Leaf spot disease adversely affects human health-promoting constituents and withanolide biosynthesis in Withania somnifera (L.) Dunal.

AIMS: The present work investigates the implication of leaf spot disease on the antioxidant potential and commercial value of pharmaceutically important constituents of Withania somnifera, a high-valued medicinal plant. METHODS AND RESULTS: Leaf spot disease was induced in W. somnifera by inoculating Alternaria alternata (Fr.) Keiss. pathogen. Total polyphenolic content and antioxidant potential showed a significant decrease during leaf spot disease. Evaluation of pharmaceutically active constituents withaferin A, withanone and withanolide A utilizing high-performance liquid chromatography showed a significant decrease in diseased samples as compared to healthy ones. Quantitative expression of major genes involved in withanolide biosynthesis also showed down-regulation in diseased samples. Alterations in the ultra-structure of chloroplasts were also analysed under transmission electron microscopy to get a better insight into the changes of withanolide biosynthesis in leaf during disease infestation. CONCLUSIONS: The present work suggests that when the pathogenic fungus invades the host plants, it evokes multiple responses, which could be studied at various levels. The knowledge gained from this work will provide appropriate rationale for controlling the bio-deterioration of the pharmaceutically active metabolites in W. somnifera and development of suitable strategies against leaf spot disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate the effect of leaf spot disease on the human health-promoting constituents and withanolide biosynthesis in this high-valued medicinal plant.

In Vitro Expression and Production of Antibody Against Cymbidium mosaic virus Coat Protein.

Polyclonal rabbit antisera were produced using coat protein of Cymbidium mosaic virus (CymMV) Indian isolate expressed in E. coli as GST fusion. The expressed protein was purified by GST-fusion protein purification kit for use as an immunogen in rabbits. Antisera prepared in this manner reacted in double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) with extract from CymMV-infected tissue. The results indicate that polyclonal antisera prepared from expressed CymMV coat proteins were useful for the detection of CymMV in an array of assays. The detection system developed is highly effective for detection of Indian strain of the virus in comparison to kits available in the international market.

Complete nucleotide sequence of cherry virus A (CVA) infecting sweet cherry in India.

Cherry virus A (CVA) is a graft-transmissible member of the genus Capillovirus that infects different stone fruits. Sweet cherry (Prunus avium L; family Rosaceae) is an important deciduous temperate fruit crop in the Western Himalayan region of India. In order to determine the health status of cherry plantations and the incidence of the virus in India, cherry orchards in the states of Jammu and Kashmir (J&K) and Himachal Pradesh (H.P.) were surveyed during the months of May and September 2009. The incidence of CVA was found to be 28 and 13% from J&K and H.P., respectively, by RT-PCR. In order to characterize the virus at the molecular level, the complete genome was amplified by RT-PCR using specific primers. The amplicon of about 7.4 kb was sequenced and was found to be 7,379 bp long, with sequence specificity to CVA. The genome organization was similar to that of isolates characterized earlier, coding for two ORFs, in which ORF 2 is nested in ORF1. The complete sequence was 81 and 84% similar to that of the type isolate at the nucleotide and amino acid level, respectively, with 5' and 3' UTRs of 54 and 299 nucleotides, respectively. This is the first report of the complete nucleotide sequence of cherry virus A infecting sweet cherry in India.

Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein.

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91-100% and 70-98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.

Coat protein gene diversity among Chrysanthemum virus B isolates from India.

The complete coat protein (CP) sequences from 29 Indian isolates of Chrysanthemum virus B (CVB) were determined and analysed in relation to other previously characterized carlaviruses. The CP genes of the Indian CVB isolates were highly heterogeneous, sharing nucleotide sequence identities of 74-98%. Based on phylogenetic analyses, the isolates formed three groups potentially representing either two or three major CVB strain groupings. Recombination analysis revealed at least one definite recombination event involving the exchange of sequences between members of different groups. To our knowledge this is the first reported evidence of homologous recombination in carlaviruses.

Wild Himalayan Cherry (Prunus cerasoides) as a Natural Host of Prunus necrotic ringspot virus in India.

