Virus Genes (2011) 43:93–101 DOI 10.1007/s11262-011-0605-9
Complete nucleotide sequence of Croton yellow vein mosaic virus and DNA-b associated with yellow vein mosaic disease of Jatropha gossypifolia in India S. K. Snehi • M. S. Khan • S. K. Raj V. Prasad
•
Received: 19 February 2011 / Accepted: 24 March 2011 / Published online: 9 April 2011 Ó Springer Science+Business Media, LLC 2011
Abstract A severe yellow vein mosaic disease was noticed on several Jatropha gossypifolia plants growing nearby agriculture fields at Lucknow, India. Diseased plants exhibited yellow vein mosaic, leaf deformation, vein swelling and stunting. A population of whiteflies (Bemisia tabaci) was also noticed in the vicinities; therefore, begomovirus infection was suspected. To confirm begomovirus association, total DNA was isolated from symptomatic leaf samples and subjected to PCR using DNA-A, DNA-B and DNA-b-specific primers. DNA-A and DNA-b was successfully amplified but several attempts failed to amplify DNA-B indicating monopartite nature of the begomovirus. The sequence analysis of amplicons revealed the presence of 2757 nucleotides of DNA-A genome (EU727086) and 1315 nt of DNA-b molecule (EU604296). The sequence analysis of DNA-A (EU727086) revealed the highest 96% identities and closest relationship with Croton yellow vein mosaic virus (CYVMV, AJ507777) infecting Croton bonplandianum in India. The DNA-b (EU604296) showed the highest 96% sequence identity and closest phylogenetic relationship with CYVMV-associated DNA-b (AM410551) isolated from Croton sp. in Pakistan. Based on the highest sequence identities and closest phylogenetic relationships of the DNA-A genome and DNA-b molecule with respective sequences of various isolates of Croton yellow vein mosaic virus, the begomovirus associated with yellow vein mosaic S. K. Snehi M. S. Khan S. K. Raj (&) Department of Plant Molecular Virology, CSIR-National Botanical Research Institute, Lucknow 226 001, Uttar Pradesh, India e-mail:
[email protected] V. Prasad Virology Laboratory, Department of Botany, University of Lucknow, Lucknow 226 007, Uttar Pradesh, India
disease of J. gossypifolia was identified as an isolate of Croton yellow vein mosaic virus. Keywords Jatropha gossypifolia Yellow vein mosaic disease Whitefly transmission Croton yellow vein mosaic virus DNA-b
Introduction The genus Begomovirus is a member of family Geminiviridae, which is the second largest group of plant viruses [1]. Begomoviruses are transmitted by whitefly (Bemisia tabaci) and cause important diseases in many dicotyledonous crops including important vegetable crops and weeds [2]. They have monopartite (DNA-A) or bipartite (DNA-A and DNA-B) genome. DNA-A have six open reading frames (ORFs): AV1 (coat protein, CP) and AV2 (pre-coat protein) on the virion-sense strand, required for virus accumulation and symptom development [3] and AC1 (replication initiation protein, Rep), AC2 (transcriptional activator protein, TrAP), AC3 (replication enhancer protein, REn) and AC4 (C4 protein) on the complementary-sense strand. AC4 is found to be involved in host range determination, symptom severity and virus movement [4–6]. Whilst DNA-B has two ORFs encoding: BV1 (nuclear shuttle protein, NSP) on the virion-sense strand and BC1 (movement protein, MP) on the complementarysense strand, both of which are essential in efficient systemic spread and symptom expression [7, 8]. A novel satellite molecule, called DNA-b, has been found to be associated with some monopartite begomoviruses [9, 10]. DNA-b is a circular single-stranded DNA of approximately half the size (1350 nt) of the begomovirus DNA-A component [9, 11]. The bC1 gene is located on the
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complementary-sense strand of all DNA-b species [12]. DNA-b depends on the helper virus for replication and encapsidation, and its presence is essential for symptom expression [9]. Most of the studies have revealed that DNA-b is involved in symptom development but few of them have minimum role in disease development [13, 14]. Jatropha gossypifolia L. of family Euphorbiaceae commonly grows in road sides, railway tracts, barren and agricultural fields in India. It is commonly known as Chandrajyot due to its medicinal properties and used in fever, carbuncles, eczema, itches, tongue sores, swollen mammae, stomach ache, blood purifier, antibacterial, anticancer and venereal disease [15]. The natural infection of begomovirus has been reported in Jatropha spp. across the world: Jatropha mosaic virus on J. gossypifolia in Jamaica [16, 17]; African cassava mosaic virus on Jatropha multifida in India and East and West Africa [18] and a begomovirus closely related to Indian cassava mosaic virus and Sri Lankan cassava mosaic virus on Jatropha curcas in India [19–23]. We report here complete nucleotide sequence of Croton yellow vein mosaic virus and DNA-b associated with yellow vein mosaic disease of J. gossypifolia for the first time in India.
