Identification of the pathogen causing gray mold and comparative analysis of physiological resistance among different germplasm resources in blueberry

【Objective】Blueberry gray mold, caused by Botrytis cinerea, is a serious disease in blueberry production. Under greenhouse cultivation conditions, high humidity, poor ventilation, and insufficient resistance in cultivars exacerbate the prevalence of the disease, more prevalent and severe, leading to significant economic losses. Currently, control of gray mold relies primarily on chemical agents, but this can lead to the development of resistance in the pathogen, complicate fruit quality management, and pose potential environmental risks. Therefore, breeding disease- resistant blueberry germplasm is the most cost-effective approach to disease control. This study aimed to identify the pathogen of blueberry gray mold and screen out highly pathogenic representative strains. Furthermore, the study sought to identify high-quality disease-resistant germplasm and explore the differences in its physiological re-sponses during infection.【Methods】Eureka blueberry plants displaying typical gray mold symptoms were collected from three blueberry cultivation bases in Yunnan, Changchun, and Dandong, and disease symptoms were observed. The pathogen was isolated, purified, and cultured using tissue isolation method. Fungal genomic DNA from single colonies was extracted using a modified CTAB method, and PCR reactions were performed using universal fungal primers ITS1/ITS4. After purification and sequencing, sequences were aligned using DNAMAN software and NCBI database BLAST analysis. A phylogenetic tree was constructed in MEGA 12.0 with the NJ method. Based on the clustering results, molecular identification of the isolated strains was performed to confirm their taxonomic status. Hyphal morphology of the selected gray mold strains based on molecular identification was observed using conventional lactophenol cotton blue staining. Healthy intact leaves from six blueberry cultivars were inoculated in vitro, and the pathogenicity of the strains was assessed by comparative analysis four days after infection. Representative highly pathogenic gray mold strains were selected and evaluated for resistance to the disease in common evergreen blueberry cultivars grown in greenhouses using the same in vitro inoculation method. Leaf lesion area was quantitatively analyzed using ImageJ software to compare resistance differences among different blueberry cultivars. Resistance was graded based on the percentage of lesion area, allowing for the identification of representative resistant and susceptible cultivars. References to relevant literature on plant physiology and pathology were used to determine cell wall enzyme activity indices associated with pathogen infection response. Dynamic changes in the activities of these enzymes in resistant and susceptible varieties under infection with B. cinerea were compared to further assess the relationship between cell wall physiological responses and disease resistance.【Results】Based on diseased samples collected, gray mold was found to damage flowers, stems, leaves, and fruit of blueberries, with the flowers being more severely affected. Seven pathogen strains were isolated and purified using tissue separation methods and designated YN1, YN2, YN3, CC1, CC2, CC3, and DL1. Following PCR amplification of ITS sequences, strains were aligned in the NCBI sequence library and a phylogenetic tree was constructed. Strains CC1, YN1, DL1, and YN3 were identified as members of the genus Botrytis and clustered closely with several known reference strains of B. cinerea. Strains CC2, YN2, and CC3, on the other hand, belong to the genus Cladosporium and are more distantly related to Botrytis. Morphological comparison of four gray mold strains following molecular identification revealed CC1 to be the fastest growing, with dense and robust hyphae, white tomentose colony, and botryose conidiophores. Pathogenicity assays revealed significant differences among the strains, with CC1 exhibiting more pronounced tissue destruction and possessing stronger infection and spread capabilities than the other strains, making it a representative inoculum strain. An in vitro inoculation method was used to evaluate the resistance of 13 common greenhouse blueberry cultivars. Based on lesion area statistics and a resistance grading standard, no blueberry germplasm was found to be completely immune to gray mold. However, the moderately resistant cultivar Y42 and the susceptible cultivar F6 were identified, with significant differences in resistance (P＜0.05), making them representative cultivars for subsequent physiological response studies. Changes in cell wall-related enzyme activities during gray mold infection revealed distinct differences in the response mechanisms of the two cultivars. Polygalacturonase (PMG) activity in F6 peaked at 48-72 h post-inoculation and was significantly higher than that in the control (P＜0.05), indicating that the pathogen induced a strong response in cell wall-degrading enzymes, promoting tissue softening and disease spread. Y42 activity remained low, close to that of the control, suggesting that it inhibited cell wall decomposition and enhanced structural defense. Carboxymethycellolose (CMC) activity decreased in both varieties after inoculation, but was higher in F6, re-flecting more severe cell wall damage. β-1,3-glucanase (GLU) activity in F6 initially declined rapidly before significantly increasing at 48-72 h (P＜0.05), demonstrating a typical delayed response. Y42 increased at 24-48 h, suggesting that it may effectively inhibit pathogen spread early on. Chitinase (CHI) activity decreased rapidly in both varieties, with the greater decrease in Y42 suggesting that their defenses do not rely on sustained CHI accumulation and may limit disease through early physical barriers and immune responses. Overall, while F6 had high chitinase activity, it was lower than the control, in contrast to Y42, indicating weaker defense and disease resistance.【Conclusion】This study identified the pathogen of blueberry gray mold using morphological and molecular biological methods and screened CC1 as a highly pathogenic representative strain. F6 (susceptible) and Y42 (moderately resistant) were identified as typical materials for blueberry resistance to gray mold. The different physiological response characteristics revealed the differences in the defense mechanisms of blueberries under gray mold stress, providing an applicable material basis for the identification of blueberry gray mold resistance and the utilization of resistant germplasm. It also provides clearer research targets for further analysis of the resistance mechanism under gray mold stress and for the breeding of resistant varieties.