Coat Protein Gene Specific Primers Research Articles

Identification of stable sources of resistance to mungbean yellow mosaic virus (MYMV) disease in mungbean [Vigna radiata (L.) Wilczek

AbstractYellow mosaic disease (YMD) caused by mungbean yellow mosaic virus (MYMV) is one of the most destructive biotic production constraints in mungbean. Development and introduction of resistant cultivars are considered as the most economical and eco-friendly option to manage YMD, for which availability of stable sources of resistance is a pre-requisite. A set of 14 mungbean genotypes including a susceptible check were evaluated for responses to YMD under natural infection across three seasons and under challenged inoculation in glasshouse for one season. None of the genotypes were immune to YMD and produced different degrees of response to MYMV in terms of yellow mosaic symptoms (YMS). Based on the delayed appearance of initial YMS, and lower estimates of per cent disease index and area under disease progressive curve (AUDPC) in response to natural infection and challenged inoculation, five genotypes namely AVMU 1698, AVMU 1699, AVMU 16100, AVMU 16101 and KPS 2 were identified as resistant to YMD. Failure of detection of MYMV through polymerase chain reaction (PCR) using MYMV coat protein gene-specific primer and successful detection of the same through rolling circle amplification-PCR suggested latent infection of MYMV in resistant genotypes. The resistance response of the five genotypes could be attributed to enhanced activities of enzymes such as peroxidase, polyphenol oxidase and phenylalanine ammonia lyase and increased concentration of total phenols. These results are discussed in relation to strategies to breed mungbean for resistance to YMD.

Biological, Pathological, and Molecular Characteristics of a New Potyvirus, Dendrobium Chlorotic Mosaic Virus, Infecting Dendrobium Orchid.

Dendrobium smillieae is one of the popular orchids in Taiwan. This report describes a new potyvirus tentatively named Dendrobium chlorotic mosaic virus (DeCMV) causing chlorotic and mosaic symptoms in D. smillieae. Enzyme-linked immunosorbent assay (ELISA) tests using six antisera against orchid-infecting viruses revealed that only a monoclonal antibody against the potyvirus group reacted positively with crude saps prepared from a symptomatic dendrobium orchid. Potyvirus-like, flexuous, filamentous particles were observed under an electron microscope, measuring approximately 700 to 800 nm in length and 11 to 12 nm in diameter. Sequence analyses revealed that DeCMV coat protein gene shared 59.6 to 66.0% nucleotide sequence identity and 57.6 to 66.0% amino acid sequence identity, whereas the DeCMV complete genome shared 54.1 to 57.3% nucleotide sequence identity and 43.7 to 49.5% amino acid sequence identity with those other known potyviruses. These similarity levels were much lower than the criteria set for species demarcation in potyviruses. Thus, DeCMV can be considered a new potyvirus. The whole DeCMV genome contains 10,041 nucleotides (GenBank accession no. MK241979) and encodes a polyprotein that is predicted to produce 10 proteins by proteolytic cleavage. In a pathogenicity test, results of inoculation assays demonstrated that DeCMV can be transmitted to dendrobium orchids by grafting and mechanical inoculation, as verified by ELISA and western blot analyses using the DeCMV polyclonal antiserum and by reverse transcription polymerase chain reaction using the coat protein gene-specific primers. The inoculated orchids developed similar chlorotic and mosaic symptoms. In conclusion, DeCMV is a novel orchid-infecting potyvirus, and this is the first report of a new potyvirus that infects dendrobium orchids in Taiwan.

First report of association of begomovirus in yellow mosaic disease of bur cucumber in India

