Biological and molecular characterization of cucumber mosaic virus isolate causing severe mosaic in Gherkin (Cucumis Anguria L.) in India.
Gherkin (Cucumis anguria L.) is an important cucurbitaceous vegetable crop grown in southern states of India like Andhra Pradesh, Karnataka and Thamil Nadu for slicing and pickling. Not only the gherkins are consumed in India, but also exported to other countries like Russia, followed by USA, Canada and Europe valued upto Rs.502 crores. Bottled gherkins pickled in vinegar contribute nearly 50% of the exports (Sukumaran, 2007). They are usually picked when 4 to 8 cm in length and pickled in jars or cans with vinegar or brine to resemble a pickled cucumber. The term can also be used to refer to the West Indian Burr Gherkin, a related species, originally from West Africa and introduced to the West Indies, probably by the Portuguese (Mugadur and Nittur, 2011). Viruses are the most common causes of diseases affecting cucurbits. These diseases result in losses through reduction in growth and yield and are responsible for distortion and mottling of fruit, making the product unmarketable. More than 25 viruses belonging to genera Cucumo, Como, Tobamo, Poty and Ilarvirus are known to infect cucurbits world wide (Lovisolo, 1980). The mosaic disease in cucurbits was reported to cause by several viruses including members of genera Cucumo, Como, tobamo and potyvirus (Mukhopadhyay, 1985). The genus Cucumovirus is the major virus group infecting cucurbits, of which Cucumber Mosaic virus (CMV) is one of the most wide spread virus in the World infecting over 1000 plant species belonging to more than 85 families (Rossinck, 2002) causing yield losses as high as 40-60% (Varma and Giri, 1998). Although the occurrence of CMV on gherkin have been reported (Rashmi, 2005), but exact identification of the causal virus was remained unaddressed. In this article, we report the occurrence of CMV on the basis of biological and coat protein (CP) gene sequence-based properties, a new record on Cucumis anguria L. in India.
MATERIALS AND METHODS
A roving survey was conducted in the districts of Southern Karnataka viz., Bangalore urban, Bangalore rural, Chikkaballapura, Kolar, Tumkur and also in Chittoor district of Andhra Pradesh to study the incidence of cucumber mosaic virus disease in Gherkin crop. Among the different districts surveyed, the maximum disease incidence was recorded in Kolar (51.27%) followed by Chittoor (40.27%), Tumkur (35.62%), Chikkaballapura (31.45%), Bangalore urban (23.34%) and Bangalore rural (17.15%). Among the varieties grown in the surveyed area, cv. Green long was found highly susceptible to CMV followed by Sparta and Azax and the results were tabulated in Table 1.
The infected plants exhibiting symptoms like mosaic pattern of light and dark green or yellow and green on the leaves, vein banding, malformation of leaves, yellow spotting and stunting were collected and subjected to Double Antibody Sandwich enzyme linked immunosorbent assay (DAS-ELISA) using antisera specific to genus CMV (Fig. 2), a few of them reacted positively with CMV antiserum. Such samples were maintained through mechanical inoculation by extracting sap in chilled 0.1 M phosphate buffer (pH 7) containing 0 x 1% 2-mercaptoethanol in a mortar kept in an ice tray. The extracted sap was rubbed on the leaves of test plants dusted with celite, which were then washed off with tap water after 2-3 min.
The Study was undertaken to know the infectivity of CMV to other host plants. Healthy gherkins and other plant species i.e., Cucumber (Cucumis sativus L.), Ash gourd (Benincasa hispida Thumb.), Ridge gourd (Luffa acutangula L.), Bush squash (Cucurbita pepo var. Clypeata), Muskmelon (Cucumis melo L.), Pumpkin (Cucurbita maxima Duchesne), Round melon (Praecitrullus fistulosus), Watermelon (Citrullus lanatus Thumb.) and bottle gourd (Lagenaria siceraria Standl.) belongs to family Cucurbitaceae, Tobacco (Nicotiana tobaccum L. and Nicotiana glutinosa L.), Tomato (Solanum lycopersicum L.), Chilli (Capsicum annuum L.) and Brinjal (Solanum melongena L.) belongs to Solanaceae, Papaya (Carica papaya) belongs to Caricaceae, Bhendi (Abelmoschus esculentus (L.)) and Cotton (Gossypium hirsutum) belongs to Malvaceae, Castor (Ricinus communis) belongs to Euphorbiaceaae, Lima bean (Phaseolus lunatus) belongs to Fabaceae and Radish (Raphanus sativus) belongs to the family Crucifereceae were raised in polyethylene bags in insect proof glasshouse. Seedlings at two leaf stage were inoculated with standard extract of virus by mechanical sap inoculation as described earlier. In each plant species, ten plants were inoculated and one set of un-inoculated plants were maintained as control. The inoculated plants were kept in the insect proof glass house and examined periodically for symptom expression. The standard extract was also inoculated to local lesion hosts viz., Chenopodium amaranticolor L. belongs to Chenopodiaceae and cowpea (Vigna unguiculata L. Walp.) belongs to Legminosae to confirm the presence of virus. The symptoms expressed by the different plant species were recorded.
