Search

In this study, the full-length components of mungbean yellow mosaic India virus (MYMIV) DNA-A (MW590720 & MW600934) and DNA-B (MW659819 & MW659820) from a soybean isolate were cloned and sequenced. Nucleotide sequence analysis of both MYMIV components revealed > 96% identity and close ancestry with MYMIV isolates from legumes in Southeast Asia. Furthermore, dimeric infectious clones of MYMIV were generated in the pCAMBIA1302 vector, and a seed infiltration protocol was established for mungbean, soybean, and Nicotiana tabacum . Agroinfiltration induced yellow mosaic symptoms in mungbean and N. tabacum plants 3 weeks post-infiltration, which were further confirmed by PCR using MYMIV-specific DNA primers., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03778-7., Competing Interests: Conflict of interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

During our previous study, the mixed infection of canna yellow mottle virus (CaYMV), bean yellow mosaic virus (BYMV), and cucumber mosaic virus (CMV) was identified in a Black Knight cultivar of canna exhibiting severe yellow streak and mottling symptoms. Before the development of the virus-free plants, the ability of callogenesis and organogenesis from the ovary, stalk, and rhizome explants was tested on different concentrations and combinations of TDZ, NAA, BAP, and Ads growth regulators. The performance of rhizome explants was above all the explant types and 33.33 ± 1.67 rhizomes (out of 50 placed) showed callus development on ME medium (MS supplemented with 0.8 mg/L TDZ and 0.25 mg/L NAA) and further on a refined M4 medium (MS supplemented with 4.0 mg/L BAP, 1.0 mg/L NAA and 50 mg/L Ads) produced 4.06 ± 0.16 shoots per explant. The development of virus-free plants was attempted by in vitro chemotherapy using ribavirin. Not only in callogenesis and shoot development but also in the ribavirin treatments, rhizomes developed about 3.78 ± 0.68 shoots per explant on 40 mg/L ribavirin in the ME medium. These optimizations suggested that ME medium for callogenesis, M4 medium for shoot development and the treatment of 40 mg/L ribavirin for 30 days at M4 medium was effective. The elimination of coinfection of all three viruses from rhizome explants of 0.5 cm 2 of the Black Knight cultivar was attempted. Consequently, a total of 53.33% of plants free from all three viruses (48 out of the 90 plants developed) were obtained when screened by RT-PCR and PCR for their absence., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Narcissus (Narcissus tazetta) is a bulbous ornamental plant propagated vegetatively from bulbs. The Cyrtanthus elatus virus-A (CyEV-A) had been reported to cause a severe mosaic and yellow stripe disease in narcissus. Therefore, this study aimed to develop a protocol for the elimination of CyEV-A from infected bulblets by in vitro chemotherapy (30-50 mg/L ribavirin for 30 days) and electrotherapy (10-30 mA for 20 min), individually and in combination, to produce virus-free plants. The regenerated plants obtained from these treatments were screened for the absence of the CyEV-A by reverse-transcription polymerase chain reaction assays using a set of degenerate primers specific for a potyvirus coat protein gene. The results showed that in vitro chemotherapy (30 mg/L ribavirin for 30 days) alone produced 46.0 % (14/30) of virus-free plants, while electrotherapy (20 mA for 20 min) alone produced 40.0 % (12/30) of virus-free plants. In comparison, a combination of chemotherapy (30 mg/L ribavirin for 30 days) and electrotherapy (20 mA for 20 min) produced 50.0 % (15/30) of virus-free plants. The virus-free plants obtained from this combination treatment exhibited better growth and produced more bulbs compared to the other treatments and control. The protocol may be used for the control of the virus disease in narcissus., (Copyright © 2021. Published by Elsevier B.V.)

Potyvirus species associated with yellow leaf stripe disease of Indian narcissus ( Narcissus tazetta L.) var. Paperwhite has been studied by sequence analyses of ~ 1.5 kb genomic fragments obtained from seven RT-PCR amplifications of infected samples. Sequence analysis revealed the occurrence of three potyvirus species: cyrtanthus elatus virus- A (CEVA: KF430815, KF430816, KM066973, KM066974); narcissus yellow stripe virus (NYSV: KM066972, JQ686724) and narcissus degeneration virus (NDV: MK572806). The existence of three potyvirus species: CEVA, NYSV and NDV are being reported in Indian narcissus., Competing Interests: Conflict of interestThere is no conflict of interest., (© King Abdulaziz City for Science and Technology 2020.)