In November- December, 2016–2017 Bur cucumber (Sicyos angulatus) plants samples showing severe symptom of yellowing, mosaic and leaf curling were collected from Horticulture research centre (HRC), Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut. Polymerase chain reaction using coat protein gene specific primer (ToLCVAV1F and ToLCVAV1R) gave an amplification of ~ 750 bp, similar to leaf curl virus reported on different crops and weeds. The gene was sequenced and submitted to GenBank with MF471344. BLAST analysis showed the association of Begomovirus with 99% nucleotide sequence identity with Tomato leaf curl Palampur virus (ToLCPalV) strain India: UP: Matera: Tomato [KF157552]. Phylogenetic analysis also supported the relationship of the obtained sequence with other begomoviruses as they clustered in a separate monophyletic cluster with ToLCPalV-Karnataka, India (KY564204, KY564205), India; ToLCPalV-Varanasi, India (FJ931537, GQ225738); ToLCPalV-Matera (KF157552), India and ToLCPalV-Allipur, India (HQ848383). Sequence Identity matrix with other begomovirus sequences was found to be in range between (78 and 99%). To our knowledge, this is the first confirmed evidence of a begomovirus infecting weed bur cucumber (Sicyos angulatus) in India.

Prevalence of severe Citrus tristeza virus infection in Khasi mandarin throughout the north-eastern states of India: an evidence study based on coat protein gene analysis

Citrus tristeza virus (CTV) infected plant samples from the North Eastern states of India namely Arunachal Pradesh (AR1), Assam (AS1), Nagaland (NL1), Meghalaya (M1), Sikkim (SK1) and Tripura (TR1) were examined through double antibody sandwich enzyme linked immunosorbent assay and reverse transcriptase polymerase chain reaction using coat protein (CP) gene specific primers, yielding amplicon of 672 base pair for all the isolates. The sequence analysis revealed nucleotide (nt) and amino acid (aa) sequence similarity with other exotic CTV isolates. AR1 and AS1 shared similarity with T3 isolate (Florida) at 98–99% and 95–99% for nt-aa respectively, whereas, M1 showed 99% similarity with stem pitting isolate B165 (Bangalore). SK1 and NL1 were 95–97% and 97–99% similar with Hawaiian isolate HA at nt-aa level. TR1 showed 97% homology with seedling yellowing Israeli isolate VT. The CP gene sequence based phylogenetic analysis grouped present isolates into four clades with reference severe isolates (i.e. VT, B165, HA and T3), indicating their probable evolution. TR1 and NL1 are the putative recombinant isolates and ASI and SK1 were the major and minor parents respectively. This might be due to the conserved nature of CP gene. Moreover, the CP gene sequences of present isolates showing nucleotide identity to the VT, T3 and B165 isolates also suggest the prevalence of severe CTV isolates in Khasi mandarin growing areas comes under North East (NE) region of India.

First Report of Soybean yellow mottle mosaic virus on Soybean (Glycine max) in India

Soybean (Glycine max) is one of the most important crops as a source of high-quality protein and oil. In India, soybean area has expanded with the introduction of high-yielding varieties with wide adaptability, which also favored the development of new diseases with increased incidence. During 2016, 38 samples from initial varietal trials of soybean cultivars from India exhibiting symptoms such as mosaic, mottling, necrosis, and puckering were collected from the fields of the Indian Agricultural Research Institute, New Delhi, India, and tested for the presence of viruses, namely, Cowpea mild mottle virus, Groundnut bud necrosis virus, Soybean mosaic virus, Soybean vein necrosis virus, Soybean yellow mottle mosaic virus (SYMMV), and Tobacco streak virus. Crude sap from 3 of the 38 samples strongly reacted with the polyclonal antibodies raised against SYMMV in direct antibody coating ELISA (A₄₀₅ₙₘ= 1.2 to 1.8) (Sandra et al. 2015). To confirm the SYMMV infection, total RNA was extracted from the ELISA-positive soybean leaf samples showing mosaic and mottling symptoms using the SV Total RNA isolation system (Promega, Madison, WI) followed by reverse-transcription polymerase chain reaction (RT-PCR) with coat protein (CP) gene specific primers (forward, 5′-ATGAATTCATGAATGGAACAATGCTCACCGT-3′; reverse, 5′-CAAAGCTTAGTGTGTGGGTTTTAACTGGCGTTGTTA-3′) (Sandra et al. 2015). The RT-PCR product of 1,065 base pairs was cloned into TA cloning vector (Real Biotech Corporation), and the clones were sequenced in both orientations and deposited in GenBank (accession no. MG768974). Sequence analysis of the CP gene showed 98 to 100% identity with Indian isolates (KR260903, KR260902, and KP259608) followed by 82 and 92% similarity with Korean isolates of SYMMV at nucleotide and amino acid levels, respectively. Identity at the nucleotide and amino acid level of 29 to 42% and 16 to 25% was observed with the corresponding CP region of other carmoviruses. For further confirmation, French bean seedlings (cv. Pusa Parvati) were mechanically sap inoculated with an extract from an ELISA-positive soybean leaf sample, and these seedlings showed systemic chlorotic blotches after 10 to 12 days postinoculation, which were then confirmed by ELISA and RT-PCR as above. Previously, SYMMV was reported on soybean from South Korea and North America (Li et al. 2009; Nam et al. 2009). More recently, SYMMV was reported on mungbean and urdbean from India (Sandra et al. 2015). To our knowledge, this is the first report of natural infection of soybean by SYMMV in India and suggests that this newly identified SYMMV may be adapting to other leguminous host plant species.

