Occurrence and molecular detection of bipartite begomovirus on tomato in Southern India

Abstract

Begomoviruses are major constraint in tomato production across the world. In India, tomato production is affected by both monopartite and bipartite begomoviruses. Monopartite were reported to infect tomato crop across India. However, bipartite were reported to infect tomato in Nothern India. In the current study, seventy begomovirus symptomatic tomato samples were collected from southernstates of India and analysed using PCR-based specific primer. The analysis revealed that 45 of them were monopartite, 23 of them were bipartite and 8 bipartite also showed the presence of betasatellite. This is the first report of bipartite begomovirus infecting tomato in Southern India.     

PCR Based Diagnosis of Begomoviruses Associated with Tomato Leaf Curl Disease in India

This paper reports development of a set of common primers and four sets of virus specific primers to detect ToLCNDV, ToLCBV, ToLCGV and ToLCKV, which are associated with tomato leaf curl disease (TomLCD) in India. The primer sets were first validated by amplification of desired genomic fragments from the cloned genomes of the four begomoviruses and then were tested for the detection of these viruses in field samples collected from different locations in India. Of the 26 TomLCD samples tested by PCR, 22 tested positive with the set of common primers and 16 tested positive for the target begomoviruses. ToLCNDV was detected in four samples, ToLCBV in six, ToLCKV in four, and ToLCGV in five samples. This limited survey shows that ToLCNDV and ToLCBV are distributed in northern and southern India, whereas ToLCKV and ToLCGV have a wider distribution. Mixed infection by two target viruses was observed in three TomLCD samples collected from Maharashtra, Punjab and Uttar Pradesh; two of these samples were infected with ToLCKV, which is prone to recombination. The results of the present study are indicative of association of uncharacterized variants or new begomoviruses with TomLCD in India, as (a) 27% of the samples found positive by the set of common primers did not amplify with species specific primers, and (b) 16% of the samples tested negative by PCR using common primers, although all the samples were collected from plants showing typical TomLCD symptoms. These plants might have been infected by some uncharacterized virus(es).     

Complete Nucleotide Sequence of Ageratum enation virus and an Alphasatellite Infecting a New Host Glycine max in India

During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine max) in Himachal Pradesh, India. PCR‐based detection confirmed the presence of a begomovirus. The viral genome was amplified by rolling circle amplification, cloned and sequenced. The complete nucleotide sequence of DNA‐A showed highest nucleotide identity to an isolate of Ageratum enation virus infecting a weed Ageratum conyzoides. In addition, a DNA molecule was found which shared 95% nucleotide identity with an alphasatellite infecting ageratum. Neither beta satellite nor DNA‐B was detected in the infected samples.     

Book Review

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.     

A Leaf Curl Disease in Germplasm of Rapeseed‐Mustard in India: Molecular Evidence of a Weed‐Infecting Begomovirus–Betasatellite Complex Emerging in a New Crop

Evaluation of 130 accessions of rapeseed‐mustard germplasm grown at the National Bureau of Plant Genetic Resources, New Delhi, India during the winter season (2011–2012) revealed the occurrence of a leaf curl disease in seven accessions. The occurrence of the disease was observed in another 62 of 525 accessions evaluated during 2012–2013. The association of a monopartite begomovirus and betasatellite was established with the symptomatic plants by whitefly transmission and PCR amplification. The complete nucleotide sequences of the begomovirus (JX270684, 2745 nucleotides), obtained by rolling circle amplification, showed the highest sequence identity (98.1%) with the weed‐infecting begomovirus, Croton yellow vein mosaic virus. Analysis of recombination indicated the probable occurrence of many overlapping inter‐ and intraspecific recombination events. The sequence of betasatellite (JX270685, 1355 nucleotides) showed the highest sequence identity (95.7%) with Croton yellow vein mosaic betasatellite. Begomoviruses were not previously known to naturally infect rapeseed‐mustard. This is the first report of the emergence of a weed‐infecting begomovirus–betasatellite complex in rapeseed‐mustard germplasm in India and raises the concern on utilization of such susceptible germplasm in crop improvement programmes.     

Molecular characterization of ‘Clover proliferation’ phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches’ broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of ‘clover proliferation’ (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of ‘clover proliferation’ subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.     

A New PCR Method: One Primer Amplification of PCR-CTPP Products

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.     