Materials and methods Virus source and whitefly transmission
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enhancer protein (AC3, REn) and partial replicase protein, Rep (AC1) PARIv722/PALIc1960 [26]. PCRs were set up in a 50 ll reaction mixture containing: template DNA (100 ng), dNTPs (10 mM each), primers (each 25 pmol), Pfu DNA polymerase (1.5 U, Bangalore Genei Pvt. Ltd) and assay buffer (1X, Bangalore Genei Pvt. Ltd) in a Peltier thermal cycler PTC200 engine (MJ Research, Waltham, MA, USA). The PCR was done with the conditions: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, specific annealing temperatures for 1 min according to the primers used (47°C for CPIT-I/CPIT-T primers, 52°C for PALIv1978/PARIc496 primers, and 52°C for PALIv722/ PALIc1960 primers) and extension at 72°C for 1.5 min. The final extension cycle was for 5 min at 72°C. To confirm, whether the virus isolate is monopartite or bipartite, the PCR was also performed using BC1F and BC1R primers located in DNA-B [27]. The PCR reactions were conducted as: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1.5 min and a final extension at 72°C for 5 min. The DNA-b was amplified using the b01 and b02 primers [28]. The PCR reactions were conducted as: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1.5 min and final extension at 72°C for 5 min.
For virus transmission, adult non-viruliferous whiteflies (B. tabaci) maintained on Clitoria terenta (in an insect proof cage) were starved for 2 h, and then allowed to feed on infected J. gossypifolia leaves showing yellow vein mosaic symptoms for an acquisition access period of 24 h. These viruliferous whiteflies were transferred onto healthy test seedlings of J. gossypifolia, J. curcas, Lycopersicon esculentum cv. Pusa Ruby, Cajanus cajan and Datura stramonium (8–10 whiteflies per plant) and were allowed for 24-h inoculation access period. Two plants from each test species were kept as negative controls which were fed by similar non-viruliferous whiteflies. The test plants were monitored for symptoms for 35 days in the insect proof glass house.
Cloning and sequencing of PCR amplicons
DNA extraction and PCR
The sequence data of the virus isolate were analysed by the Entrez program using BLASTn (http://www.ncbi.nlm.nih. gov/BLAST) and compared with existing sequences of begomovirus strains available in the GenBank database. The matrix for pair-wise alignment of selected begomovirus strains was obtained using the Genomatix DiAlign 2 program (http://www.genomatix.de/cgi-bin/dialign/dialign.pl). ORFs, either in the virion or complementary sense were translated by ExPASy Computational tool (http://www.expasy.org/ tools/dna.html). Phylogenetic analyses were perused using
The total DNA was extracted from young leaf samples of symptomatic and asymptomatic J. gossypifolia plants by the method described earlier [24]. PCR was performed using three sets of begomovirus primers: specific to coat protein (AV1) gene CPIT-I/CPIT-T [25]; pre-coat protein (AV2) and AC4 and partial replicase protein (AC1) PALIv1978/PARIc496 [26] and coat protein (AV1), transcriptional activator protein (AC2, TrAP), replication
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The PCR amplicons were subjected to agarose gel electrophoresis, purified from the gel using the Qiaquick Kit (Qiagen) and cloned into the pGEM-T Easy Vector System-1 (Promega Life Corporation, USA), and E. coli strain DH5a was transformed. Potential containing clones were screened for the presence of insert (gene) by restriction analysis. Three positive clones of each amplicon were sequenced in both orientations, and consensus sequence data obtained was deposited in GenBank database. Analyses of sequence data
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the Molecular Evolutionary Genetics Analysis tool (MEGA v. 4.2) [29] with 1,000 replicates bootstrapping, and a phylogenetic trees were generated with the Neighbour joining method and viewed by the NJ plot program.
