Significance and Impact of the Study: In India, a new virus, apple necrotic mosaic virus (ApNMV), has been found to infect the apple trees along with ApMV, apple stem pitting virus (ASPV) and apple stem grooving virus (ASGV). This study was designed to detect the ApNMV along with three other viruses (ApMV, ASPV and ASGV) simultaneously using multiplex RT-PCR in a single tube. The assay developed in this study will be an effective detection tool for large-scale surveys and indexing of large apple germplasm for the production of virus-free planting material in temperate fruits. This will also help in studying the distribution of all the four viruses in apple growing regions of India and abroad.

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Grapevine (Vitis labrusca L.), a member of the family Vitaceae and native of North America, is grown as a table grape. During the survey for the grapevine diseases in May 2022, we noticed numerous yellow pustules of rust on lower side of leaves of ’Bangalore Bule’ in Nandi village (13°22'59.7"N77°42'33.4"E), Chikkaballapur district of Karnataka state of India. The crop was at maturity stage and the rust disease severity was determined using the scale given by Angelotti et al. (2008), which was up to 10%. The disease symptoms were numerous small raised yellow pustules on the abaxial surface corresponding to adaxial surface chlorotic spots. In severe conditions, spots cover the entire leaf and defoliation occurs. Similar disease symptoms were reported by Ono (2000); Weinert et al. (2003); and Primiano et al. (2017). The pathogenicity test was performed on cuttings of ’Bangalore Bule’ grapevine in a glasshouse at 25±1°C. The urediniospores were collected from diseased leaves using a brush, 3×104 ml-1 suspension in distilled water was used for inoculation on the abaxial surface of leaves. Control plants were sprayed with distilled water. The leaves developed symptoms in 15–17 days after inoculation, and the pathogen was confirmed by symptomatology and microscopic observation of urediniospores. Urediniospores were short-pedicellate, sessile, obovoid to obovoid-ellipsoid, and uniformly echinulate with 42.98-32.54 x 31.37-25.15 µm in size. The aecial stage of the Phakopsora has been reported on an alternate host, Meliosma simplicifolia (Hosagoudar 1988). As the internal transcribed spacer (ITS) region offers some utility in the molecular detection of the Phakopsora genus (Rush et al. 2019), the pathogen was confirmed by studying the different regions in the ITS such as ITS1, 5.8S rRNA, and ITS2. Total DNA was extracted from urediniospore mass using the Macherey-Nagel kit (Duren, Germany) by following the manufacturer's protocol. The quantity of isolated DNA was checked using an Qubit 3.0 fluorometer (Invitrogen) before being subjected to polymerase chain reaction (PCR) amplification in a thermocycler (Eppendorf-vapo.protect) using ITS1 and ITS4 primers (IDT, Singapore) targeting ITS1, 5.8S rRNA, and ITS2 regions, and the obtained amplicon (~700 bp) was purified using Macherey- Nagel Nucleospin gel and PCR clean-up kit (Duren, Germany) as per the manufacturer’s protocol and sequenced by Sanger’s dideoxy chain-termination method [ABI 3730 (48 capillaries) electrophoresis]. The sequence was edited in BioEdit (https://bioedit.software.informer.com/7.2/) and aligned in MUSCLE, and the phylogenetic tree was constructed in MEGA 11 using the neighbor-joining method by following the maximum likelihood criterion (Kumar et al. 2018). The sequence data was deposited at NCBI (accession number OP221661). The basic local alignment search tool (BLAST) search sequence of the isolate Nandi-KA in GenBank revealed 97.91% homology with sequence of Phakopsora sp. (accession number KC815548.1) and 96.87% with Phakopsora euvitis (accession number AB354790.1). Based on disease symptoms, fungal morphology, the pathogenicity test, and ITS sequence, the fungus was identified as Phakopsora euvitis, the pathogen causing grapevine leaf rust disease. Though there are similar disease symptoms observed on grapevine in India (EPPO 2016); the pathogen was not confirmed. To our knowledge, this is the first report of Phakopsora euvitis causing leaf rust disease in grapevine (V. labrusca) in India.

Besides apple mosaic virus (ApMV), apple necrotic mosaic virus (ApNMV) has also been found to be associated with apple mosaic disease. Both viruses are unevenly distributed throughout the plant and their titer decreases variably with high temperatures, hence requiring proper tissue and time for early and real-time detection within plants. The present study was carried out to understand the distribution and titer of ApMV and ApNMV in apple trees from different plant parts (spatial) during different seasons (temporal) for the optimization of tissue and time for their timely detection. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) was carried out to detect and quantify both viruses in the various plant parts of apple trees during different seasons. Depending on the availability of tissue, both ApMV and ApNMV were detected in all the plant parts during the spring season using RT-PCR. During the summer, both viruses were detected only in seeds and fruits, whereas they were detected in leaves and pedicel during the autumn season. The RT-qPCR results showed that during the spring, the ApMV and ApNMV expression was higher in leaves, whereas in the summer and autumn, the titer was mostly detected in seeds and leaves, respectively. The leaves in the spring and autumn seasons and the seeds in the summer season can be used as detection tissues through RT-PCR for early and rapid detection of ApMV and ApNMV. This study was validated on 7 cultivars of apples infected with both viruses. This will help to accurately sample and index the planting material well ahead of time, which will aid in the production of virus-free, quality planting material.