Litchi anthracnose caused by Colletotrichum karstii in Guangxi, China.

Abstract

Litchi (Litchi chinensis Sonn.), a native fruit tree from southern China, has been planted in many subtropical and tropical countries for its fruit which are considered delicious and of medicinal value (Anderson et al. 2013). Anthracnose, one of the most important diseases on litchi, can cause flower drop, fruit drop, and fruit rot. Infected leaves form dark brown spots which turn to reddish brown with gray-white edges. Infected fruits formed dark brown spots which developed eventually to entire black rotted fruits. On both tissues, small dots of acervuli appeared with high humidity (Lai et al. 2004). On 20 April 2019, two leaf spots samples of litchi from different plants were collected from a 2 ha litchi orchard in Xintang Town (N 22.38˚, E 108.61˚), Qinzhou City, Guangxi province. The incidence of leaf spots in the orchard was above 20%. Each sample was cut into multiple pieces targeting zone between symptomatic and healthy plant tissues, disinfected with 75% ethanol for 10 s and 1% sodium hypochlorite (NaClO) for 1 min, and then washed three times with sterilized distilled water. The sterilized leaf tissues were placed on potato dextrose agar (PDA) and incubated at 28°C in darkness for one week. The growing hyphae from each sample was transferred to fresh PDA. The pieces from each leaf yielded a similar fungal morphotype over 75% of the time, and a representative one from each leaf was retained and called LZ1-1 and LZ3-1. The resulting colonies were incubated on the PDA for 7 days with gray to white aerial tufted hyphae, and abundant colorless to pale orange conidia in center of colony. The conidia were smooth, apex obtuse, base truncate, straight, cylindrical, and the contents remained granular. The conidial size of LZ1-1 was 10.6 to 21.4 × 4.5 to 9.1 μm (n=100) and that of LZ3-1 was 12.7 to 16.7 × 5.5 to 8.0 μm (n=100). Appressoria of LZ1-1 (6.9 to 14.9 × 6.0 to 11.1 μm) (n=100) and LZ3-1 (6.5 to 15.4 × 5.4 to 11.4 μm) (n=100) were pale to medium brown, ovoid to bullet-shaped, not nodose, and smooth-walled to undulate. DNA was extracted from two isolates, followed by PCR amplification and sequencing using primers for the rDNA internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) (Damm et al. 2012). The resulting sequences were deposited in GenBank (ITS: MW494453 and MW494454, ACT: MW495034 and MW495035, CAL: MW495036 and MW495037, CHS-1: MW495038 and MW495039, GAPDH: MW495040 and MW495041, TUB2: MW495042 and MW495043). The concatenated sequences comprised of six genomic regions of LZ1-1, LZ3-1 and other sequences of Colletotrichum obtained from GenBank were used to construct a Neighbor-Joining (NJ) tree with 1000 bootstrap replicates using MEGA4 (Tamura et al. 2007). The results revealed both LZ1-1 and LZ3-1 were clustered with type strain of C. karstii with high bootstrap value. The pathogenicity of the two isolates was determined by inoculating on leaves of 1-year-old litchi saplings in the greenhouse. Slight scratches were made on the surface of healthy leaves and 10 μL of spore suspension (106 conidia/mL) in 0.1% Tween 20 were inoculated onto each wounded spot. The blank control groups were inoculated with 10 μL 0.1% Tween 20. Each isolate was inoculated onto at least 27 leaves of three saplings, with each leaf wounded at spots. The inoculated saplings were placed in a greenhouse (12 h/12 h light/dark, 25 ± 2°C), and humidity maintained by covering plastic bags. The leaves inoculated with spore suspension showed reddish-brown spots after one week, while no symptoms were observed in the control. Each fungal isolate was consistently reisolated from inoculated leaves, thus fulfilling Koch's postulates. It was reported that members of the C. acutatum species complex and the C. gloeosporioides species complex could cause anthracnose on litchi (Ling et al. 2019), including C. gloeosporioides, C. siamense, C. fioriniae, and C. simmondsii (Ling et al. 2019; 2020). To our knowledge, this is the first report of anthracnose on litchi in China caused by C. karstii, a member of the C. boninense species complex. This study expands the understanding of the pathogen of anthracnose on litchi which can lead to improved management and control.