Kajal Kumar Biswas

Molecular characterization of Citrus tristeza virus isolates from the Northeastern Himalayan region of India

Citrus tristeza virus (CTV) isolates from the Darjeeling hills of the Northeastern Himalayan region of India were characterized by biological indexing, multiple molecular marker (MMM) analysis, heteroduplex mobility assay (HMA) and sequence analysis. Variability was studied using the CP gene and a 5′ ORF1a fragment of the CTV genome. HMA and sequence analysis of the 5′ ORF1a fragment classified Darjeeling isolates into two groups, whereas CP gene analysis provided evidence for three different groups. Darjeeling CTV isolates shared nucleotide sequence identities of 89–97 and 91–92% in the 5′ ORF1a fragment and CP gene, respectively, suggesting extensive diversity among CTV isolates from this Indian region.

Complete genome sequence of mandarin decline Citrus tristeza virus of the Northeastern Himalayan hill region of India: comparative analyses determine recombinant

The complete genome sequence of a mandarin (Citrus reticulata) decline CTV isolate, Kpg3, of the Darjeeling hills of the Northeastern Himalayan region of India is reported for the first time. The complete Kpg3 genome has 19253 nt, and its nucleotide sequence identity ranged from 79% with the Florida CTV isolate T36 to 94% with the Israel isolate VT, whereas its identity to B165, the other Indian isolate, was 89%. Phylogenetic analysis indicated that the Kpg3 genome is closely related to isolate VT and distantly to T36 and B165. Recombination analysis indicated that Kpg3 is recombinant and originated through multiple recombination events in which parts of the genome were exchanged between divergent CTV sequences.

Intra-farm diversity and evidence of genetic recombination of Citrus tristeza virus in Delhi region of India

Infection of Citrus tristeza virus (CTV) in different citrus orchards of New Delhi was detected by direct antigen coated-ELISA and RT-PCR. Sweet orange (Citrus sinensis) orchards were found to be susceptible to CTV with estimated disease incidence up to 39%. Kagzi kalan (C. lemon), Pumello (C. paradisi) and Kinnow mandarin (C. reticulata) orchards did not show CTV infection. Three CTV isolates, D1, D7 and D15 randomly selected from infected sweet orange orchards were considered for biological and molecular characterization. In the host range study, all the Delhi isolates infected Darjeeling mandarin (C. reticulata), Kagzi lime (C. aurantifolia), sour orange (C. aurantium) and sweet orange but not Kinnow mandarin. A fragment of 5′ORF1a and complete coat protein (CP) gene of these three isolates were cloned, sequenced and compared with other Indian and international CTV isolates. Delhi isolates shared 85–92% sequence identity for 5′ORF1a fragment and 89–91% for CP gene among them. Phylogenetic analysis segregated three Delhi isolates into three genogroups for each of 5′ORF1a fragment and CP gene, however phylogenetic relationships for both the genomic regions was incongruent. Recombination detecting program RDP3 detected CTV isolate D7 as recombinant, indicating genetic variability in CTV isolates might be the outcome of recombination events between divergent CTV sequences. An attempt was made in present study to characterize CTV isolates biologically and at genetic level, and to determine genetic diversity at farm level and study the recombination of CTV isolates in Delhi region.

Diagnosis of symptomless Yellow mosaic begomovirus infection in pigeonpea by using cloned Mungbean yellow mosaic India virus as probe.

One isolate of Mungbean yellow mosaic India virus (MYMIV) of mungbean plants from Sri Ganganagar, Rajasthan, designated as MYMIV-Mg was isolated and DNA-A and DNA-B, the two full length bipartite genomic components of this virus, were cloned. The [α-32P] labeled diagnostic probes specific to these cloned DNA-A and -B of MYMIV-Mg were used to detect the virus infection in infected plants by nucleic acid spot hybridization (NASH) test. The NASH tests detected the MYMIV infection and concentration of viral titre in susceptible, moderately susceptible, resistant and symptomless genotypes of pigeonpea (Cajanus cajan) plants. Fourteen genotypes of pigeonpea were tested against five naturally occurring MYMIV variants viz.,.MYMIV Bg, -MgD, -MoL, -Mg and -Pp1 through viruliferous whitefly (Bemisia tabaci) transmission in greenhouse condition. Disease incidence and severity of MYMIV in different pigeonpea genotypes varied with the variants of MYMIV. Many genotypes of pigeonpea did not produce visible yellow mosaic symptoms after inoculation with MYMIV variants MYMIV-Bg, -MbD and -MoL, although, majority of the symptomless genotypes were found to be infected by MYMIV, as viral DNA was detected by NASH test.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

