Coat Protein Gene Specific Primers Research Articles

First Report of Natural Infection of Watermelon mosaic virus (WMV) Infecting Bottle Gourd and Snake Melon

Cucurbitaceous crops, one of the main crops of agriculture, are sensitive to many plant viruses. In August 2019, virus-like symptoms were observed on some cucurbit plants grown in private home gardens in Antalya and Denizli provinces (Turkey). A total of 53 leaf samples were sampled from plants with the most symptoms (melon (Cucumis melo L.), watermelon (Citrullus lanatus L.), bottle gourd (Lagenaria siceraria (Molina) Standl.), and snake melon (Cucumis melo var. flexuosus) and tested by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) against possible watermelon mosaic potyvirus (WMV) infection. The coat protein gene (CP) specific primer sets amplified a gene product of nearly 820 bp fragment from symptomatic plants. WMV infections were detected in 31 individual cucurbit plants, including 11 melons, 8 watermelons, 7 snake melons and 5 bottle gourds. The presence of viral infection was found only in ornamental squash plants in Antalya province and in all cucurbits sampled in Denizli province. To better comprehend the molecular characteristics of virus isolates, the amplified viral DNA fragments were cloned in a proper prokaryotic plasmid, sequenced by Next Generation Sequencing (NGS) and recorded to GenBank. Bioinformatic analyses using the Basic Local Alignment Search Tool (BLAST) showed that the identified CP gene sequences exhibited significant nucleotide homogeneity, supported by a high nucleotide similarity index with that of other isolates around the world. In addition, Turkish isolates isolated from Antalya and Denizli regions showed approximately 94% nucleotide similarity among themselves. For phylogenetic inference, WMV sequences were subjected to multiple alignments with isolates from different geographic origins of the same viruses. Molecular phylogeny showed that all WMV isolates are closely related to other world WMV isolates at variable rates. WMV is wide host range viruses in cucurbit crops, however, this work is the first scientific report of WMV isolates detected in bottle gourd and snake melon from the South and West Regions of Turkey all over the world.WMV are broad spectrum viruses in cucurbit crops. This work is the first scientific report of Watermelon mosaic potyvirus isolates detected in bottle gourd and snake melon from the South-West Region of Turkey in all over the world.

Distribution of Papaya Ring Spot Virus Infecting Papaya in Kerala, India

In the midst of the rising interest of papaya cultivation in Kerala, it is inevitable to study the current status of the Papaya ring spot disease in the state. Knowing the severity and distribution of PRSV isolates, causing ring spot disease, helps in the better formulation of effective management strategies against it. Purposive sampling surveys carried out in 2021-22 across five agro ecological units (AEUs) of Kerala, recorded papaya ring spot disease incidence (PRSD) ranging from 50 to 100 per cent in the cultivated areas with vulnerability index of 33.54 to 98.22. Highest disease incidence was recorded from AEU 8- Southern Laterites: Thiruvananthapuram (100.0%) and highest vulnerability index was recorded from Kalliyoor (98.22). Twenty symptomatic samples tested positive for PRSV in double antibody sandwich- ELISA (DAS-ELISA) using PRSV polyclonal antiserum. RT-PCR using coat protein gene specific primers RKJ 52 and 3 yielded amplicons of size approximately ~890bp in all the samples. Virulence of the Kerala isolates was evaluated based on the symptom expression, disease incidence and vulnerability index, upon mechanical transmission of PRSV on two months old papaya plants (variety- Red Lady). PRSV isolate from Kalliyoor (TVM1) inoculated on papaya plants expressed chlorosis, mottling, malformation of leaves and stunting with 96.80 vulnerability index. Maximum vulnerability index and severe symptoms including stunting in the inoculated plants were observed in isolates from southern and central Kerala, which include Thiruvananthapuram, Alappuzha and Thrissur had the most virulent PRSV isolates compared to north Kerala.

