Colletotrichum aeschynomenes Causing Anthracnose in Combretum indicum in Guangxi, China.

ABSTRACT

Combretum indicum（L.）Jongkind, distributed in Southeast Asia, is widely planted in southern China for its ornamental and medicinal value. In February 2023, anthracnose symptoms were observed on C. indicum leaves in Nanning Garden Expo (N22°43', E108°28'), Guangxi, China, causing severe defoliation of infected plants with a foliar disease incidence ranging from 40 to 60% (n = 100) in a 2 ha field. Disease symptoms began with small red spots (2 to 3 mm by 2 to 3 mm) on the leaves and gradually enlarged to larger irregular light grey lesions with yellowish halos (3 to 5 mm by 2 to 8 mm). In the late stage, spots merged into larger irregular lesions (5 to 15 mm by 6 to 13 mm) and the necrotic lesions abscised. Three diseased samples in total were collected from plants in three different locations. Symptomatic leaves were cut into small pieces (3×3 mm), disinfected with 75% ethanol solution for 10 s, 2% NaClO for 1 min followed by three washes in sterile distilled water. Tissue pieces were separately plated on potato dextrose agar (PDA) and incubated at 25°C for five days. One representative isolate from each sample (SJ-1, SJ2-1 and SJ3-1) were chosen for further analysis. Colonies were villiform, initially white, later turning gray in 7 days on PDA at 25℃. The average diameter for colonies were 8.1 cm, 8.0 cm and 8.1 cm for SJ1-1, SJ2-1 and SJ3-1, respectively. Conidia were aseptate, hyaline, cylindroid, and averaged 11.94 µm × 5.04 µm, 11.78 µm × 5.14 µm and 11.74 µm × 4.59 µm (n=90) for SJ1-1, SJ2-1 and SJ3-1, respectively. The characteristics were close to the descriptions of Colletotrichum spp. (Weir et al. 2012). Genomic DNA was extracted from 7-day-old aerial mycelia of these isolates. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB2), chitin synthase (CHS-1), calmodulin (CAL) and the intergenic region between apn2 and MAT1-2-1 (ApMat) were amplified using primers ITS1/ITS4 (White et al. 1990), GDF/GDR, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-354R, CL1C/CL2C (Weir et al. 2012) and AM-F/AM-R (Silva et al. 2012), respectively. Sequences were deposited in GenBank (ITS OR540240-OR540242; GAPDH PP328968-PP328970; ACT PP328959-PP328961; TUB2 PP328971-PP328973; CHS-1 PP328965-PP328967; CAL PP328962-PP328964 and ApMat OR548253-OR548255). A phylogenetic analysis was made via Bayesian inference based on the concatenated sequences (ITS, GAPDH, ACT, TUB2, CHS-1, CAL and ApMat). According to morphology and phylogenetic analysis, SJ1-1, SJ2-1 and SJ3-1 were identified as C. aeschynomenes. Pathogenicity was confirmed on leaves with and without wounds of 24 one-year-old C. indicum plants in a greenhouse in Nanning, Guangxi Province. The wound was made with a sterilized needle. Wounded and unwounded leaves were inoculated with 20 µl of conidial suspension (106 spores/ml in 0.1% sterile Tween 20) of the three isolates and control plants were inoculated with water containing 0.1% sterile Tween 20 (6 leaves/plant, 3 plants/treatment). All plants were covered with plastic bags to maintain a high humidity environment and placed in a 28°C growth chamber with constant light. After 7 days of incubation, necrotic lesions were observed on inoculated wounded leaves, whereas unwounded leaves and control plants showed no symptoms. The fungi were re-isolated from symptomatic leaves, completing Koch's postulates. These species can cause severe diseases in a variety of plants worldwide, such as Manihot esculenta, Theobroma cacao and Myrciaria dubia (Sangpueak et al. 2018; Nascimento et al. 2019; Matos et al. 2020). To our knowledge, this is the first report of C. aeschynomenes causing C. indicum leaf anthracnose in China. The results will provide valuable information for management of anthracnose in C. indicum.