biological and molecular variability of the begomoviruses ... - SIPaV

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Journal of Plant Pathology (2009), 91 (3), 637-647

Edizioni ETS Pisa, 2009

637

BIOLOGICAL AND MOLECULAR VARIABILITY OF THE BEGOMOVIRUSES ASSOCIATED WITH LEAF CURL DISEASE OF KENAF IN INDIA S. Paul1, R. Ghosh1, S. Chaudhuri2, S. K. Ghosh1 and A. Roy1 1 Plant

Virus Laboratory and Biotechnology Unit, Division of Crop Protection, Central Research Institute for Jute and Allied Fibres, Barrackpore, Kolkata-700120, West Bengal, India 2 Department of Botany, University of Kalyani, Kalyani, West Bengal, India

SUMMARY

Kenaf (Hibiscus cannabinus L.) is a malvaceous bast fibre crop and the most promising source of “tree-free” newsprint in the world. For the last few years a begomovirus associated leaf curl disease of kenaf, characterized by upward leaf curling and stunted growth, has been observed at different locations in India. In this study biological and molecular analyses of kenaf leaf curl disease-associated begomovirus isolates collected from three different locations were carried out. Whitefly transmission efficiency was different for the three geographical isolates which possessed very narrow host ranges. Except kenaf, the isolates from northern India could not infect other experimental hosts studied. Cloning and sequencing of the complete genomes of these isolates revealed the presence of three different monopartite begomoviruses. In eastern and southern India Mesta yellow vein mosaic virus (MeYVMV) and Tomato leaf curl Joydebpur virus (ToLCJV) were associated with the disease, respectively, while in northern India a new begomovirus species was involved, tentatively named Kenaf leaf curl virus (KLCuV). Nucleic acid spot hybridization tests using non-radiolabeled probes developed against these viruses detected the presence of begomoviruses from symptomatic samples collected from the fields at the different locations. Keywords: Begomovirus, betasatellite, whitefly, host range, kenaf leaf curl disease, phylogenetic relationship.

INTRODUCTION

Geminiviruses are placed in four genera, Mastrevirus, Curtovirus, Topocuvirus and Begomovirus of the family Geminiviridae, based on the number of genome components, type of insect vector, host range and sequence relatedness (Fauquet et al., 2008). Begomovirus (type species Bean golden mosaic virus) is the only genus with Corresponding author: A. Roy Fax: +913325350415 E-mail: [email protected]

a bipartite genome composed of circular, single-stranded DNA designated as DNA-A and DNA-B of size ~2.7 kb (Harrison, 1985; Lazarowitz, 1992; Briddon and Markham, 1994; Rybicki et al., 2000). Begomoviruses are transmitted by the whitefly Bemisia tabaci. They are emerging plant viruses, considering their increasing incidence and the severity of the diseases they cause in a number of economically important crops, mostly in tropical and subtropical regions (Polston and Anderson, 1997; Faria et al., 1999). Some of the Old World begomoviruses can dispense with the DNA-B genome component and are thus grouped as monopartite begomoviruses. This could be due to the presence of ORF AV2 in the DNA-A, which encodes a protein that participates in virus movement (Padidam et al., 1996). Recently, some satellite DNA molecules, known as DNA β or betasatellites, have been found associated with such monopartite begomoviruses and they depend upon their helper viruses for encapsidation, replication, movement, and insect-transmission (Briddon et al., 2008). Kenaf is cultivated primarily for obtaining ligno-cellulosic bast fibers from its stem. Besides its common use for making rope, cordage, canvas, sacking, carpet backing, and fishing nets, recently, the fibre from this crop is also being used as a non-woody source of paper pulp. Kenaf is grown in different states of India like West Bengal, Uttar Pradesh, Andhra Pradesh, and Orissa. Average yield is quite low due to infection by a number of diseases, of which virus diseases are particularly important (Brunt et al., 1996). Among these virus diseases, kenaf leaf curl disease (KLCuD) (Fig. 1), characterized by upward curling of the leaves followed by stunting of the plant, has been spreading quite consistently in the last few years in different parts of India, thus it has become important to understand the etiology. An earlier report showed the association of monopartite begomoviruses and betasatellite molecules (Paul et al., 2006) with such symptomatic samples. The coat protein genes of the associated begomoviruses were cloned and sequenced (accession No. EF620563, EF620561) from symptomatic samples collected from northern and eastern India. These results suggested the presence of two different begomoviruses.