Vasconcellea cauliflora – a new natural host of cucumber mosaic virus

Vasconcellea cauliflora plants exhibiting chlorotic spots, mottling and mosaic with yellowing on leaves were observed in Pune, India in October 2020. The disease incidence was 16%. The samples were screened for cucumber mosaic virus (CMV), papaya ringspot virus, tobacco mosaic virus and zucchini yellow mosaic virus using ELISA. Sap inoculation on healthy V. cauliflora plants using CMV positive DAS-ELISA samples reproduced field symptoms. The transmission upon mechanical inoculation was 100%. For further confirmation, RT-PCR was carried out using CMV coat protein gene specific primers. Amplicons of expected size (c. 650 bp) were obtained in all symptomatic and sap inoculated samples. No amplification was observed in the asymptomatic samples. BLASTn analysis revealed that the amplicon sequence shares more than 99% identity with CMV isolates and in phylogenetic analysis it clustered with CMV IB isolates. This is the first record of V. cauliflora as a natural host of CMV worldwide.

Molecular Identification of Tomato Leaf Curl New Delhi Virus Associated with Mosaic Disease of Pumpkin from Central India

The Tomato leaf curl New Delhi virus is an emerging disease of pumpkin associated with mosaic disease in central, India. Symptomatic pumpkin leaf samples from different locations in the Bhopal district were collected and confirmed for begomovirus disease by PCR using a begomovirus coat-protein gene-specific primer. All five samples were amplified, expecting ~800 bp of amplicons of associated begomovirus; these amplicons were purified and sequenced for identification of begomovirus species associated with mosaic disease on the pumpkin. The consensus nucleotide sequence data were submitted to the NCBI database under the accession numbers OQ320768, OQ320770, OQ320774, OQ116977, and OQ116978. The understudy begomovirus isolates showed the highest 95–97% nucleotide sequence identities and close phylogenetic relationships with several isolates of Tomato leaf curl New Delhi virus. To our knowledge, this was the first record of the Tomato leaf curl New Delhi virus associated with the mosaic disease of pumpkin in Central India.

Molecular detection of rice tungro bacilliform virus (RTBV) on weed host in South Sulawesi

Rice tungro bacilliform Virus (RTBV) is one of the viruses that cause tungro disease in rice plants causing leaf yellow and plant stunting. Some weed species have also been described as potential hosts for RTBV in addition to rice. However, RTBV cannot be diagnosed in weeds only by looking at their morphological symptoms because weeds infected with RTBV do not show typical symptoms. Hence this research was conducted to detect the presence of RTBV molecularly on several weed hosts in rice fields in South Sulawesi. The polymerase Chain Reaction technique with coat protein gene-specific primers was used to detect RTBV. A total of 29 leaf samples consisting of 12 weed species collected from Sidrap, Pinrang, Maros, and Gowa were then extracted genomic DNA from the leaf samples for PCR analysis. The results of PCR analysis showed that weed samples that tested positive for RTBV were Panicum repens (Lawawoi/Sidrap), Echinochloa crusgalli (Lawawoi/Sidrap), Cyperus difformis (Lanrang/Sidrap), Echinochloa crusgalli (Palangga/Gowa), Fimbristilys miliacea (Lanrang/Sidrap), and Eleusine indica (Lawawoi/Sidrap) which were indicated by the presence of RTBV virus DNA fragments with 430 bp in length in all these weeds. Weeds infected by RTBV formed 2 clusters. The first cluster consisted of RTBV in P.repens and E.crusgalli from Lawawoi/Sidrap, RTBV in C.difformis and F.miliacea from Lanrang/Sidrap, and RTBV in E.indica from Palangga/Gowa. At the same time, the second cluster consisted of RTBV in E.indica from Lawawoi/Sidrap.

First Report of Cucurbit Chlorotic Yellows Virus Affecting Squash and Pumpkin in Florida

A field survey was conducted in Florida during 2020-2021 in squash and pumpkin research fields to identify the viruses associated with leaf symptoms of yellowing, crumpling, and vein yellowing. The symptoms were similar to previously reported whitefly transmitted viruses such as cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV), and squash vein yellowing virus (SqVYV). Another potential virus of interest was the cucurbit chlorotic yellows virus (CCYV) that was recently reported on watermelon in Florida. Symptomatic leaves were tested by RT-PCR with coat protein (CP) gene-specific (GS) primers for CuLCrV, CYSDV, CCYV, and SqVYV with nuclear inclusion protein (NIa) GS primers. Amplifications for CuLCrV, CYSDV, and CCYV were detected in squash and pumpkin samples. The CCYV amplicons were further sequenced and compared with available CCYV sequences. The NCBI BLAST analysis revealed similarities of the RdRP (100%), HSP70h (99.75%), and CP (99.75%) to the Shanghai CCYV isolates (RNA KY400636 and RNA2 KY400633). To our knowledge, this is the first report of CCYV on squash and pumpkin in Florida.