Bean yellow mosaic virus (BYMV) is a prevalent virus and major threat to gladiolus cultivation the world over. In the gladiolus repository at CSIR-NBRI, Lucknow, several plants (82-88%) of three economically important cultivars were found infected by BYMV showing severe mosaic and stripe symptoms. Affected plants exhibit diminished quality and quantity of florets and corms, thus reducing their value. Attempts were made to eliminate BYMV from the infected gladiolus cormel explants in vitro through thermotherapy (37 °C for 30 days), chemotherapy (30 mg/L ribavirin for 30 days), and electrotherapy (30 mA for 20 min), either alone and in different combinations. The in vitro regenerated plants were free from BYMV infection when checked by RT-PCR using BYMV-specific primers. The combination of electro- and chemotherapies has given the best response as compared to other treatments. Among the individual therapies, electrotherapy (30 mA/20 min) was found to be the best for and production of BYMV-free gladiolus plants (44-46%) with moderate regeneration efficiency (54-58%) followed by chemotherapy and thermotherapy. However, the cormels obtained from a combination of electro- and chemotherapy treatment (30 mA/20 min + 30 mg/L) has given highest virus free (46-52%) and highest therapy efficiency indices (56%) as compared to other treatments. Further, these cormels showed better developed root systems and produced more cormels which were larger in size as compared to the other treatments and control when grown in tissue culture media., Competing Interests: Compliance with ethical standardsThe authors declare that they have no conflict of interest.

Parthenium hysterophorus plants exhibiting severe leaf curl and stunting symptoms were observed near agriculture fields in Lucknow, India. The association of a begomovirus, β-satellite, and α-satellite with these symptoms of a Parthenium disease was investigated by sequence analyses of virus and satellite DNA amplified by rolling circle amplification and polymerase chain reaction. The highest sequence identities and closest phylogenetic relationships for the begomovirus, β-satellite, and α-satellite detected in P. hysterophorus plants were to Tomato leaf curl virus (ToLCV), papaya leaf curl β-satellite (PaLCuB), and Ageratum yellow vein India α-satellite (AYVIA), respectively. These findings identified the virus and satellites infecting the Parthenium sp. as ToLCV, PaLCuB, and AYVIA, respectively. P. hysterophorus and tomato seedlings infected with cloned ToLCV, PaLCuB, and AYVIA by agroinoculation developed leaf curl symptoms, whereas plants infected with ToLCV alone or with ToLCV and AYVIA developed mild yellowing. The results show that this complex infects and causes disease in P. hysterophorus and tomato. P. hysterophorus is an invasive weed commonly found around agricultural fields and along roadsides in India. These results indicate that P. hysterophorus plants infected with ToLCV and associated satellite DNA act as an alternate host (reservoir), and that could lead to increased incidence of tomato leaf curl disease.

Cucumber mosaic virus (CMV) is an important virus since it causes severe losses to many economically important crops worldwide. Five new isolates of CMV were isolated from naturally infected Hippeastrum hybridum, Dahlia pinnata, Hemerocallis fulva, Acorus calamus and Typhonium trilobatum plants, all exhibiting severe leaf mosaic symptoms. For molecular identification and sequence analyses, the complete coat protein (CP) gene of these isolates was amplified by RT-PCR. The resulting amplicons were cloned and sequenced and isolates were designated as HH (KP698590), DP (JF682239), HF (KP698589), AC (KP698588) and TT (JX570732). For study of genetic diversity among these isolates, the sequence data were analysed by BLASTn, multiple alignment and generating phylogenetic trees along with the respective sequences of other CMV isolates available in GenBank Database were done. The isolates under study showed 82-99% sequence diversity among them at nucleotide and amino acid levels; however they showed close relationships with CMV isolates of subgroup IB. In alignment analysis of amino acid sequences of HH and AC isolates, we have found fifteen and twelve unique substitutions, compared to HF, DP and TT isolates, suggesting the cause of high genetic diversity.

During a survey in June 2011, severe leaf yellow mosaic disease was observed on about 45 % plants of Jatropha curcas growing in the Katerniaghat wildlife sanctuary in India. An association of a begomovirus with disease was detected in 15 out of 20 samples by PCR using begomovirus genus-specific primers and total DNA isolated from symptomatic leaf samples. For identification of the begomovirus, the complete genome was amplified using a Phi-29 DNA-polymerase-based rolling-circle amplification kit and total DNA from five representative samples and then digested with BamHI. The linearized RCA products were cloned and sequenced. Their GenBank accession numbers are JN698954 (SKRK1) and JN135236 (SKRK2). The sequences of the two begomovirus isolates were 97 % identical to each other and no more than 86 % to those of jatropha mosaic India virus (JMIV, HM230683) and other begomoviruses reported worldwide. In phylogenetic analysis, SKRK1 and SKRK2 clustered together and showed distant relationships to jatropha mosaic India virus, Jatropha curcas mosaic virus, Indian cassava mosaic virus, Sri Lankan cassava mosaic virus and other begomoviruses. Based on 86 % sequence identities and distant phylogenetic relationships to JMIV and other begomoviruses and the begomovirus species demarcation criteria of the ICTV (<89 % sequence identity of complete DNA-A genome), the begomovirus isolates associated with leaf yellow mosaic disease of J. curcas were identified as members of a new begomovirus species and provisionally designated as jatropha leaf yellow mosaic Katerniaghat virus (JLYMKV). Agroinfectious clones of the DNA molecule of the begomovirus isolate were also generated, and the fulfillment of Koch's postulates was demonstrated in J. curcas plants.

