RESEARCH COMMUNICATIONS

Diagnosis, monitoring and transmission characteristics of Cotton leaf curl virus

J. A. Khan* and J. Ahmad
Plant Virus Laboratory, National Botanical Research Institute, Lucknow 226 001, India

Cotton leaf curl disease (CLCuD), a major threat to the cotton industry, has a complex etiology. Despite the identification and involvement of Cotton leaf curl virus (CLCuV, genus Begomovirus) and additional different satellite DNA molecules termed as DNA and DNA 1 with CLCuD, the precise causative agents appear to be unclear. A cotton plant (Gossypium hirsutum) exhibiting symptoms of CLCuD was collected from Punjab and investigated for the presence of CLCuV in its hosts and vector. It was transmissible through whitefly (Bemisia tabaci) causing CLCuD symptoms in cotton. Employing begomovirus-specific primers located in the conserved regions of the coat protein gene, total DNA isolated from infected tissues of cotton leaf was subjected to PCR. It yielded a DNA fragment of about 550 base pairs, which was cloned and sequenced. In multiple sequence alignment with other CLCuV isolates, it revealed up to 99% nucleotide sequence identity in the corresponding regions. PCR and Southern hybridization assays were applied to monitor CLCuV in its hosts and to study transmission characteristics. A time course experiment demonstrated the presence of CLCuV in its alternate host (Lycopersicon esculentum), 12 days before the appearance of symptoms. Further, it could be detected in a single whitefly vector. Transmission studies showed that CLCuV could be acquired by whitefly within 4 h and was transmitted to test plant (Nicotiana benthamiana) within 1 hr of feeding. Two whiteflies were sufficient to induce symptoms. This study will help in understanding the epidemiology of CLCuV and designing effective management strategies to control this devastating disease of national importance. COTTON, an important commercial crop, is extensively cultivated in India. It is infected by several pests and pathogens inducing different diseases. Among them, cotton leaf curl disease (CLCuD) is the most important, causing enormous losses to the crop13. During the last decade, CLCuD has caused substantial losses to cotton crop in Pakistan that later spread into India38. The disease was believed to be caused by cotton leaf curl geminivirus4,9. Recent studies have shown that it is a complex disease with the involvement of Cotton leaf curl virus (CLCuV, genus Begomovirus) and satellite molecules, viz. DNA 1 (genus Nanovirus) and DNA . Further DNA 1, a circular single-stranded DNA of 1.4 kb associated with the diseased cotton plants, reveals some sequence homology to genomic components of nanoviruses
*For correspondence. (e-mail: jawaidkhan14@yahoo.co.in) CURRENT SCIENCE, VOL. 88, NO. 11, 10 JUNE 2005