First report of a begomovirus infecting Ocimum kilimandscharicum in India

Ocimum kilimandscharicum, commonly known as Camphor basil, is a perennial evergreen shrub in the family Lamiaceae. Its essential oil possesses a wide range of pharmacological properties with anti-malarial, antioxidant, anti-microbial, and wound healing attributes (Soni et al., 2012). During a survey in February 2020, an infestation of whiteflies and viral symptoms, including leaf mottling, yellowing, mosaic, curling and deformation (Fig. 1), were observed on the leaves of O. kilimandscharicum in the experimental fields of the CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India. The disease was observed in patches of plants in different parts of the field (an area of c. 80 m2 in total) and disease incidence was estimated to be approximately 20%. The disease was not transmissible via mechanical inoculation with sap obtained from infected leaf tissues to O. kilimandscharicum, Nicotiana benthamiana or N. glutinosa. Eighteen samples (13 diseased and five asymptomatic) were collected from different locations of the field and total genomic DNA was isolated. PCR was done using begomovirus coat protein gene-specific primers (Hallan et al., 1998). All 13 diseased samples yielded an amplicon of c. 770 bp while no amplification was observed from the five asymptomatic samples. The positive samples were subjected to PCR using two sets of overlapping primers K1F/K1R and K2F/K2R for complete genomic characterisation of the DNA-A segment (Kumar et al., 2011). The samples yielded amplicons of c. 1,200 bp and 1,700 bp, respectively. The PCR products were cloned and sequenced. BlastN analysis revealed 97.6% identity with the DNA-A segment of Tomato leaf curl New Delhi virus (ToLCNDV) from lentil isolated in Kanpur, India (Naimuddin et al., 2016). The sequence was submitted to GenBank (Accession no. OP589304). Phylogenetic analysis was performed with different begomovirus isolates (Table 1) using MEGA 7 software (Tamura et al. 2004). The isolate in the current study and ToLCNDV ex lentil share a common ancestor and fall in the same clade (Fig. 2). Ongoing increases in the incidence of begomovirus infection in economically important crops are becoming a major concern globally. To the best of our knowledge this is the first record of a begomovirus infecting O. kilimandscharicum. Camphor basil is commonly grown in kitchen gardens and commercial fields, and infected plants may serve as a virus reservoir for other hosts, therefore, management strategies are required. We are thankful to the Director, CIMAP, Lucknow for the necessary facilities and DST-New Delhi for INSPIRE-SRF to Soumya Sinha.

Seed Transmission Behaviour of Bean Common Mosaic Virus in Green Gram

Background: The virus-distribution and bimodal seed transmission of BCMV in green gram was studied. The Bean common mosaic virus (BCMV) infecting leguminous crops have been found to be the most devastating Potyvirus as they cause considerable yield loss. Emergence of symptom in the first trifoliate leaf stage of the plants under the natural condition confirms that the virus may be seed borne, which has been investigated in the present study. Methods: The distribution of the virus in various parts of the seeds of mung bean (Vigna radiata) plants naturally infected in the field was determined by ELISA, polymerase chain reaction (PCR) and sequencing. Result: Nucleotide sequencing of the PCR amplicons from the seed parts from groups of ten seeds revealed the presence of Bean common mosaic virus (BCMV) in the seed coat, cotyledon and embryonic axes. The grow out test performed with the same batch of seeds, there was symptom development after 3 weeks of sowing and the presence of virus was detected in all the seedlings through ELISA test. RT-PCR amplification of viral cDNA from whole seed, seed coat, cotyledons and embryo generated an amplicon of ~1300 with BCMV coat protein gene specific primer. The genomic sequence (GenBank accession No. ON944468) showed highest nucleotide identity of 90-91% sequence similarities with NL-1 strain (KF114860.1) and 94% sequence similarities with MY15-014 strain (MW079241.1) of previously available BCMV- CP virus gene sequence.