Identification of Elm Yellows Phytoplasma in Plum Trees in China

Plum plants (Prunus cerasifera Ehrh) with small and rolled leaves resembling symptoms of phytoplasma infection were observed during 2008 and 2009 in the ornamental garden of Northwest A&F University (Republic of China). Nested polymerase chain reaction (PCR) using a combination of phytoplasma‐specific universal primer pairs (R16F2m/R16R1m‐R16F2n/R16R2) amplified 16S rDNA with the expected size (1.2 kb) from all samples of symptomatic plum plants. Sequencing results and restriction fragment length polymorphism (RFLP) analysis of the 1248 bp R16F2n/R16R2 products showed that the phytoplasma belongs to group 16SrV. Phylogenetic analysis showed that the phytoplasma had a close relation to JWB phytoplasma. This is, we believe, the first report of elm yellows phytoplasma infecting plum plants in China.     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).     

Detection and Characterization of Phytoplasma Associated with Big Bud Disease of Tomato in China

Symptomatic tomato plants exhibiting big bud, proliferation and small leaves of lateral shoots, purplish top leaves, phyllody, enlarged pistils, hypertrophic calyxes and small and polygonal fruit were collected in Yunnan Province of China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants by transmission electron microscopy. The presence of phytoplasma in collected samples was further analysed and identified by PCR and virtual computer‐simulated restriction fragment length polymorphism (virtual RFLP). A 1.2 kb product was amplified by PCR with universal primers R16F2n/R16R2. Sequence comparisons revealed that the tested strains shared 99% 16S rRNA gene sequence similarity with members of ‘Candidatus Phytoplasma aurantifolia’ (16SrII group). Phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences confirmed that the phytoplasma is a member of the 16SrII group. This is the first report of 16SrII group phytoplasma infecting tomato in China.     

Molecular characterizations of two begomoviruses infecting Vinca rosea and Raphanus sativus in India

Dear Editor Samples of Vinca rosea and Raphanus sativus leaves showing typical leaf curling were collected from gardens and fields of Bhatinda,Punjab (India).An expected product of ～550 bp in size was amplified from total DNA extracts of symptomatic leaf samples with universal primers on the coat protein region of begomoviral DNA-A component.Moreover,DNA β were also detected in both V.rosea and R.sativus using β satellite universal primers.This is the first report of a β satellite associated with V.rosea in India.The presence of begomoviruses was also confirmed by Southern blot analysis using cloned DNA-A probe of Papaya leaf curl virus.Sequence analysis of viruses infecting V.rosea (Vinca yellow vein virus) and R.sativus (Raphanus sativus leaf curl Bhatinda virus) showed 74％ and 84％ nucleotide sequence identity with Papaya leaf curl virus,respectively.     

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.     

Infection of tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus with betasatellites, results in enhanced level of helper virus components and antagonistic interaction between DNA B and betasatellites

Tomato leaf curl New Delhi virus (ToLCNDV) (Geminiviridae) is an important pathogen that severely affects tomato production. An extensive survey was carried out during 2003–2010 to study the diversity of begomoviruses found in tomato, potato, and cucurbits that showed symptoms of leaf puckering, distortion, curling, vein clearing, and yellow mosaic in various fields in different regions of India. Ten begomovirus isolates were cloned from infected samples and identified as belonging to the species ToLCNDV. A total of 44 % of the samples showed association of betasatellites, with CLCuMuB and LuLDB being the most frequent. The ToLCNDV cloned component DNA A and DNA B were agroinoculated on Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with or without betasatellites, CLCuMuB or LuLDB. The viral genome levels were then monitored by real-time polymerase chain reaction at different time points of disease development. Plants co-inoculated with betasatellites showed enhanced symptom severity in both N. benthamiana and tomato, as well as increases in helper viral DNA A and DNA B levels. The DNA B and betasatellites acted antagonistically to each other, so that the level of DNA B was 16-fold greater in the presence of betasatellites, while accumulation of betasatellites, CLCuMuB and LuLDB, were reduced by 60 % in the presence of DNA B. DNA B-mediated symptoms predominated in CLCuMuB-inoculated plants, whereas betasatellite-mediated leaf abnormalities were prominent in LuLDB-co-inoculated plants. Inoculation with the cloned components will be a good biotechnological tool in resistance breeding program.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