FIRST REPORT OF CUCURBIT APHID-BORNE YELLOWS VIRUS AFFECTING SUMMER SQUASH AND MELON IN MONTENEGRO

The phytosanitary status of cucurbit crops in the central and coastal regions of Montenegro was evaluated with a survey in 2015. Leaf samples of summer squash, zucchini squash (Cucurbita pepo L.) and melon (Cucumis melo L.) showing mosaic, interveinal mottling or yellowing, bright yellowing, blisters and distortions were collected from 101 plants. All samples were screened by DAS-ELISA (Bioreba, Switzerland) for Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Moroccan watermelon mosaic virus (MWMV), Zucchini yellow mosaic virus (ZYMV), Zucchini yellow fleck virus (ZYFV) and Papaya ringspot virus (PRSV). Serological analysis showed that 91.1% of the samples were infected with at least one of three viruses, the most prevalent of which was CMV (62.4%), followed by WMV (50.5%) and ZYMV (12.9%). Samples from 20 plants showing symptoms resembling those caused by poleroviruses were analyzed by RT-PCR using the coat protein (CP) gene-specific primers CE9/CE10 of Cucurbit aphid-borne yellows virus (CABYV) (Juarez et al., 2004) An amplicon of the expected size (600 bp) was generated from six samples of summer squash and one of zucchini squash. Single infections by CABYV were found in two of seven positive samples, while other five were co-infected with CMV, WMV and/or ZYMV. Nucleotide sequences of the complete CP gene from four Montenegrin isolates (accession Nos. KX398662-KX398665) were identical to one another, sharing the highest nucleotide identity (99.1%) with an isolate from France (X76931). A previous survey in the former Yugoslavia did not reveal the presence of CABYV (Dukic et al., 2002). Afterwards, the virus has been reported in several neighboring countries (Tomassoli and Meneghini, 2007; Vucurovic et al., 2011), but to our knowledge, this is the first report of CABYV infecting C. pepo in Montenegro.

OCCURRENCE OF CUCUMBER MOSAIC VIRUS IN OLEANDER (NERIUM OLEANDER) IN HUNGARY

In June 2016 mosaic symptoms characteristic of viral in- fection were observed in a ten year-old potted oleander ( Ner- ium oleander ) maintained in Velence (Hungary). Inoculation of test plants ( Chenopodium , Cucumis and Nicotiana spp.) with extracts of diseased oleander leaves demonstrated the presence of a mechanically transmitted plant virus marked Nol. The Nol isolate caused local lesions on Chenopodium quinoa , mosaic on Cucumis sativus and local etched ring pat- tern followed by vein clearing and recovery on the top leaves of Nicotiana tabacum cv. Xanthi-nc. These symptoms sug- gested the presence of B pathotype (subgroup II strain) of Cucumber mosaic virus (CMV). CMV was identified in the oleander leaves using CMV specific lateral flow immunoas- says (Bioreba AgriStrip). To confirm the presence of CMV, t otal RNA was extract- ed from N . clevelandii infected with CMV-Nol and used in RT-PCR with subgroup I and II CMV coat protein (CP) gene specific primers, respectively (Salanki et al ., 1997). The DNA fragment of the expected size (1460 bp) was amplified from tobacco leaves using subgroup II specific primers solely. The PCR product was cloned into pGEM-T Easy Vector (Pro- mega, USA), sequenced (Biomi, Hungary) and the complete CP gene nucleotide sequence was deposited in GenBank (accession No. KX951465). The complete CP gene of CMV- Nol showed 99% nucleotide and 100% amino acid sequence identity with a Serbian CMV isolate (232Mrg, accession No. KP034963.1). This is the first record on the natural occur- rence of CMV in oleander in Hungary.

