First Report of Colletotrichum cliviae Causing Anthracnose of Rubber Tree in China.

Abstract

Rubber tree (Hevea brasiliensis) is the most important species producing commercially viable natural rubber latex, which is an important raw material for many industrial uses. Anthracnose caused by Colletotrichum is one of the most severe diseases of rubber tree, especially in China. However, the diversity of Colletotrichum from rubber trees is complex and needs further identification (Liu et al. 2018). In January 2021, anthracnose-like lesions were observed on the leaves of rubber seedlings at a plantation in Qiongzhong City (19.08 N, 109.54E), Hainan Province, China. The lesions initially produced dark brown spots on the leaves of rubber trees, and then gradually evolved into larger necrotic spots. The diseased leaves shrank and died until they fell off. Small pieces of symptomatic leaf tissues were soaked in 0.5% mercuric chloride for 40 s, washed with sterile water for three times, and plated on potato dextrose ager (PDA). The plates were incubated at 28°C with 12-h light/dark regime for 10 days. Two isolate HN16 and HN18 from different leaves were subcultured on PDA using the single-spore method. The center of the colony is brown or grayish brown with the leading edge of growth in culture being white. Conidia were hyaline, septate, and cylindrical, with obtuse ends (14-20 μm × 4-6 μm).These morphological features confirmed the identity of the two isolated fungi as Colletotrichum cliviae (Yang et al. 2009). To confirm the morphological identification, five loci informative for identification of Colletotrichum spp. were amplified and sequenced, including the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), beta-tubulin (TUB), actin (ACT), an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (CHS-1), using the primer pairs ITS-1F/ITS4,T1/Bt-2b, ACT-512F/ACT-783R, GDF/GDR and CHS-79F/CHS-345R, respectively (Weir et al 2012). Sequences were deposited in NCBIs GenBank database (MW971911-MW971920). A multilocus phylogenetic analysis revealed that the sequences of the two isolates and published sequences of C. cliviae S37 clustered into one clade with a 100% confidence level. The two isolates were determined as C. cliviae by combining morphological and molecular characteristics. To verify pathogenicity, six leaves of rubber plants at the light green phase were sprayed with drop of spore suspension (106 conidia/ml) and sterile distilled water as control. After inoculation, all leaves were maintained in a climate box at 28°C with 100% relative humidity. After 3 days, lesions similar to those observed with dark brown spots in the field appeared on the inoculated leaves, while the controls remained symptomless. The fungus was reisolated from inoculated leaves and confirmed as C. cliviae by morphological and molecular identification, fulfilling Koch's postulates. The pathogen has been mainly reported on soybean in Brazil and China. To our knowledge, this is the first report of anthracnose on rubber tree caused by C. cliviae in China. However, Colletorrichum gloeosporioides and Colletorrichum acutatum are the two major causatives agent of anthracnose of rubber trees in China (Cai et al. 2016; Shi et al. 2019). The disease caused by C. cliviae was observed only at one rubber plantation with its distribution needing further investigation.