Abstract：【Objective】 Kiwifruit canker is a severe bacterial disease that poses significant threat to the kiwifruit industry casusing by Pseudomonas syringae pv. actinidiae (Psa). This disease affects various parts of kiwifruit, including the trunk, leaves, flowers, and roots. Among these, the impact on the flowers is particularly critical, as it directly leads to yield loss in the affected year. Psa can damage the buds, petals, and peels, causing the buds fail to bloom, turn brown, till fall off. Flower organs provide a conducive environment for microbial colonization. Consequently, microorganisms residing inside or on the surface of flower play a protective role against pathogen infection. Numerous studies have shown that environmental factors influence microbial community composition of plants. Among these factors, external pathogen invasion acts as the major selection pressure, significantly affecting floral microbial community structure. This study aims to investigate changes in microbial diversity and community structure of floral bacteria and fungi under varying canker disease severity using high-throughput sequencing. The results laid a theoretical foundation for understanding the interaction mechanisms between kiwifruit and Psa, as well as for developing biological control strategies for this disease. 【Methods】In April May 2022, during the flowering period of the 'Donghong' kiwifruit, which is also a high-infected period for kiwifruit canker disease, samples were collected from kiwifruit experimental ochrard in Xijiadian Town, Danjiangkou City, Hubei Province. Three disease severity levels were set: healthy, moderate, and severe, respectively. High-throughput sequencing technology was employed to analyze the diversity and structure of bacterial and fungal microflora in these samples. Specifically, 16S rRNA and ITS1 gene sequencing were used to study bacterial and fungal communities, respectively. 【Results】A total of 584,580 high-quality 16S rRNA sequences and 520,169 high-quality ITS1 sequences were obtained after quality control. Clustering of these sequences revealed 3,410 bacterial operational taxonomic units (OTUs) and 12,986 fungal OTUs. Among the samples with three different disease severities, there were 37 common bacterial OTUs and 383 common fungal OTUs. The proportion of unique bacterial OTUs was 37.5% in healthy samples, 27.0% in moderately diseased samples, and 18.6% in severely diseased samples. For fungi, the unique OTUs accounted for 18.3%, 25.6%, and 19.4%, respectively. Species classification analysis identified as 27 phyla, 55 classes, 134 orders, and 210 families in the bacterial community, as well as 16 phyla, 52 classes, 118 orders and 252 families in the fungal community. As disease severity increased, the number of bacterial taxa at each classification level decreased. Conversely, the number of fungal OTUs initially increased and then decreased, which is consistent with the trend of OTUs quantity changes in samples with different degrees of disease incidence. Both the diversity index and the number of OTUs decreased as disease severity increased. Significant differences in bacterial diversity indices were observed among the different disease severities (P ＜ 0.05). The diversity index and the number of OTUs initially increased with moderate disease severity then decreasing with severe disease severity. Notably, the fungal diversity index in severely diseased samples was slightly higher than in healthy samples. Fungal diversity differed significantly between moderately diseased samples and other levels (healthy and severe) (P ＜ 0.05). In the bacterial community, samples with the same disease severity clustered closely, indicating good repeatability. Samples with different disease severities were significantly separated, suggesting distinct differences in bacterial microbial communities (P ＜ 0.05). In the fungal community, healthy samples were significantly separated from infected samples (P ＜ 0.05). Among diseased samples, those with moderate and severe disease severity showed some overlap but also distinct differences, indicating similarities and differences in their microbial communities. Among the samples of different severity, the dominant bacteria genera were Pseudomonas and Cyanobacteria; the dominant fungal genera are Mortierella, Alternaria, Cladosporium and Fusarium. The relative abundances of Pseudomonas, Cladosporium, Alternaria, and Filobasidium increased with disease severity, while the relative abundances of Cyanobacteria and Mortierella decreased. Significant differences in the relative abundances of Pseudomonas, Alternaria, and Mortierella were observed among different samples (P ＜ 0.05). 【Conclusion】The findings of this study reveal significant changes in the microbial community structure of kiwifruit flowers in response to Psa invasion. The diversity and abundance of specific bacterial and fungal taxa were markedly influenced by the severity of the disease. The reduction in bacterial diversity and the initial increase followed by a decrease in fungal diversity suggested that disease severity exerts selective pressure on microbial communities. This pressure beneficial for the proliferation of certain pathogenic microorganisms while inhibiting others. The increased relative abundances of Pseudomonas, Cladosporium, Alternaria, and Filobasidium in severely diseased samples highlight their potential roles in the disease progression. The significant differences in microbial communities between healthy and diseased samples underscore the potential for utilizing microbial indicators as diagnostic tools for early disease detection.
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