English

Soybean (Glycine max L.), the most popular oil seed crop, grown in the experimental fields of Tamil Nadu Agricultural University and adjoining areas exhibited symptoms of necrosis and stunting. The symptoms were similar to bud blight of soybean caused by the Tobacco streak virus (TSV) and the disease is most prevalent throughout the country. To study the nature of infection, sap inoculation of the soybean strain induced local as well systemic infection on cowpea plants cv. C 152 and resulted in the production of circular necrotic lesions and death of plants. The samples were also serologically positive in DAC-ELISA and it has also yielded a protein band of approximately 29 kDa corresponding to coat protein of TSV in Western blot assay. For the characterization of virus, RT-PCR was carried out with a newly designed coat protein gene specific primer, which resulted in amplification of the expected 929 bp size. Sequence analysis of the CP gene had nucleotide similarity of 80.6 to 99.3% with known isolates of TSV. The multiple sequence alignment revealed that CP gene showed single unique variation and some of deletion and addition mutation was found in nucleotide and amino acid sequences against the isolates of other soybean Brazil and USA isolates and produced single unique variation at position 344 where adenine was substituted with guanine. There was no deletion and addition between nucleotide sequences in the group of Indian isolates, further confirms the placement of the soybean isolate of TSV in a single subgroup. Key words: Soybean, Tobacco streak virus, coat protein gene, diversity analysis.

Deteksi Virus Tungro pada Gulma Padi Sawah Menggunakan Teknik PCR

Virus tungro disease is a serious problem to rice crop in a certain area of rice production in Indonesia. The disease is caused by a combined infection of Rice Tungro Bacilliform Virus (RTBV) and Rice Tungro Spherical Virus (RTSV). Both viruses were reported to infect ratoon rice plants, weeds, and wild rice. The study was conducted to detect RTBV and RTSV on some weeds. Weed samples were collected from rice fields in West Java, Bali, West Nusa Tenggara, Papua, and West Sumatera. The detection of RTBV and RTSV were carried out using Polymerase Chain Reaction (PCR) and Reverse Transcription (RT) – PCR, employing coat protein gene specific primers. RTBV specific DNA fragment of ~1400 bp size was successfully amplified from various weed species including: F. miliacea, C. iria, M. vaginalis, L. adscendens, S. zeylanica, D. sanguinalis, and E. crusgalli. RTSV specific DNA fragment of ~787 bp size was successfully amplified from weed species of F. miliacea, L. octovalvis, and D. sanguinalis. RTBV or RTSV specific DNA fragment was not amplified from L. flava and P. distichum. Weed samples infected by both viruses did not show any tungro symptom. Virus detection based on molecular technique was able to determine the status of weed whether it is as an alternate host of viruses. Weeds sanitation prior to rice planting, therefore, should be considered as an integral part of virus disease management.

Molecular Detection and Identification of Begomovirus Isolate on Tomato from Central Region of India

The natural occurrence of leaf curl and blisters disease on tomato was observed at Bhopal, India in the January, 2015. The begomovirus was amplified on symptomatic tomato by polymerase chain reaction (PCR) using coat protein gene specific primers. The purified PCR product (~800 bp) was sequenced and submitted in GenBank database (KU760803) and identified by their sequence analyses. The isolate under study (KU760803) showed 97% to 99% sequence identities and closest phylogenetic relationships with various isolates of Tomato leaf curl New Delhi virus (ToLCNDV), therefore, the isolates under study were identified as isolates of ToLCNDV associated with leaf curl and blister disease on tomato in the first time from central region of India.

Seed-borne nature of a begomovirus, Mung bean yellow mosaic virus in black gram.