Double antibody sandwich ELISA
Double antibody sandwich (DAS) ELISA was done on polystyrene plates using the protocol described by Clark et al. (1986). Wells were initially coated with CMV IgG at 1 1/4g per ml of coating buffer. Antigen was prepared by grinding leaf tissues in 5 vol of PBS-T containing 2% polyvinyl pyrrolidone (PVP) and 0 x 2% BSA followed by centrifugation at 8000 rpm for 1 min. Supernatant obtained was used to load onto ELISA plates. CMV specific alkaline phosphatase conjugate was used at 1 : 200 dilution. One hour after the addition of substrate (p-nitrophenyl phosphate, Genei, Bangalore), the ELISA reactions were read at 405 nm by using an ELISA reader.
RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR)
RNA was extracted using RNA extraction kit (Shrimpex Biotech services Pvt. Ltd.). RT-PCR was performed in the same tube without any buffer changes in between as described by Pappu et al (1993). The primers designed for the CP gene sequences of CMV (based on multiple sequence alignments of CP sequences available in GenBank) were used to prime the amplification. Genome sense primer 5 '-ATGGACAAATCTGAATCAAC-3 ' was derived from the beginning of the first 20 bases of the coding region while antisense primer, 5'TCAAACTGGGAGCACCC-3 ' represented last 17 bases of the coding region of the CP gene. The PCR reaction (25.0/) contained 2.5 1/4l of 10X PCR buffer (Supplied with the enzyme), 2.0/l 25mM Mg[Cl.sub.2], 2.0/l 2mM dNTPs mixture, 0.5 1/4l Forward primer (20pmol/[micro]l), 0.5/l Reverse primer (20pmol/ [micro]l), 0.5 1/4l Taq Polymerase(3U/[micro]l), 5.0/l c-DNA and 12.0 1/4l deionised nuclease free water. The PCR amplification was carried out in a thermal cycler (Eppendorf) with the following conditions; initial denaturation at 94[degrees]C for 3 min. followed by 35 cycles having the following parameters 1 min. of denaturation at 94[degrees]C, 1 min. of annealing at 53[degrees]C and extension for 2 min. at 72[degrees]C followed by a final extension for 10 min. at 72[degrees]C. Amplified DNA fragments were electrophoresed in 0.8 per cent agarose gel.
Sequencing of the CMV CP gene
After successful confirmation for the presence of expected coat protein gene, the DNA was isolated in large scale using RNA extraction kit (Shrimpex Biotech services Pvt. Ltd.) and sequenced using the automated sequencing facility at Chromous Biotech Pvt. Ltd., Bangalore. Sequencing was done in both directions using M 13 forward and reverse primers.
The CP gene sequence obtained was subjected to Basic Local Alignment Search Tool (BLAST) in National Centre for Biotechnological Information (NCBI) Database and was compared with various CMV strains belonging to subgroup I (A and B) and II and also with the CP gene sequences of CMV Indian isolates (Table 2) and were edited by using DNASTAR programme.
Sequence data were compiled using MEGA 6.06 version programme. Multiple sequence alignments were made using Clustal W. The aligned files created by ClustalW were bootstrapped 1000 times for generating neighbour-joining phylogenetic tree using Tree Explorer. Tomato aspermy virus (TAV) CP gene (accession No. AJ550020) was used as outsource (Verma et al., 2006).
In CMV inoculated gherkin cv. Green long, characteristic symptoms of mosaic was developed within 8 to 10 days after inoculation. Initially, alternate green and yellow patches and reduction in leaf size was observed. Later, the infected plants developed symptoms such as vein-banding, severe mosaic and slight puckering with downward curling of the leaves. Often stunting, leaf distortion and wrinkled foliage and mottling were also observed (Fig. 1).
