First report of Colletotrichum grevilleae causing fruit anthracnose of pomegranate in Taiwan.

Pomegranate (Punica granatum L.), native to northern India, is a popular fruit crop. In Taiwan, pomegranate is mainly cultivated in the mid and southern regions including Waipu, Taichung and Neipu, Pingtung. However, diseases affecting production and quality of pomegranate are largely understudied in Taiwan where gray mold and Cercospora leaf spot of pomegranate were ever reported (Hsieh, 1978; Hsieh and Wu, 1989). Pomegranate fruit (cv. Tunisia) showing anthracnose symptoms were found in a commercial pomegranate farm of 0.4 ha in Waipu (N24.33111, E120.68978) in August 2020. Around 40% fruit were infected and displayed symptoms of brown sunken lesions with indistinct edges typically occurred at the fruit ripening stage (supplementary Fig. S1). To isolate the pathogen, diseased exocarps were surface sterilized with 1% NaOCl followed by 70% ethanol. Surface-sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. A fungus was consistently isolated from diseased fruit, and five isolates (PGCo1, WP1, WP2, WP3, and WP4) were obtained. When cultured on PDA 25℃ under near UV light (360 nm) with a 12-h:12-h light/dark cycle for 10 days, the isolates initially produced white hyphae and subsequently formed dark gray to olivaceous black colonies with white edge, and no conidiomata was observed (supplementary Fig. S1). Conidia were usually aseptate, hyaline, cylindrical to ellipsoid and obtuse at either one or both ends (supplementary Fig. S1), and measured 16.0 ± 1.6 (11.4-18.9) × 5.6 ± 0.5 (4.5-6.9) μm (n = 100). The isolates were further identified by sequence comparison and phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of the rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), chitin synthase 1 (CHS-1), actin (ACT) and calmodulin (CAL) genes (Weir et al., 2012). The sequences were deposited in the GenBank under accession numbers ON849040-ON849044 and ON862106-ON862130. BLASTn searches showed the ITS sequences of the pomegranate isolates were 100% identical to that of C. grevilleae CBS 132879T (NR_163525). The ACT, GAPDH and TUB2 sequences of the isolates were 100%, 100% and 99.57%, respectively, identical to those of C. grevilleae CBS 132879T (KC296941, KC297010 and KC297102). The CHS-1 sequences were 99.67-100% identical to that of C. grevilleae CBS 132879T (KC296987). The CAL sequences showed 91.50% and 100% identity with those of C. grevilleae CBS 132879T (KC296963) and C. grossum CAUG7T (KP890147), respectively, both members of the C. gloeosporioides species complex. Multilocus sequence analysis of the combined data was conducted using MEGA X software (Kumar et al., 2018). The five isolates were clustered with C. grevilleae CBS 132879T, indicating that the pomegranate isolates are C. grevilleae (Supplementary Fig. S2). For pathogenicity test, 6 symptomless ripe pomegranate fruit (cv. Tunisia) were surface sterilized, wounded by stabbing a sterile needle, and inoculated with a conidial suspension (10 μl each, 1×106 conidia/ml) of PGCo1. Sterile water was used as control. The inoculated fruit were kept in a moist plastic chamber (>95% relative humidity, 25 ± 2°C) in darkness. The experiment was performed twice. Seven days post inoculation, the artificially inoculated fruit showed anthracnose symptoms of sunken brown lesions indistinguishable from the natural infection. The control remained symptomless. The same fungus was repeatedly isolated from the symptomatic fruit, fulfilling Koch's postulates. Prior to this study, only the ex-type strain of C. grevilleae was collected from root and collar rot of Grevillea sp. (Liu et al., 2013). This is the first report of C. grevilleae causing anthracnose on pomegranate in Taiwan. Since no fungicide has been approved for pomegranate cultivation in Taiwan, future study is necessary to develop a management strategy against this disease.