First report of Phakopsora euvitis causing leaf rust disease on grapevine (Vitis labrusca L.) in India

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.