First Report of Anthracnose on Kadsura coccinea Caused by Colletotrichum fructicola in China.

Abstract

Kadsura coccinea (Lem.) A. C. Smith, an evergreen liana, is widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown numerous biological activities, such as anti-tumor, anti-HIV, and anti-oxidant (Yang et al. 2020). In June 2020, a leaf spot disease on K. coccinea was observed in a plantation in Longan (23°3´N, 107°54´E), Guangxi, China. Disease incidence was observed on about 15% (43/283) of the plants. Symptoms began as small brown spots that expanded into approximately irregular spots (Fig. 1A-1B). To isolate the pathogen, diseased leaves were collected. The leaves were sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 90 s, then rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed into potato dextrose agar (PDA) plates. The plates were incubated in an incubator at 25°C in dark for 3 days. Fungal colonies with similar morphology of 28 isolates were consistently isolated from the 30 infected tissues. Five representative isolates (Y1 to Y5) were selected to study for further characterization. Fungal colonies were initially cottony, pale white to grayish-green with abundant aerial hyphae (Fig. 1E). The mature conidia were hyaline, cylindrical, and unicellular, with rounded ends, 11.41-20.03 × 4.52-7.97 μm (average 15.64 × 5.86 µm; n =100) (Fig. 1D). Perithecia were brown, globose, 87.92-199.54 × 71.08-189.63 μm (average 147.56 × 118.50 µm; n =100). Asci were unitunicate, thin-walled, clavate, 28.99-65.85 × 5.27-13.07 μm (average 48.01 × 9.54 µm; n =100). Ascospore were hyaline, one-celled, 10.61-20.71 ×3.47-7.5 μm (average 17.09 × 5.54 µm; n =100). Appressoria were brown, ovoid, sometimes clavate, 5.83-10.53 × 3.92-6.38 μm (average 8.23 × 5.35 µm; n =100). The DNA sequences of actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS), and β-tubulin (TUB2) were amplified by PCR using the primer pairs ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-354R, GDF/GDR, ITS1/ITS4, and T1/Bt2b (Wang et al. 2020), respectively. Sequences were submitted to GenBank (Accession nos. MZ724634 to MZ724638 for ACT, MZ724639 to MZ724643 for CAL, MZ724649 to MZ724653 for CHS-1, MZ724654 to MZ724658 for GAPDH, MZ596330 to MZ596334 for ITS, and MZ724644 to MZ724648 for TUB2). These sequences were 98.88%-100% identical to sequences of reference isolates FJ907426, FJ917508, JX009866, JX010033, JX010165, and JX010405 of Colletotrichum fructicola ICMP 18581 ex-type recorded in GenBank. Phylogenetic analysis of combined ACT, CAL, CHS-1, GAPDH, ITS, TUB2 genes with 32 sequences obtained from GenBank using the maximum likelihood method showed that the five isolates clustered with three reference isolates of Colletotrichum fructicola as a distinct clade (Fig. 2). Pathogenicity tests were performed on young, fully expanded leaves of 1-year seedlings. A 10 μl conidial suspension (1×106 conidia/ml) was inoculated on each wound on the left-half leaf and a 10 μl sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 3 days and plants were maintained in a growth chamber at 28°C, 80% relative humidity, and a 12-h photoperiod. Anthracnose spots formed 3 to 4 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as C. fructicola, completing Koch's postulates. The pathogen was previously reported to cause anthracnose on strawberry (Zhong et al. 2021), preharvest fruit rot in apple (Nodet et al. 2019), leaf spot disease in Pouteria campechiana (Yang et al. 2021). Anthracnose on Kadsura coccinea caused by Colletotrichum siamense has previously been reported (Jiang et al. 2021). However, to our knowledge, this is the first report of C. fructicola causing leaf spot on K. coccinea in China.