Host Range studies
Among the test plant species inoculated with the crude sap, Ash gourd, bottle gourd, Bush squash, Cucumber, Muskmelon, Pumpkin, Ridge gourd, Round melon and Chilli showed systemic symptoms like mosaic mottling and vein banding and Tomato showed shoestring symptoms upon inoculation. Localized chlorotic followed necrotic lesions were observed on leaves of Chenopodium amaranticolor and Vigna unguiculata L. Walp., four to five days after inoculation (Table 3 & Fig. 3).
Detection of Cucumber Mosaic Virus in Gherkin through DAS-ELISA
DAS-ELISA technique was used for the detection of cucumber mosaic virus of gherkin. Infected samples showed strong positive reaction to Alkaline phosphatase labelled CMV specific antisera. Absorbance values of duplicate wells were recorded at 30 minutes after adding substrate at 405nm. O.D. values of infected samples were 2 to 3 times higher than that of healthy/buffer control samples, which is indicated by high contrast yellow colour (yellowish green) developed by the CMV infected gherkin plants in ELISA (Fig. 2). Amplification of coat protein gene of CMV
The total RNA was isolated from CMV infected gherkin leaf sample and cDNA was synthesized using reverse transcriptase enzyme. The cDNA obtained in RT-PCR step was used as template for converting double stranded DNA by using gene specific forward and reverse primers in the presence of taq DNA polymerase. The PCR successfully amplified the CP gene of ~700 bp from infected gherkin leaf samples confirmed with positive control (Banana). The amplicon of CMV Guntur isolate CP genes were confirmed by electrophoresis and ~700 bp band was found confirming the presence of CP gene (Fig. 4) and no amplification obtained in healthy leaf samples.
Sequence analysis of coat protein gene of CMV
PCR products were purified and sequenced. The size of the product obtained was 631 bp. The coat protein nucleotide sequence of cucumber mosaic virus infecting gherkin was compared with sequences of other CMVs obtained from the NCBI database. Sequence analysis of CP of CMV-Gherkin revealed 79-80 per cent nucleotide homology with CMV strains of subgroup II. However with subgroup IA it showed 89-92 per cent nucleotide homology. CMV-Gherkin showed a high nucleotide homology with the strains of CMV subgroup IB (95-99%). It clearly indicated that test virus (CMV-Gherkin isolate) belong to CMV subgroup IB. Additionally, test virus showed a very high homology and sequence conservation (99%) in terms of nucleotide with the AN strain (subgroup IB) as mentioned in Table 2.
A phylogenetic tree (Fig. 5) constructed using CP gene nucleotide alignment of various strains of CMV and TAV as an out-group also favours the results of sequence similarity of CMV-Gherkin with CMV subgroup IB (AJ810260.1|AN isolate). Clear clusters of CMV subgroups IA, IB and II formed in the dendrogram.
The results presented reveal the occurrence and identification of CMV on gherkin on the basis of biological and CP sequence similarities. The virus was identified as a member of subgroup IB. DAS-ELISA method standardized in the present study could be used to detect CMV infection in gherkin as well as other hosts. This would help in identifying and certifying planting material to check spread of the virus.
In host range studies, Cucumber (Cucumis sativus L.), Ash gourd (Benincasa hispida Thumb.), Ridge gourd (Luffa acutangula L.), Bush squash (Cucurbita pepo var. Clypeata), Muskmelon (C. melo L.), Pumpkin (C. maxima Duchesne), Round melon (Praecitrullus fistulosus), Watermelon (Citrullus lanatus Thumb.) and bottle gourd (Lagenaria siceraria Standl.) belongs to family Cucurbitaceae, Chenopodium amaranticolor L. belongs to Chenopodiaceae, Tobacco (Nicotiana tobaccum L. and N. glutinosa L.), Tomato (Solanum lycopersicum L.), Chilli (Capsicum annuum L.) and Brinjal (Solanum melongena L.) belongs to Solanaceae, Cowpea (Vigna unguiculata L. Walp.) belongs to Legminosae, Papaya (Carica papaya) belongs to Caricaceae, Bhendi (Abelmoschus esculentus (L.)) and Cotton (Gossypium hirsutum) belongs to Malvaceae, Castor (Ricinus communis) belongs to Euphorbiaceae, Lima bean (Phaseolus lunatus) belongs to Fabaceae and Radish (Raphanus sativus) belongs to the family Crucifereceae were inoculated by sap to study the infectivity of CMV to a range of host plants.
