Recombinase Polymerase Amplification assay for rapid detection of a geminivirus associated with potato apical leaf curl disease

Journal of Plant Diseases and Protection - Recombinase Polymerase Amplification (RPA) assay was developed for specific detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV [potato]),...     

Development of simplified and rapid nucleic acid release protocol for PCR based detection of <Emphasis Type="Italic">Potato viruses</Emphasis>

A simple, cost-effective and rapid viral nucleic acid release (NAR) buffer suitable for RT-PCR based diagnostic assay was developed for the detection of potato viruses. The NAR buffer and commercially available RNA isolation kit were compared for RT-PCR based assay, where an amplicon of expected size (~380 bp) targeting PVY was observed in both isolations indicating that it can be used in RT-PCR based diagnostic assays. The same was further validated for its repeatability by running across more than hundred suspected potato leaf samples collected from different sources where, it showed consistent results for the presence of PVY indicating its reliability. The NAR buffer assay was examined for its sensitivity in comparison with the kit based isolation where both the assays were able to detect even up to 10^?5 dilution without affecting the sensitivity. NAR buffer was found stable up to 28 days at -20 °C and for 14 days at 4 °C without losing PCR sensitivity. The assay was also found effective to release the nucleic acid from potato leaves, thrips and aphids for PCR and RT-PCR based detection of DNA viruses like ToLCNDV-potato and other RNA viruses. The developed protocol is simple, less laborious, time-saving (10-15 min) and economical (1/100th of kit) as compared to kit based protocol. The assay can be adopted in diagnostic laboratories for detection of RNA/DNA viruses from potato plants and in thrips as well.     

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.     

Analysis of the coat protein gene of Indian <Emphasis Type="Italic">Potato virus X</Emphasis> isolates for identification of strain groups and determination of the complete genome sequence of two isolates

Complete coat protein (CP) gene sequences of 66 Potato virus X (PVX) isolates were sequenced and compared with other PVX isolates. The CP gene of these isolates shared 93.9–100.0 % and 97.0–100.0 % identities among them at nucleotide and amino acid sequence level, respectively. Phylogenetic analysis with isolates of known PVX strain groups showed that all 66 isolates were found in clade I (strain groups 1, 3 and 4) and none of them in Clade II (strain groups 2 and 4). The Indian isolates had the 714 bp coat protein gene and were closer to clade I isolates with 92.9–99.5 % identities and distantly related to Clade II isolates (74.2 to 80.0 % identities). Hence, these isolates may belong to either of the strain groups 1, 3 and 4. A threonine residue at position 122 and glutamine residue at position 78 were found conserved in all the Indian isolates suggesting that these isolates cannot overcome Rx1gene and Nx gene mediated resistance, characteristic of group 1 and 3. However, unique amino acid substitutions were observed in Indian isolates and further studies are required to ascertain their role in symptom expression, virulence and host range. In addition, whole genome sequences of two isolates one each from Jalandhar (Punjab) and Kufri (Himachal Pradesh) were also determined. They were 6435 nts long with five ORFs and shared 81.4–97.2 % identities to clade I isolates from USA, Russia, India, Iran, China, Japan, Taiwan and 77.0 to 77.5 % identities with clade II isolates from Peru.     

Conversion of Potato Starch and Peel Waste to High Value Nanocrystals

The present study aimed to convert starch and potato peel waste to nanocrystals. Starch nanocrystals were prepared using two methodologies: direct acid hydrolysis and enzyme pretreatment followed by acid hydrolysis. Direct hydrolysis broke down the starch granules to nanocrystals in 12 days. Enzyme pretreatment with starch hydrolytic enzymes (α-amylase and amyloglucosidase) reduced the time for preparation of starch nanocrystals by 6 days. Starch nanocrystals of optimum size were obtained with both the treatments and the resultant size ranged from 10 to 50 nm. Nanocrystals were disk-like platelets in appearance. Cellulose nanocrystals were derived from cellulosic material in the potato peel. Cellulose was isolated from peel waste with alkali treatment. Further, cellulose nanocrystals from potato peel and cellulose microcrystalline were prepared by acid hydrolysis. Microscopic images revealed that the aqueous suspension of cellulose nanocrystals derived from potato peel were single rod shaped, whereas those derived from cellulose microcrystalline were rod-like nanoparticles, agglomerated in the form of bundles including some of the rods in single units (well separated). The size of potato peel nanocrystals ranged from 40 to 100 nm (length) and cellulose microcrystalline ranged from 4 to 20 nm (diameter) by 110 to 250, given 4 to 20 nm (length), respectively. As starch nanocrystals as well as cellulose nanocrystals are derived from biopolymer, both can be considered safe for humans and the environment. Moreover, the biodegradable nature of these nanocrystals makes them superior over metallic nanoparticles, particularly in the field of nanocomposites.     

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.