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Variability of kenaf begomoviruses in India


farmers of a particular region grew kenaf in the three consecutive years so assessment was done from 40 fields randomly selected each year, and average incidence values were recorded. Disease incidence was taken as the percentage of symptomatic plants over the total number of plants assessed in a particular field. Disease severity was measured by calculating the number of symptomatic leaves out of the total number of leaves on individual plants and averaged from the data on 100 symptomatic plants observed within a particular location. Diseased plant samples, showing typical leaf curl symptoms, were collected and used as sources of virus inoculum. Nine field isolates were selected (three each from the three geographical locations) and were maintained in plants of the susceptible kenaf cv HC-583 through successive whitefly transmissions in separate chambers in an insect-proof glasshouse. Biological and molecular characterizations were conducted on these isolates.

Fig. 1. Kenaf plant showing typical leaf curl symptom.

Symptomatic samples from southern India showed the presence of an isolate of Tomato leaf curl Joydebpur virus (ToLCJoV) and its associated betasatellite (Paul et al., 2009). The betasatellite molecules from symptomatic samples obtained from eastern and northern India have also been characterized, revealing the involvement of two different isolates of Cotton leaf curl Multan betasatellite (CLCuMB) (Paul et al., 2008). However, the associated begomoviruses from eastern and northern India have not yet been studied extensively and thus the overall scenario with respect to the distribution of the disease in India and the variability of the begomovirus complexes is still not very clear. We report here the distribution of the KLCuD with special reference to its incidence and severity along with the biological and molecular variability of the associated begomoviruses.

MATERIALS AND METHODS

Disease surveys and sample collection. A survey was carried out for the last three years (2005-2007) on the incidence and severity of KLCuD in different kenafgrowing regions of northern, eastern and southern India so as to understand the distribution of the disease throughout the kenaf-producing regions. Not all the

Whitefly transmission. Tobacco whiteflies (B. tabaci) were collected from a kenaf crop grown at the Institute research farm and reared on Nicotiana tabacum in an insect-proof cage for several generations to make the culture virus-free. Adult whiteflies, which emerged from nymphs present on tobacco, were used for transmission studies. For transmission, the whiteflies were separately fed on the diseased samples (geographical isolates) and after acquisition they were released onto glasshousegrown healthy plants (10 seedlings per pot). After inoculation, inoculated plants were sprayed with 0.2% Dimethoate (Rogor) and kept in cages until symptom development. The experiment was carried out in separate cages in separate chambers for individual isolates, to avoid any cross-contamination. The transmission efficiency of the whiteflies was determined by introducing different numbers of viruliferous insects (1, 3, 5, 10, 20 and 30) per group of ten healthy plants with three replications in each set. Acquisition access periods (AAP) and inoculation access periods (IAP) of 24 h each were allowed and the number of plants developing symptoms was recorded. To determine the minimum AAP and IAP, transmission was tested with different times (2, 4, 6, 12, 18, 24 and 48 h) of either AAP or IAP separately for the three geographical isolates, keeping the corresponding times of IAP and AAP at a constant 24 h. The experiments were repeated for three times. Host range. Healthy seedlings of 16 crop species, ornamentals and weeds belonging to seven families were taken for host range study. Ten healthy adult whiteflies were allowed to have a 24 h AAP on leaf curl-infected kenaf plants (maintained under glasshouse as separate isolates) and subsequently allowed to have a 24 h IAP on healthy test plants. All plants were sprayed with