Prunus cerasoides, also known as wild Himalayan cherry, grows naturally in the Himalayas. A member of the Rosaceae family, the tree has medicinal (astringent) and other (beads, dye, wood) uses. During surveys in the northwestern Himalayan Region of India, necrotic spots were observed on leaves of P. cerasoides. Since the symptoms were typical of Prunus necrotic ringspot virus (PNRSV), preliminary detection was done by double-antibody sandwich (DAS)-ELISA (Agdia, Elkhart, IN). Positive results were obtained three times more than the negative control which is provided in the kit. To further confirm its presence, reverse transcription (RT)-PCR analysis was performed using a primer pair (upstream 5'-AACTGCAGATGGTTTGCCGAATTTGCAA-3'; downstream 5'-GCTCTAGACTAGATCTCAAGCAGGTC-3') specific for the coat protein gene (GenBank Accession Nos. AJ619984 and AJ619983). Amplification of the expected 675-bp fragment was obtained. The sequence of a cloned copy of the amplified product showed 99% similarity to the PNRSV coat protein gene (GenBank Accession No. AF170165), confirming the presence of PNRSV in P. cerasoides (sequence submitted as Accession No. AM493717). The cloned DNA has the potential for utilization as an additional tool, and an early PNRSV screening (both pollen and seed transmitted) will be highly useful to ensure healthy rootstocks are used for grafting purposes (1). PNRSV mainly infects members of the Rosaceae family, including stone fruits and ornamental plants such as peach, plum, cherry, apricot, nectarines, and roses, and was first reported in P. persica (1). Proper management of PNRSV at this level can prevent its transmission and disease development in grafted scions of commercial Prunus spp. Reference: (1) A. A. Brunt et al. Page 1047 in: Viruses of Plants. CAB International, Wallingford, UK. 1996.

Natural Occurrence of a Potyvirus on Murraya koenigii in India.

Murraya koenigii (L.) Spreng., a small, strong-smelling umbrageous tree with subcampanulate white flowers belonging to the family Rutaceae, is native to India and southeastern Asia (2). It is distributed across the Indian subcontinent excluding the higher elevations of the Himalayas. In India, the leaves are mainly used for culinary purposes. The leaves are commonly known as curry leaves or 'sweet neem'. The whole plant including bark, root, leaves, fruits, and fruit pulp is used medicinally. This plant was reported to be a host of Citrus tristeza virus (1). In a survey of potyvirus incidence in the northwestern Himalaya foothills of the Kangra and Hamirpur districts in the state of Himachal Pradesh in 2004, M. koenigii plants showing mosaic symptoms on leaves, typical of a virus infection, were frequently observed. Symptomatic leaves were tested for the presence of several viruses using enzyme-linked immunosorbent assay with specific antibodies. Positive results were obtained with potyvirus group specific antibodies (Agdia, Elkhardt, IN) in triplicate analyses of 5 of 15 leaf samples tested. To further identify the infecting virus, RNA from plants was tested using universal potyvirus primer pair P9502 and CPUP (3) and reverse transcription-polymerase chain reaction to amplify a genome fragment encoding portions of the coat protein and the 3'UTR (3). An amplification product of the expected size (~800 bp) was obtained. The product was cloned into the pGem-T easy vector (Promega, Madison, WI), and three clones were sequenced. The sequence (GenBank Accession No. AJ852504) had 92% identity to Chili vein banding mottle virus, a potyvirus infecting pepper reported from Thailand (GenBank Accession No. U72193). To our knowledge, this is the first report of a potyvirus naturally occurring on a Murraya sp. References: (1) K. Balaram and K. Ramakrishnan. Curr. Sci. 48:453, 1979. (2) J. D. Hooker. Flora British India 1:502, 1875. (3) R. A. A. van der Vlugt et al. Phytopathology. 89:148, 1999.

Occurrence of Cucumber mosaic virus in Gerbera jamesonii in India.

Gerbera jamesonii (family Asteraceae) is a popular perennial ornamental cut flower and potted plant with considerable economic importance. In a survey of gerbera grown in floriculture fields at the Institute of Himalayan Bioresource Technology (IHBT), Palampur and nearby nurseries, color break symptoms on the petals, asymmetrical ray florets, and deformed flowers were observed during 2003-2004. The virus evoked chlorotic local lesions on Chenopodium album, C. amaranticolor, and C. quinoa, while systemic mosaic was observed on Cucumis sativus, Nicotiana benthamiana, N. clevelandii, N. glutinosa, and N. tabacum cv. Samsun. The virus was transmitted nonpersistently by Myzus persicae and Aphis gossypii and was identified as Cucumber mosaic virus (CMV) using enzyme-linked immunosorbent assay (ELISA) with CMV-specific antibodies (Agdia, Elkhart, IN). Polyhedral particles approximately 29 nm were observed with electron microscopy of leaf dips from symptomatic gerbera leaves. Total RNA was isolated from the infected gerbera plants and N. glutinosa by using RNAqueous (Ambion, Austin, TX). CMV-specific primers (1) were used to detect the virus with reverse transcription-polymerase chain reaction that produced an amplicon predicted size of approximately 540 bp, but the virus was not detected in healthy controls. Sequence alignment of the amplicons (533 bp) utilizing BLAST resulted in 91 to 99% homology with the partial intercistronic region and partial coat protein gene (1042-1574 bp) (gene sequence submitted to EMBL database with Accession no. AJ634532) of CMV RNA3 in subgroup I. To our knowledge, this is the first report of CMV on gerbera in India. Reference: (1) C. De Blas et al. J. Phytopathol. 141:323, 1994.