Association of a new begomovirus with leaf crumple disease of Jatropha curcas was identified based on sequence analysis of cloned ~2.7-kb viral DNA genomes from three representative samples amplified by RCA using phi-29 DNA polymerase. Sequence analysis of these isolates SKJ1 (KM066975), SKJ2 (KM189818) and SKJ3 (KM189819)--showed the presence of a monopartite begomovirus-like genome of 2,735 to 2,738 nucleotides containing seven ORFs: V3, V2 and V1 in virion sense and C3, C2, C1 and C4 in the complementary sense. Pairwise alignment showed 99 % nucleotide sequence similarity to each other and less than 81 % identity to other begomoviruses reported worldwide. Based on begomovirus species demarcation criteria for a new species (sequence identity <89 %), the begomovirus isolates were identified as the members of a new Begomovirus species and provisionally designated as Jatropha leaf crumple India virus (JLCrIV).

A distinct strain of hollyhock yellow vein mosaic virus (HoYVMV) and Ludwigia leaf distortion betasatellite (LuLDB) were associated with yellow vein mosaic of hollyhock. The viral DNA genome (JQ911766) and betasatellite (JQ408216) shared highest nucleotide sequence identity (89.2 %) with HoYVMV (the only available sequence in GenBank) and 92 % identity with LuLDB. Agroinfiltration of HoYVMV and LuLDB induced yellow vein mosaic symptoms on hollyhock, thereby demonstrating causality of the disease.

During a survey in February 2011, severe symptoms of upward leaf curling, vein enation on lower side of the leaves, and shortening of internodes were observed on 20 out of 117 Amaranthus hypochondriacus plants (17% disease incidence) examined at breeding plots of CSIR-NBRI, Lucknow. These symptoms are typical of begomovirus infection. PCR with begomovirus-specific primers (3) produced the expected ~1.1-kb product from DNA extracts of 20 symptomatic plants but not from a non-symptomatic plant, suggesting the association of a begomovirus. The full-length begomoviral genome from a representative sample was amplified by rolling circle amplification using Ø-29 DNA polymerase and digested by BamHI, which resulted in a ~2.7 kb product when electrophoresed in 1.0% agarose gel. The product obtained was cloned, sequenced, and sequence data of 2,753 nucleotides was deposited in GenBank (Accession No. JF682242). BLASTn analysis revealed 97 to 98% nucleotide identity and forms a distinct clade with Ageratum enation virus (AEV) isolates. This shows the virus in A. hypochondriacus to be an isolate of AEV. The separate PCRs were also performed with betasatellite and alphasatellite specific primers (1,2) that resulted in ~1.3-kb amplicons from all samples, suggesting their association. The amplification products were cloned and sequenced. An analysis of betasatellite (JX512904) revealed highest 98% nucleotide identity and close phylogenetic relationship with Ageratum leaf curl betasatellite (ALCB, JQ710745). The alphasatellite (JX512905) showed highest 95% identity and close relationship with Hibiscus leaf curl alphasatellite (HLCA, FN794199). This shows the betasatellite and alphasatellite in A. hypochondriacus to be isolates of ALCB and HLCA, respectively. The partial direct repeat clones of the begomovirus (pCAM-AEV), betasatellite (pCAM-ALCB), alphasatellite (pCAM-HLCA) were generated and mobilized into Agrobacterium tumefaciens strain GV3101 and infiltrated in A. hypochondriacus seedlings. The plants inoculated with pCAM-AEV, pCAM-ALCB, and pCAM-HLCA; pCAM-AEV and pCAM-ALCB developed severe leaf curl and enation symptoms on 5/5 plant at 35 days post inoculation, which were similar to those of naturally infected plants, satisfying Koch's postulates. On the other hand, plants inoculated with pCAM-AEV alone or in combination with pCAM-HLCA developed mild symptoms. Plants inoculated with pCAM-ALCB and pCAM-HLCA did not develop symptoms. The results here show that leaf curl and enation disease of A. hypochondriacus in India is caused by AEV and ALCB and that an alphasatellite may be associated with symptomatic plants. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. E. Bull et al. Mol. Biotechnol. 23:83, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

