World.