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0.2% Dimethoate after inoculation and maintained in the glasshouse for several weeks. To detect the presence of begomoviruses in the inoculated plants, PCR was conducted with different primers as described below. DNA isolation. DNA was isolated from the nine isolates a by modified CTAB method recently developed at our lab (Ghosh et al., 2009). The modification included the use of a larger volume of extraction buffer (100 mM Tris–HCl pH 8, 10 mM EDTA pH 8, 1.4 M NaCl, 2% CTAB and 0.2% β-marcaptoethanol) and dissolving the crude nucleic acid pellet in 1 M NaCl, which markedly reduced the viscosity of the mucilage; thus the final purification step yielded more mucilage-free DNA suitable for subsequent PCR-based begomovirus detection. DNA from these nine isolates was used for molecular characterization work. DNA isolated likewise from healthy kenaf plants (cv. HC-583) was used as control. Besides these glasshouse-maintained isolates, DNA was also isolated from 130 symptomatic leaf samples collected from the fields of the three different geographical locations and the DNAs obtained were used in nucleic acid spot hybridization tests to detect the presence of begomoviruses. PCR. Two sets of abutting primers [DFL02F (5’ATGCKYTRTTRTTRTATATGGCATG-3’)/DFL02R (5’TYTCRGTRTGRTTYTCRTACTTCCC 3’)] and [NI-LC-FLF(5’-CAGAAGCCCTGATGTTCCAAG-3’) / NI-LC-FLR(5’-TACATCCTGTACAGTCTGGGC3’)], were designed from the previously characterized coat protein genes (acc no. EF620561, EF620563) of

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the begomoviruses associated with KLCuD in eastern and northern India, respectively and were used to amplify the complete genome of the begomovirus isolates from nine glasshouse-maintained isolates. In the PCR reactions, 100 ng of total plant DNA and 0.2 µM of primers were used. Along with 1X PCR buffer, 0.2 mM dNTPs and 1.5 mM MgCl2, each PCR reaction mixture contained 1 unit of High Fidelity PCR Enzyme Mix (Fermentas, Lithuania). DNA fragments were amplified in a PX2 thermal cycler (Thermo Applied Biosystem, USA) with an initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C for 1 min, 55°C for 2 min and 72°C for 3 min followed by a final extension at 72°C for 10 min. The amplified fragments were resolved after electrophoresis in 0.8% agarose gel and visualized in a UV gel documentation system (Bio-Rad, USA) after staining with ethidium bromide. For comparison, a similar study was also conducted with nucleic acid isolated from non-symptomatic leaves. Cloning and sequencing. PCR amplicons obtained from the nine isolates were cloned into pJET1.2 positive selection vector using GeneJET™ PCR Cloning Kit (Fermentas. Lithuania) following the manufacturer’s protocol. Recombinants were confirmed through colony PCR and restriction digestion using the restriction enzymes XhoI and XbaI. Clones were sequenced using the automated DNA sequencing service (Bangalore Genei Pvt., India). Sequence analysis. The sequences were assembled and a sequence identity matrix was generated using

Table 1. Average disease incidence and severity of leaf curl disease of kenaf crop at 100-120 days of crop growth in different geographical regions of India. Geographical Region

Eastern India; (State West Bengal )

Northern India; (State Uttar Pradesh)

Southern India; (State Andhra Pradesh) a b

Place (Geographical coordinate)

Average disease incidence (%)a

Average disease severity (%)b

Barrackpore (22°45'36"N 88°22'47"E) Haringhata (22°56'59"N 88°34'11"E) Bongaon (23°3'0"N 88°49'47"E)

24.52±1.36

56.25±1.45

21.44±0.95

51.0±1.65

22.20±1.10

53.42±0.97

Bahraich (27°36'12"N 81°38'19"E) Kaisarganj (27°48'35"N 78°37'47"E) Bhangha (27°43'11"N 81°55'48"E) Amadalvalasa (18°25'12"N 83°54'0"E) Ponduru (18°21'2"N 83°45'25"E)

29.45±1.89

67.23±1.57

30.50±0.96

62.76±1.94

27.29±1.22

65.14±1.43

17.06±0.92

29.63±0.87

15.85±0.67

26.2±1.28

Average disease incidence for 2005-2007 Average disease severity of 100 plants per location ± S.E.