encoding replication-associated protein10. Interestingly, DNA 1 is encapsidated in coat protein (CP) of CLCuV and is whitefly transmissible in contrast to nanoviruses, which have aphid vector. Recently, a satellite DNA, i.e. DNA has been shown to be associated with CLCuD8,11,12. CLCuV belongs to the genus Begomovirus of the family Geminiviridae. Begomoviruses are transmitted by Bemisia tabaci vector in a circulative, persistent manner and infect dicotyledonous plants. They possess a small circular, singlestranded DNA genome encapsidated in a geminate particle. While majority of begomoviruses contain bipartite genome (designated as DNA A and DNA B), an increasing number of them are being identified having only monopartite genome (equivalent to DNA A). CLCuV-infected plant contains monopartite begomovirus, i.e. DNA A as there is no evidence of DNA B10,13. It is tedious and cumbersome to prepare antisera to begomoviruses, as they are restricted to phloem tissues and occur in low concentrations in plant hosts. Therefore, serology is not ideal for their detection, identification and classification. CP is the most conserved gene of begomoviruses and International Committee on Taxonomy of Viruses has formally accepted its sequences for the provisional identification and classification of an unknown begomovirus14. Further, an arbitrary value of 90% nucleotide sequence identity has been suggested as guideline for identification of a distinct species (< 90%) or a strain (> 90%)15. Aligning the sequences of the CP (core) region for a large number of begomovirus isolates, Brown et al.16 have clearly shown that the CP (core) region is sufficient to reliably identify and classify an unknown begomovirus. This communication describes the monitoring of CLCuV in its hosts and vector. As a first step to reliably detect CLCuV DNA, it was attempted to clone and sequence the CP (core) gene of CLCuV. PCR/molecular hybridizationbased assays demonstrate its presence in the host before the appearance of symptoms, as well as in single whitefly vectors. Further, transmission characteristics of CLCuV were also studied. These investigations may help in understanding the epidemiology of CLCuV and developing effective control measures of CLCuD. Initially a cotton plant (Gossypium hirsutum) showing leaf curl disease symptoms was collected from Ludhiana, Punjab. The symptoms consisted of curling of leaves, thickening and darkening of veins. The causal agent was transmitted by B. tabaci to G. hirsutum. Healthy (virus-free) whiteflies (B. tabaci) were reared on Clitoria ternatia (immune to begomoviruses) in insect-proof cages placed in a chamber with 28C temperature and 16 h photoperiod. Healthy B. tabaci were fed on the infected cotton plant for a period of 24 h. Thereafter, viruliferous whiteflies were collected with the help of an aspirator and placed for another 24 h on healthy plants of G. hirsutum. After inoculation, plants were sprayed with an insecticide and maintained under insect-proof glasshouse conditions. Total DNA from 50 mg of infected cotton-leaf tissues was extracted essentially as described by Bendahmane et al.17.
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Finally, DNA pellets were dried under vacuum, resuspended in 50 l of TE buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA, pH 8.0) and subjected to PCR. Begomovirus-specific primers, viz. AV494 (GCC/ TATG/ATT/CAGA/GAAGCCA/C AG; viral-sense polarity) and AC1048 (GGA/GTTA/G/TGAG/AGCATGT/ ACGTACAT G; complimentary-sense polarity) corresponding to the conserved core regions of the CP genes were employed in the PCR18. A final volume of 50 l PCR mix containing 5 l DNA template (isolated from infected cotton-leaf tissues), 1.5 U Taq DNA polymerase, 175 M dNTPs and 25 pmol of each primer was taken in a PCR tube. PCR amplification was performed in a thermocycler (model Gene Amp PCR system 9700, Perkin Elmer) with the following parameters: 30 cycles with 94C for 1 min (denaturation), 48C for 1 min (annealing) and extension for 1 min. PCR product (5 l) of each sample was analysed by electrophoresis on 1% agarose gel (containing ethidium bromide) in TE buffer, pH 8.0. The gel was visualized in ultraviolet light and photographed on gel documentation system (Nighthawk, Pdi, USA). All PCR reagents and oligo primers were procured from Bangalore Genei. PCR yielded a DNA fragment of about 550 bp that was cloned into pUC 19 vector at Sma I site using SureClone Ligation kit according to the suppliers instructions (Amersham Biosciences). After transforming the ligated DNA into Escherichia coli strain DH5- cells, several white colonies were obtained on LB (luria broth) plate containing appropriate amount of antibiotic (ampicillin), X-gal (5bromo-4-chloro-3-indolyl--D-galactosidase) and IPTG (isopropyl--D-thiogalactopyranoside). One such colony was selected after preliminary screening and identity of the obtained DNA clone (henceforth referred to as pCLCP20) was confirmed after nucleotide sequence determination by an automatic sequencer (Applied Biosystems 373 DNA sequencer) and its alignment with sequences of other begomoviruses (including CLCuV isolates) available in GenBank. The sequences of the CLCuV isolate from Punjab (this study) have been submitted to GenBank (AY730590) and they were analysed using BLAST searches19 and Clustal X program20. Reference sequences for CLCuV and other begomoviruses and their respective GenBank accession numbers are given in Figure 1 and Table 1. For monitoring of CLCuV in host lysates, healthy B. tabaci were fed on infected leaves of G. hirsutum for 24 h. After acquisition access period (AAP), viruliferous B. tabaci were collected and a batch of ten insects per plant was placed on healthy seedlings of Lycopersicon esculentum var. Pusa Ruby (an alternate host of CLCuV) for an additional 24 h. Two kinds of experiments were conducted. In the first experiment, observations relied upon the appearance of symptoms in inoculated plants. In the second experiment, monitoring of virus was based on PCR amplification followed by its confirmation in Southern hybridization. To accomplish this, leaf samples were taken at 0, 3, 5, 7, 9, 11, 13, 15, 17, 19 21, 23, 25, 27, 29 and 31 days post inoculation (dpi) with viruliferous B. tabaci. Total DNA from CLCuVinfected leaf samples was isolated and subjected to PCR. The procedures for total DNA isolation and PCR amplification were the same as described earlier.