Molecular evidence for the occurrence of Pumpkin yellows virus (PuYV), a new polerovirus species associated with pumpkin plants.

In India, cucurbit aphid-borne yellows virus, a cucurbit-infecting polerovirus, is emerging in the recent past with respect to its occurrence on different crops and geographical locations. This emphasized the need for an investigation to search for the possible occurrence of the other related species Polerovirus. In this view, different cucurbit hosts exhibiting severe chlorosis, bleaching, and yellowing symptoms were collected from the Vegetable Experimental Fields of IARI, New Delhi. The samples exhibiting yellowing and bleaching symptoms were associated with small isometric virions measuring ~ 25nm under transmission electron microscope. The RT-PCR assays using generic (covering the partial RdRp, intergenic region and partial CP region) andcomplete coat protein (CP) gene-specific primers confirmed the association of a polerovirus. Further, complete CP genesequence analyses revealed the association of a distinct species of Polerovirus with the pumpkin samples. These isolates showed the CP gene sequence identities below the species demarcation limit (90%) with the corresponding gene sequences of already reported polerovirus isolates. The results of this study provide the molecular evidence for the occurrence of a new species of Polerovirus, named tentatively as Pumpkin yellows virus (PuYV) in India.

First Report of Cucurbit Chlorotic Yellows Virus Infecting Pumpkin in India.

Pumpkin (Cucurbita moschata), a member of the family Cucurbitaceae, is widely cultivated throughout the world including India. During August 2020 to January 2021, stunted pumpkin plants (cv. Pusa Vishwas), showing chlorotic patches, mosaic, and vein banding on leaves (e-Xtra Fig.1), were observed in the experimental fields of the Indian Agricultural Research Institute (IARI), New Delhi, India. Leaf-dip electron microscopy (EM) of the symptomatic plants (12 out of 37 samples) revealed the association of long flexuous virus particles measuring 650-950nm×10-12nm, suggestive of the presence of either crinivirus or potyvirus or both. Subsequently, a reverse transcription-polymerase chain reaction (RT-PCR) was performed on RNA extracted from the samples that had long flexuous virus particles using generic primers for criniviruses i.e. CriniPol-F: GCY CCS AGR GTK AAT GA and CriniPol-R: ACC TTG RGA YTT RTC AAA targeting partial RNA-dependent RNA polymerase coding region (Martin et al. 2003) and specific primers for papaya ringspot virus (PRSV) targeting a part of 3' NIb and full coat protein (CP) gene (Basavaraj et al., 2019) separately. All tested samples were positive for both crinivirus and PRSV as expected size amplicons were obtained, accounting for about 32% prevalence. As PRSV is a well-studied virus infecting cucurbits, further work was not carried on this virus and only the RT-PCR amplicon indicative of crinivirus (~515 bp) was cloned into the pGEM-T easy cloning vector (Promega, Madison, WI) and sequenced for further confirmation of the virus presence. The obtained sequence (GenBank accession No MZ318672) shared up to 90% nucleotide and 100% amino acid sequence identity with the corresponding genomic region of a cucurbit chlorotic yellows virus (CCYV) isolate from Greece (LT841297). To confirm the identity of the crinivirus species present in the same pumpkin sample, the CP gene (753bp) was amplified and sequenced using CCYV CP gene-specific primers CP-F (5'-ATG GAG AAG ACY GAC AAT AAA CAA AAT GAT GA-3') and CP-R (5'-TTA TTT ACT ACA ACC TCC CGG TGC CAA C-3') (modified from Kheireddine et al. 2020). Sequence analysis using the BioEdit tool (version 2.0) revealed that the crinivirus present in pumpkin (KC577202) shared 95 to 100% nucleotide (and 98 to 100% amino acid) sequence identity with the corresponding gene sequences of CCYV isolates originating from cucurbitaceous hosts from diverse locations. The presence of CCYV was further validated by a whitefly transmission-based bioassay followed by RT-PCR confirmation. The bioassay was performed by the whitefly species Bemisia tabaci (biotype Asia II7) using the acquisition access period and inoculation access period of 24 hours each. Six whitefly individuals per plant were used for inoculating ten pumpkin plants (cv. Pusa Vishwas) at the first true leaf stage grown in pots containing soilrite as the medium in insect-proof cages. All ten plants inoculated using whiteflies exhibited chlorosis and stunting symptoms 12-15 days post-inoculation (e-Xtra Fig.2) and were found positive for CCYV in RT-PCR assay performed using CCYV CP gene-specific primers. Though CCYV had been reported worldwide (Tzanetakis et al. 2013), its occurrence had not been reported from India. Results of the present study confirm the infection of pumpkin plants by CCYV and constitute the first report of its presence in India. Further, there is a need to investigate the extent of its spread and impact of this virus on the production of cucurbitaceous crops in the country.