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Table 2. Transmission efficiency of leaf curl disease by Bemisia tabaci. Number of whiteflys/plant 1 3 5 10 20 30

Transmission efficiency* Average Tef (%) of HC-583 Eastern India Northern India Southern India 0 0 0 0 0 0 20.0 30.0 10.0 30.0 50.0 20.0 60.0 70.0 30.0 60.0 70.0 30.0

*

Average of three replications Tef : Number of plants infected/number of plants tested.

Bioedit sequence alignment editor (version 5.0.9) (Hall et al., 1999). Multiple alignments were performed, a phylogenetic tree was constructed, and a bootstrapped consensus dendrogram was generated with 1000 replications using Neighbour Joining algorithm of MEGA 4 (version 4.0.2) (Tamura et al., 2007). To understand the recombination events, sequences were analyzed using recombination detection programme (RDP) (version beta 08) using the default value of the automated RDP command. Development of non-radioactive molecular probes and nucleic acid spot hybridization (NASH) analysis. Non-radiolabeled DNA probes were developed against the cloned begomoviruses (one isolate each from each geographical origin) by DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science, Germany) following the manufacturer’s protocol. For nucleic acid spot hybridization (NASH), DNA isolated from 130 field samples was used. After boiling and quick chilling, DNA from these samples was dotted on to nylon membrane and fixed by UV cross-linking. The hybridization was carried out using the same kit following the manufacturer’s protocol.

RESULTS

Distribution of KLCuD. The incidence and severity of KLCuD in various fields at different locations ranged from 15.8% to 30.5% (incidence) and 26.2% to 67.2% (severity), respectively (Table 1). Highest incidences were observed in northern India followed by eastern India, whereas the lowest incidence was noted in southern India. Disease transmission and host range. A minimum of 5 whiteflies was found necessary for virus transmission with 20%, 30% and 10% transmission efficiencies of isolates from eastern, northern and southern India, respectively (Table 2). After whitefly transmission, typical symptoms appeared after at least 16–20 days under glasshouse conditions. The minimum AAP was 6 h for

northern and eastern isolates while it was 12 h for southern isolates with transmission efficiency 20%, 10% and 10%, respectively. Minimum IAP was 4 h with northern and eastern isolates and 6 h with southern isolates with 10% transmission efficiency in all cases. Disease transmissibility was increased with increase in both AAP and IAP (Table 3). The control plants inoculated with non-viruliferous whiteflies did not show symptoms. Host range study revealed that the east Indian isolate produced leaf crumpling symptom in V. unguiculata with a transmission efficiency of 10%. The south Indian isolate could produce leaf crumpling in Solanum lycopersicum and leaf spot in Nicotiana tabacum with transmission efficiencies 50% and 30% respectively. The north Indian isolate did not produce symptoms in any of the se hosts studied (Table 4). Amplification of the genomes. Two different primer sets, DFL02F/DFL02R and NI-LC-FLF/NI-LC-FLR, successfully amplified the complete genome (ca 2.7 kb) of the begomoviruses (Fig. 2) from each of the nine isolates. The NI-LC-FLF/NI-LC-FLR primers pairs amplified the genomes of northern and southern isolates but not the eastern isolates. The primers DFL02F/DFL02R amplified the genomes only of the eastern isolates. No amplification was found using either of these primers with the DNA isolated from non-symptomatic leaves. Sequence analysis and molecular variability. The full length genome sequences of the begomovirus isolates were submitted to the database under accession Nos. FJ345398, FJ345399, FJ345400 (eastern isolates), EU366903, EU822321, EU822322 (northern isolates) and FJ345401, FJ345402 (southern isolates). The isolates obtained from the three geographical locations shared 76.01% nucleotide sequence identity among them whereas they shared 99.2-100% sequence identity within their respective geographical groups. As the isolates corresponding to their geographical locations were highly similar, the average sequence identity of the three isolates within a location was compared with averaged sequences from the two other groups to avoid genera-


