Identification of Colletotrichum fructicola anthrax pathogens of sea buckthorn fruits and screening of biocontrol bacteria

【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 incuba-tor 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 inoculation, and incubated in an incubator at 28 ℃ for 24 h to 48 h for observing the morphological characteristics of single colonies, and the physiological and biochemical characteristics were identified using physiological and biochemical reagent strips. The molecular identification was performed by PCR amplification of 16S rDNA and gyrB gene sequences of the strains, and a phylogenetic tree of the biocontrol bacteria was constructed using MEGA 7.0 after comparison with the NCBI database. The inoculation of biocontrol strain into protease, cellulase, β-glucanase and chitinase detection plates was carried out to detect if the biocontrol strain produces extracellular enzymes by observing the presence or absence of hyaline rings around the colonies.【Results】The sea buckthorn fruit anthracnose produced pinpoint-sized light brown spots in the middle and lower part of the fruit at the early stage of the disease, and then the spots gradually expanded into dark brown sunken round or elliptic spots, the size of the spot was usually about 5 mm, with whorls of small black spots in the center of the spots. A total of 14 representative strains were isolated and purified from sea buckthorn diseased fruits. The strain CY-8, identified as the causal agent, induced symptoms similar to the initial field observations 5 d post-inoculation. Through morphological observation, the colony was round, the front part of the aerial mycelium was tomentose, the middle part was grey-green mycelium, the edge mycelium was greyish-white, the conidium was colourless and transparent, unicellular, ellipsoidal or subellipsoid, the conidium germination produced brown appressoria in the form of horseshoe, the bristles were dark brown, and the acervulus were ellipsoidal. Combined with molecular identification, it was determined that the pathogen of sea buckthorn fruit anthracnose was Colletotrichum fructicola. Two strains of biocontrol bacteria, W32 and W22, with strong antagonistic effects, were obtained through screening, and the plate inhibition rates against C. fructicola were 66.08% and 54.76%, respectively. The inhibition rates were calculated from three independent replicates, with standard deviations of ±0.60% and ±1.19%, respectively. According to the morphological characteristics, the strain W22 colonies were milky white, flattened, irregular in morphology, with rough and opaque surface, crumpled, Gram-positive, and rod-shaped. The strain W32 colonies were yellowish and opaque, with untidy edges, moist and slightly elevated surface, with slightly sticky texture, Gram- stained positive, and rod- shaped, and the results of molecular and biological identification showed that the strain W32 was Bacillus velezensis, the strain W22 was B. subtilis. Both strains of biocontrol bacteria were able to produce protease and β- glucanase, and neither was able to produce cellulase or chitinase.【Conclusion】The pathogen causing anthracnose on the sea buckthorn fruits in Chaoyang City, Liaoning Province, was identified as C. fructicola, and two strains of biocontrol bacterial strains, both belonging to the Bacillus spp., were screened out from the soil with good control effects. It was assumed that the preventive bacterial strains could degrade the cell wall of the pathogenic bacteria through the production of extracellular enzymes such as protease and β-glucanase, thus exerting preventive effects. This study would provide a basis for field identification of anthracnose on sea buckthorn fruits, and provide new strain resources with application potential for biological control of the disease.