Top working of apple (Malus domestica Borkh.) trees of old, unproductive, and less preferred cultivars with the newly introduced spur type commercial cultivars has become a common practice with many growers in the northwestern Himalayan region of India. Typical viral symptoms of curling, puckering, and necrosis on leaves were observed with an incidence of 80% on Red Chief, Super Chief, Scarlet Spur, Schillet Spur, Washington Red Delicious, and many other newly introduced cultivars during surveys conducted in May and June 2009. Leaf samples from top worked trees were tested for the presence of Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) by employing biological detection (herbaceous and woody indicators), double antibody sandwich (DAS)-ELISA), and reverse transcriptase (RT)-PCR based detection. Mechanical transmission to herbaceous indicators produced chlorotic lesions on Chenopodium quinoa and C. amaranticolor, whereas marginal necrosis was induced on Phaseolous vulgaris within 9 to 21 days after sap inoculations. All three viruses, i.e., ASGV, ASPV, and ACLSV, were detected from these herbaceous indicators in DAS-ELISA (BIOREBA AG, Switzerland). Furthermore, symptoms similar to those observed in orchards were produced when the test budwood was inoculated onto the woody indicator (M. pumila ‘Spy 227’) plant by double grafting, grafting cum budding, and double budding methods within time periods ranging from 4 months in double grafting, 5 months in double budding, to 1 year 4 months in the grafting cum budding method. The presence of all three viruses was confirmed by DAS-ELISA again in Spy 227 woody indicator. PCR detection was carried out by using the coat protein gene specific primers (ASGV5641 [forward], ASGV6396 [reverse]; ACLSV6784 [forward], ACLSV7365 [reverse] [2]; ASP-C [sense], ASP-A [anti-sense] [1]) of all the viruses detected through ELISA. The amplified products were cloned, sequenced, and deposited in NCBI (GenBank Accessions KC110892 for ASGV, KC154859 for ASPV, and KC154862 for ACLSV). BLASTn analysis showed the ASGV isolate had 97 to 98% sequence identity with Indian (FM204881) and Brazilian (AF438409) ASGV isolates. The ASPV and ACLSV isolates had 98% and 99% sequence identity with Chinese (JF895517) and Japanese (AB326230) isolates, respectively. To the best of our knowledge, this is the first report of apple top working disease associated with ASGV, ASPV, and ACLSV infection in commercial cultivars of apple from India and seems to be a serious threat for growing virus-free healthy stocks in orchards. Top working disease in apple associated with ASGV, ASPV, and ACLSV viruses has been reported from Japan (3,4). References: (1) J. K. Kundu et al. Plant Prot. Sci. 39:88, 2003. (2) O. Nickel et al. Fitopatol. Brasil. 26:655, 2001. (3) H. Yanase. Bull. Fruit Tree Res. Stn., Japan Ser. C 1:47, 1974. (4) H. Yanase et al. Acta Hortic. 44:221, 1975.
The application of RT-PCR assay for the detection of Apple stem pitting virus and Apple stem grooving virus in four apple cultivars