Distribution and molecular characterization of potato virus Y (PVY) strains infecting potato (Solanum tuberosum) crop in Kashmir (India).

The online version contains supplementary material available at 10.1007/s13337-021-00722-2.

Co-existence of chlorosis inducing strain of Cucumber mosaic virus with tospoviruses on hot pepper (Capsicum annuum) in India

Cucumo- and tospoviruses are the most destructive viruses infecting hot pepper (chilli). A diagnostic survey was conducted to assess the prevalence of cucumo and tospoviruses in chilli growing tracts of Tamil Nadu. Infected plants showing mosaic with chlorotic and necrotic rings, veinal necrosis, mosaic mottling, leaf filiformity and malformation were collected. Molecular indexing carried out through reverse transcription polymerase chain reaction (RT-PCR) with coat protein gene specific primer of Cucumber mosaic virus (CMV) and tospovirus degenerate primer corresponding to the L segment (RdRp). Ostensibly, amplifications were observed for both CMV and tospoviruses as sole as well for mixed infections. The sequence analysis indicated that the Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) to be involved with CMV in causing combined infections. The co-infection of CMV with CaCV was detected in 10.41% of the symptomatic plant samples and combined infection of CMV with GBNV was recorded in around 6.25% of the symptomatic plants surveyed. The amino acid substitution of Ser129 over conserved Pro129 in coat protein of CMV implies that CMV strain involved in mixed infection as chlorosis inducing strain. Further, the electron microscopy of symptomatic plant samples explicated the presence of isometric particles of CMV and quasi spherical particles of tospoviruses. This is the first molecular evidence for the natural co-existence of chlorosis inducing CMV strain with CaCV and GBNV on hot pepper in India.

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah, Saudi Arabia.

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

The molecular characterization of the coat protein sequence and differentiation of CMV- subgroup I on tobacco from native flora in Turkey

Cucumber mosaic virus (CMV) has a broad plant-host range and a wide ecological zone distribution. Virus-like symptoms were observed on tobacco fields of Adiyaman province (Turkey) showing conspicuous mottling, greenish mosaic patterns and severe malformations of leaves. A total of forty tobacco samples tested positive against CMV by reverse transcription polymerase chain reaction (RT-PCR) using coat protein gene specific primers. Five randomly chosen CMV isolates were cloned into pGEM T-Easy vector and transformed into Escherichia coli JM109 strain. The recombinant bacterial clones containing insert-DNA were further purified and sequenced bidirectionally. In multiplex-RT-PCR studies carried out, it was found that all 40 CMV isolates belong to Subgroup I by resulting a 593 bp long DNA fragments. CMV subgroup IA was found to predominate in 4 out of 5 tobacco samples and CMV subgroup IB was found in 1 out of 5 CMV-positive samples by comparing the isolates with CMV reference isolates in phylogenetic tree. However, no Subgroup II sequences were found by multiplex RT-PCR using discriminating primers. The nucleic acid sequences were analyzed for the investigation of diversity of coat protein (CP) sequences of 5 CMV isolates. The sequence similarity ranged from 94.2-100% with the CMV subgroup I isolates infecting diverse plants in other regions of the world. The evolutionary tree revealed that the CMV IA Adiyaman isolates exhibited a genetic affinity with Australian and Spanish isolates. However, the CMV IB Adiyaman isolate showed a close genetic relationship with only the Australian isolates. To our knowledge, this study shows for the first time the occurrence of CMV IA and IB isolates infecting cultured tobacco plants in Adiyaman province.

