Identification of the pathogen causing kiwifruit soft rot and the control effect of fungicides

【Objective】Kiwifruit (Actinidia chinensis) is a deciduous species of the Actinidiaceae family. Zhongmi 2, a mid-maturing cultivar, exhibits high yield potential but is highly susceptible to soft rot disease in postharvest storage. This study aimed to identify the causal pathogens of soft rot in Zhongmi 2 kiwifruit, and to evaluate the efficacy of fungicides against the disease, for providing a reference for its effective management.【Methods】Firstly, we investigated the occurrence situation and observed the symptoms of kiwifruit soft rot in the field. Then, the pathogens were isolated by the tissue isolation method. They were disinfected with 0.5% NaClO for 1 min. Small pieces of the diseased tissue edge were placed on potato dextrose agar (PDA) (200 g potato extracts L-1 , 2% glucose, and 2% agar) medium. After culturing for 5 days at 25 ℃, a mycelial plug was collected from the growing edge of each colony and transferred to a new PDA plate followed by incubation at 25 ℃ for 7 days. Emerging colonies were transferred several times by the hyphal tip method until and pure cultures were obtained. The morphological characteristics of the isolates were observed, and the size of conidia was measured to complete the morphological identification. Genomic DNA extraction kit was used to extract the DNA of the pathogenic fungi. The rDNA internal transcribed spacer (ITS), elongation factor 1-α (EF-1α) and beta-tubulin (TUB) were amplified and sequenced. The obtained multi-gene splicing sequence was spliced according to the order of ITS-EF-1α-TUB, and the phylogenetic tree was constructed using the neighbor-joining method in MEGA 11.0 software for molecular biological identification. And the pathogenicity of the isolates was tested based on Koch's postulates. The selected isolates were screened for their pathogenicity on kiwifruits according to their morphological and molecular data. The pathogenicity of the isolates was assessed by growing these isolates on kiwifruit fruit under laboratory conditions. Briefly, kiwifruit were washed two times using sterile dH2O and dried in a transfer hood. From each isolate, mycelial plugs with a diameter of 5 mm were obtained from the edges of 5-day-old colonies and added to the surface of each kiwifruit, with control fruit being inoculated using fungi-free PDA. Three replicates each composing 10 fruit were prepared for each treatment. Following the inoculation, fruit were transferred to a sterilized plastic box and were incubated at 25 ℃ . After the typical symptoms were formed, the diseased parts were separated again. Finally, thirteen fungicides were selected for field trials on the prevention and control of soft rot in Zhongmi 2 kiwifruit in 2023 and 2024. The fruit were collected from each treatment and stored at room temperature, and the disease incidence and control efficiencies were calculated after 15 and 30 days of storage.【Results】A total of 79 isolates were obtained from 82 diseased samples. GroupⅠ included 44 isolates that showed cultural and conidial morphology typical of Diaporthe sp. on PDA plates. They formed white colonies with thin mycelia and produced two types of conidia. The α- conidia were fusiform, hyaline, and single- celled, with lengths ranging from 5.6 to 8.7 μm and widths from 2.4 to 3.1 μm. The β-conidia were filiform, hyaline, and singlecelled, with lengths ranging from 18.7 to 33. 6 μm and widths from 1.1 to 2.1 μm. Group Ⅱ included 35 isolates that displayed cultural and conidial morphology typical of Botryosphaeria sp. on PDA plates. They formed white to gray colonies that developed moderate aerial hyphae, and conidia were fusiform, hyaline, and aseptate, with lengths ranging from17.3 to 27.6 μm and widths from 5.3 to 9.1 μm. Combined with the morphological characteristics of the pathogen and the phylogenetic analysis based on ITS, EF-1α and TUB gene sequences, two species were identified as the pathogens causing kiwifruit soft rot in the study. They were Diaporthe eres and Botryosphaeria dothidea. Pathogenicity tests confirmed that re-isolation from inoculated symptomatic tissues yielded strains identical to the original inoculants. In 2023 field trials, five fungicides (10% difenoconazole water dispersible granules (WG), 75% trifloxystrobin · tebuconazole WG, 25% pyraclostrobin suspension concentrate (SC), 40% flusilazole emulsifiable concentrate (EC) and 43% tebuconazole SC) showed control effect exceeding 85% after 15 days of storage and maintained efficacy above 75% after 30 days of storage. Similar results were observed in 2024: under non-individually packaged storage, these five fungicides achieved control effect of ＞84% at 15 days and ＞74% at 30 days. However, when kiwifruits were stored with individual packaging, the effect improved to ＞87% at 15 days and ＞80% at 30 days, demonstrating slightly superior performance compared to non- packaged storage.【Conclusion】The study indicated that the pathogens causing kiwifruit soft rot were identified as D. eres and B. dothidea. Five fungicides (10% difenoconazole WG, 75% trifloxystrobin ·tebuconazole WG, 25% pyraclostrobin SC, 40% flusilazole EC and 43% tebuconazole SC) exhibited significant field control effect. Furthermore, individual packaging during storage enhanced fungicide performance, suggesting its potential as a complementary strategy for postharvest disease management. These findings provide critical insights for optimizing integrated control measures against kiwifruit soft rot.