First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Abstract

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 μm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 μm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.