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Table 3. Transmission efficiency of leaf curl disease of kenaf with various acquisition access period (AAP) and inoculation access period (IAP). Acquisition access period (hour)

Inoculation access period (hour)

Average transmission efficiencya,b (%) North Indian isolates

East Indian isolates

2 24 0.0 4 24 0.0 6 24 20.0 12 24 30.0 18 24 60.0 24 24 60.0 48 24 60.0 24 2 0.0 24 4 10.0 24 6 20.0 24 12 30.0 24 18 50.0 24 24 60.0 24 48 70.0 a Five Bemisia tabaci per plant were used, bAverage of three replications.

tion of voluminous repetitive information. Analysis of pair-wise sequence identity showed that the eastern and northern isolates shared average 80.4% identity, eastern and southern isolates shared average 72.2% identity, and northern and southern isolates shared an average 75.3% nucleotide sequence identity. Sequence identities of our begomovirus isolates with other begomoviruses are presented in Table 5. The isolates from eastern, southern and northern India shared highest average nucleotide sequence identity with MeYVMV-[India: Barrackpore2:2006] (94.5%, EF428256), ToLCJoV-[Bangladesh] (95.4%, AJ875159) and MaYVM-[China:Yunnan206:2004] (88.2%, AJ744881), respectively. According to the latest begomovirus nomenclature (Fauquet et al., 2008), isolates obtained from eastern and southern India were observed to be the isolates of MeYVMV and ToLCJoV, respectively. However, the sequence identity values of the isolates obtained from northern India are just below the threshold cut-off value of 89% that has been chosen as the species demarcation criterion for begomoviruses (Fauquet et al., 2008). Such a small difference is not enough to establish separate identity. But examination of biological properties showed that the northern isolates could not produce symptoms and were unable to multiply in malvastrum and cotton as evidenced from whitefly transmission followed by PCR. Based on these points, we are proposing that the isolates obtained from north India might be members of a new species, for which we have chosen the descriptor Kenaf leaf curl virus-[India:Bahraich:2007] (KLCuV-

0.0 0.0 10.0 20.0 50.0 50.0 50.0 0.0 10.0 10.0 30.0 40.0 50.0 60.0

South Indian isolates 0.0 0.0 0.0 10.0 30.0 30.0 30.0 0.0 0.0 10.0 20.0 30.0 30.0 30.0

[Ind:Bah:07]). Phylogenetic analysis showed that the eastern, northern and southern isolates clustered with different isolates of MeYVMV, MaYVV and tomato in-

Fig. 2. PCR amplicons of begomoviruses from glasshousemaintained isolates of northern, southern and eastern India (Lane 1-3). DNA from healthy control plant did not yield any amplicon (Lane 4). Lane M, 1kb DNA ladder.

East Indian isolate

South Indian isolate B

A

B

0/10

-

0/10

-

Gossypium hirsutum (Malvaceae)

0/10

-

1/10 (Leaf crumpling)

25

0/10

Abelmoschus esculentus (Malvaceae)

0/10

-

0/10

-

0/10

-

Sida cordifolia (Malvaceae)

0/10

-

0/10

-

0/10

-

V. unguiculata (Fabaceae)

0/10

-


28

0/10

-

Cajanas cajan(Fabaceae)

0/10

-

0/10

-

0/10

-

Luffa acutangula (Cucurbitaceae)

0/10

-

0/10

-

0/10

-

N. tabacum(Solanaceae)

0/10

-

0/10

-

3/10 (Leaf spot)

25-28

Capsicum annuum (Solanaceae)

0/10

-

0/10

-

0/10

-

Solanum lycopersicum (Solanaceae)

0/10

-

0/10

-


20-22

Corchorus capsularis (Tiliaceae)