Table 1.

Per cent nucleotide sequence identity of the coat protein (core) gene of Cotton leaf curl virus, Punjab isolate with related begomoviruses Reference (GenBank accession number) AJ002449 AY45683 AF363011 AJ0022458 AJ002447 AF240675 AF188481 AJ437618 AJ558121 AF314531 AJ457824 AF336806 U38239 AF295401 AF428255 AY456684 AJ579307 AJ002459 AF314144 AJ314739 Percentage nucleotide sequence identify 99 98 98 82 82 91 90 89 89 89 86 85 84 82 82 82 82 82 83 83

Begomovirus/origin

Cotton leaf curl virus Kokhran (806b, Pakistan) Cotton leaf curl virus Dabawali (Pakistan) Cotton leaf curl virus Rajasthan Cotton leaf curl virus Multan (26, Pakistan) Cotton leaf curl virus Multan (62, Pakistan) Tobacco curly shoot virus (Y1, China) Tomato leaf curl virus Bangladesh Ageratum enation virus Nepal Euphorbia leaf curl virus (G35, China) Pepper leaf curl virus Bangladesh Malvastrum yellow vein virus (Y47, China) Chilli leaf curl virus (Multan, Pakistan) Tomato leaf curl virus Bangalore II Tomato leaf curl virus Bangalore (Ban5) Tomato leaf curl virus Bangalore (Kolar) Tomato leaf curl virus Bangalore Cotton (Fatehabad) Cassava mosaic virus Sri Lanka (Adivaram) Cotton leaf curl virus Multan (Okra, Pakistan) Ageratum yellow vein virus Sri Lanka Indian cassava mosaic virus Maharashtra

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Figure 1. Nucleotide sequence alignment of PCR-amplified CP gene fragment of CLCuV isolate (accession no. AY370590; this study) with other closely related begomoviruses. The origin and accession of the CP gene sequences are as follows: AJ437618 (Ageratum enation virus Nepal), U38239 (Tomato leaf curl virus Bangalore II), AF314531 (Pepper leaf curl virus Bangladesh), AF363011 (Cotton leaf curl virus Rajasthan), AJ002449 (Cotton leaf curl virus Kokhran, Pakistan). Asterisks show identical nucleotides. Nucleotide positions are given according to AF363011.

The specificity and authenticity of PCR products was checked in Southern hybridization. To this end, a hybridization probe representing the CP gene of CLCuV was prepared by amplifying the clone, viz. pCLCP-20 in PCR employing begomovirus-specific primers (AV496 and AC1048). The PCR product was run on 0.7% agarose gel and the desired amplified DNA fragment was cut-off the gel and purified using Sephaglas BandPrep kit (Amersham Biosciences). The eluted DNA was manipulated for preparing hybridiCURRENT SCIENCE, VOL. 88, NO. 11, 10 JUNE 2005