Closterovirus in India: Distribution, Genomics and Genetic Diversity of Citrus Tristeza Virus

Only one closterovirus species (Family: Closteroviridae), Citrus tristeza virus (CTV) is known to occur in India. CTV is one of the most important plant viruses in India and extensive studies have been conducted over the last 60 years. The failure of Malta sweet orange on sour orange root stocks provided the evidence of tristeza disease in India. CTV infects nearly all the citrus species and citrus relatives and hybrids showing variables biological symptoms. Most citrus species and cultivars are susceptible to infection but some are tolerant inducing no obvious symptoms. Citrus orchards in Northeast India are severely affected by citrus decline, and several orchards in this region have been wiped out and many Sweet orange orchards in South and Central India are facing problem of decline. Toxoptera citricida is an efficient vector for the local natural spread of CTV in India. Stem pitting symptoms caused by CTV are not common in India. The Indian CTV isolates are genetically diverse and seven to ten genetic variants have been recognized in India. The complete genome (19,253 nt) of a mandarin decline inducing CTV strain, Kpg3 from the Darjeeling hills was sequenced. This chapter presents the work conducted on CTV in India.

Detection of Cotton leaf curl-begomovirus and betasatellite DNA in Weed and other hosts in and around Cotton fields

In the present study, effort was made to identify alternative host of CLCuD-begomovirus and betasatellite based on PCR, using specific primers targeting partial C1 and CP gene of CLCuDbegomovirus and #C1 gene of betasatellite. A total number of 30 weeds and other hosts under 15 host species under 10 plant families were collected from cotton field and its surrounding of Sri Ganganagar and Hanumangarh districts of Rajasthan, and cotton fields of ICAR-IARI, New Delhi. The CP gene was detected in Blumea sp. and brinjal; #C1 gene in Abutilon theophrasti, Croton sp., Ipomoea triloba and brinjal; and partial C1 gene in Abutilon theophrasti, Croton sp., Ipomoea triloba, Ageratum conyzoides, wild cucurbit and brinjal. These weed and other hosts can act as reservoirs of CLCuD and its associated satellite molecules.

Multiple and mixed infections with yellow mosaic, leaf crinkle and bud necrosis disease complex in mungbean: A threat to cultivation of mungbean in India

The occurrence, incidence, yield losses and diagnostics of yellow mosaic disease (YMD) caused Mungbean yellow mosaic India virus (MYMIV), bud necrosis disease (BND) caused by Groundnut bud necrosis virus (GBNV) and Urdbean leaf crinkle disease complex (ULCD) were studied in mungbean (Vigna radiata) in Delhi condition. Eighteen mungbean cultivars were grown for successive four years from 2006 to 2009. Overall disease incidence of 11.6-18.5% for YMD; 14.4-20.5% for ULCD and 9.4-14.5% for BND were estimated. The mixed infection was studied in kharif season and overall it was 10.5% for YMD+ULCD, 8.3% for YMD+BND, 6.8% for ULCD+BND and 4.3% for YMD+ULCD+BND. None of the mungbean cultivars exhibited resistance to any of the three diseases. Yield loss estimated in susceptible mungbean cv. PS 16 was up to 61.1, 91.0 and 76.8% over control for YMD, ULCD and BND, respectively. Mixed infection with YMD and BND caused 92.8% yield losses. Biological and molecular detection using enzyme linked Immuno-sorbent assay (ELISA), polymerase chain reaction (PCR) and nucleotide analysis determined that MYMIV is the causal agent of YMD and GBNV of BND. Although etiology of ULCD was not confirmed, however, seed and sap transmission study showed that it is caused by an infectious agent.