The complete RNA3 genome of Cucumber mosaic virus (CMV) was amplified by RT-PCR from three infected gerbera (Gerbera jamesonii) leaf samples exhibiting severe chlorotic mosaic and flower deformation symptoms. The amplicons obtained were cloned sequenced and deposited in GenBank under the accessions JN692495, JX913531 (from cv. Zingaro) and JX888093 (from cv. Silvester). These sequences shared 98-99 % identities to each other and with a strain of CMV-Banana reported from India, and 90-95 % identities with various strains of CMV reported worldwide. Phylogenetic analysis revealed their closest affinity with CMV-Banana strain, and close relationships with several other strains of CMV of subgroup IB. This study provides evidence of subgroup IB CMV causing severe chlorosis and flower deformation in two cultivars (Zingaro and Silvester) of G. jamesonii in India.

Amaranthus hypochondriacus L. infected with Ageratum enation virus (AEV) was investigated for identifying alteration in the anatomical structures, sap translocation and metabolomic variations using light microscopy, magnetic resonance imaging, NMR spectroscopy and GC-MS, respectively. Combination of GC-MS and NMR spectroscopy identified 68 polar and non-polar metabolites that were present in different levels in healthy and virus-infected A. hypochondriacus. Contrast of T₁ and T₂ weighted MR images showed significant differences in the spatial distribution of water, lipids and macromolecules indicating alterations in the cortical region and disruption of vascular bundles in virus-infected stem tissues. MRI observations are supported by light microscopic examination. Microscopic examination of AEV infected stem revealed severe hyperplasia with a considerable reduction in size of stem cells. The NMR spectroscopy and GC-MS analysis indicated that viral infection significantly affected the plant primary and secondary metabolism resulting in decreased glucose and sucrose content and increase in the concentration of β-sitosterol and stigmasterol. Higher accumulation of TCA cycle intermediates such as citric acid and malic acid in AEV infected plants indicated enhanced rate of respiratory metabolism. The viral stress significantly increases the concentration of erythritol and myo-inositol as compared to healthy ones. Lower concentration of glucose and sucrose in viral-infected stem tissues suggests decreased translocation of photosynthates in the plants. The results demonstrated potential of MRI, NMR spectroscopy and GC-MS for studying anatomical and metabolic variations in virus-infected plants., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

An isolate of Cucumber mosaic virus (CMV) retrieved from tomato in India was characterized based on its transmission by sap inoculations, Western blot immuno-assay and sequence analysis of RNA3 and 2b genome. The RNA3 genome was of 2,220 nucleotides (nt) which contained two ORFs: movement protein of 852 nt translating 283 amino acids and coat protein of 657 nt translating 218 amino acids. The complete sequence of RNA3 geneome (Acc. EF153734) shared highest 98-99% identities with P1-1, Tfn, and Nt9 strains of CMV infecting tomato reported from abroad. The 333 nucleotides long RNA2b gene (Acc. EF710773) also showed highest 98% identities with P1-1 and 97% with Tfn and NT9 strains of CMV but only 83-84% identities with Indian strains of CMV. Therefore, the isolate under study was identified as a new isolate of CMV of subgroup IB based on highest sequence similarities and closer affinity to European or East Asian isolates of CMV.

Yellow mosaic disease was observed on Jatropha gossypifolia plants growing in Kathaupahadi, Madhya Pradesh, India, and whiteflies (Bemisia tabaci) were found in the vicinity. Association of a new begomovirus with yellow mosaic disease of J. gossypifolia has been detected by PCR using begomovirus DNA-A-specific primers. The complete DNA-A genome (~2.7 kb) of this virus isolate was amplified by rolling-circle amplification (RCA) followed by digestion with Bam HI. The ~2.7-kb amplicons was cloned and sequenced, and the data obtained were submitted to GenBank under accession numbers FJ177030. The genome of the virus isolate consisted of six open reading frames (ORFs): V2 (pre-coat protein) and V1 (coat protein) in the virion sense and C3 (REn protein), C2 (TrAP protein), C1 (replication-associated protein) and C4 (C4 protein) in the complementary sense. BLASTn analysis of the nucleotide sequence (2757 nt) of the viral genome (FJ177030) showed 84-85% identity and a distinct phylogenetic relationship with DNA-A of tomato leaf curl virus-Bangalore II (U38239) and tomato leaf curl Karnataka virus (AY754812). Based on its 85% sequence identity to all other begomoviruses known to date and ICTV species demarcating criteria (< 88% identity), the name Jatropha yellow mosaic India virus (JYMIV) is proposed. JYMIV is considered to be monopartite, as neither DNA-B nor DNA-β components associated with begomoviruses were detected.