The virus produced systemic symptoms viz., chlorotic lesions, severe mosaic mottling and leaf distortion on all cucurbitaceous crops except watermelon at seven to ten days after inoculation. Localized chlorotic lesions followed by necrotic were observed on leaves of C. amaranticolor and Cowpea (V unguiculata L. Walp.) at four to five days after inoculation. However, virus failed to infect solanaceae family species N. tabaccum L., N. glutinosa L., Brinjal, Bhendi and other crops belonging to families Caricaceae, Malvaceae, Euphorbiaceae, Fabaceae and Crucifereceae. The results obtained on host range of CMV are in conformity with the findings of Chandankar et al. (2013), host range of CMV on thirty one crops of different families; eleven crops belonging to Cucurbitaceae, Compositae, Chenopodiaceae, Amaranthaceae, Leguminosae and Solanaceae family showed viral disease expression. However, inoculum failed to induce symptoms on crops belonging to Crucifereae, Caricaceae and Malvaceae.
Use of Alkaline-phosphatase labelled cucumovirus specific antisera was greatly facilitated for identification of the virus. The Double Antibody Sandwich enzyme linked immunosorbent assay (DAS-ELISA) resulted in positive reaction to cucumovirus specific antisera. Several researchers used ELISA for identification of cucumber mosaic virus viz., Korbin and Kaminska (1998), Krstic et al. (2002), Dukic et al. (2002), Sevik et al. (2003), Gholamalizadeh et al. (2008) and Shomaila Iqbal et al. (2012).
Attempts have been made to characterize the CMV at molecular level by amplifying the coat protein gene by using specific primers. The total RNA was isolated from leaf sample of gherkin infected with CMV and cDNA was synthesized using reverse transcriptase enzyme. The cDNA obtained was used as template for converting double stranded DNA using gene specific forward and reverse primers in presence of taq DNA polymerase. The PCR successfully amplified the CP-gene, the amplicons of CP gene of test virus (CMV-gherkin isolate) was estimated electrophoretically as ~700 bp confirming the presence of CP gene. PCR products were purified and sequenced with specific primer to a size of 631 bp.
The gherkin CMV showed sharing 99% homology with CMV isolate AN infecting chilli. Phylogenetic analysis of CP genes of CMV indicated that CMV isolates fall into three subgroups viz., Subgroup IA, IB and II. In the present study, the test virus shares 95-99 per cent nucleotide sequence homology with the subgroup IB, and clustered with the AN isolate belongs to subgroup IB. It clearly indicated that test virus (CMV-Gherkin isolate) belong to CMV subgroup IB.
Similarly, Madhubala et al. (2005) characterized CMV causing mosaic, leaf distortion and stunting in vanilla (Vanilla planifolia Andrews) on the basis of biological and coat protein (CP) nucleotide sequence properties. CP gene of the virus was amplified using RT-PCR and were cloned and sequenced. The sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB.
Hareesh et al. (2006) reported the natural infection of CMV in Indian long Pepper (Piper longum L.) and Betel vine (Piper beetle L.) was detected by RT-PCR. The amplicons of CP gene sequences were cloned and sequenced using the genome sense primer 5' ATGGACAAAT CTGAATCAAC 3' derived from beginning of the first 20 bases of the coding region and the antisense primer, 5' TCAAACTGGGAGCACCC 3' represented last 17 bases of the coding region of the CP gene. Zitikaite and Staniulis (2006) identified the 400bp DNA fragment of CMV infecting cucumber based on cDNA amplified product through PCR using virus-specific oligonucliotides.
The Complete coat protein (CP) gene of CMV infecting banana was sequenced by Shahanavaj Khan et al. (2011). The sequenced regions were found to contain single open reading frame of 657 nucleotides, potentially coding 219 amino acids. Phylogenetic analysis of nucleotide and amino acid sequence of CP gene revealed that CMV infecting banana belonging to IB subgroup.
The authors are thankful to the Department of Plant Pathology, UAS, GKVK, Bangalore for providing the research facilities.
