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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 a-satellite with these symptoms of a Parthenium disease was investigated by sequence analyses of vims 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 a-satellite detected in P. hysterophorus plants were to Tomato leaf curl virus (ToLCV), papaya leaf curl β-satellite (PaLCuB), and Ageratum yellow vein India a-satellite (AYVIA), respectively. These findings identified the vims 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. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.