The yellow mosaic viruses (YMV) infecting legumes are considered to be the most devastating begomoviruses as they incite considerable yield loss. The yellow discoloration of pods and seeds of infected plants and symptom emergence in the very first trifoliate leaf of the plants in the field were suggestive that the virus may be seed borne, which was investigated in the present study. The distribution of the virus in various parts of the seeds of black gram (Vigna mungo L. Hepper) plants naturally infected in the field was determined by polymerase chain reaction (PCR), Southern blot analysis, and sequencing. Nucleotide sequencing of the PCR amplicons from the seed parts from groups of ten seeds revealed the presence of mung bean yellow mosaic virus (MYMV) in the seed coat, cotyledon, and embryonic axes. The presence of virion particles was confirmed through double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) even in a single whole seed. In confocal microscopy, positive fluorescent signals were obtained using coat protein gene-specific primers in the embryonic axes. However, in the growth tests performed with the same batch of seeds, there was no symptom development in the seedlings though the virus (both DNA A and B components) was detected in 32 % of tested seedlings. In this study, the MYMV was detected in seed coat, cotyledon, and embryo. This study revealed that the MYMV is a seed-borne virus.

New Record of Catharanthus yellow mosaic virus and a Betasatellite Associated with Lethal Leaf Yellowing of Kalmegh (Andrographis paniculata) in Northern India

Andrographis paniculata (Family Acanthaceae), also called Kalmegh, is a medicinal herb in India well-known for its various pharmaceutical properties (1). In August 2012, during a survey in the northern parts of India, several Kalmegh plants in Barabanki District of Uttar Pradesh Province showed typical virus-like symptoms along with prominent lethal leaf yellowing. The infected plants initially showed some chlorotic streaks, which later turned completely yellow, ultimately leading to premature death. Mechanical/sap inoculation failed to transmit the pathogen. Based on the symptomology, a heavy infestation of whiteflies (Bemisia tabaci) in the infected fields, and lack of mechanical transmission, the association of a begomovirus was suspected. The disease incidence was calculated to be about 15 to 20% on the basis of plant population. Twenty samples from naturally infected plants of A. paniculata were collected from various field locations. Total genomic DNA from the symptomatic and non-symptomatic samples was isolated by the modified CTAB method (4). The initial PCR-based detection was performed using begomovirus coat protein gene specific primers (forward 5'-ATGGCGAAGCGACCAG-3' and reverse 5'-TTAATTTGTGACCGAATCAT-3'), which generated an amplicon of 771 bp in most of the (17/20) symptomatic samples. No amplification was obtained in healthy or non-symptomatic plant samples. The full-length genome was amplified via rolling-circle amplification (RCA) according to the manufacturer's instructions using random hexamer primers and φ29 DNA polymerase. A portion of the RCA product (1 μl) was subjected to digestion with different restriction enzymes, out of which BamHI yielded DNA fragments of approximately 2.7 and 1.3 kb, corresponding to DNA-A and β satellite molecules, respectively. These fragments were eluted from the gel and cloned into the suitable restriction site of pGreen0029 vector. The positive clones were checked by restriction digestion. Twelve out of 20 clones were found to be positive and sequenced. The complete genome sequences of DNA A (2,754 bp) and β (1,366 bp) satellites were deposited in the GenBank database with the accession numbers KM359406 and KM359407, respectively. The absence of DNA-B molecule was ascertained, as no PCR amplification was detected with DNA-B-specific primers. Sequence analysis showed highest nucleotide identity (90%) with Catharanthus yellow mosaic virus (CYMV) (HE580234) and ≤85% identity with other begomoviruses of the database. Sequence analysis of the associated betasatellite showed 96% identity with Andrographis yellow vein leaf curl betasatellite (KC967282). CYMV was first reported on Catharanthus roseus with no associated betasatellite from Pakistan (2). However, this is the first report of CYMV along with a betasatellite infecting A. paniculata in India. Recently a begomovirus (Eclipta yellow vein virus) infection was reported on A. paniculata in association with Andrographis yellow vein leaf curl betasatellite from India for the first time (3); now the crop has also become a host of CYMV. Thus, this study highlights the spread of CYMV from its preliminary host to a new host plant (A. paniculata), across the South Asian countries. Therefore, it is important to take measures for the management of its transmitting vector so as to curtail the spread of the virus to new economically and commercially important crops.