(1.) Chandankar, V.D., Mondhe, M.K., Bhoyar, P.R., Ninawe, B.N., Jadesha, G. Biophysical characterization, host range and transmission studies of Cucumber mosaic virus. The Bioscan., 2013; 12(8) 437-441.
(2.) Clark, M.F., Lister, R.M., Bar Joseph, M. ELISA Techniques. Methods Enzymol., 1986; 118(9) 742-766.
(3.) Dukic, N., Branka Krstic, Ivana Vico, Katis, N.I., Chryssa Papavassiliou et al. Biological and serological characterization of viruses of summer squash crops in Yugoslavia. J. of Agril. Sci., 2002; 47(12) 149-160.
(4.) Gholamalizadeh, R., Vahdat, A., Keshavarz, T., Elahinia, A., Bananej, K. Occurrence and distribution of ten viruses infecting cucurbit plants in Guilan province, Iran. Acta Virol., 2002; 52(28) 113-118.
(5.) Hareesh, P.S., Madhubala, R., Bhat, A.I. Characterization of Cucumber mosaic virus infecting Indian long pepper (Piper longum L.) and betel vine (Piper betle L.) in India. Indian J. of Biotech., 2006; 5(26) 89-93.
(6.) Hsu, H.T., Barzuan, L., Hsu, Y.H., Bliss, W., Perry, K.L. Identification and subgrouping of Cucumber mosaic virus with mouse monoclonal antibodies. Phytopathol., 2000; 90(2) 615-620.
(7.) Korbin, M., Kaminska, M., Characterization of cucumber mosaic cucumovirus isolates, Phytopathologia-Polonica, 1998; 16(8) 71-84.
(8.) Khan, A.A., Sharma, R., Afreen, B., Naqvi, Q.A., Kumar, S., Snehi, S.K., Raj, S.K. Molecular identification of a new isolate of Cucumber mosaic virus subgroup II from basil (Ocimum sanctum) in India. Phytoparasitica, 2011; 39(21) 199-203.
(9.) Krstic B. Branka, Natasa D. Dukia, Nikolaos I. Katis, Janos B. Berenji, Ivana M. Vico, et al, Identification of viruses infecting pumpkins (Cucurbita pepo L.) in Serbia. Proceedings for Natural Sci., 2002; 103(2) 67-79.
(10.) Lovisolo, O., Virus and viroid diseases of cucurbits. Acta Hortic., 1980; 88(10) 33-82.
(11.) Madhubala, R., Bhadramurthy, V., Bhat, A .I., Hareesh, P.S., Retheesh, S.T., et al, Occurrence of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India. J. Biosci., 2005; 30(5) 339-350.
(12.) Mugadur, N.S., Nittur, D.S., Gherkin cultivation in Haveri district of Karnataka: An economic analysis. Contemporary Res. in India, 2011; 1(21) 42-50.
(13.) Mukhopadhyay, S. In Prospectives in Virology, edited by I B M Gupta, B P Singh, H N Verma & K M Srivastava (Print House, Lucknow) 1985; 75-104.
(14.) Palukaitis, P., Garcia Arenal, F. Cucumoviruses. Adv. Virus Res, 2003; 62(6) 241-323.
(15.) Palukaitis, P., Roossinck, M.J., Dietzgen, R.G., Francki, R.I.B., Cucumber mosaic virus. Adv. Virus Res., 1992; 41(2) 281-348.
(16.) Pappu, S.S., Brand, R., Pappu, H.R., Rybicki, E., Gough, K.H., et al, A polymerase chain reaction method adapted for selective cloning of 30 non translated regions of potyviruses: application to dasheen mosaic virus. J. Virol. Methods, 1993; 41(13) 9-20.
(17.) Rashmi, C.M. Characterization of viruses causing mosaic diseases on gherkin (Cucumis anguria L.). M Sc Thesis, University of Agricultural Sciences, Bangalore, India 2005.
(18.) Rossinck, M.J. Evolutionary history of cucumber mosaic virus deduced by phylogenetic analysis. J. Virol., 2002; 76(2) 3382-3387.
(19.) Roossinck, M.J. Cucumoviruses (Bromoviridae)-general features, In Encyclopedia of Virology, 2nd edn, edited by L Granoof & R G Webster (SanDiego: Academic Press) 1999; 315-320.
(20.) Sevik, M.A., Ondokuz Mayis universitesi, Ziraat F akultesi, Bitki Koruma Bolumu. Viruses infecting cucurbits in Samsun, Turkey. Pl. Dis., 2003; 87(3) 341-344.