Evidence for the association of mastrevirus, cucumovirus and phytoplasma with chickpea stunt disease and their putative insect vectors in India

Symptoms of severe stunting, little leaf, yellowing and axillary shoot proliferation were recorded on chickpea plants in three northern states of India (New Delhi, Uttar Pradesh and Rajasthan) during January–March, 2017–2018 with disease incidences of 6–56%. Nested PCR assays with phytoplasma-specific universal primer pairs (P1/P7 and 3Far/3Rev) and 16S rRNA gene sequence analysis confirmed the association of ‘Candidatus Phytoplasma trifolii’ in the symptomatic chickpea variety Pusa Bheema at New Delhi. The in silico RFLP and phylogeny analyses of 16S rRNA sequence of the chickpea stunt (CpS) affected samples confirmed the association of 16SrVI-D subgroup phytoplasma. Similar phytoplasma strain (16SrVI-D subgroup) was detected in the leafhopper species, Hishimonus phycitis collected from symptomatic chickpea fields at New Delhi. Association of icosahedral virus particles of ~25–30 nm diameters were seen under electron microscope in CpS affected samples as well as in the aphids, Myzus persicae (Sulzer) collected from the symptomatic CpS samples at New Delhi. Both the plant and aphid samples were tested positive with the polyclonal antibodies (pAbs) of cucumber mosaic virus (CMV) in DAS-ELISA and ISEM tests. PCR assays using coat protein (CP) gene specific primers for CMV and the resultant sequence comparison also confirmed the association of CMV with the CpS affected symptomatic chickpea plants and M. persicae. The CpS affected samples collected from all the three states were further tested positive with chickpea chlorotic dwarf virus (chickpea chlorotic dwarf virus, CpCDV) CP gene specific primers in PCR assays. The 2.7 kb DNA sequence resulted from the rolling circle amplification (RCA) from the CpS affected chickpea samples of New Delhi revealed a highest sequence similarity of 99% with the CpCDV isolate from Pakistan. These results suggested a wider occurrence of CpCDV in chickpea crops in all the three states and a mixed infection of phytoplasma [‘Ca. P. trifolii’ (16SrVI-D subgroup)] and two plant viruses (CMV and CpCDV) associated with CpS disease at New Delhi.

RT-PCR Based Cloning and Sequencing of Potato Leaf Roll Virus-Coat Protein (PLRV-CP) Gene for Characterization as a Bangladeshi PLRV Isolate and Its Phylogenetic Analysis

An experiment was conducted in Molecular Biology and Plant Virology Laboratory under the Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh. Total RNA was extracted from Potato leaf roll virus (PLRV) positive leaves and complementary DNA (cDNA) were synthesized from total RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) based detection conditions were optimized by using coat protein (CP) gene specific primers. In PCR amplification cDNA and in nucleotide sequencing PCR product was used as a template. A 346 bp amplicon of PLRV- CP gene was amplified and amplified gene region was sequenced. The expected nucleotide sequence of amplified PLRV-CP gene showed 95 to 98% homology when compared with the isolates sequences reported in Gene Bank database. This explored novel PLRV-CP gene was characterized as a PLRV Bangladeshi isolate (Accession number, Bankit 2274496, MN605963). PLRV-CP gene protein modeling was carried out using Expert Protein Analysis System (ExPaSy), DNASTAR’s protein tools server used for 3D protein modeling. Phylogenetic analysis was also carried out, the tree was made by using MEGA 4.0 software and maximum parsimony method was selected to construct phylogenetic tree. The RT-PCR based molecular technique optimized in this study, would be a useful for early detection, epidemiological studies of PLRV as well as in seed tubers certification program and the novel hyper variable sequenced region of PLRV-CP gene will be useful in pathogen derived resistance breeding program against the PLRV local strain.