Phylogenetic analysis of American hop latent virus in Humulus lupulus surveyed in Tasmania and Victoria, Australia

During July to August 2013 hop (Humulus lupulus) buds growing in Tasmania (n = 280) and Victoria (n = 200) from nine varieties were sampled and tested for the presence of American hop latent virus (AHLV) by enzyme linked immunosorbent assay and reverse transcription polymerase chain reaction using coat protein gene specific primers. Immunological results showed 153/480 bud samples were positive. RT-PCR resulted in an additional 19 positive samples. Coat protein gene sequence analysis showed 98–99 % identity with AHLV isolates from the USA.

First report of mixed infection of Papaya ringspot virus and phytoplasma in papaya in India.

Papaya plants exhibiting symptoms such as multiple axillary shoots, leaf reduction, mosaic, chlorosis, flattened petiole, oily spots on stem and virescence of flowers were observed in Pune (India). Electron microscopy observations of leaf dip preparations revealed flexuous filamentous particle of 760x12 nm, suggesting the presence of a potyvirus, which was identified as Papaya ringspot virus (PRSV) in ELISA using a specific antiserum. The presence of PRSV was confirmed by RT-PCR using coat protein gene-specific primers that amplified a fragment of ca. 950 bp in size. Sequence analysis of the RT-PCR product (GenBank accession No. KJ142142) showed 87 to 100% identity at the nucleotide level with other Indian PRSV isolates (Jain et al., 2004). The presence of a phytoplasma was tested in symptomatic papaya by PCR using universal P1/P7 and R16mF2/R16mR1 primer pairs. BLAST analysis of the consensus sequence obtained by sequencing PCR products (GenBank accession No. JQ346525) revealed 99% nucleotide sequence identity with Candidatus Phytoplasma aurantifolia, a member of group 16Sr II phytoplasma. Transmission of the phytoplasma to healthy papaya grafted onto infected papaya was obtained four weeks after grafting with the development of multiple axillary shoots. The presence of a phytoplasma and a virus in papaya has previously been reported in Puerto Rico and Cuba (Licha, 2002; Arocha et al., 2009). To our knowledge, this is the first evidence of mixed infection of PRSV and a phytoplasma in papaya from India, although these two pathogens were reported separately earlier (Jain et al., 2004; Rao et al., 2011).

Molecular epidemiology of begomoviruses occurring on some vegetables, grain legume and weed species in the Terai belt of north India

Four vegetable crops (chilli, tomato, potato and sponge gourd), one grain legume (black gram) and two weed species (Ageratum conyzoides and Croton bonplandianum) showing prominent symptoms of Begomovirus were sampled during 2011–2012 from the Bahraich district of the Terai belt in northern India. Total genomic DNA isolated from two of each infected samples were analyzed by PCR analysis using begomovirus coat protein gene specific primers. The amplified products (~775 bp) were cloned and sequenced. Tomato leaf curl New Delhi virus (ToLCNDV) was detected in sponge gourd, chilli, potato and tomato; and Ageratum enation virus (AEV) in black gram, A. conyzoides and C. bonplandianum samples. In phylogenetic analysis, ToLCNDV, and AEV isolates clustered in two distinct clades with their respective begomovirus isolates. This is the first record of tentative identification of AEV infecting black gram and C. bonplandianum in India. The study also revealed the wide occurrence of ToLCNDV in northern India.

