Isolation and identification of Athelia bombacina causing postharvest fruit rot of Honeycrisp apple in Xinjiang

Abstract:【Objective】The apple postharvest disease seriously affect the development of apple industry. In 2021, our research team found that dark brown spots occurred on the stored Honeycrisp apples in the cold storage of Shuanghe city, Xinjiang Uygur Autonomous Region (XUAR), and the fruit damage rate reached 50%. This study identified the pathogenic bacterium which would cause brown spots on Honeycrisp apples.【Methods】The purified strains were obtained by single spore isolation, and were inoculated on apple fruits in vitro to identify the pathogenicity. The classification status of strains was determined by morphological characteristics and molecular technology. According to Koch's postulates, the fruits were inoculated through wound inoculation and no wound inoculation and were placed in a sterile fresh-keeping box. The inoculum was removed 24 h after inoculation and the lesion diameter was used to measure and record by the vernier caliper from the 3rd d to the 18th d. The strains were reisolated after the same lesions appeared, then we observed and recorded the morphology, color, growth rate and spore production of colonies on the second day. The mycelial diameter, spore morphology and size were observed and recorded under the optical microscope to clarify its morphological characteristics. The Ezup column fungal genome DNA extraction kit was used to extract DNA. The ITS1 and ITS4 were used to amplify the internal transcribed spacer (ITS) sequence, the NL1 and NL4 were used to amplify the ribosomal large subunit (LSU). The products were detected by 1% agarose gel electrophoresis. The primers and products were sequenced by Shanghai Sangon Bioengineering Co., Ltd (Shanghai, China).The sequences were spliced with seqman v.7.1.0 software and blast alignment. we constructed phylogenetic trees by Maximum parsimony (MP), Maximum likelihood (ML) and Bayesian (BI) using combined polygenic fragments. The bootstrap support rate of MP and ML were greater than or equal to 50%, and BI was greater than or equal to 90%. Finally, the phylogenetic tree was used to clarify the taxonomic status of pathogens.【Results】A total of 27 strains were isolated from the 45 diseased fruit tissue blocks with typical lesions. Among them, 14 strains were white colonies, and the highest isolation rate was 31.11%. There were 13 other strains, including 7 bacteria, 3 Alternaria fungi and 3 unknown fungi. The isolation rates of the strains with white colonies were 20%, 46.67% and 26.67% respectively when the disinfection time was 3, 5 and 7 min. After inoculating the strain into the fruit, the fruits of the control and no injury treatment had no disease. After 48 hours of treatment, yellowish brown micro round spots were formed. The hyphae penetrated and adhered to the fruit surface. When the culture time was extended to 15 days, the lesion color changed from yellowish brown in the initial stage to yellowish brown in the middle and dark brown at the edge. The average expansion rate of the diseased spots was 0.63 mm·d^-1. The basidiospores and colonies basically consistent with the morphological characteristics of the original inoculated strain XJAU- PG-1 could be obtained from the inoculated diseased apple spots. Therefore, XJAU-PG-1 was further determined to be the pathogenic strain of apricot rot of Honeycrisp apple. The colony growth rate of strain XJAU-PG-1 was 15 mm· d-1 at 25 ℃ on PDA medium. The colony was always white and expanded radially and the initial hyphae were loose and thick. The basidiospores are oval or pear shaped, with slightly sharp and slightly skewed base, colorless and transparent, size (2.4-3.6) μm×(1.7-2.4) μm. The phylogenetic analysis of polygenic sequences included 26 sequences, including 1590 total characters, 1135 constant characters, 82 variable characters and 373 parsimony-informative characters. The heuristic searched bootstrap method constructed an MP tree, TL = 896, CI = 0.762, RI = 0.902, RC = 0.688, HI = 0.238 for analysis. The ML tree and BI tree were consistent with the MP tree topology. The phylogenetic tree showed that strains XJAU-PG-1, XJAU-PG-2 and A. bombacina were clustered together with a support rate of 100/100/100. Combined with the morphological characteristics, strains XJAU- PG- 1 and XJAU- PG- 2 were identified as Athelia bombacina. 【Conclusion】Strain XJAU- PG- 1 was isolated from the postharvest diseased Honeycrisp apple fruits. According to the morphological characteristics and the comprehensive analysis of molecular phylogeny of multi gene (ITS, LSU) fragments, strains XJAU-PG-1 and XJAU-PG-2 were identified as A. bombacina. Through the determination of its pathogenicity and the verification of Koch’s rule, A. bombacina was determined to be the pathogen of apple fruit rot. This is the first time to report that A. bombacina was the pathogen of apricot rot occurred on Honeycrisp apple during storage.