First Report of Postharvest Fruit Rot on Loquat Caused by Didymella coffeae-arabicae in China.

Abstract

Loquat (Eriobotrya japonica Lindl.) popular fruit crop in Southwest China. In July 2023, a postharvest fruit rot with about 5% disease incidence was observed in two supermarkets of Ya'an City, Sichuan Province, China. The fruits exhibited circular, light brown to brown, rotten, slightly sunken lesions, covered with a grey-white mycelial mass. Isolations were made from infected tissues from the margins of the lesions of 12 symptomatic fruits which were surface-sterilized in 75% ethanol for 15 s and 1% sodium hypochlorite for 30 s, then rinsed in sterile water and air dried. The samples were plated on potato dextrose agar (PDA) and incubated at 25℃ for 3 days. A total of 10 isolates were obtained. The colonies on PDA were circular, with smooth margins, white to gray green, with abundant aerial mycelia. Conidia were ellipsoidal, smooth, transparent, and measured approximately 4.4 to 7.2 × 2.2 to 3.3 µm (n = 30). The isolates were initially identified as Didymella spp. The identity was of two representative isolates (JFRL 03-1688, JFRL 03-1689) was further confirmed using the internal transcribed spacer (ITS) region, 28S nrDNA (LSU), β-tubulin 2 (TUB2) and DNA-directed RNA polymerase II core subunit (RPB2), which were amplified and sequenced using primers V9G/ITS4, LR0R/LR7, Btub2Fd/Btub-4Rd and RPB2-5F2/RPB2-7cR, respectively (Carbone and Kohn 1999, Aveskamp et al. 2009, Chen et al. 2017). These sequences were deposited into GenBank with accession numbers: OR649239 and OR649240 for ITS, OR649248 and OR649249 for LSU, OR672208 and OR672209 for TUB2, OR672215 and OR672216 for RPB2. A BLASTn homology search for these nucleotides showed 99-100% identity to ITS (MH863293), LSU (MH863293), TUB2 (FJ427104) and RPB2 (KT389603) sequences of ex-type strain of Didymella coffeae-arabicae CBS 123380. The maximum likelihood analyses were performed for the combined ITS, LSU, TUB2 and RPB2 using the IQtree web server (Trifinopoulos et al. 2016). The phylogenetic tree showed that these two isolates clustered with D. coffeae-arabicae in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. coffeae-arabicae based on morphological and molecular characteristics. To evaluate pathogenicity, four healthy loquat fruits were surface-sterilized and wounded, then 50 μL conidial suspension (1×106 conidia/mL) of the isolate JFRL 03-1688 was inoculated on the wound. Another set of four fruits was inoculated with sterile water as a control. All treated fruits were incubated at 25 ℃ with 80% humidity, and the experiment was repeated twice. Five days later, all the wounded fruit inoculated with D. coffeae-arabicae showed similar symptoms to those observed in the field, while the control fruits remained healthy. D. coffeae-arabicae was consistently reisolated from infected fruit and confirmed by morphological and molecular data, fulfilling Koch's postulates. D. coffeae-arabicae was reported as a saprobic or pathogenic on Coffea arabica, Lagerstroemia indica and Leucaena species (Aveskamp et al. 2009; Chen et al. 2017; Jayasiri et al. 2019; Vu et al. 2019), but to our knowledge, this is the first report of Didymella coffeae-arabicae causing postharvest fruit rot on loquat in China. The study expands the host range of D. coffeae-arabicae and will be helpful to develop efficient management strategies on fruit rot disease of loquat.