Results Disease symptoms and virus transmission During the survey in February 2008, natural infection of a severe yellow vein mosaic disease was observed on a large
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number of J. gossypifolia plants growing nearby agriculture sites in Lucknow, Uttar Pradesh, India (Fig. 1). The disease incidence was about 40% in the area surveyed. Naturally infected plants exhibited yellow vein mosaic symptoms accompanied with reduction in leaf size and height of plants (Fig. 2). A population of whiteflies (B. tabaci) was also observed in the growing area; therefore, an association of begomovirus with the disease was suspected. The disease was successfully transmitted to all the test species except D. stramonium. The transmission rate was 100% in J. gossypifolia, 83.3% in L. esculentum and 66.6%
Fig. 1 The geographical map for the area in India from where Croton yellow vein mosaic virus isolated
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Amplification of virus DNA-A, DNA-B and DNA-b molecules
Fig. 2 Naturally infected plants showing yellow vein mosaic symptoms and reduction in leaf size
in J. curcas and C. cajan (Table 1). However, negative control plants and D. stramonium did not develop any symptom. The yellow vein mosaic symptoms similar to naturally infected J. gossypifolia were observed on inoculated J. gossypifolia after 25–30 days of inoculation (Fig. 3a). The begomovirus infection in these plants was confirmed by PCR (Table 1) using coat protein gene of begomovirus-specific primers: CPIT-I/CPIT-T [25].
As expected, bands of *800 bp, *1.2 and *1.2 kb were consistently amplified from (3/3) symptomatic samples (Fig. 4 a, b and c) but not from (1/1) asymptomatic sample during PCR using three sets of DNA-A specific primers: CPIT-I/CPIT-T [25]; PALIv1978/PARIc496 and PARIv722/PALIc1960 [26]. However, the PCR carried out with DNA-B-specific primers did not result positive amplification in any of the plant samples, confirming the virus isolate to be monopartite. During PCR with b01/b02 primers [28], an expected size *1.3 kb amplicon of DNA-b was also amplified from (3/3) symptomatic samples but not from (1/1) asymptomatic sample (Fig. 4d). Based on the positive amplification of expected sized amplicons by begomovirus DNA-A and DNA-b-specific primers, the associations of begomovirus and DNA-b molecule were confirmed with the disease. Cloning DNA-A and DNA-b molecules of virus The *800 bp, 1.2 and 1.2 kb amplicons of DNA-A and *1.3 kb amplicon of DNA-b obtained were cloned into the pGEM-T Easy Vector System-1 (Promega Life Corporation,
Table 1 PCR analyses of samples from experimentally inoculated test plants using coat protein gene of begomovirus-specific primers: CPIT-I/ CPIT-T [25] S. no.