zation probe labelled with 32P dCTP following primer extension method21. PCR products from total DNA isolated from infected leaf tissues of L. esculentum at different dpi were subjected to Southern hybridization. They were run in agarose gel and transferred by capillary action to Hybond N membrane (Amersham Biosciences), and then UV-cross-linked for 15 min22. The membrane was prehybridized at 42C (1 h) followed by hybridization with the radiolabelled probe at
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65C overnight. It was washed twice each in 2X SSC, 0.1% SDS and 1X SSC, 0.1% SDS for 5 and 15 min respectively, at room temperature followed by another wash in 1X SSC, 0.1% SDS at 65C for 15 min. The blot was exposed to Kodak diagnostic film for autoradiography. For the detection of CLCuV in whitefly vector, healthy B. tabaci were fed on CLCuV-infected cotton plants for 24 h. After AAP, they were collected and total DNA was isolated. Insects in groups of 1, 2 and 3 were taken in separate eppendorf tubes and ground in 100 l of 0.4% SDS, 1 mg/ml proteinase K followed by incubation at 55C. The mixture was extracted twice with an equal volume of phenolchloroformisoamylalcohol (25 : 24 : 1). The aqueous phase was collected and DNA was ethanol pelleted overnight at 20C. Total DNA was pelleted and subsequently washed with 70% alcohol and dissolved in TE buffer (10 mM TrisHCl, pH 7.5, 1 mM EDTA, pH 8.0) after drying in vacuum. PCR-amplified DNA fragments obtained from viruliferous or healthy B. tabaci were electrophoresed and transferred to Hybond N membrane. The blot was subjected to Southern hybridization using radiolabelled probe prepared from the CP gene (clone pCLCP-20) of CLCuV. Procedure for labelling DNA probe and its hybridization to PCR amplified DNA fragment was the same as described earlier. Let us consider acquisition of CLCuV by whiteflies. The minimum AAP required for CLCuV transmission was determined by allowing healthy B. tabaci to feed on CLCuVinfected Nicotiana benthamiana for a period of 0, 1, 2, 3, 4, 6, 12, 24, 30, 42 and 48 h. Ten whitefiles were collected from infected plants, and five from healthy virusfree plants for each AAP. Total DNA was isolated from B. tabaci and PCR was performed. PCR product (1 l) of each AAP was spotted on Hybond N membrane and subjected to dot-blot hybridization with radiolabelled probe prepared from the CP gene (clone pCLCP-20) of CLCuV. Protocols for isolating total DNA from whiteflies, PCR amplification, labelling of DNA probe and its hybridization to PCR products were the same as described earlier. For inoculation access period experiments, healthy B. tabaci were fed on CLCuV-infected plant for a period of 24 h. Thereafter, viruliferous B. tabaci were collected with the help of an aspirator and ten whiteflies per plant were allowed to feed on individual healthy N. benthamiana for 0, 1, 2, 4, 5, 8, 16, 24, 36 and 48 h. Plants were sprayed with an insecticide and maintained in insect-proof glasshouse. Symptoms were recorded after 4 weeks. Let us consider relationship between number of viruliferous whiteflies and appearance of symptoms. The transmission efficiency of CLCuV was determined by allowing B. tabaci to feed on CLCuV-infected plants for a period of 24 h. Thereafter, viruliferous B. tabaci in batches of 1, 2, 3, 4, 5, 6 and 7 numbers were transferred to individual healthy N. benthamiana and allowed a 24 h IAP. After IAP, plants were sprayed with an insecticide and maintained in insect-proof glasshouse. Symptoms were recorded after 4 weeks.
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The causal agent of the disease could be transmitted to G. hirsutum by B. tabaci. The symptoms consisted of curling (upward) of leaves, thickening and darkening of vein on the undersurface of leaf forming a network of secondary and tertiary veins. In some cases, veins could develop into cup-like enations on the undersurface (Figure 2). Let us consider the cloning and sequencing of CLCuV CP gene. PCR yielded a DNA fragment of expected size (about 550 bp from the core region of the CLCuV CP gene) with DNA template isolated from CLCuV-infected leaf tissues (Figure 3). The authenticity of PCR fragment was confirmed by its cloning (resulting in clone pCLCP-20) and sequencing. The ends of the sequences close to the primers were not included for analyses. The sequences of our clone have been submitted to GenBank (accession no. AY730590). They were aligned with sequences of other closely related begomoviruses (including CLCuV isolates) available in GenBank (Figure 1). Further, a comparison of nucleotide sequence identity with other closely related begomoviruses is presented in Table 1. Among others, it shared 99% nucleotide sequence identity with CLCuV Kokhran strain (Pakistan), 98% with CLCuV Dabawali strain (Pakistan) as well as CLCuV Rajasthan strain. CLCuV strain from Multan (Pakistan; accession nos AJ002458 and AJ002447) showed relatively significant differences, having 82% identity. For the monitoring of CLCuV in host plants, PCR was performed on total DNA isolated from the leaf tissues of CLCuV-infected L. esculentum (var. Pusa Ruby) at various dpi. This resulted in PCR fragment of about 550 bp from DNA isolated at 17 dpi and later (Figure 4 (top), lanes 47). The specificity of PCR-amplified DNA fragment was confirmed by its hybridization with the DNA probe prepared from the CP gene of CLCuV. The PCR fragment obtained with DNA of 17 dpi or later, hybridized with the DNA probe in Southern hybridization. It, however, did not give any positive signal with DNA of 15 dpi or earlier (Figure 4 (bottom) lanes 13).

