Molecular evidence for Apple stem pitting virus infection in India

Apple stem pitting virus (ASPV; genus Foveavirus) is a major virus of pome and stone fruits (Martelli & Jelkmann, 1998), and thus its reliable diagnosis is a crucial step in the production of virus-free planting material. A serious disease was observed in apple and cherry trees in the Rohru, Solan and Chamba regions of Himachal Pradesh (HP) and in the Srinagar region of Jammu & Kashmir (J&K). Surveys were conducted in fruit growing areas from 2007 and diseased trees were observed in May (Srinagar) and September 2008 (Rohru, Chamba and Solan). Samples were collected from trees showing symptoms: 15 cherry samples (five each of varieties Mishri, Stella and Sour cherry) and 45 apple samples (varieties Gold Spur, Well Spur, Red Chief, Red Fuji, Royal Delicious, Vance Delicious, Star Crimson, Scarlet Gala, Silver Spur, Oregon Spur, Tydemann, Gala and Spartan). Affected apple trees showed symptoms of inner bark pits (Fig. 1), fruit deformation, early leaf fall and decline, while cherry trees showed vein yellowing symptoms. All 60 samples were tested for ASPV by ELISA (Bioreba, Switzerland), out of which 20 were found positive. Interestingly, variation in ASPV titre was observed, as trees that tested negative in October were found to be positive in the month of January upon retesting. These samples were also indexed by ELISA for Apple stem grooving virus, Apple chlorotic leaf spot virus (Agdia, USA) and Apple mosaic virus (Sediag, France). All samples tested negative for these other viruses. RT-PCR was performed from the 20 ELISA positive samples from five different regions with RNA isolated using RNeasy Plant mini kit (Qiagen, Germany with minor modifications) and primer pair with target sequences in the coat protein gene of ASPV genome (Menzel et al., 2002). An amplicon of ~370 bp was obtained, which was cloned into pGEM-T Easy Vector (Promega, USA) and sequenced. A sequence of 366 bp was obtained (GenBank Accession no. FM863704). The sequence was found to be 99% identical to the CP gene sequence of a Chinese ASPV isolate (ACD43643). Based on the ELISA, RT-PCR and sequence data, the virus was identified as Apple stem pitting virus. To the best of our knowledge, this is the first report of ASPV from India.