(21.) Shomaila Iqbal, Muhammad Ashfaq, Hussain Shah, Inam Ul haq M., Aziz Ud Din. Prevalence and distribution of cucumber mosaic virus (CMV) in major chilli growing areas of Pakistan. Pak. J. Bot, 2012; 44(32) 1749-1754.
(22.) Sukumaran, A. Gherkin exports may slow on lower demand from Russia. Live Mint and the Wallstreet J., 2007.
(23.) Varma, A., Giri, B.K. Virus diseases, in Cucurbits. edited by N M Nayar & T A More (Oxford and IBH Publishing Co. Pvt. Ltd, New Delhi) 1998; 225-245.
(24.) Verma, N., Mahinghara, B.K., Ram, R., Zaidi, A.A. Coat protein sequence shows that Cucumber mosaic virus isolate from geraniums (Pelargonium spp.) belongs to subgroup II. J. Biosci, 2006; 31(6) 47-54.
(25.) Zitikaite, I., Staniulis, J. The use of RT-PCR for detection of viruses infecting cucumber. Agronomy res., 2006; 4(2) 471-474.
VV. Kavyashri, Anil Pappachan, A.S. Padmaja, N. Nagaraju and K.T. Rangaswamy
Department of Plant Pathology, UAS, GKVK, Bangalore--560 065, India.
(Received: 10 January 2016; accepted: 09 February 2016)
* To whom all correspondence should be addressed.
Caption: Fig. 1. Mechanically inoculated gherkin plants showing CMV symptoms
Caption: Fig. 2. Detection of CMV from symptomatic gherkin plants through DAS-ELISA. Yellow colour--positive; No colour development--negative
Caption: Fig. 3. Plants exhibiting different kinds of symptoms upon mechanical inoculation with Gherkin isolate of CMV under green house condition
Caption: Fig. 4. Amplification of CP gene from CMV infected gherkin leaf samples
Caption: Fig. 5. Phylogenetic relationship of CMV-Gherkin with the strains of CMV subgroups I (A and B), II and Indian strains based on the amino acid alignment using ClustalW through TreeExplorer. Tomato aspermy virus (TAV) (Acc. No. AJ550020) was used as an outgroup. The numbers below the joining lines are bootstrapping values
Table 1. Incidence of Cucumber mosaic virus on gherkin in different districts of Southern Karnataka Sl. Districts & Name of the Variety/ Disease No. Taluk Location Line * Incidence (%) * 1 Bangalore urban ZARS, GKVK Green long 23.34 Bengalore north 2 Bangalore rural Challahalli Azax 16.00 Doddaballapur Konenahalli Azax 17.33 Linganahalli Sparta 18.12 3 Chikkaballapura Poshottahalli Azax 33.59 Chikkaballapura Rayamakalahalli Green long 44.32 Doddamarali Azax 16.98 Chikkaballapura Gorthapalli Green long 44.32 Baagepalli Bagepalli rural Green long 34.52 Gollapalli Azax 18.48 Chikkaballapura Bommashettihalli Sparta 36.57 Gowribidanuru Machenahalli Sparta 21.63 Kudumalakunte Azax 32.65 4 KolarKolar Gadadasanahalli Green long 56.35 rural Sugatur Green long 68.79 Doddabommanahalli Azax 28.67 5 TumkurSira Baladevarahatti Green long 59.65 Dasegowdanahatti Azax 27.64 Agrahara Azax 19.58 6 Additional Venkatagirikota Green long 50.73 Chittoor Santhipuram Green long 43.65 (Andhra Pradesh) Nagalapuram Sparta 26.42 Sl. Districts & Name of the PDI No. Taluk Location (mean) 1 Bangalore urban ZARS, GKVK 23.34 Bengalore north 2 Bangalore rural Challahalli 17.15 Doddaballapur Konenahalli Linganahalli 3 Chikkaballapura Poshottahalli 31.63 Chikkaballapura Rayamakalahalli Doddamarali Chikkaballapura Gorthapalli 32.44 Baagepalli Bagepalli rural Gollapalli Chikkaballapura Bommashettihalli 30.28 Gowribidanuru Machenahalli Kudumalakunte 4 