Figure 2. A cotton (Gossypium hirsutum) plant showing typical symptoms of leaf curl disease of cotton: leaf-like outgrowth on the underside of leaf; darkening of veins showing net-like structures. CURRENT SCIENCE, VOL. 88, NO. 11, 10 JUNE 2005

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Symptoms were regularly observed in the inoculated plants. CLCuV symptoms started appearing at 29 dpi. These results showed that CLCuV DNA could be detected by PCR 12 days before the appearance of symptoms. For the detection of CLCuV in whitefly vector, total DNA isolated from whiteflies in groups of 1, 2 and 3 was subjected to PCR amplification. It yielded a faint DNA fragment of about 550 bp from 1, 2 and 3 numbers of viruliferous whiteflies. Its presence was confirmed by Southern hybridization, where it gave strong signals when hybridized to a probe prepared from the CP gene of CLCuV (Figure 5, lanes 24). For acquisition access time course experiments, acquisition of CLCuV in B. tabaci vector was studied by detecting CLCuV DNA at various AAP in dot-blot hybridization. CLCuV could be detected in B. tabaci as early as 4 h after access to the infected plant (Figure 6). For inoculation access time course experiments, viruliferous B. tabaci had 0, 1, 2, 4, 5, 8, 16, 24, 36 and 48 h of IAP. B. tabaci were able to transmit the virus and could develop symptoms in test plants when the IAP was 1 h or more. The number of plants infected was 0/10, 2/10, 2/10, 4/10, 5/10, 6/10, 6/10, 7/10, 7/10 and 7/10 respectively.

Figure 3. PCR amplification of CLCuV CP gene fragment of about 550 base pairs (lane 2) from nucleic acid extracted from CLCuVinfected leaves. Lane 1, EcoRI and HindIII digested -DNA marker.

Figure 5. Detection of CLCuV DNA in a batch of 1, 2 and 3 B. tabaci. They were given a 24 h acquisition on CLCuV-infected cotton plant (G. hirsutum). Total DNA was isolated from healthy and viruliferous whiteflies and subjected to PCR. The PCR products were run in agarose gel, transferred to Hybond N membrane and hybridized with 32 P-labelled DNA probe representing the CP gene of CLCuV. CLCuV DNA was detectable in 1, 2 and 3 numbers of whiteflies (lanes 24), and was absent in healthy whitefly (lane 1).

Figure 4. Monitoring of CLCuV DNA in Lycopersicon esculentum at various dpi with viruliferous Bemisia tabaci. (Top) Total DNA was isolated from CLCuV-infected leaf tissues of L. esculentum at 11, 13, 15, 17, 19, 21, 23 dpi and subjected to PCR. The PCR products were run in agarose gel containing ethidium bromide. CLCuV DNA was detectable at 17, 19, 21, 23 dpi (lanes 47), while it was absent at 11, 13, 15 dpi (lanes 13), (Bottom) Southern hybridization of PCR-amplified DNA fragments with 32P-labelled DNA probe prepared from the CP gene of CLCuV. CURRENT SCIENCE, VOL. 88, NO. 11, 10 JUNE 2005