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-β-specific primers. DNA-A and DNA-β 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-β 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-β (EU604296) showed the highest 96% sequence identity and closest phylogenetic relationship with CYVMV-associated DNA-β (AM410551) isolated from Croton sp. in Pakistan. Based on the highest sequence identities and closest phylogenetic relationships of the DNA-A genome and DNA-β molecule with respective sequences of various isolates of Croton yellow vein mosaic virus, the begomovirus associated with yellow vein mosaic disease of J. gossypifolia was identified as an isolate of Croton yellow vein mosaic virus.

Alterations in the anatomical structures, sap translocation and metabolic profiles in Jatropha curcas L. (Euphorbiaceae), infected with Jatropha mosaic virus (JMV) have been investigated using MRI and HR-MAS NMR spectroscopy. The contrast of MRI images distinguishes abnormalities in anatomical structures of infected and healthy stem. The HR-MAS NMR spectroscopic analysis indicated that viral infection significantly affected the plant metabolism. Higher accumulation of TCA cycle intermediates, such as citrate and malate, in JMV-infected plants suggested a higher rate of respiration. The respiration rate was more than twofold as compared to healthy ones. The viral stress also significantly increases the concentrations of alanine, arginine, glutamine, valine, GABA and choline as compared to healthy ones. Microscopic examination revealed severe hyperplasia caused by JMV with a considerable reduction in the size of stem cells. Lower concentration of glucose and sucrose in viral-infected stem tissues indicates decreased translocation of photosynthates from leaves to stem due to hyperplasia caused by JMV. The MR images distinguished stele, cortical and pith regions of JMV-infected and healthy stems. Contrast of T(1)- and T(2)-weighted images showed significant differences in the spatial distribution of water, lipids and macromolecules in virus-infected and healthy stem tissues. The results demonstrated the value of MRI and HR-MAS NMR spectroscopy in studying viral infection and metabolic shift in plants. The present methodology may help in better understanding the metabolic alterations during biotic stress in other plant species of agricultural and commercial importance.

Coat protein (CP) -mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

Jatropha curcas L. is a major commercial biodiesel fuel crop grown on 98 million acres (39.66 million ha) in India. Severe mosaic disease accompanied by yellow spots was noticed on 15% of J. curcas growing in the experimental plots of NBRI, Lucknow, India, during October of 2006. Inoculations with sap from symptomatic plants resulted in systemic mosaic on three of seven J. curcas seedlings. Gel diffusion tests were performed with antiserum to Cucumber mosaic virus (CMV), Tobacco ringspot virus, and Chrysanthemum virus B (PVAS-242a, PVAS-157, and PVAS-349, respectively; ATCC, Manassas, VA). Leaf sap of infected plants reacted only with PVAS-242a, indicating the presence of CMV. Reverse transcription (RT)-PCR assays with CMV coat protein gene specific primers (Genbank Accession Nos. AM180922 and AM180923) and total nucleic acid extracted from symptomatic J. curcas leaf tissue yielded the expected ~650-bp amplicon, which was cloned and sequenced (GenBank Accession No. EF153739). BLAST analysis indicated 98 to 99% nucleotide identity with CMV isolates (GenBank Accession Nos. DQ914877, DQ640743, AF350450, AF281864, X89652, AF198622, DQ152254, DQ141675, and DQ028777). Phylogenetic analysis showed that the J. curcas isolate was more closely related to Indian isolates of CMV belonging to subgroup Ib. Literature surveys revealed records of Jatropha mosaic virus on J. gossypiifolia in Puerto Rico (1) and on J. curcas in India (2). To our knowledge, this is the first report of CMV on J. curcas. References: (1) J. K. Brown et al. Arch. Virol. 146:1581, 2001. (2) D. S. A. Narayana et al. Curr. Sci. 91:584, 2006.

