Identification and pathogenicity of Colletotrichum species associated with Guanximiyou pomelo anthracnose

[Objective] Guanximiyou pomelo (Citrus grandis) is a famous and popular Citrus species for its sweet and excellent nutrients native to Pinghe county of Fujian province, China. In Guanximiyou pomelo, anthracnose caused by Colletotrichum spp. is a serious disease limiting its production. In 2018, the disease seriously affected over 60% of Guanximiyou pomelo trees in an orchard in Pinghe county. The Guanximiyou pomelo anthracnose mainly damages leaves, twigs and fruits. In the edge, tip or middle of damaged leaves develop brown spots, which form a “V” shape then withering and falling off after the diseased leaves die. The damaged twigs show spots from the petiole and the base of axillary bud to the bottom of twigs. On fruit, symptoms appear as green, irregular and sunken lesions in the young stage, that turn to brown rot and then falling off in mature stage. This study aimed to clarify the species of Colletotrichum spp. associated with Guanximiyou pomelo anthracnose and determine the pathogenic characteristics of the pathogens, so as to provide a better acknowledge for the diversity of the pathogen species and scientific basis for the prevention and control of the disease in Fujian province. [Methods] A survey of anthracnose disease was conducted in 13 Guanximiyou pomelo orchards from 2019 to 2021 in Pinghe county, Fujian province. The leaves, twigs and fruit with symptoms were collected from the pomelo trees and were used as disease samples. The Colletotrichum spp. were isolated by the plant tissue isolation method. 4 × 4 mm diseased tissues were surface-sterilized with 70% ethanol for 30 s, 0.1% mercuric chloride for 60 s, washed three times in sterile water and dried on sterilized filter paper, then placed onto potato dextrose agar (PDA) plates and incubated under 2 8 ℃ in the dark. The single mycelium was used for purifying strains, and pure cultures were stored in PDA at 4 ℃. The colony characteristics, conidia morphology and appressorium for representative strains of the identified Colletotrichum spp. were recorded. The Colletotrichum spp. genomic DNA was extracted using a fungus genomic DNA extraction kit, which was identified through partial rDNA-ITS (ITS), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glutamine synthetase (GS) region sequence. Phylogenetic tree based on the combined ITS-ACT-TUB2-GAPDH-GS sequences was constructed by maximum like-lihood method with MEGA 7.0. The conidia suspension of 58 representative Colletotrichum spp. were used in the pathogenicity tests to inoculate twigs and leaves of Guanximiyou pomelo according to Koch's postulate. Correlation analysis of mycelial growth rate, appressorium formation rate and pathogenicity were determined by Pearson method. [Results] A total of 350 strains with the similar morphology to Colletotrichum spp. were isolated from the collected samples infected by the Guanximiyou pomelo anthracnose disease. C. gloeosporioides species complexes, C. boninense species complexes, C. truncatum species complexes, C. magnum species complexes and C. orchidearum species complexes were identified based on morphological characteristics and ITS sequencing. Colony observation showed that there were significant differences in aerial and substrate mycelia's color among the five Colletotrichum species complexes. C. gloeosporioides species complexes, C. boninense species complexes, C. truncatum species complexes, C. magnum species complexes and C. orchidearum species complexes had thirteen, thirty, one, nine, five different colony characteristics, respectively. Conidia of C. truncatum, and C. orchidearum species complexes were significant different from the other three Colletotrichum species complexes. Conidia of C. orchidearum species complexes was curve and rounded at both ends, which was sickle and acuminate at both ends by C. truncatum species complexes, and the other three Colletotrichum species complexes were cylindrical, rounded at both ends or top end rounded, base end raised. To classify the taxonomic status of the Colletotrichum spp., the multi-genes (ITS, ACT, TUB2, GAPDH and GS) were used to build phylogenetic tree of 58 Colletotrichum isolates with different colony characteristics. Among the 58 Colletotrichum isolates, one was identified to be C. gloeosporioides, twelve were C. fructicola, thirty were C. karstii, nine were C. brevisporum, five were C. cliviicola and one was C. truncatum. Pathogenicity tests revealed that the above five Colletotrichum spp. induced lesions on both twigs and leaves of Guanximiyou pomelo, with C. cliviicola being the sole non-pathogenic exception. The strains isolated from infected sites were identical to the strains inoculated. The pathogenicity of C. gloeosporioides was slightly stronger than C. fructicola, but significantly stronger than the other three species. BZMYTJ20 demonstrated significantly stronger pathogenicity than JFMYTJ53, while DXMYTJ5 had no pathogenicity in leaf infection assays, although they all belonged to C. karstii. The mycelial growth rates of those pathogens ranged from 6.27 to 13.53 mm·d-1. C. gloeosporioides was the fastest, followed by C. fructicola, C. brevisporum and C. truncatum, while C. karstii was the slowest. The appressorium formation rate of those pathogens ranged from 33.65% to 82.52%; C. gloeosporioides was the highest, followed by C. brevisporum, C. fructicola and C. karstii, and C. truncatum was the lowest. By analyzing the correlation between mycelium growth rate and appressorium formation rate and pathogenicity, the correlation coefficient r=0.373 3 and 0.364 1, so it was clear there were some positive correlation between mycelial growth rate, appressorium formation rate and pathogenicity. [Conclusion] Based on colony and morphological characteristics, phylogenetic analysis of the multiple genes (ITS, ACT, TUB2, GAPDH and GS) and pathogenicity, the pathogens of Guanximiyou pomelo anthracnose disease in Fujian were identified as 5 species, including C. gloeosporioides, C. fruticola, C. karstii, C. brevisporum and C. truncatum, among which C. gloeosporioides was the dominant. C. fruticola, C. karstii, C. brevisporum and C. truncatum were first identified as the Guanximiyou pomelo anthracnose pathogen in Fujian province, which confirmed that the pathogens of Guanximiyou pomelo anthracnose tended to be diversified and differentiated in Fujan province. There were great differences in the growth rate of mycelium, appressorium formation rate and pathogenicity among different Colletotrichum species, and there was some positive correlation between mycelial growth rate, appressorium formation rate and pathogenicity. This study can provide theoretical data for the diversity research and sustainable prevention and control of Guanximiyou pomelo anthracnose in Fujan province.