Figure 6. Acquisition of CLCuV in B. tabaci during 48 h of AAP. Healthy whiteflies in groups of ten were given AAP on CLCuVinfected and healthy Nicotiana benthamina plants. Insects were collected after 0, 1, 2, 3, 4, 6, 12, 24, 30, 42 and 48 h of AAP. Total DNA was extracted from whiteflies and PCR was performed. One-fiftieth of PCR products were spotted on Hybond N membrane and hybridized with 32P-labelled DNA probe representing the CP gene of CLCuV. CLCuV DNA was detectable in whitefly lysates at 4, 6, 12, 24, 30, 42 and 48 h of AAP (lanes 511), and was absent at 0, 1, 2, 3 h AAP (lanes 14). 1807

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In our experiment, the acquisition and inoculation access thresholds for CLCuV transmission by B. tabaci were 4 and 1 h, respectively. To find the number of whiteflies inducing CLCuV symptoms, healthy B. tabaci were given AAP for 24 h. Viruliferous whiteflies in batches of 1, 2, 3, 4, 5 and 10 numbers had inoculation access to healthy N. benthamiana. The number of plants that showed symptoms was 0/10, 3/10, 3/10, 5/10, 5/10 and 7/10 respectively. A minimum of two whiteflies could induce CLCuV symptoms. This communication describes the monitoring of CLCuV in its potential hosts and whitefly vector. Viral DNA could be detected as early as 17 days after inoculation or 12 days before the appearance of symptoms in its alternate host, L. esculentum. It is possible that at this stage the concentration of the virus is not sufficient to be effectively spread by whitefly. If this is the case, it might be feasible to take advantage of this time lag to destroy infected plants in order to slow down or stop CLCuV epidemics. In general, epidemiological studies can be conducted either by monitoring the spread of viruliferous vectors (B. tabaci) or by identifying virus-infected plants. A better understanding of the virusvectorhost relationship may be an important aspect in developing means or designing strategies to prevent CLCuV infection. As CLCuD of cotton is not seed-borne, both the causal agents and whitefly vector must survive on alternate hosts. In India, among others, crops such as tomato (L. esculentum), okra (Abelmoschus esculentus), pepper (Capsicum annuum), guar (Cyanopsis tetragonoloba), sunn hemp (Crotalaria juncea) and bitter gourd (Mormordica charantia) show typical leaf curl symptoms caused by begomoviruses. The association of begomoviruses with many crops is evident by serological tests7,23, PCR and Southern hybridization assays9,2326. However, these reports do not provide conclusive evidence on the identity of the causative agent. Authentic identity of associated begomovirus in alternate hosts is of utmost importance for understanding the epidemiology. Recently, PCR and nucleic acid spot hybridization assays were applied to detect Cotton leaf curl Rajasthan virus (CLCuRV) DNA A and CLCuRV DNA in their potential hosts and whitefly vector8. The virus investigated in our studies was identified as an isolate of CLCuV. This is supported by its transmission through whitefly, expression of characteristic symptoms of CLCuD, and PCR-based amplification of diagnostic DNA band of about 550 bp with begomovirus-specific primers. Further, after sequencing PCR amplicon revealed a high percentage of nucleotide sequence identity (up to 99) with CLCuV isolates from India and Pakistan. In the present study, CLCuV DNA could be detected in extracts of individual B. tabaci using PCR followed by Southern hybridization. This has helped in studying the transmission properties of CLCuV. In our experimental conditions, the minimum AAP and IAP was 4 and 1 h, respectively. These findings, however, differ from those of
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Nateshan et al.7, who found that in Karnataka (India), B. tabaci required a minimum of 1 h AAP and 5 min IAP. This difference could be due to the different feeding behaviour or specificity of insect vector in different hosts. Further, the titre of virus in different hosts could influence vector acquisition. For our routine whitefly transmission of CLCuV, 24 h each of AAP and IAP was used to achieve maximum transmission. Detection and diagnosis of geminiviruses have mainly depended upon biological characteristics such as symptomatology and transmission to test plants. In this communication, an application of DNA-based virus-detection approach has been extended to study the monitoring and transmission characteristics of CLCuV in its hosts and vector. The sensitive and rapid assay developed in the study to detect CLCuV in its hosts and vector may facilitate research in this area.

