Identification of Colletotrichum fructicola anthrax pathogens of sea buckthorn fruits and screening of biocontrol bacteria
摘要
[Objective] In 2023, it was found in the planting base of sea buckthorn in Chaoyang City, Liaoning Province that the fruit of sea buckthorn variety Snowy Yellow showed symptoms of anthracnose, and the incidence rate reached more than 20%. The aim of this study was to identify the pathogens causing anthracnose in sea buckthorn fruits, and to screen out the biocontrol bacteria that can effectively inhibit the pathogenic bacteria, in order to provide assistance for prevention and control of anthracnose of sea buckthorn. [Methods] Diseased fruits with typical symptoms were collected and the pathogenic fungi were obtained by tissue isolation method. After single-spore purification in PDA medium, the pathogenicity of the purified strains was verified by the back-joining method according to Koch's rule. After the strain was cultured on PDA medium for 7 d, the morphology and colour of the colonies were observed and recorded, and the morphology of spores and conidial discs were observed under a light microscope after continue culture for 14 d. The slide with conidia was placed into the incubator at 28℃ for 36 h, and then taken out for observation of adherent spores. The six genes of ITS, CAL, CHS-1, GAPDH, TUB2 and ACT were amplified by PCR. The PCR amplification was performed under the following conditions: Initial denaturation at 95℃ for 4 min, followed by 35 cycles of 95℃ for 30 s, at the 52℃ (ITS, TUB2), 58℃ (CHS-1, ACT), 59℃ (CAL, GAPDH) annealing for 30 s, and 72℃ extension for 7 min. The sequencing results were compared with the NCBI database, and the phylogenetic tree of the pathogen was constructed by MEGA 7.0. Beneficial microorganisms were isolated from the rhizospheric soil of the healthy sea buckthorn fruit trees using the dilution coating method, and antagonistic bacteria were screened using the plate standoff method with the anthracnose pathogen of the sea buckthorn fruit as the target strain. The screened bacterial strains were inoculated on LB plates by line