环境样本中微型和微微型浮游植物高通量测序的引物优化

分别以18Sr DNA的V4区和V9区为目标基因,采用高通量测序平台和生物信息学方法,分析海水样品中微型和微微型浮游植物多样性。利用在线分析软件对V4(F/R)、V9(F/R)和C4(F/R)3对引物的敏感性、特异性进行了评估和比较,发现自行设计的引物V4(F/R)对真核藻类的扩增特异性高于V9(F/R)和C4(F/R)。高通量测序结果显示,检测样品平均获得68834条原始序列,高质量数据占99%以上,获得基因注释的序列数达94%以上。3对引物V4(F/R)、V9(F/R)、C4(F/R)鉴定的平均微型/微微型浮游植物OTUs数分别为78、42、58,引物V4(F/R)鉴定效率相对较高,同时对细小微胞藻(Micromonas pusilla)、(金牛微球藻Ostreococcus tauri)、密球藻(Pycnococcus provasolii)、抑食金球藻(Aureococcus anophagefferens)、赤潮异弯藻(Heterosigma akashiwo)等优势种检出频率高于引物V9(F/R)。相对已发表的2对引物,设计的引物V4(F/R)对海洋微型和微微型藻类多样性检测更为高效。     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

First Report of a Croton yellow vein mosaic virus (CYVMV) Associated with Tomato Leaf Curl Disease in India

Leaf curl disease symptoms were observed in tomato crop grown in a tomato field at Matera district of Bahraich, India, in March 2013 with an 85% disease incidence. The infected plants exhibited leaf curl symptoms accompanied with puckering, vein swelling and stunting of the whole plant. PCR carried out with begomovirus coat protein gene and DNA beta‐specific primer sets resulted in positive amplification of ~775 bp and 1.35 kbp, respectively, with all symptom‐bearing plant samples. BLASTn and phylogenetic analyses of CP gene sequences showed highest and close relationship with Croton yellow vein mosaic virus (CYVMV) isolates, while the phylogenetic study of betasatellite sequence showed distinct relationships with other begomovirus associated betasatellites reported from India and abroad. This is a first report of a CYVMV associated with tomato leaf curl disease in India.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

A mixture of begomoviruses in leaf curl-affected tobacco in Karnataka, South India

Tobacco leaf curl is widespread in several states in India including Andhra Pradesh, Gujarat, Karnataka, Bihar and West Bengal. Tobacco leaf curl virus (TbLCV) isolates collected from five different parts of India induced four distinct symptom phenotypes (group I, II, III & IV) on tobacco cultivars Samsun and Anand 119 (Valand & Muniyappa, 1992). PCR was performed on DNA extracted from group I and IV leaf curl‐affected tobacco from Karnataka, India using degenerate begomovirus‐specific primers. Subsequent cloning and sequencing of PCR products revealed preliminary evidence for the presence of at least three begomoviruses in the affected material following alignment of a 333 bp region of the coat protein gene (CP). The complete CP and common region (CR) of two putative begomoviruses, Tobacco leaf curl virus‐Karnataka1 (TbLCV‐Kar1) and Tobacco leaf curl virus‐Karnataka2 (TbLCV‐Kar2), were sequenced using PCR clones obtained with designed sequence‐specific primers. Phylogenetic analysis of the CP and CR of TbLCV‐Kar1 and TbLCV‐Kar2 placed them in the Asian Old World begomovirus cluster. The two viruses differed from each other significantly in both the CP gene and the CR (< 90% nucleotide sequence identity). This difference, in conjunction with distinct iterative sequences strongly suggests that these begomoviruses are distinct from one another. Group I and IV tobacco were also found to harbour a possible third begomovirus following the 333 bp CP alignment. Comparison of TbLCV‐Kar1 and TbLCV‐Kar2 with other geminiviruses, showed that both sequences shared high nucleotide sequence identity (> 90%) with other begomoviruses in either the CP or CR, thereby suggesting these viruses to be possible strains of other reported begomoviruses. Combined comparison of the CP and CR sequences however, suggests that the two viruses are not strains of other reported begomoviruses, but may be distinct begomoviruses that could have arisen through recombination events during mixed infections. Phylogenetic comparison demonstrated no significant homology between the Indian tobacco begomoviruses and a tobacco‐infecting begomovirus from Zimbabwe, again showing that as with other geminiviruses, there is a geographic basis for phylogenetic relationships rather than an affiliation with tobacco as a host.