Rauvolfia serpentina, family Apocynaceae, is widely cultivated in India and adjoining countries for the production of roots used in several herbal formulations (1). Severe mosaic and stunting of the whole plant was observed on R. serpentina growing in experimental plots of NBRI, Lucknow, in 2006. The causal pathogen was transmitted by sap inoculation on Nicotiana tabacum cv. White Burley, N. rustica, and N. glutinosa, which produced necrotic local lesions and systemic mosaic. The virus reacted positively with antiserum of Cucumber mosaic virus (CMV; PVAS 242a, ATCC, Manassas, VA) in gel double diffusion tests, indicating the presence of CMV. Total RNA was isolated from infected and healthy leaf tissues of R. serpentina, and reverse transcription (RT)-PCR was performed using CMV specific primers AM180922/AM180923. RT-PCR resulted in an expected size (~650 bp) amplicon in infected but not healthy samples. The amplicon was cloned, sequenced, and the data was submitted to GenBank (Accession No. DQ914877). BLAST search analysis of the virus isolate showed highest (99%) sequence identity with CMV isolates (GenBank Accession Nos. DQ640743, AF350450, X89652, and AF281864). The virus isolate showed a close phylogenetic relationship with Indian isolates of CMV belonging to subgroup IB. A literature survey revealed that there is no report of occurrence of any virus on R. serpentina except some fungal infections (2). To our knowledge, this is the first record of natural occurrence of CMV on R. serpentina. References: (1) Anonymous. Wealth of India 8:376, 1969. (2) R. S. Shukla et al. EPPO Bull. 36:11, 2006.

Soybean, Glycine max (L.) Merr., is a protein- and oil-rich crop cultivated in India and abroad. A yellow mosaic disease was observed on soybean with 80 to 90% disease incidence during August 2005 at fields of the National Botanical Research Institute, Lucknow, in northern India. Soybean plants were found to be infested with whiteflies (Bemisia tabaci) suggesting begomovirus etiology. The disease agent was transmitted experimentally by whiteflies, and symptoms developed after 23 days. Total DNA was isolated from 51 leaf samples collected from 42 symptomatic and 9 asymptomatic plants. Polymerase chain reaction was performed using begomovirus coat protein-specific primers 5'-ATGGCGAA GCGACCAG-3' and 5'-TTAATTTGTGACCGAATCAT-3' (AM180920/ AM180921). An amplicon of the expected size (~800 bp) was obtained in all 42 symptomatic leaves but not from any of the nine asymptomatic leaf samples. The amplicon was cloned, and the identical sequence of three clones was submitted to GenBank (Accession No. DQ343283). BLAST search of nucleotide sequences revealed 95% identity with Cotton leaf curl Kokhran virus (CLCKV) (GenBank Accession Nos. AJ002449, AJ002448, AJ496286, and AY456683) and 57% identity with Mungbean yellow mosaic India virus (MYMIV-Sb, GenBank Accession No. AY049772). Results indicated that the virus associated with yellow mosaic disease of soybean is an isolate of CLCKV rather than MYMIV-Sb (1) reported earlier on soybean from northern India. To our knowledge, this is the first report of soybean as a new host of Cotton leaf curl Kokhran virus. Reference: (1) K. S. Usharani et al. Curr. Sci. 86:845, 2004.

Natural occurrence of yellow mosaic disease was observed on bitter gourd (Momordica charantia). Association of geminivirus with the disease was investigated through polymerase chain reaction using geminivirus-specific primers and Southern hybridization with a probe prepared from the cloned DNA of a known geminivirus. The fruits, leaves and stem of infected and healthy plants were studied for phytochemical composition. The amounts of protein were 49%, 50% and 66% higher, total carotenoids were 36%, 33% and 40% lower, vitamin C were 23%, 48% and 50% lower, total phenols were 28%, 31% and 43% lower, and antioxidant activity were 36%, 48% and 43% lower in the severely virus infected fruits, leaves and stem, respectively, as compared with healthy plants. The loss in the quantity of these phytochemicals was also observed even in mild infected plants, which further increased with the severity of the symptoms. Similarly, ethanol and 50% ethanol soluble extractive were also 25-43% lower in the fruits, leaves and stem of infected plants as compared with the healthy plants. A 45% and 54% lower caffeic acid, and 78% and 59% lower amounts of ferulic acid in the fruits and stem, respectively, and a 25% loss of gallic acid were noticed in the leaves of the severely infected plants.

Cucumber mosaic virus (CMV) A strain (CMV-A) isolated from Amaranthus tricolor was partially characterized at molecular level. Complete coat protein (CP) and movement protein (MP) ORFs were cloned and sequenced. The 657 bp region of CP gene and the 840 bp region of MP gene encode 218 and 276 amino acids, respectively. CP, at nucleotide level, showed 90-98% sequence identity with the CMV subgroup I and less than 80% with the CMV subgroup II, it showed at amino acid level 92-96% identity with the subgroup I and 74-87% with the subgroup II. The nucleotide and amino acid sequence identities of MP ranged in 91-94% and 92-96%, respectively with the subgroup I but in 81-83% with the subgroup II. Phylogenetic trees generated from nucleotide and amino acid sequences of both CP and MP genes identified the virus strain as a member of the subgroup IB. CMV-A CP also displayed a remarkably higher homology with Indian strains of CMV than with other CMV strains and formed a separate cluster within the subgroup IB.