Test plants
No. of plants showed symptoms after whiteflies transmission/total number of plant taken (%)
Symptoms developed after 25–30 dpi
Positive amplicon of *800 bp obtained during PCR in samples
1
J. gossypifolia
6/6 (100)
Yellow vein mosaic
6/6
2
J. curcas
4/6 (66.6)
Mild yellowing, leaf curling and chlorosis
6/6
3
L. esculentum cv. Pusa Ruby
5/6 (83.3)
Mild yellowing and leaf curling
6/6
4
C. cajan
4/6 (66.6)
Mild yellowing and mosaic
6/6
5
D. stramonium
0/6 (0.0)
No symptoms
3/6
Fig. 3 The yellow vein mosaic symptoms obtained after whitefly transmission on J. gossypifolia (a), mild yellowing and leaf curling symptoms on J. curcas (b), L. esculentum (c) and C. cajan (d) after 25–30 days of inoculation
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Fig. 4 PCR amplification of various components of DNA-A genome and DNA-b molecule of virus isolate associated with yellow vein mosaic disease of J. gossypifolia. The positive amplicons of *800 bp, *1.2, *1.2 and *1.3 kb were obtained from all three symptomatic samples by primers: CPIT-I/CPIT-T [25]; PALIv1978/PARIc496 and PARIv722/PALIc1960 [26] and b01/b02 [28] shown in lanes 1–3 of the panel a, b, c and d, respectively. Lane H: asymptomatic healthy sample and lane M: Lambda DNA digested with EcoR I and Hind III (Genei, Bangalore, India) as marker
USA) and transformed into competent E. coli strain DH5a. Three independent clones of each amplicon were sequenced in both orientations. The consensus sequence data of three identical sequences were combined to complete DNA-A genome and DNA-b. The sequence resulted in the presence of 2757 and 1315 nucleotides of DNA-A and DNA-b, respectively, which were deposited in the GenBank database under Accession Nos. EU727086 and EU604296, respectively. Sequence and phylogenetic analysis of DNA-A genome The analysis of complete DNA-A (EU727086) revealed the presence of six ORFs encoding for AV2 (145–501 nt, precoat protein), and AV1 (305–1075 nt, coat protein) genes in the virion sense. The AC3 (1078–1482 nt, replication enhancer protein, REn); AC2 (1223–1627 nt, transcriptional activator protein, TrAP); AC1 (1530–2615 nt, replication associated protein and AC4 (2201–2458 nt, C4 protein) genes were located in the complementary sense. The two non-translated regions were located from 1–144 nt at 50 end and 2616–2757 nt at 30 end positions (Fig. 5).
Fig. 5 DNA-A genome of CYVMV (EU727086) associated with yellow vein mosaic disease of J. gossypifolia
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BLASTn analysis of the complete sequence of DNA-A showed the highest 96% identity with Croton yellow vein mosaic virus (CYVMV, AJ507777) isolated from Croton bonplandianum in New Delhi, India. The identities were 93–92% with CYVMV isolates from Acalypha sp. (FN645926, FN645898, FN645901 and FN645902) and from radish (FJ593629), Cyamposis tetragonoloba (FN645915) reported from India, and 87% with CYVMV isolate of Croton glandulosus (FN543112) from Pakistan. During Genomatix DiAlign analysis of complete nucleotide sequence of DNA-A (EU727086) with the selected begomoviruses (which showed 96–87% identities in BLASTn and also some other begomovirus isolates of Jatropha spp.), the virus isolate showed a maximum 93% identities with CYVMV (AJ507777) isolate of C. bonplandianum. The similarities were 91–89% with other isolates of CYVMV (FN645926, FN645901, FJ593629, FN645898, FN645902 and FN645915); 82% with CYVMV (FN543112); ToLCNDV (DQ629102); 81–79% with PLCV (FM955601, AJ436992 and Y15934) and minimum 66-56% with JYMIV (FJ177030); JLCV (GU451249 and EU798996); JMIV (HM230683) and JCMV (GQ924760) isolates from Jatropha spp. (Table 2). Pairwise similarities of six ORFs encoded in DNA-A of virus isolate under study (EU727086) were also analysed at nt basis with respect to the selected begomovirus isolates. The AV2 ORF showed 98–93% similarities with isolates of CYVMV, 91–88% with isolates of ToLCNDV, PLCV and JYMIV, and less than 88% with rest of the begomovirus isolates taken for study. The AV1 gene showed 97–76% with isolates of CYVMV and less than that with other