Mungbean yellow mosaic India virus is associated with yellow mosaic disease of blackgram (Vigna mungo L.) in Andhra Pradesh, India

To study the variability and to identify the species of Begomovirus associated with yellow mosaic disease of blackgram in Andhra Pradesh, India, infected blackgram samples were collected from six districts belonging to three regions of Andhra Pradesh. The total DNA was isolated by modified CTAB method and amplified with coat protein gene-specific primers (RHA-F and AC abut) resulting in 900 bp gene product. The PCR products were cloned, sequenced and deposited in GenBank. The sequence analysis of six clones showed that the size of amplified CP gene of YMV was 920 bp. Based on nucleotide sequence identity of six isolates representing three regions of Andhra Pradesh, the isolates from Rayalaseema and Telangana region are the same variant of YMV (>99.5% identity) and isolate from coastal Andhra is another variant of YMV (>95.4%) when compared with other region isolates. Comparison of CP gene sequence of YMV-TPT isolate with 27 other isolates in database revealed more than 93.2 and 86.2% identity with MYMIV isolates and less than 80 and 64% identity with MYMY isolates that originate from Indian sub-continent and South-East Asia at nucleotide and amino acid level, respectively. Phylogenetic tree based on CP gene sequences of six isolates with other isolates from GenBank formed unique cluster with MYMIV. Hence the YMV infecting blackgram in Andhra Pradesh is caused by MYMIV rather than MYMY as reported in Tamil Nadu which is adjoining state in southern India.

First Report of Chilli leaf curl India virus Infecting Mentha spicata (Neera) in India.

Mint (Mentha spp.; family Lamiaceae) is an important essential oil-bearing crop cultivated on the Indian subcontinent as a cash crop for the international market and industrial purposes. Since May 2010, typical symptoms such as yellow vein, leaf yellowing, mosaic, crinkling, and cupping were observed, which led to significant yield loss in spearmint (M. spicata var. Neera) at CIMAP experimental fields and farmers' fields of Badaun, Rampur, and Moradabad regions of Uttar Pradesh province, India. Disease incidence was recorded in the range of 40 to 50%. Mentha spp. has been reported to be affected by many viral diseases (3). Due to the absence of fungal/bacterial infection, lack of mechanical transmission of the pathogen, and presence of whiteflies in the fields, the causal pathogen was suspected to be a begomovirus. Total genomic DNA was extracted from the leaves of naturally infected and healthy samples of Mentha by the CTAB protocol. Eighteen symptomatic samples were collected from different location of fields and screened for the presence of begomovirus. DNA from these samples was used as PCR template to amplify a 771-bp fragment using begomovirus coat protein (CP) gene specific primers. Eleven of 18 (61.1%) samples were found positive. PCR products were cloned into the pGEM-T Easy (Promega) and sequenced using the universal M13F/M13R primers showed sequence similarity with Chilli leaf curl India virus. To amplify the full-length DNA-A/B and a possible β-satellite, a second detection method was used: rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA products were digested independently with various restriction enzymes: BamHI, EcoRI, EcoRV, HincII, HindIII, SacI, and KpnI. Digested products were resolved on 1% agarose gel and the bands corresponding to ~2.7 and ~1.3 kb were purified using Nucleospin Gel and PCR Clean-up Kit and cloned into the respective sites of pGreen0029 vector. The sequence of full-length DNA-A (2,749 bp) and β-satellite component (1,347-bp) were obtained and deposited in NCBI GenBank with accession nos. KF312364 and KF364485, respectively. The sequence analysis showed maximum nucleotide identity (99%) with Chilli leaf curl India virus (FM877858) and distant affinities (≤88%) with other begomoviruses. The sequence analysis of isolated β-satellite showed 93% identity with Ageratum yellow vein virus satellite (AJ252072.1). No presence of DNA-B was detected using the universal primer PBL1v2040/PCRc1 (2), thus confirming it to be a monopartite begomovirus (1). Viruliferous whiteflies (Bemisia tabaci) proved Koch's postulation by inducing similar symptoms on healthy plants while aphids (Myzus persicae) failed to transmit the virus. To our knowledge, this is the first report of Chilli leaf curl India virus infecting M. spicata var. Neera in India. Mint is widely grown together with other reported hosts of begomoviruses, and thus could pose a serious threat as future expansion of begomovirus to new crops. Hence, the development of resistant varieties coupled with the implementation of adapted integrated pest management strategies would be essential for successful production of mint crops.