1. Dickson, R. C., Johnson, M. M. and Laird, E. F., Leaf crumple, a virus disease of cotton. Phytopathology, 1954, 44, 479480. 2. Brown, J. K. and Nelson, M. R., Geminate particles associated with cotton leaf crumple disease in Arizona. Phytopathology, 1984, 74, 987990. 3. Briddon, R. W. and Markham, P. G., Cotton leaf curl virus disease. Virus Res., 2000, 71, 151159. 4. Varma, A. et al., Occurrence of leaf curl of cotton and okra in northern India. In 6th International Congress of Plant Pathology, 28 July6 August 2003, Montreal, Canada, 17.5.14. 5. Ajmera, B. D., Occurrence of leaf curl virus on American cotton (G. hirsutum) in north Rajasthan. In National Seminar on Cotton Production: Challenges in 21st Century, 1820 April 2004, Hisar. 6. Ali, M., Ahmad, Z., Tanveer, M. and Mahmood, T., Cotton leaf curl virus in the Punjab. Current situation and review of work. Central Cotton Research Institute, Multan/CLCV Project, Ministry of Food, Agriculture and Livestock, 1995, p. 117. 7. Nateshan, H. M., Muniyappa, V., Swanson, M. M. and Harrison, B. D., Host range, vector relations and serological relationships of cotton leaf curl virus from southern India. Ann. Appl. Biol., 1996, 128, 233244. 8. Radhakrishnan, G., Malathi, V. G. and Varma, A., Detection of DNA A and DNA associated with cotton leaf curl and some other plant diseases caused by whitefly transmitted geminiviruses. Indian Phytopathol., 2004, 57, 5360. 9. Mansoor, S., Bedford, I., Pinner, M. S., Stanley, J. and Markham, P. G., A whitefly transmitted geminivirus associated with cotton leaf curl disease in Pakistan. Pak. J. Bot., 1993, 25, 105107. 10. Mansoor, S. et al., Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan. Virology, 1999, 254, 290299. 11. Briddon, R. W. et al., Identification of DNA components required for induction of cotton leaf curl disease. Virology, 2001, 285, 234 243. 12. Briddon, R. W. et al., Diversity of DNA beta, a satellite molecule associated with some monopartite begomoviruses. Virology, 2003, 312, 106121. 13. Liu, Y., Robinson, D. J. and Harrison, B. D., Defective forms of cotton leaf curl virus DNA-A that have different combinations of sequence deletion, duplication, inversion and rearrangement. J. Gen. Virol., 1998, 79, 15011508. 14. Mayo, M. A. and Pringle, C. R., Virus Taxonomy 1997. J. Gen. Virol., 1998, 79, 649657. CURRENT SCIENCE, VOL. 88, NO. 11, 10 JUNE 2005