begomovirus isolates (Table 2). The AC3, AC2, AC1 and AC4 ORFs showed 86–79, 94–87, 92–85 and 96–91%, respectively, with the isolates of CYVMV and less than that with rest of the begomovirus isolates studied (Table 2). The phylogenetic analysis of the virus isolate (EU727086) showed more close relationship with CYVMV (AJ507777) reported from India and also shared close relationships with other isolates of CYVMV (FN645902, FN645915, FJ593629, FN645901, FN645898 and FN645926) but distinct relationship with CYVMV (FN543112), ToLCNDV (DQ629102) and PLCV (Y15934, AJ436992 and FM955601). The other begomoviruses reported on Jatropha spp. viz JLCV (GU451249 and EU798996), JMIV (FJ177030), JCMV (GQ924760) and JMIV (HM230683) reported from India did not show any relationship with the isolate under study (Fig. 6). Sequence and phylogenetic analysis of DNA-b molecule BLASTn analysis of DNA-b (EU604296) of J. gossypifolia isolate showed the highest 96% sequence identity with CYVMV-associated DNA-b, beta C1 gene (AM410551) isolated from Croton spp. from Pakistan. The identities were 88–90% with CYVMV-associated DNA-b, isolates of radish (FJ593630), C. bonplandianum (EF597245) and Crotalaria juncea (GQ183866, EU557375 and GQ183865) from India. The multiple sequence alignment of DNA-b (EU60 4296) showed maximum 94% similarities with CYVMV (AM410551), 88–84% with CYVMV isolates (FJ593630,
Table 2 Sequence similarity at nucleotide (nt) and amino acid (aa) of DNA-A and six ORFs of begomovirus isolate from J. gossypifolia (EU727086) with the selected begomoviruses analyzed by Genomatix DiAlign program Acc. no.
Virus
Host
Location
DNA-A
AV2 nt/aa
AV1 nt/aa
AC3 nt/aa
AC2 nt/aa
AC1 nt/aa
AC4 nt/aa
AJ507777 FN645926
CYVMV CYVMV
C. bonplandianum Acalypha sp.
New Delhi, India India
93 91
98/100 97/98
97/97 97/97
85/88 85/86
94/89 92/86
92/96 87/93
96/92 94/91
FN645901
CYVMV
Acalypha sp.
Haryana, India
91
96/97
97/97
84/85
91/86
88/93
94/92
FJ593629
CYVMV
Radish
India
90
96/98
96/97
85/82
92/87
88/93
94/91
FN645898
CYVMV
Acalypha sp.
Haryana, India
90
97/98
97/97
85/86
91/87
85/88
91/88
FN645902
CYVMV
Acalypha sp.
Haryana, India
89
97/98
97/97
86/87
92/90
87/93
92/97
FN645915
CYVMV
C. tetragonoloba
Haryana, India
89
93/94
95/96
82/82
88/82
87/93
94/91
FN543112
CYVMV
C. glandulosus
Pakistan
82
96/98
76/82
79/79
87/79
86/92
91/91
DQ629102
ToLCNDV
Tomato
New Delhi, India
82
91/89
74/80
74/79
92/86
86/85
91/58
FM955601
PLCV
Legume
Pakistan
81
88/73
73/63
81/81
87/79
86/91
93/92
AJ436992
PLCV
Cotton
Pakistan, India
80
90/87
76/80
82/79
88/82
85/91
91/89
Y15934
PLCV
Papaya
Lucknow, India
79
88/85
74/80
80/79
84/74
85/88
93/91
FJ177030
JYMIV
J. gossypifolia
M. P., India
66
88/84
75/77
76/72
67/62
65/68
55/49
CYVMV Croton yellow vein mosaic virus, ToLCNDV Tomato leaf curl New Delhi virus, PLCV Papaya leaf curl virus, JYMIV Jatropha yellow mosaic India virus
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Fig. 6 Phylogenetic analysis of the DNA-A genome of CYVMV isolate (EU727086) associated with yellow vein mosaic disease of J. gossypifolia showing relationships with isolates of CYVMV and other begomoviruses
EF597245, GQ183866, EU557375 and GQ183865) and minimum 56–53% with Tomato leaf curl virus (AJ542492, EF068245, EF095958 and AY438559). Phylogenetic analysis of the DNA-b (EU604296) showed the closest relationship with CYVMV (AM410551) and also shared close relationships with other isolates of CYVMV (EF597245, FJ593630, GQ183865, GQ183866 and EU557375) but distinct relationship with Radish leaf curl virus (EU31117 and EF175734) and Tomato leaf curl virus (AJ542492, AY438559, EF068245 and EF095958) (Fig. 7).
