Brakta A, Thakur P D, Handa A
Plant Virology Laboratory, Department Plant Pathology, College of Horticulture, Dr. Y.S. Parmar University of Horticulture & Forestry, Nauni Solan (H.P.) 173230, India.
Plant Dis. 2013 Jul;97(7):1001. doi: 10.1094/PDIS-11-12-1082-PDN.
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.
在印度喜马拉雅地区西北部，许多种植者已经普遍采用将老旧、低产且不太受欢迎的苹果（Malus domestica Borkh.）品种的树，改造成新引进的短枝型商业品种这种做法。2009年5月和6月进行的调查中，在红首领、超级首领、猩红短枝、席勒短枝、华盛顿红元帅以及许多其他新引进的品种上，观察到典型的叶片卷曲、起皱和坏死等病毒症状，发病率达80%。通过生物学检测（草本和木本指示植物）、双抗体夹心（DAS）-酶联免疫吸附测定（ELISA）以及基于逆转录酶（RT）-聚合酶链反应（PCR）的检测方法，对高接换种树的叶片样本进行检测，以确定是否存在苹果茎沟病毒（ASGV）、苹果褪绿叶斑病毒（ACLSV）和苹果茎痘病毒（ASPV）。汁液接种后9至21天内，机械接种到草本指示植物上，藜麦和苋色藜出现褪绿病斑，而菜豆出现边缘坏死。在DAS-ELISA（瑞士BIOREBA AG公司）检测中，从这些草本指示植物中均检测到了ASGV、ASPV和ACLSV这三种病毒。此外，当通过双嫁接、嫁接结合芽接以及双芽接方法，将测试接穗在4个月（双嫁接）、5个月（双芽接）至1年4个月（嫁接结合芽接）的时间段内接种到木本指示植物（苹果‘斯派227’）上时，产生了与果园中观察到的类似症状。再次通过DAS-ELISA在斯派227木本指示植物中确认了这三种病毒的存在。利用通过ELISA检测到的所有病毒的外壳蛋白基因特异性引物（ASGV5641 [正向]，ASGV6396 [反向]；ACLSV6784 [正向]，ACLSV7365 [反向] [2]；ASP-C [正义链]，ASP-A [反义链] [1]）进行PCR检测。扩增产物进行克隆、测序，并保存在美国国立生物技术信息中心（NCBI）（ASGV的GenBank登录号为KC110892，ASPV为KC154859，ACLSV为KC154862）。BLASTn分析表明，该ASGV分离株与印度（FM204881）和巴西（AF438409）的ASGV分离株具有97%至98%的序列同一性。ASPV和ACLSV分离株分别与中国（JF895517）和日本（AB326230）的分离株具有98%和99%的序列同一性。据我们所知，这是印度苹果商业品种中与ASGV、ASPV和ACLSV感染相关的苹果高接换种病的首次报道，似乎对果园中培育无病毒健康苗木构成严重威胁。日本曾报道过与ASGV、ASPV和ACLSV病毒相关的苹果高接换种病（3,4）。参考文献：（1）J. K. Kundu等人，《植物保护科学》39:88，2003年。（2）O. Nickel等人，《巴西植物病理学》26:655，2001年。（3）H. Yanase，《日本果树试验场通报》C辑1:47，1974年。（4）H. Yanase等人，《园艺学报》44:221，1975年。
