Optimized loop-mediated isothermal amplification assay for <Emphasis Type="Italic">Tomato leaf curl New Delhi virus-[potato]</Emphasis> detection in potato leaves and tubers

Apical leaf curl disease of potato is caused by a whitefly transmitted begomovirus, Tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) in India. Detection of this virus is essential to manage the disease, particularly in healthy potato seed production systems. Large scale testing of micro-plants demands a simple, rapid and sensitive assay. Hence, loop-mediated isothermal amplification (LAMP) method was developed for specific detection of ToLCNDV-[potato]. Six primers that recognize the coat protein gene sequence of ToLCNDV-[potato] were designed and LAMP assay was optimized using different concentrations of magnesium sulphate, betaine, dNTPs, Bst DNA polymerase and temperature. The results were assessed by visual observation of turbidity, colour change using SYBR green dye and also by gel electrophoresis. The assay successfully detected the virus in infected plants collected from potato fields whereas no cross-reactions were observed with healthy plants and other potato viruses. The optimized assay was as sensitive as PCR assay and could detect up to 0.002 pg of total DNA. The assay could detect the virus in infected potato tubers and also in asymptomatic plants. Print-capture LAMP assay was developed and its application could reduce the cost and time of the assay in large scale testing under seed production.     

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R.?solanacearum has been classified into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.     

Potato apical leaf curl disease: current status and perspectives on a disease caused by tomato leaf curl New Delhi virus

Journal of Plant Diseases and Protection - Potato apical leaf curl disease (PALCD) caused by a unique bipartite virus [tomato leaf curl New Delhi virus (ToLCNDV)] has emerged as a global threat....     

Diversity analysis of Tomato leaf curl New Delhi virus-[potato], causing apical leaf curl disease of potato in India

The complete coat protein (cp) gene sequence of eighty Tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) isolates collected from eleven states were determined. Phylogenetic analysis based on cp gene grouped the isolates into two major clades (I & II) and they shared 95.9–100.0% identity. The DNA A and DNA B of eight representative isolates (six from clade I and two from clade II) were 2739–2740 and 2692–2694 nts long and shared 94.6–99.4% and 97.2–99.5% homology within the isolates, respectively. Among the eight isolates, the DNA A of two isolates (Clade II), GWA-5 and FAI-19 had 94.6–95.3% sequence identity to other six isolates and formed a sub-clade within the ToLCNDV-[potato] isolates. Similar grouping was also revealed with AC1 and AC4 genes of these eight isolates. The DNA A components shared more than 90.0% identity with the DNA A of ToLCNDV isolates from cucurbitaceous crops, tomato, bhendi, 89.0–90.0% with ToLCNDV-papaya isolates and 70.4–74.0% with other tomato leaf curl viruses. Hence, the begomovirus infecting potatoes are the ToLCNDV isolates, designated as ToLCNDV-[potato]. Whereas, the DNA B components shared 86.6–91.7% identity with ToLCNDV isolates from cucurbits, tomato and bhendi. Evidence for intra-species recombination was detected only in DNA A with a maximum of three events in GWA-5 and FAI-19 isolates. Analysis of cp gene, DNA A, iterons and recombination events clearly indicate that two groups of ToLCNDV-[potato] infects potato in India.