Discussion A number of J. gossypifolia growing nearby agricultural fields in Lucknow, India were found to be infected with yellow vein mosaic disease, and population of whiteflies (B. tabaci) was noticed in the growing area; therefore, an association of begomovirus with the disease was suspected. The transmission studies proved Koch’s postulates as disease could be successfully transmitted through whiteflies from naturally infected J. gossypifolia to inoculated
J. gossypifolia plants which exhibited similar disease symptoms. The association of the begomovirus with yellow vein mosaic disease of J. gossypifolia was determined by PCR from total DNA isolated from symptomatic leaf samples using primers specific to begomovirus genus [25] and DNA-b [28] which revealed positive amplification of the expected size bands in the symptomatic leaf samples of J. gossypifolia. During the PCR using DNA-A, DNA-B and DNA-bspecific primers, DNA-A genome and DNA-b molecule were successfully amplified. However, our several attempts failed to amplify DNA-B genome during the PCR using DNA-B specific primers [27], which indicated the presence of a monopartite begomovirus. It has been worked out earlier that some of the begomoviruses do not consist DNA-B genome [30, 31]. Sequence analysis of the amplicons revealed the presence of 2757 nucleotides of DNA-A genome (EU727086) and 1315 nt of DNA-b molecule (EU604296) which shared the highest 96% identities and closest phylogenetic relationship with respective sequence of CYVMV and CYVMV-associated DNA-b, respectively. On the basis of
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Fig. 7 Phylogenetic analysis of the DNA-b molecule associated with yellow vein mosaic disease of J. gossypifolia (EU604296) showing relationships with isolates of CYVMV and other begomoviruses
the highest sequence identities and closest phylogenetic relationships of the DNA-A genome and DNA-b molecule of the virus under study with respective sequences of various isolates of CYVMV reported from India (AJ507777) and Pakistan (AM410551), the begomovirus associated with yellow vein mosaic disease of J. gossypifolia was identified as an isolate of CYVMV. CYVMV was reported for the first time on C. bonplandianum from India in 1963 [32]. Later on, occurrence of CYVMV was reported on C. bonplandianum weed in India [33]. Serological detection and antigenic variation of Tobacco leaf curl virus and CYVMV have also been determined [34]. J. gossypifolia has neither been reported as a natural host nor an experimental host of CYVMV in India and abroad. Therefore, natural occurrence of CYVMV and its DNA-b molecule on J. goosypifolia seems to be the first report from India. Since J. gossypifolia grows as weed in India and abroad nearby agricultural fields and most of the plants were found to be infected with yellow vein mosaic disease and its transmission has been proved through whiteflies from naturally infected J. gossypifolia to J. gossypifolia, J. curcas (a biodiesel yielding plant), L. esculentum (important staple crop) and C. cajan (agriculturally important plant), the association of CYVMV and its
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DNA-b molecule with yellow vein mosaic disease of J. gossypifolia may be considered as a serious threat to the cultivation of other economically important crop plants. Acknowledgments The authors are thankful to the Director of the National Botanical Research Institute, Lucknow, U. P., India for providing the facilities and the Department of Biotechnology, New Delhi, India for funding.
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