First Report of Anthracnose Fruit Rot Caused by Colletotrichum fioriniae on Litchi in China.

Abstract

Anthracnose fruit rot of litchi (Litchi chinensis Sonn.), caused by Colletotrichum spp., has been mainly associated with the C. acutatum species complex and C. gloeosporioides species complex (Farr and Rossman 2020). In June 2010, isolates of the C. acutatum species complex were isolated together with the C. gloeosporioides species complex from anthracnose lesions on litchi fruits (cv. Nuomici) obtained from a litchi orchard in Shenzhen (N 22.36°, E 113.58°), China. The symptoms typically appeared as brown lesions up to 25 mm in diameter, causing total fruit rot and sometimes fruit cracking. Based on the number of isolates we collected, the C. acutatum species complex appears less frequently on infected fruit compared to the C. gloeosporioides species complex. Since only the C. gloeosporioides species complex has been reported in China (Qi 2000; Ann et al. 2004), we focused on the C. acutatum species complex in this study. Pure cultures of fungal isolates were obtained by single-spore isolation. The isolate GBLZ10CO-001 was used for morphological characterization, molecular and phylogenetic analysis, and pathogenicity testing. Colonies were cultured on potato dextrose agar (PDA) at 25 ℃ for 7 days, circular, raised, cottony, gray or pale orange, with reverse carmine, and 39.6 to 44.7 mm in diameter. Conidia were 13.5 to 19 × 4 to 6 µm (mean ± SD = 15.9 ± 1.1 × 5.2 ± 0.3 µm, n = 50) in size, hyaline, smooth-walled, aseptate, straight, fusiform to cylindrical with both ends acute. Appressoria were 5.5 to 13.5 × 4.5 to 7.5 µm (mean ± SD = 7.6 ± 1.6 × 6.0 ± 0.7 µm, n = 50) in size, subglobose to elliptical, sometimes clavate or irregular, smooth-walled, with entire edge, sometimes undulate, pale to medium brown. These morphological characteristics were consistent with the descriptions of several Colletotrichum species belonging to the C. acutatum species complex, including C. fioriniae (Shivas and Tan 2009; Damm et al. 2012). For molecular identification, genomic DNA was extracted and the ribosomal internal transcribed spacer (ITS), partial sequences of the β-tubulin (TUB2), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and histone3 (HIS3) genes were amplified and sequenced using the primer pairs ITS4/ITS5, T1/Bt2b, ACT512F/ACT783R, GDF1/GDR1, CHS-79F/CHS-354R, and CYLH3F/CYLH3R, respectively (White et al. 1990; Damm et al. 2012). The resulting sequences were submitted to GenBank (ITS: MN527186, TUB2: MT740310, ACT: MN532321, GAPDH: MN532427, CHS-1: MT740311, HIS3: MT740312). BLAST searches showed 98.70%-100% identity to the sequences of the C. fioriniae ex-holotype culture CBS 128517. The phylogram reconstructed from the combined dataset using MrBayes 3.2.6 (Ronquist et al. 2012) showed that isolate GBLZ10CO-001 clustered with C. fioriniae with high posterior probability. Koch's postulates were performed in the field to confirm pathogenicity. Isolate GBLZ10CO-001 was grown on PDA (25 ℃ for 7 days) to produce conidia. In June 2014, litchi fruits (cv. Nuomici) were sprayed with conidial suspensions (106 conidia/ml), with sterile water as blank controls, and each treatment inoculated at least 15 fruits. Inoculated fruits were covered by an adhesive-bonded fabric bag until the trial ended. After 31 days, typical symptoms were observed, while control fruits remained asymptomatic. The fungus was re-isolated from diseased fruits and identified as C. fioriniae according to the methods described above. To our knowledge, this is the first report of anthracnose fruit rot on litchi caused by C. fioriniae, one species of the C. acutatum species complex, in China. For the difficulty in distinguishing anthracnose caused by C. fioriniae from the C. gloeosporioides species complex just by the symptoms, and mixed infection usually occurring in the field, further investigations are required to reliably assess the potential threat posed by C. fioriniae for litchi production in China.