A. Jeevalatha

Uniplex and duplex PCR detection of geminivirus associated with potato apical leaf curl disease in India

Apical leaf curl disease has emerged as a new disease in potato during the last decade in India due to a change in planting date and an increased whitefly population. Its incidence is on the rise threatening the cultivation of potato across the country. Hence, a PCR assay was developed for the detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV-Potato) which is the causal agent of apical leaf curl disease in potato. Primers specific to the coat protein (AV1) and replicase (AC1) gene regions were designed and used for standardization of the PCR. Some of the primers (LCVCPF1/LCVCPR1, LCVREPF2/LCVREPR2, LCrep1F/LCrep2R) could detect the virus in 2.4-0.24pg of total DNA of infected plant. A duplex PCR assay was optimized with the selected coat protein gene specific primers and primers specific to potato urease gene, a housekeeping gene served as an internal check. The suitability of these primers was examined for detection of the virus in 80 potato apical leaf curl disease samples from 11 different potato growing states of India and also from micro-plants grown in tissue culture. The selected coat protein primer pair (LCVCPF1/LCVCPR1) was found to be conserved in all 80 isolates except for a few isolates, which had a single nucleotide substitution in the forward primer sequence. These substitutions did not interfere with amplification of the coat protein gene. The primers could detect the virus using a print-capture PCR assay both in the presence and absence of an internal control. These results indicate the robustness of the PCR assay for virus indexing of mother stocks in the seed production system.

An insight into differentially regulated genes in resistant and susceptible genotypes of potato in response to tomato leaf curl New Delhi virus-[potato] infection

Apical leaf curl disease, caused by tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) is one of the most important viral diseases of potato in India. Genetic resistance source for ToLCNDV in potato is not identified so far. However, the cultivar Kufri Bahar is known to show lowest seed degeneration even under high vector levels. Hence, microarray analysis was performed to identify differentially regulated genes during ToLCNDV-[potato] infection in a resistant (Kufri Bahar) and a susceptible cultivar (Kufri Pukhraj). Under artificial inoculation conditions, in Kufri Pukhraj, symptom expressions started at 15days after inoculation (DAI) and then progressed to severe symptoms, whereas no or only very mild symptoms were observed in Kufri Bahar up to 35 DAI. Correspondingly, qPCR assay indicated a high viral load in Kufri Pukhraj and a very low viral load in Kufri Bahar. Microarray analysis showed that a total of 1111 genes and 2588 genes were differentially regulated (|log2 (Fold Change)|>2) in Kufri Bahar and Kufri Pukhraj, respectively, following ToLCNDV-[potato] infection. Gene ontology and mapman analyses revealed that these altered transcripts were involved in various biological & metabolic processes. Several genes with unknown functions were 5 to 100 fold expressed after virus infection and further experiments are necessary to ascertain their role in disease resistance or susceptibility. This study gives an insight into differentially regulated genes in response to ToLCNDV-[potato] infection in resistant and susceptible cultivars and could serve as the basis for the development of new strategies for disease management.

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.

GENETIC DIVERSITY OF RALSTONIA SOLANACEARUM STRAINS CAUSING BACTERIAL WILT OF POTATO IN THE MEGHALAYA STATE OF INDIA

Bacterial wilt or brown rot caused by Ralstonia solanacearum is one of the most damaging diseases of potato worldwide. Strains of R. solanacearum have conventionally been classified into five races (r) and six biovars (bvs). Based on variation of DNA sequences, R. solanacearum strains are now classified into four phylotypes that broadly reflect the ancestral relationships and geographical origin of the strains. These phylotypes can be further subdivided into sequevars based on phylogenetic analysis of the endoglucanase (egl) gene sequences. In the present study, bacterial wilt-infected potato plants/tubers were collected from various potato-growing regions of the East Khasi Hills and West Khasi Hills districts of Meghalaya (India) during summer and autumn of 2012 and 2013. A total of 114 bacterial isolates were recovered, whose identity was confirmed as R. solanacearum by PCR with the species-specific primers 759/760. Using the biovar (bv) classification scheme, 67 isolates (58.8%) were categorized as bv 2T, 41 (35.9%) as bv 2 and six as bv3 (5.3%). A phylotype- specific multiplex PCR assigned 67 (58.8%) isolates to phylotype IV (bv2T), 41 (35.9%) to phylotype II (bv2) and six (5.3%) to phylotype I (bv3). A phylogenetic analysis of partial sequences of the egl gene of all isolates clustered 59 phylotype IV (bv 2T) isolates closely to sequevar 8 (GenBank accession No. DQ657634). The remaining 8 phylotype IV (bv 2T) isolates did not cluster with any of the four sequevar, i.e. 8, 9, 10 and 11 that are defined with phylotype IV and form a distinct sequevar group. All 41 phylotype II (bv 2) isolates clustered with sequevar 1 (EF371844); three phylotype I (bv 3) isolates with sequevar 30 (AB508598), and three phylotype I (bv 3) isolates with sequevar 47 (GU295014). This is the first study that demonstrates a considerable biovar and phylogenetic diversity among R. solanacearum that cause potato bacterial wilt in the Meghalaya state of India.

Optimized loop-mediated isothermal amplification assay for Tomato leaf curl New Delhi virus-[potato] 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.

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd) along with and without internal control from potato. The amplification of the specific targets was identified by their melting points viz., 85.93±0.22, 85.62±0.34 and 82.07±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVd-NFP/PSTVd-NRP and PSTVd-QFP1/PSTVd-QRP1, respectively. The realtime RT-PCR assay was 1×104 times more sensitive than RT-PCR assay and the assay could detect up to 20 copies of the target using serially diluted plasmid and up to 0.025 fg of total RNA from infected tissues diluted in healthy plant RNA. Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with elongation factor 1- α (ef-1α) gene, a stable housekeeping gene of potato. Duplex realtime RT-PCR assay showed two melt peaks indicating the successful amplification both the PSTVd RNA and internal control RNA. The developed assays could consistently detect PSTVd and proved to be highly sensitive and rapid for the detection of PSTVd in post entry quarantine testing.