An attempt was made to detect various viruses of Piper betle grown at Mahoba and Banthara in India. DAC-ELISA and RT-PCR tests were performed in leaf sap samples of betelvine for detection of a cucumovirus (Cucumber mosaic virus) and potyvirus (Bean yellow mosaic virus) using specific antibodies and universal primers of respective viruses. DAC-ELISA could detect only CMV. However, RT-PCR detected both cucumovirus and potyvirus infection in betelvine samples. Association of CMV with betelvine was observed for the first time in the present study.

Pumpkin (Cucurbita maxima) is an economically important vegetable crop of the family Cucurbitaceae. Yellow mosaic in C. maxima was observed in and around Lucknow, India, during the winter season of 1999-2000. High populations of whiteflies (Bemisia tabaci) were associated with diseased plants. Whiteflies were allowed to feed on diseased C. maxima plants and placed on healthy C. maxima seedlings. Symptoms of yellow mosaic disease were successfully reproduced in the seedlings. These results suggested a possible involvement of a geminivirus with the disease. Total DNA was extracted from infected and uninfected C. maxima seedlings by the CTAB method. DNA samples were used as templates in polymerase chain reaction (PCR) using degenerate primers A (5'-TAATATTACCKGWKGVCCSC-3') and B (5'-TGGACYTTRCAWGGBCCT TCACA-3'), which have been designed to amplify a region of about 500 to 600 nt from DNA-A of a majority of begomoviruses (1). The PCR products obtained were analyzed on a 1.5% agarose gel. The major amplicon obtained from the DNA of infected plants was about 550 bp as expected from these primers. However, no such amplicon was observed in DNA samples from uninfected plants. The amplicon obtained from PCR was cloned in a suitable cloning vector. Three separate clones were sequenced and the corrected sequence data (GenBank Accession No. AF381977) was compared with the corresponding sequences of other known geminiviruses. The highest identity (88%) was with a strain of monopartite Tomato leaf curl virus (TLCV) from southern India (GenBank Accession No. U38239). The first 34 nucleotides showed significant identity (91% and 97%, respectively) with Althea rosea enation mosaic virus (GenBank Accession No. AF014881) and TLCV strain D2 reported from Australia (GenBank Accession No. AF084006), both of which are monopartite begomoviruses. These results establish the provisional identity of the pathogen causing yellow mosaic disease on pumpkin in India as a closely related strain of the monopartite TLCV. The yellow leaf disease of muskmelon in Thailand and watermelon leaf curl disease in Pakistan also were recently reported to be caused by an Indian strain of TLCV (2,3). However, to our knowledge this is the first report of detection of a monopartite geminvirus causing yellow mosaic disease on pumpkin in India. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) S. H. Khan et al. Plant Dis. 84: 102, 2000. (3) K. Sarmretwanich, Plant. Dis. 84:707, 2000.

A cucumber mosaic virus isolate was found to be associated with mottle crinkle and severe mosaic disease of Egyptian henbane (Hyoscyamus muticus L.). The virus has been characterized as an Indian isolate of cucumber mosaic virus (CMV) based on non-persistent transmission by aphid, presence of 28-nm isometric particles, capsid protein of 26 K and single-stranded tripartite RNA genome with a subgenomic RNA (RNA 4). There was no evidence of satellite RNA genome. The isolate showed a strong serological relationship with S and A strains of CMV (CMV-S and CMV-A) in double diffusion test. A band of the 26 K capsid protein was also detected by Western blot analysis using antibodies specific to CMV-S.

Lethal necrosis or systemic stem necrosis followed by death of Nicotiana benthamiana, severe leaf deformations of N. tabacum cv. white burley and blister formations on N. tabacum cv. samsun NN symptoms were induced by experimental inoculations of CMV RNA preparations containing satellite RNA (sat-RNA). Inoculations of RNA preparations without sat-RNA did not induce that severe symptoms on these plants, only late mild mosaic was observed. It is suggested that sat-RNA of CMV isolate has a certain role for enhancing severity of symptoms in tobacco plants. Local and systemic lethal necrosis of N. benthamiana is due to sat-RNA present with genome of CMV isolate. It is the first report of lethal necrosis induced in N. benthamiana by CMV satellite.