First Report of Tomato Leaf Curl New Delhi Virus Infecting Crossandra infundibuliformis in India

Crossandra infundibuliformis (L.), commonly known as firecracker flower, is a common flowering plant belonging to the family Acanthaceae. It is commonly cultivated in southern India for its attractive flowers. Symptoms of leaf yellowing, leaf mottling, leaf curling, and distortion as well as stunted growth and flower abortion were observed in C. infundibuliformis plants grown in Dharmapuri district (Tamil Nadu, India). In 2011, we observed the first symptom of leaf curl disease, and a partial sequence of tomato leaf curl New Delhi virus (ToLCNDV) was deposited in GenBank (JN410650). Further, in a survey conducted from 2013 to 2015, a total of 30 symptomatic samples suspected of begomovirus infection were collected along with a nonsymptomatic sample. In particular, intact twin icosahedral geminivirus particles (∼18 × 30 nm) were observed by high-resolution transmission electron microscopy in a partially purified extract from symptomatic leaves. Total DNA was extracted and subjected to PCR using coat protein gene-specific degenerate primers (FP/RP) (Riyaz et al. 2013) and replication-associated protein gene-specific degenerate primers ToLCNDVRepFP (5′-ATGAAGTAWGAACAGCCRCAC-3′) and ToLCNDVRepRP (5′-CCATCCGAACATTCAGGGAG-3′). PCR products of the expected sizes of ∼500 and ∼850 bp were obtained from 25 out of 30 plants, and respective PCR products were eluted from gels and sequenced. In sequence analysis, the isolate from C. infundibuliformis (CI1) showed 89 to 92% nucleotide identity with the ToLCNDV isolates reported earlier from India (KM269361, KM269355, and KP868764). Rolling circle amplification (RCA) was performed using a TempliPhi kit (GE Healthcare). RCA products were subjected to PCR amplification employing abutting primers AKKCI1F (5′-ACCTAATGGCAGAATTGTAATTTTG-3′) and AKKCI1R (5′-AATATTATACGAATGGCCGCTTTAG-3′) for DNA-A and BKKCI1F (5′-ACCGAAAGGCCGCGAAAATTTATCCTG-3′) and BKKCI1R (5′-AATATTAGACGAAAGGCCGCTATGA-3′) for DNA-B. The amplified PCR products were ligated into pJET1.2/blunt vector and cloned using a CloneJet PCR cloning kit (Thermo Scientific, U.S.A.). The clones were sequenced by primer walking, and the corresponding sequences assembled to DNA-A and DNA-B fragments were deposited under GenBank accessions MK395548 and MK395549, respectively. DNA-A (2,742 bp) showed 96% sequence identity with the DNA-A component of a ToLCNDV isolate infecting the bitter gourd crop in India (KP868764), whereas DNA-B (2,694 bp) showed 87% identity with the DNA-B component of a ToLCNDV isolate from India infecting chrysanthemum (MG758147). In addition, association of a betasatellite was also confirmed through PCR assay using universal primer pair Beta01/Beta02 (Briddon et al. 2002), followed by sequencing. The nucleotide sequence of the associated betasatellite (1,352 bp) shared 93.7% identity with the betasatellite component of tomato leaf curl New Delhi betasatellite (ToLCuNDB) reported from New Delhi (JX679002) on tomato. The betasatellite sequence was deposited in GenBank (MK040727). This study records the coexistence of bipartite ToLCNDV and ToLCuNDB in C. infundibuliformis with the evidence of their full-length genome for the first time from India.

Disease incidence and molecular indexing of viruses infecting King chilli (Capsicum chinense Jacq) in North East India

A survey was conducted in King chilli growing areas of four Northeast states of India namely, Assam, Nagaland, Manipur and Meghalaya for virus disease incidence particularly Cucumber mosaic virus (CMV), Chilli veinal mottle virus (ChiVMV) and Chilli leaf curl virus (ChiLCV). The virus disease incidence based on symptomatology ranged from 15 to 65% with highest incidence recorded in Golaghat district of Assam and lowest in the Ukhrul district of Manipur. The polymerase chain reaction based detection using coat protein gene specific primers for the three viruses revealed the presence of CMV and ChiVMV in all the four states whereas ChiLCV was recorded only in one location from Assam. In two of the samples collected for CMV and ChiVMV, mixed infection of the two viruses was confirmed. Based on molecular method based detection, the incidence of CMV infection in King chilli among all the samples tested for the virus was 32.5% whereas it was 25.97% for ChiVMV.

First Report of Cucurbit Aphid-Borne Yellows Virus in Cucurbita pepo and Cucurbita maxima in Slovenia

First Report of Cucurbit Aphid-Borne Yellows Virus in <i>Cucurbita pepo</i> and <i>Cucurbita maxima</i> in Slovenia