0/10

-

0/10

-

0/10

-

C. olitirius (Tiliaceae)

0/10

-

0/10

-

0/10

-

Croton bonplandianum (Euphorbiaceae)

0/10

-

0/10

-

0/10

-

Acalypha indica (Euphorbiaceae)

0/10

-

0/10

-

0/10

-

Rhaphanus sativus (Brassicaceae)

0/10

-

0/10

-

0/10

-

Ageratum conyzoides (Asteraceae)

0/10

-

0/10

-

0/10

-

A: Number of symptomatic plants/total number of plants inoculated, B: Incubation period (days)

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0/10


A Malvastrum coromandelianum (Malvaceae)

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North Indian isolate


Name of the plant species (family)


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Table 4. Host range study of three geographical isolates under experimental glasshouse condition.


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Table 5. Sequence identity of the examined begomovirus isolates with other begomoviruses. Highest values are indicated in bold letters. Sequence identity with examined begomovirus isolates (in %) Northern Eastern Southern

Begomovirus name (accession No.)

India

India

India

Malvastrum yellow vein virus-[China:Yunnan206:Ageratum:2003] (AJ744881)

88.2

79.4

76.4

Malvastrum yellow vein virus-[China:Yunnan 47:2001] (AJ457824)

87.9

78.6

75.7

Malvastrum yellow vein Yunnan virus-[China:Yunnan160:2003] (AJ786711)

87.1

81.9

74.2

Cotton leaf curl Bangalore virus-[India:Bangalore:2004] (AY705380)

83.5

87.4

74.1

Bhendi yellow vein mosaic virus-[India:Maharastra:08] (EU482411)

82.6

75.9

75.1

Mesta yellow vein mosaic virus -[India:Barrackpore2:2006] (EF428256)

81.4

94.5

72.1

Tomato leaf curl Karnataka virus-Janti [India:Janti:2005] (AY754812)

80.2

72.9

83.6

Tomato yellow leaf curl virus-Oman [Oman:Al-Batinah:2005] (DQ644565)

75.8

69.8

74.3

Malvastrum yellow mosaic virus-[China:Hainan 37:2004](AM236756)

81.3

80.6

74.7

Cotton leaf curl Multan virus-Bhatinda [India:Bhatinda](DQ191160)

80.0

83.6

74.7

Cotton leaf curl Multan virus-Hisar [Pakistan:Multan 311:Okra:1996](AJ002459)

79.6

80.8

73.7

Cotton leaf curl Multan virus-Rajasthan [India:New Delhi2:2003](AY795605)

79.9

81.7

75.1

Cotton leaf curl Multan virus-Rajasthan [India:Abohar:2003](AY795606)

79.9

81.7

75.0

Mesta yellow vein mosaic Bahraich virus-[India:Bahraich:2007](EU360303)

79.0

80.3

72.2

Okra yellow vein mosaic virus-[Pakistan:Faisalabad 201:1995](AJ002451)

79.4

79.5

74.3

Tomato leaf curl Karnataka virus-Bangalore [India:Bangalore:1993]( U38239)

79.9

72.7

84.3

Tomato leaf curl Gujarat virus-[Nepal:Panchkhal:2000](AY234383)

76.6

74.1

81.7

Tomato leaf curl Gujarat virus-[India:Vadodara:1999](AF413671)

76.6

74.3

82.2

Tomato leaf curl Joydebpur virus-Bangladesh [Bangladesh](AJ875159)

75.6

72.0

95.4

fecting begomoviruses, respectively (Fig. 3). The genome organization of the isolates characterized in this study indicated the presence of all the six open reading frames (ORFs) (AV1 and AV2 in the virion sense and AC1, AC2, AC3 and AC4 in the complementary sense) reported to be present in the genome of Old World begomoviruses. All the ORFs of eastern and southern isolates shared maximum sequence identity with different isolates of MeYVMV (98-100%) and with ToLCJoV (99.5-100%) respectively. Interestingly, ORF-wise nucleotide sequence identity analysis of northern isolates revealed that while AV1, AC1, AC2 and AC3 shared highest identity (90-95%) with different isolates of MaYVV reported from China, the AC4 shared highest identity (94.6%) with an isolate of Tomato yellow leaf curl virus-Oman [Oman:Al-Batinah:2005] (TYLCV-OM[OM:Alb:05],DQ644565) and the AV2 ORF shared highest identity (95.9%) with Cotton leaf curl Bangalore virus-[India:Bangalore:2004] (CLCuBV[IN:Ban:04], AY705380) (Table 6), thus indicating possible recombination in the genome of these northern