Launaea acaulis (Roxb.) Babc. Aerr. and L. procumbeus (Roxb.) Ramayya & Rajgopal are weeds that grow near agricultural fields in India during the rainy season. Yellow net symptoms were observed in both species, but not in crops, in 1998 and 1999. Nicotiana tabacum cv. White Burley developed leaf curl symptoms after virus transmission from L. acaulis by whiteflies (Bemisia tabaci). Thus, the involvement of a geminivirus was suspected. Total DNA from infected and uninfected tobacco and Launaea plants was extracted by the cetyl trimethyl ammonium bromide method and precipitated by ethanol. Polymerase chain reaction (PCR), using DNA from infected and uninfected plants as a template and degenerate oli-gonucleotide primers designed from DNA-A of whitefly-transmitted African cassava mosaic virus (ACMV) (1) was performed. An expected ≈500-bp fragment was observed after agarose gel electrophoresis of the PCR product. Total DNA from infected plants gave positive signals and was homologous with the probe from cloned DNA-A of Indian tomato leaf curl virus (2) in high-stringency Southern hybridization tests. No PCR amplification and positive signals were obtained from DNA from uninfected plants. PCR amplification of the 500-bp fragment with primers from DNA-A of whitefly-transmitted ACMV resulted in positive signals, and homology with the probe indicated that L. acaulis and L. procumbens harbor whitefly-transmitted geminivirus infections. This is the first report of geminivirus infection in these Launaea spp. in India. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) K. M. Srivastava, et al. J. Virol. Methods 51:297, 1985.

Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed., (Copyright 1999 Academic Press.)

Recent advances in biotechnology and molecular biology have played a significant role in development of rapid, specific and sensitive assays for detection of plant viruses. Production of monospecific polyclonal antibodies, monoclonal antibodies have enabled to group isolates of viruses and distinction of closely related strains. In cDNA hybridization applications, there is an increasing interest to employ non-radioactive probes for detection of nucleic acids. Detection limit of nucleic acid is remarkably comparable to those of radioactive labelled probes. Application of polymerase chain reaction (PCR) has made it possible to amplify the low numbers of viral RNA/DNA molecules and their subsequent detection. Underlying principles, their advantages and disadvantages for application of monospecific polyclonal antibodies, hybridoma technology, molecular hybridization and PCR technology with reference to detection of plant viruses have been discussed in this review.

A diagnostic method based on Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed for detection of CMV genome directly in crude extracts of both healthy and infected gladiolus tissue using primers from the conserved sequences of CMV RNA-3. RT-PCR resulted in the amplification of 540 bp long fragment of CMV coat protein gene (CMV-CP), as expected in most of the plant parts of infected gladiolus samples. Positive signals in Southern hybridization of amplified fragments with cloned CMV-CP cDNA probe also confirmed the presence of CMV genome in gladiolus.

Virus strains isolated from Ocimum sanctum and Zinnia elegans were identified as cucumber mosaic virus (CMV) strains on the basis of non-persistant aphid transmission, 28 nm particles, 26 kDa coat protein subunits and serological relationships with CMV and chrysanthemum aspermy virus. The strains showed some biological, serological and satellite RNA based differentiation with other CMV strains isolated earlier from chrysanthemum, petunia and tobacco.

Geminivirus association with yellow mosaic disease of C. sativus has been investigated by dot/slot blot hybridization tests using nucleic acid probe derived from DNA-B of Indian Tomato Leaf Curl geminivirus. The disease was transmitted experimentally on C. sativus by whitefly, Bemisia tabaci. Some weeds were also found to harbour geminivirus infection in dot/slot blot hybridization tests.

Virus causing severe chlorosis/mosaic disease of banana was identified as a strain of cucumber mosaic virus (CMV). Association of CMV with the disease was established by Western immunoblot using polyclonal antibodies to CMV-T and slot blot hybridization with nucleic acid probe of CMV-P genome.

RNA isolation from purified cucumber mosaic virus, CMV-U strain and cDNA synthesis was carried out. The coat protein gene (RNA4) region was amplified selectively by polymerase chain reaction (PCR) with CMV RNA4-specific primers. Double-stranded cDNA was cloned in PRT103 vector at Xho1/Kpn1 site and about 1kb insert obtained. The insert was partly sequenced which showed 50% sequence homology with CMV-Q, C and WL strains.

Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; 100 and 150 mg/l) incorporated into brinjal explant culture medium induced in complete elimination of eggplant mottled crinkle virus - Indian isolate (EMCV-1) from infected explant cultures of S. melongena L. and production of virus free plant progeny. Acridine orange and ethidium bromide (150 ml/l) were also potent antiviral agents to some extent.