Leaf Spot Caused by Colletotrichum siamense, C. fructicola, and C. aeschynomenes on Ixora chinensis in Guangxi, China

Abstract

Ixora chinensis Lam., an important ornamental flower, has become more and more popular in the southwest and southeast regions of China for its bright and abundant flowers (Li et al. 2019). In March 2022, 100% I. chinensis plants showed typical anthracnose symptoms on leaf in Nanning, Guangxi, China (108°22′ N, 22°48′ E). The central areas of lesions were grayish white with small black particles arranged in a wheel pattern, and the edges of lesions were light red to brown. Three diseased leaf samples were collected from three gardens, respectively. 5×5 mm tissues were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on PDA at 25°C under 12/12 h light/darkness. Eighty-three morphologically similar colonies were observed on PDA after 5 days, and eight isolates G1-3, G2-1, G3-3, W-1, W-2, LCH2-1, LCH3-3, and LCH4-1 were selected for further study. Genomic DNA of these isolates were extracted from 7-day-old mycelia. Primer pairs ITS1/ITS4, GDF1/GDR1, T1/βt2b, CHS Ⅰ-79F/CHS Ⅰ-354R, CL1/CL2, ACT-512F/ACT-783R, and MAT1-2-1/apn2 were used to amplify ITS loci and GAPDH, CHS-Ⅰ, CAL, ACT, ApMAT genes, respectively (Yang et al. 2009; Silva et al. 2012; Liu et al. 2015). Sequences have been deposited in GenBank (ITS: OQ771884 to OQ771891, GAPDH: OQ759576 to OQ759583, TUB2: OQ759584 to OQ759591, CHS-1: OQ759568 to OQ759575, CAL: OQ759560 to OQ759567, ACT: OQ759552 to OQ759559, ApMat: OQ759544 to OQ759551). Phylogenetic analysis was performed with raxmlGUI v.2.0.0. based on combined sequences of ITS, GAPDH, TUB2, CHS-1, CAL, ACT, and ApMAT using maximum parsimony analysis. The results revealed that isolates G2-1 and W-2 were clustered with Collectrichum fructicola, G3-3, W-1, G1-3, LCH2-1, and LCH3-3 were clustered with C. siamense, and LCH4-1 was clustered with C. aeschynomenes. Three representative isolates W-2, G3-3, and LCH4-1 were selected for morphology and pathogenicity observation. On PDA, the colonies of three isolates presented white to grey cottony mycelia，from the margin to the center, W-2 was white, grey, and light gray, G3-3 showed light gray, white, and grey, LCH4-1 was white and light gray, respectively. Conidia were all hyaline, one-celled, cylindrical, and straight. The conidial sizes of W-2, G3-3, and LCH4-1 were 11.03 to 17.53 × 4.93 to 8.42 μm (n=100), 10.63 to 19.06 × 3.73 to 6.92 μm (n=100), and 11.61 to 20.39 × 3.65 to 6.67 μm (n=100), respectively. Pathogenicity tests of three isolates were conducted on leaves of 1-year-old I. chinensis plants with and without wounds, three plants for each treatment, and five leaves inoculated for each plant. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site. Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton, and all plants were bagged and placed in the greenhouse to maintain humidity at 25℃. After 10 days, all wounded and inoculated leaves showed leaf spot, whereas unwounded and control leaves remained asymptomatic. Koch’s postulates were fulfilled by re-isolating the causal agents from diseased leaves. C. siamense and C. aeschynomenes could cause anthracnose of I. chinensis in China (Liu et al. 2016, Li et al. 2021). However, to our knowledge, this is the first report of C. fructicola infecting I. chinensis in China. This study may provide reference for further epidemiological study and prevention of anthracnose on I. chinensis.