isolates. In fact further analysis of recombination through RDP software showed the occurrence of multiple overlapping recombination events with different parental combinations (Table 7). Nucleic acid spot hybridization (NASH) analysis. Among the 130 symptomatic samples collected from eastern (55), northern (47) and southern (28) India, 98.18%, 100% and 92.85% samples, respectively, gave positive hybridization signals with their corresponding begomovirus specific probes. But these probes gave cross-reaction and hence no species specific detection was possible.

DISCUSSION

In the tropics and subtropics, begomoviruses are emerging as a major threat due to their increasing incidence and the severity of the diseases they cause in many economically important crops (Polston and Anderson,


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Fig. 3. Rectangular cladogram presenting the phylogenetic grouping of different begomoviruses based on alignments of the complete nucleotide sequences. Dendrogram was constructed with Neighbor-Joining method. Numbers at nodes indicate bootstrap scores (1,000 replicates).

1997). Evolution of newer viruses and variants of the existing ones and occurrence of mixed infections with different begomoviruses and satellite molecules has evinced the adaptability to new hosts by these begomoviruses which has posed such a serious threat (Varma and Malathi, 2003). Among the newly emerging begomovirusassociated diseases in India, KLCuD is spreading rapidly and seriously threatens this crop in future. The importance of the disease stems from the fact that it causes reduction in plant height and thus the disease probably has an adverse effect in the fibre yield and quality. Any broad-spectrum strategy for virus disease management needs a thorough understanding on the variability of different isolates of a particular virus. For be-

gomoviruses, studies on host range and vector transmission could indicate the variability among different species, but for more precise identification, molecular characterization is essential for in-depth understanding on their genetic variability. Among the molecular tools, PCR-based approaches, like amplification of targeted viral genome or genomic component(s) followed by DNA sequencing has proved to be more accurate and easy for studying variability among different isolates of begomoviruses (Briddon and Markham, 1994; Sharma et al., 2005). In the present study, we have shown the association of three different begomoviruses with KLCuD from three geographical locations in India. All these viruses


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Table 6. Sequence identity of present north Indian begomovirus isolates with other closely related begomoviruses and comparison of ORFs wise sequence identity at the nucleotide level. Highest values are indicated in bold letters.

Begomovirus name

Abbreviation (accession No.)

Malvastrum yellow vein virus[China:Yunnan206: Ageratum:2003]

MaYVV[CN:Yn206:Age:03] (AJ744881)

Malvastrum yellow vein virus[China:Yunnan 47:2001]

MaYVV-[CN:Yn47:01] (AJ457824)

Malvastrum yellow vein Yunnan virus-[China:Yunnan160:2003]

MaYVYV[CN:Yn160:03] (AJ786711)

Cotton leaf curl Bangalore virus[India:Bangalore:2004]

CLCuBV-[IN:Ban:04] (AY705380)

Tomato yellow leaf curl virusOman [Oman:AlBatinah:2005]

TYLCVOM[OM:Alb:05] (DQ644565)

Overall nt seq. (% identity)

ORFs AC1

AC2

AC3

AC4

AV1

AV2

88.2

90.2

95.1

93.0

92.3

89.3

89.6

87.9

89.7

94.9

92.0

92.6

90.1

88.7

87.1

93.5

93.1

92.3

93.6

78.3

87.3

83.7

82.3

90.7

89.1

69.9

89.1

95.9

76.0

84.1

64.6

70.8

94.6

73.3

70.9

Table 7. Analysis on recombination of northern Indian begomovirus isolate using RDP beta 8 version. Nucleotide coordinate