Molecular identification of Cucumber mosaic virus isolates of subgroup IB associated with mosaic disease of eggplant in India

Association of Cucumber mosaic virus (CMV) with severe mosaic disease of eggplant (Solanum melongena L.) collected from Lucknow and Kanpur, India was initially detected by host reaction and serological assay and confirmed by RT-PCR employing coat protein gene specific primers. Further, molecular identification of the virus isolates was done by cloning and sequence analysis of the complete RNA3 genome. Based on 97-99% identities and phylogenetic relationships, the virus isolates infecting eggplant were identified as members of CMV subgroup IB.

Serological Identity of Potato Virus X (PVX) and PCR Characterization of Its Coat Protein (CP) Gene

Potato virus X (PVX) is among top ten most economically damaging plant viruses in the world and its increasing incidence is getting an alarming situation in potato crop of Pakistan. During two consecutive years (2010-11 and 2011-12), the incidence of PVX was recorded in potato fields at Rawalpindi, Islamabad, Faisalabad and Sahiwal. The samples were collected and subjected to Double Antibody Sandwiched (DAS) Enzyme Linked Immunosorbant Assay (ELISA) and average incidence of PVX was determined about 16.86% (OD405nm 1.38) during 2010-11 and 27.10% (OD405nm 0.479) in 2011-12. The infectivity of the virus was assayed through mechanical inoculation on Nicotiana tabacum cv. Samsun, N. rustica, Datura stramonium, Chenopodium sp. Gomphrena and Capsicum annuum producing local lesion, mosaic and mottling symptoms. Coat protein (CP) gene specific sense and antisense primer successfully amplified a 750bp fragments through Polymerase Chain Reaction (PCR) assay.

First Report of Apple Top Working Disease Caused by Viruses (Apple stem grooving virus, Apple chlorotic leaf spot virus, and Apple stem pitting virus) in Apple in India.

Top working of apple (Malus domestica Borkh.) trees of old, unproductive, and less preferred cultivars with the newly introduced spur type commercial cultivars has become a common practice with many growers in the northwestern Himalayan region of India. Typical viral symptoms of curling, puckering, and necrosis on leaves were observed with an incidence of 80% on Red Chief, Super Chief, Scarlet Spur, Schillet Spur, Washington Red Delicious, and many other newly introduced cultivars during surveys conducted in May and June 2009. Leaf samples from top worked trees were tested for the presence of Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) by employing biological detection (herbaceous and woody indicators), double antibody sandwich (DAS)-ELISA), and reverse transcriptase (RT)-PCR based detection. Mechanical transmission to herbaceous indicators produced chlorotic lesions on Chenopodium quinoa and C. amaranticolor, whereas marginal necrosis was induced on Phaseolous vulgaris within 9 to 21 days after sap inoculations. All three viruses, i.e., ASGV, ASPV, and ACLSV, were detected from these herbaceous indicators in DAS-ELISA (BIOREBA AG, Switzerland). Furthermore, symptoms similar to those observed in orchards were produced when the test budwood was inoculated onto the woody indicator (M. pumila 'Spy 227') plant by double grafting, grafting cum budding, and double budding methods within time periods ranging from 4 months in double grafting, 5 months in double budding, to 1 year 4 months in the grafting cum budding method. The presence of all three viruses was confirmed by DAS-ELISA again in Spy 227 woody indicator. PCR detection was carried out by using the coat protein gene specific primers (ASGV5641 [forward], ASGV6396 [reverse]; ACLSV6784 [forward], ACLSV7365 [reverse] [2]; ASP-C [sense], ASP-A [anti-sense] [1]) of all the viruses detected through ELISA. The amplified products were cloned, sequenced, and deposited in NCBI (GenBank Accessions KC110892 for ASGV, KC154859 for ASPV, and KC154862 for ACLSV). BLASTn analysis showed the ASGV isolate had 97 to 98% sequence identity with Indian (FM204881) and Brazilian (AF438409) ASGV isolates. The ASPV and ACLSV isolates had 98% and 99% sequence identity with Chinese (JF895517) and Japanese (AB326230) isolates, respectively. To the best of our knowledge, this is the first report of apple top working disease associated with ASGV, ASPV, and ACLSV infection in commercial cultivars of apple from India and seems to be a serious threat for growing virus-free healthy stocks in orchards. Top working disease in apple associated with ASGV, ASPV, and ACLSV viruses has been reported from Japan (3,4).