First Report of Colletotrichum truncatum Causing leaf spot on Clausena lansium in China.

Abstract

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, leaf spot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 2 ha). Leaf spots were round or irregular with a clear yellow halo around a brown, necrotic lesion. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C in the darkfor 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (CLCT-1, CLCT-2, and CLCT-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. The colonies on PDA were gray white at first, subsequently turning grayish to dark gray, with numerous black microsclerotia and setae. Conidia were hyaline, aseptate, falcate with pointed ends, and 16.5 to 22.3 × 2.5 to 3.2 μm (n = 30). Morphological characteristics of the isolates were consistent with the description of Colletotrichum truncatum (Schwein.) Andrus & W. D. Moore (Sawant et al. 2012). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequences were submitted to GenBank under accession numbers OP740964 to OP740966 (ITS), OP800837 to OP800839 (GAPDH), and OP800843 to OP800845 (ACT). The sequences of the three isolates were 100% identical (ITS, 547/547 bp; GAPDH, 290/290 bp; and ACT, 266/266 bp) with those of C.truncatum (accession nos. GU227869, GU228261, and GU227967) through BLAST analysis.. In addition, a phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that nested within the clade containing C. truncatum (the type strain CBS 112998) by the maximum likelihood method. From the combination of the morphological and molecular characteristics, the isolates were determined to be C.truncatum. A pathogenicity test was performed in a greenhouse at 24 to 30°C with 80% relative humidity. Wampee plants (cv. JIXIN, n =5, 1-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water. Leaf spots were observed on the inoculated plants after 10 days while the control ones remained healthy. The pathogen re-isolated from all the symptomatic leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C.truncatumhas also beenreportedto be thecausalagent of anthracnose disease in multiple crops (Diao et al. 2014;Villafana et al. 2018; Stella de et al. 2021), thus, this is the first to report C.truncatum causing leaf spot on C. lansium in China. This study provides an important reference for the control of the disease due to the high host range ofC.truncatum.