Number of event

Major parent

Minor parent

Average p value

49-578

90 90

583-956

76

1996-95

1192

2022-2680

1192

2131-2695

1192

1605-2562

1192

1060-1704

12

1139-1841

638

1448-2373

1192

2104-2708

1192

CLCuBV[IN:Ban:04](AY705380) CLCuBV[IN:Ban:04](AY705380) ToLCKVBan[IN:Ban:93](U38239) CLCuKVMan[IN:Dab](AY456683) CLCuKV-Fai[PK:K72b:95] (AJ002448) ToLCGV[IN:Vad:99](AF413671) CLCuKV-Fai[PK:Fai1] (AJ496286) MaYMV[CN:Hn36:04](AM236755) CLCuMVHis[IN:ND:99](AY765256) CLCuMVFai[PK:Fai2](AJ496287) ToLCGV[IN:Vad:99](AF413671)

1.317x10-02

38-585

CLCuMVBha[IN:Bha](DQ191160) CLCuMVHis[IN:His:99](AY765253) CLCuBV[IN:Ban:04](AY705380) CLCuBV[IN:Ban:04](AY705380) MeYVMV-[IN:Bar2:06] (EF428256) CLCuBV[IN:Ban:04](AY705380) CLCuMVRaj[IN:Sir:99](AY765254) OYVMV[PK:Fai201:95](AJ002451) ToLCKVBan[IN:Ban:93](U38239) CLCuBV[IN:Ban:04](AY705380) CLCuAV[PK:K802a:96](AJ002455)

1.317x10-02 9.680x10-03 7.999x10-04 7.999x10-04 7.999x10-04 7.999x10-04 1.317 x10-02 1.365 x10-03 7.999x10-04 7.999x10-04


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are transmitted by whiteflies with different transmission efficiencies (lowest for southern isolates) and have narrow host ranges. Disease incidence was also lowest in southern India. This lower incidence of the disease in the south could be connected with the lower transmission rate of the ToLCJoV in kenaf but thorough investigation in this regard in future may provide more definite evidence. The present investigation together with the previous study on the variability of betasatellites (Paul et al., 2008) has revealed the variability of the begomovirus complexes associated with KLCuD in India. It is apparent that, in eastern India, the begomovirus complex is composed of MeYVMV and CLCuMV while in southern India, ToLCJoV and its associated betasatellite constitute the complex. In northern India, a proposed new species of begomovirus, tentatively named as Kenaf leaf curl virus, and an isolate of CLCuMV were found associated with the disease. Infectivity analysis with these begomovirus complexes could be a potential area of future research for understanding the role of these begomovirus complexes in causing symptoms. Such region-specific variability in begomoviruses is common in tomato and cotton, where the same leaf curl symptom has been reported to be caused by different begomoviruses at different locations (Kirthi et al., 2004; Mansoor et al., 2003; Reddy et al., 2005). In-silico recombination studies indicate that the north Indian isolate could have evolved by recombination. Using statistical methods, Padidam et al. (1999) showed that genomic recombination is very frequent with geminiviruses, between variants of the same virus and also between species. The experimental evidence for such natural recombination could be another challenging topic in future research in the context of understanding the evolution of such newer begomoviruses. As the three begomovirus species characterized in the study shared average 76% nucleotide sequence identity among themselves, the probes developed against each species cross reacted with the others and hence it was not possible to specifically detect one particular species of the virus. So at this point of time, it is not possible to unravel the distribution pattern of each virus in different locations. However, the molecular probes developed in this investigation could be used as general diagnostic tools to detect the presence of begomovirus in field samples.

ACKNOWLEDGEMENTS

We thank the Director of Central Research Institute for Jute and Allied Fibers for providing infrastructural support. S. Paul is indebted to the Indian Council of Agricultural Research, New Delhi, for providing financial assistance to carry out the work.

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Received December 6, 2008 Accepted July 20, 2009

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