Leaf Spot Disease on Punica granatum Caused by Alternaria alternata in China

Pomegranate (Punica granatum L.), which is native to central Asia, is considered as one of the most renowned commercial fruit trees in the world. The planting area in China is roughly 120 thousand hectares. In June 2020, symptoms of leaf spot on P. granatum appeared in Nanyang City (32º40´34˝N, 111º44´20˝E), Henan Province, with an incidence rate of 35% in several 3.3-hectare orchards. Initially, the lesions showed as round or subrounded brown spots on affected leaves. The spots then progressively developed into irregular lesions with distinct yellow halos surrounding them. Parts of the lesions were weakly zonate, which finally led to leaf withering and falling off. Diseased tissues were cut into 5×5 mm2 pieces, which were surface sterilized with 75% ethanol solution for 30 s, washed 3 times in sterilized water, and put on potato dextrose agar (PDA) plates supplemented with 50 μg ml-1 streptomycin. A total of 16 purified fungal isolates with similar phenotypic features were obtained. Three randomly chosen isolates SLY11, SLY24, and SLY25 were utilized for the investigation. Fungal colonies on PDA were first white to gray and later mycelium became olive green to blackish brown. To examine the morphological properties of conidia, we utilized potato carrot agar (PCA) culture medium and incubated it at 23°C under a 12-hour light/dark alternation. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa and 1 to 4 longitudinal septa. Conidiophores were septate, solitary, and measured 22.7 (±4.64) × 10.6 (±2.15) μm (n=50), with a conical beak length of 0 to 5.5 μm. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (TEF1), β-tubulin gene (TUB), and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and GDF1/GDF2 from genomic DNA. Sequences were submitted to GenBank with accession numbers OL840230, OL840231, OL840232 for ITS, OL982540, OL982541, OL982542 for TEF1, OL982543, OL982544, OL982545 for TUB, OL862608, OL862609, OL862610 for GAPDH sequences of isolates SLY11, SLY24, and SLY25, respectively. BLASTn analysis of ITS (OL840230), TEF1 (OL982540), TUB (OL982543), GAPDH (OL862608) sequences indicated 100, 99.59, 99.68, and 100% similarity to the sequences of Alternaria alternata strain HC-2 (MT644140), BJFA-1 (MK895958), CS36-5 (KY814630), and ag1 (KP057228) in GenBank. Isolates SLY11, SLY24, and SLY25 formed a clade with the type strains CBS 130265 and CBS 130258 of A. alternata in phylogenetic trees established, clearly seperating from other Alternaria spp. The morphological features and molecular analyses supported the isolates as members of A. alternata. To validate the pathogenicity of the isolates, ten healthy leaves of 3-year-old potted pomegranate trees were utilized for testing and inoculated with conidial suspension (106 conidia ml-1), 20 µl each leaf. Control plants were inoculated with sterilized water. An additional pathogenicity test was repeated on wounded leaves. The inoculated plants were placed at 28°C in a greenhouse (12 h light per day and 90% relative humidity) for 5 days. The pathogenicity testing was conducted three times. Distinct lesions were found on the nonwounded and wounded leaves of inoculated plants after 3 to 5 days. The morphology and ITS sequences of the fungi that were reisolated from each of the inoculated plants were similar to that of the inocula, fulfilling Koch’s postulates. Fruit rot of pomegranate induced by A. alternata was not identified in our investigation. A. alternata is reported to induce leaf spot disease on P. granatum in India (Zakir et al. 2009), Israel (Ezra et al. 2010), Spain (Garcia-Jimenez et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot disease on P. granatum in China. Severe leaf disease caused by A. alternata can lead to reduced pomegranate yields in the harvest stages. This note will aid in pathogen identification and disease control.