Star anise (Illicium verum) has been cultivated for centuries in southern China, and its fruit is an important seasoning spice, and can be used as a medicine (Wang et al. 2011). It is grown mainly in Guangxi, Guangdong, Guizhou, and Yunnan provinces, in China. Anthracnose is one of the important diseases of star anise, which seriously affects the yield and quality by infecting twigs, pedicels, fruit stalks and fruits (Liao et al. 2017). When leaf spots first appear, they are round, water-stained, small, dark brown spots, which expands into round separated spots, then the spots become yellowish brown with small black acervuli arranged in a circular pattern. On 22 August 2019, four leaf spot samples of star anise were collected, with two each from Shanglin County and Jinxiu County in Guangxi Province. The plantations in this area of around 8 ha had more than 80% leaf spot incidence. Small pieces of tissues (5 mm × 5 mm) were taken from the zone between symptomatic and healthy plant tissues, surface-disinfected in 75% ethanol for 10 s and 1% NaClO (sodium hypochlorite) for 1 min, and washed three times in sterilized distilled water. The sterilized leaf tissues were placed on potato dextrose agar (PDA) and incubated at 28°C in darkness for a week. Hyphae growing from tissue pieces were subcultured onto fresh PDA. Three of the four leaves yielded cultures resembling Colletotrichum spp. Four fungal isolates were obtained by a single-spore isolation method. The isolates JX1-2 and JX1-5 were collected from Jinxiu County while SL1-2 and SL2-1 were collected from Shanglin County. Genomic DNA was extracted from these four fungal isolates, followed by PCR amplification and sequencing of the rDNA internal transcribed spacer (ITS), actin (ACT), Apn2-Mat1-2 intergenic spacer, partial mating type (Mat1-2) (ApMat), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012). The sequences have been deposited in GenBank (ITS: MW301215 to MW301218; ACT: MW348965 to MW348968; ApMat: MW348973 to MW348976; CAL: MW348957 to MW348960; CHS-1: MW348969 to MW348972; GAPDH: MW348961 to MW348964). For phylogenetic analysis, MEGAX (Kumar et al. 2018) was used to produce a Maximum Likelihood (ML) tree with 1000 bootstrap replicates, based on a concatenation of the sequenced genomic regions for each of the four isolates from this study as well as sequences of other Colletotrichum species obtained from GenBank. The results revealed that isolates JX1-2, JX1-5, and SL1-2 were C. horii, and SL2-1 was C. fructicola (Weir et al.2012). The resulting colonies were initially white with abundant aerial hyphae, and white-gray after three days at 28°C on PDA. Isolate SL2-1 eventually turned greenish-grey after 14 days, while the center of C. horii isolates turned iron-gray with white-gray marginal. Both species of Colletotrichum had hyaline conidia that were terete, smooth, apex obtuse, base truncate, and there were no significant differences (P>0.05) in conidial size between C. horii (10.5 to 33.6 × 3.6 to 9.3 μm) (n=300) and C. fructicola (13.1 to 16.2 × 4.7 to 7.1 μm) (n=100). Pathogenicity tests were conducted in the greenhouse on 1-year-old star anise seedlings, and performed with a conidial suspension (10 µL of 106 conidia/mL) containing 0.1% Tween 20 placed onto lightly wounded sites on healthy leaves. Light cross-shaped wounds were made with sterilized toothpicks, gently scratching the surface without piercing the leaf. Each isolate was inoculated onto three seedlings, with at least eight leaves per seedling inoculated in two spots after light wounding. Control seedlings were inoculated with water containing 0.1% Tween 20. All inoculated seedlings were maintained in the greenhouse (12 h/12 h light/dark, 25±2°C), and covered with plastic bags to maintain high humidity throughout. The wounded sites inoculated with C. horii darkened to greenish-brown after 24 h, and C. fructicola gave similar symptoms after 36 h. Then the wounds turned to light brown round spots, and after 5 days, the spots expanded to water-stained spots with dots of acervuli arranged in a circular pattern. No symptoms were observed for the non-inoculated control. Each fungal isolate was consistently re-isolated from inoculated leaves, thus fulfilling Koch's postulates. There were significant differences (P<0.05) in aggressiveness between the two species, with C. horii showing larger diameter lesions (averaging 10.2 mm) than C. fructicola (averaging 8.4 mm). Anthracnose of star anise caused by C. horii (Liao et al. 2017) and C. coccdes (Wu et al. 2003) has been previously reported in China; however, to our knowledge, this is the first report of C. fructicola infecting star anise in China. This study may provide reference for further epidemiological study and prevention of anthracnose on star anise.
Experimental study on essential anise star oil purification in vacuum batch distillation