Strawberry latent ringspot virus Infecting Roses in India.

Rose is an economically important crop of India and the world. A survey of rose plantations in and near the Kangra Valley of Himachal Pradesh, India, showed virus-like symptoms, including yellow flecking in young leaves and reduction in leaflet size, while some were symptomless. These symptoms are similar to those for Strawberry latent ringspot virus (SLRSV) (1). Sap inoculation from symptomatic and some symptomless leaves to Chenopodium amaranticolor resulted in chlorotic local lesions followed by systemic chlorosis. SLRSV was detected in this indicator host and six rose cultivars (Happiness, Iceberg, First Prize, Ganga, Pink Panther, and Oklahoma) showing characteristic symptoms of SLRSV using enzyme-linked immunosorbent assay (ELISA) with ELISA kit (DSMZ, Braunschweig, Germany). Reverse transcription-polymerase chain reaction was performed with SLRSV-specific primers (2), and a product of the expected size of ˜181 bp was amplified. The authenticity of the fragment was confirmed by sequencing. Isolated SLRSV was also inoculated to seed-grown rose seedlings and after 20 days postinoculation the same symptoms (yellow flecking in young leaves) were observed. These results established the identity of the virus that caused yellow flecking on rose leaves in India as SLRSV. To our knowledge, this is the first report of SLRSV infecting rose in India. References: (1) A. F. Murant. Strawberry latent ringspot virus. No. 126 in: Description of Plant Viruses, CMI/AAB, Surrey, U.K., 1974. (2) E. Bertolini et al. J. Virol. Methods 96:33, 2001.

Molecular characterization of a carnation etched ring virus isolate from India.

Incidence of the Carnation etched ring virus (CERV), the only DNA virus reported to date on carnation, was investigated by a bioassay using a partially purified virus as inoculum and then by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Out of 61 carnation cultivars analyzed 41 (67%) were found positive. The virus positivity was verified by polymerase chain reaction (PCR) and nucleotide sequencing. The amplified 1349 bp fragment was by about 98% and 96% identical with respect to coat protein (CP) and enzymatic polyprotein genes, respectively, as compared to the sequences available in the database. In terms of amino acid sequence similarity, the homology values were 99% and 97%, respectively. Comparison with other caulimoviruses revealed that CERV is most closely related to the Cauliflower mosaic virus (CaMV). High genetic stability of CERV may be attributed to the fact that it has evolved from the same initial sequence in an original host. Because of global market of cut flowers and vegetative propagation it has been dispersed around the world.

Tomato yellow leaf curl virus (TYLCV) capsid protein (CP) subunit interactions: implications for viral assembly.

Tomato yellow leaf curl virus (TYLCV) capsid protein (CP) forms the capsule that encapsidates the viral genomic ssDNA. We have analyzed the homotypic interaction capacity of full-length and mutated CP. We found that full-length CP interacts with itself. Truncation of the protein from the C-terminal led to diminution of the self-interaction process. Also, the N-terminal region of the CP seemed to be necessary for the interaction. As the two deletion mutants interacted successfully with the wildtype protein, while they failed to self-interact, we suggest that the N-terminal amino acids interact with amino acids of the C-terminal region. Changes in CP homotypic interaction capacity were detected when mutations in the middle portion of the protein were introduced.

Nucleotide sequence and intergeminiviral homologies of the DNA-A of papaya leaf curl geminivirus from India.

Coat protein gene, rep protein gene and intergenic region of the genome of a whitefly transmitted geminivirus (WTG) causing severe leaf curl in papaya plants were PCR amplified, cloned and sequenced. Comparison of the amino acid sequence of the putative coat protein product of papaya leaf curl virus (PLCV) with some other mono and bipartite WTGs revealed a maximum of 89.8% homology with Indian cassava mosaic virus. The genomic organization of PLCV-India is similar to other WTGs with bipartite genomes. Comparison of the coat protein N-terminal 70 amino acid sequence (and other biological features) of PLCV with other geminiviruses shows that PLCV is a distinct geminivirus from India and is related to WTGs from the old world.

Reverse transcription-polymerase chain reaction (RT-PCR) for direct detection of cucumber mosaic virus (CMV) in gladiolus.

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.

Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative form of viral DNA.

DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolation of plasmid DNA from Escherichia coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses. Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono- and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus.