Molecular Detection of Latent Apple chlorotic leaf spot virus in Elite Mother Plants of Apple.

Apple chlorotic leaf spot virus (ACLSV; family Betaflexiviridae genus Trichovirus) is one of the economically important latent virus infecting apple (Malus × domestica Borkh.). Reverse transcriptase polymerase chain reaction (RT-PCR) procedures were used to amplify coat protein gene of ACLSV. Among 5 primer sets used, two primer sets (1F1R and 1F2R) amplified fragments of expected size (432 bp). Products visible on agarose gel were produced using templates extracted from apple leaves. The results were further validated by sequencing fragment of 432 bp which was amplified from leaf of apple by using primer set 1F 1R. Comparisons with published sequences indicated that the isolate have very high 91 % identity values to the corresponding region of ACLSV isolate from apple. Selected primer pair (1F1R) was further used for screening 42 elite mother plants collected from apple growing areas of Himachal Pradesh, India, where in 17 were found free from ACLSV. Use of NAD5 gene in mitochondrial mRNA of the apple as an internal control, reduced the risk of false negative results that may occur with routine RT-PCR assays.