In surveys of apple (Malus domestica) orchards in various parts of Himachal Pradesh, samples from trees showing necrotic symptoms on the leaves were collected and tested for detection of Prunus necrotic ringspot virus (PNRSV) initially by ELISA followed by RT-PCR using coat protein gene primers. Positive results were obtained in samples from Kullu and Kalpa regions. The virus gene sequences showed 88-97% similarity to corresponding sequences of other PNRSV isolates deposited in the GenBank database using ncbi.nih.nlm.gov. Although the similarity was high, there were some distinct differences with the Spanish isolate. This is the first report of PNRSV in apple from India.

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.

Himalayan wild cherry (Prunus cerasoides), widely distributed in the Himalayas, was found to exhibit mild virus-like symptoms. Leaf samples from affected plants were subjected to enzyme-linked immunosorbent assay indexing for known pome and stone fruit viruses, viz., Apple mosaic virus, Apple chlorotic leaf spot virus (ACLSV), Prunus necrotic ring spot virus and Apple stem grooving virus. Results showed the presence of ACLSV. For further characterization, the coat protein gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Sequencing of the amplicon confirmed the presence of ACLSV. The isolate shared 98% amino acid sequence identity with P1R9D9 (Bulgarian) and C-2 (Hungarian) isolates of ACLSV. This study reports the presence of ACLSV in Himalayan wild cherry.

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Apple mosaic virus (ApMV) is an important virus of apple worldwide. Surveys were conducted in the states of Himachal Pradesh (HP) and Jammu & Kashmir (J&K), India, to determine the prevalence of ApMV and to develop ways for its early diagnosis. Symptomatic leaf samples, bud and bark were collected during spring and late summer. DAS-ELISA revealed the presence of ApMV in 13/198 samples from four districts in HP, indicating a disease incidence of approximately 6.5%. The sequence of amplicons produced by reverse transcription-polymerase chain reaction (RT-PCR) using virus-specific primers confirmed the ELISA results. Comparison of the sequences of three amplicons with nine complete available sequences of ApMV coat protein (CP) (from apple) at amino acid level revealed a maximum of 96% homology to a Korean isolate of ApMV (AY125977). Comparison of the amino acid sequence of the CP of the Indian isolate with the amino acid sequence of the CP from different hosts showed that the Indian isolate clustered most closely with an isolate found in pear and originating from Czechoslovakia. Cloned DNA was reliably used for diagnosis of the virus and was a useful tool for screening at nursery level using slot blot hybridization. This study confirms the presence of ApMV at the molecular level in India and reveals sequence information.