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15. Rybicki, E. P., A proposal for naming geminiviruses: A reply by the Geminiviridae study group chair. Arch. Virol., 1998, 143, 421 424. 16. Brown, J. K., Idris, A. M., Torres-Jerez, I., Banks, J. K. and Wyatt, S. D., The core region of the coat protein is highly useful for establishing the provisional identification and classification of begomoviruses. Arch. Virol., 2001, 146, 15811598. 17. Bendahmane, M., Schalk, H.-J. and Gronenborn, B., Identification and characterization of wheat dwarf virus from France using a rapid method for geminivirus DNA preparation. Phytopathology, 1995, 85, 14491455. 18. Wyatt, S. D. and Brown, J. K., Detection of Subgroup III geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction. Phytopathology, 1996, 86, 12881293. 19. Altschul, S. F., Thomas, L. M., Alejandro, A. S., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J., Gapped BLAST and PSIBLAST, a new generation of protein data base search programs. Nucleic Acids Res., 1997, 25, 33893402. 20. Thompston, J. D., Higgins, D. G. and Gibson, T. J., CLUSTAL W: Improving the sensitivity of multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res., 1994, 22, 46734680. 21. Feinberg, A. P. and Vogelstein, B., A technique for radio-labelling DNA restriction endonuclease fragments to high specific activity. Ann. Biochem., 1983, 150, 612. 22. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, 2nd edn. 23. Harrison, B. D., Liu, Y. L., Khalid, S., Hameed, S., Otim-Nape, G. W. and Robinson, D. J., Detection and relationships of cotton leaf curl virus and allied whitefly transmitted geminiviruses occurring in Pakistan. Ann. Appl. Biol., 1997, 130, 6175. 24. Khan, J. A., Siddiqui, M. K. and Singh, B. P., The association of begomovirus with bitter melon in India. Plant Dis., 2002, 86, 328. 25. Khan, J. A., Siddiqui, M. K. and Singh, B. P., The natural occurrence of a begomovirus in sunn hemp (Crotalaria juncea) in India. Plant Pathol., 2002, 51, 398. 26. Khan, J. A., Sohrab, S. S., Aminuddin and Gupta, R. K., Detection of a begomovirus affecting guar [Cyamopsis tetragonoloba (L.) Taub.] in India. Z. Pflanzenkr. Pflanzenschutz., 2002, 109, 68 73.

Inheritance of thermosensitive genic male sterility in rice (Oryza sativa L.)

Li Rongbai1, M. P. Pandey2,* and Pankaj Sharma2
1

Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization and Guangxi Crop Genetic Improvement and Biotenology Lab, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi 530 007, China 2 Department of Genetics and Plant Breeding, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar 263 145, India

Thermosensitive genic male sterility (TGMS) in rice is a useful trait for exploitation of heterosis, especially of intersubspecific kind using two-line system. Three pairs of independent recessive (tms) genes with additive effects were involved in TGMS expression in UPRI 95-140 TGMS. Expression of the trait in F2 generation involving 44 different genetic backgrounds indicated monogenic (3F : 1S), digenic (15 : 1S) and trigenic (63F : 1S) inheritance with frequencies of 18.2, 52.3 and 29.5% respectively. No single pair of genes was capable of causing complete male sterility. Two pairs of major tms genes in UPRI 95-140TGMS, non-allelic to any of the known tms genes, were located on chromosomes 3 and 7, and tentatively designated as tms6(t) and tms7(t) respectively. THERMOSENSITIVE genic male sterility (TGMS) is a useful genetic tool for the development of two-line hybrids in rice. At the thermosensitive stage of panicle development, the TGMS gene(s) cause/s male sterility under high environmental temperatures and result/s in fertility under low temperatures. A TGMS line can therefore be used for hybrid seed production as well as for its seed multiplication under different growing environments. The system has advantages in much simpler and economic hybrid seed production and broader choice of male parents for enhancing yield potential as the maintainer and restorer lines employed in the currently used three-line hybrid breeding system based on male sterility, are not required1. The trait has shown monogenic inheritance and three independent genes, tms1, tms2 and tms3 were reported24. Few other TGMS sources have also been reported5,6. Recently, a new TGMS source, UPRI 95-140TGMS was identified in our hybrid breeding programme7. The trait in the line has shown digenic inheritance8. However, further intensive investigations of the line revealed more complicated inheritance of the trait and therefore, these findings are reported in this communication. The TGMS line, UPRI 95-140TGMS, a spontaneous mutant with known fertilitysterility transformation behaviour under different temperatures7, was studied for inheritance of its TGMS expression. Forty-four male fertile indica rice lines/ varieties (Table 1) were used as male parents to cross the
*For correspondence. (e-mail: matappandey@rediffmail.com)

ACKNOWLEDGEMENTS. We thank D. P. Pushpangadan, Director, NBRI, Lucknow for providing necessary research facilities.

Received 13 September 2004; revised accepted 27 January 2005

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