KolarKolar Gadadasanahalli 51.27 rural Sugatur Doddabommanahalli 5 TumkurSira Baladevarahatti 35.62 Dasegowdanahatti Agrahara 6 Additional Venkatagirikota 40.27 Chittoor Santhipuram (Andhra Pradesh) Nagalapuram Rabi 2013-14; * Stage of the crop: 30-45 days Table 2. Coat protein gene sequences of various Cucumber mosaic virus strains used for comparison Subgroup Strain Accession number Origin % Identity IA Kor L36251 Korea 89 FT D28487 Japan 90 C D00462 USA (NY) 91 KM AB004780 Japan 91 Sny U66094 Israel 92 Ny U22821 Australia 92 Fny D10538 USA (NY) 92 FC D10544 USA 92 IB D AF281864 India 95 Phym X89652 India 95 H AF350450 India 95 CWL2 JN054635 Malaysia 96 Oxalis JQ779842 India 96 KS44 AJ810259 Thailand 96 Rauvolfia DQ914877 India 96 Kerala AY754359 India 97 Jasmine KF129062 India 98 AN AJ810260 India 99 II Trk7 L15336 Hungary 79 S AF063610 USA 79 SP103 U10923 USA 79 Q M21464 Australia 80 LS AF127976 USA 80 m2 AB006813 Japan 80 Table 3. Host range of cucumber mosaic virus disease of gherkin * Sl. Name of Scientific Transmission No. the host name (%) Cucurbitaceae 1 Cucumber Cucumis sativus L. 70.0 2 Ash gourd Benincasa hispida Thumb. 30.0 3 Ridge gourd Luffa acutangula L. 80.0 4 Bush squash Cucurbitapepo var. Clypeata 90.0 5 Muskmelon Cucumis melo L. 80.0 6 Pumpkin Cucurbita maxima Duchesne 40.0 7 Round melon Praecitrullus fistulosus 50.0 8 Watermelon Citrullus lanatus Thumb. -- 9 Bottle gourd Lagenaria siceraria Standl. 60 Solanaceae 10 Tobacco Nicotiana tabaccum L. -- 11 Tobacco Nicotiana glutinosa L. -- 12 Tomato Solanum lycopersicum L. 70.0 13 Brinjal Solanum melongena L. -- 14 Chilli Capsicum annuum L. 50 Chenopodiaceae 15 Chenopodium Chenopodium amaranticolor L. 100.0 Leguminosae 16 Cowpea Vigna unguiculata L. Walp. 100.0 Caricaceae 17 Papaya Carica papaya -- Malvaceae 18 Bhendi Abelmoschus esculentus (L.) -- 19 Cotton Gossypium hirsutum -- Euphorbiaceae 20 Castor Ricinus communis -- Fabaceae 21 Lima bean Phaseolus lunatus -- Cruciferaceae 22 Radish Raphanus sativus -- Sl. Name of Symptoms Confirmation No. the host observed by DAS- ELISA Cucurbitaceae 1 Cucumber GYP, BP, SG, RF + 2 Ash gourd CS, GYP, SG, RF + 3 Ridge gourd GYP, BP, SG, RF + 4 Bush squash GYP, BP, SG, RF + 5 Muskmelon GYP, BP, SG, RF + 6 Pumpkin CS, GYP, SG, RF + 7 Round melon GYP, BP, SG, RF + 8 Watermelon No symptoms - 9 Bottle gourd GYP, BP, SG, RF + Solanaceae 10 Tobacco No symptoms - 11 Tobacco No symptoms - 12 Tomato GYP, fern leaf + 13 Brinjal No symptoms - 14 Chilli Mosaic with + downward curling Chenopodiaceae 15 Chenopodium Chlorotic local lesions + followed by necrosis Leguminosae 16 Cowpea Chlorotic local lesions + followed by necrosis Caricaceae 17 Papaya No symptoms - Malvaceae 18 Bhendi No symptoms - 19 Cotton No symptoms - Euphorbiaceae 20 Castor No symptoms - Fabaceae 21 Lima bean No symptoms - Cruciferaceae 22 Radish No symptoms - * Experiment conducted twice ** No. of plants inoculated: 10 GYP-Green yellow patches BP-Blistering and Puckering SG-Stunted growth RF-Reduced flowering CS-Chloratic spots
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|Author:||Kavyashri, V.V.; Pappachan, Anil; Padmaja, A.S.; Nagaraju, N.; Rangaswamy, K.T.|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Sep 1, 2016|
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