Identification, host range and fungicide screening of the pathogen causing leaf spot on Malus‘Donald Wyman’

【Objective】Donald crabapple (Malus‘Donald Wyman’) has experienced widespread outbreaks of leaf spot disease within four consecutive years in Dalian region, resulting in substantial defoliation and plant mortality. This disease poses a serious threat to the cultivation, management, and ornamental value of Donald crabapple. The primary objective of this study was to identify the fungal pathogen responsible for the leaf spot disease. Furthermore, a series of chemical fungicide screenings were conducted to evaluate the efficacy of different fungicides against the pathogen, with the aim of providing a scientific basis for the effective prevention and control of Malus‘Donald Wyman’leaf spot disease.【Methods】The field sampling and pathogen isolation field surveys were conducted in multiple locations across Dalian city, Liaoning Province, China, including roadsides and scenic areas with concentrated plantings of Malus‘Donald Wyman’. Symptomatic leaves were collected during the summer and autumn seasons of 2023 and 2024 from Tongniuling and Daheishan scenic areas in Jinpu New District, Dalian city. The collected samples were placed in sterile plastic bags and transported to the laboratory for pathogen isolation. Leaf tissues with clear lesion boundaries were selected, and 5 mm × 5 mm sections were excised from the junction between diseased and healthy tissues. The samples were surfacesterilized by immersion in 75% ethanol for 30 seconds, followed by 3% sodium hypochlorite solution for 30 seconds, and subsequently rinsed 2-3 times with sterile distilled water. The sterilized tissues were placed into potato dextrose agar (PDA) medium and incubated at 25 ℃ in complete darkness for 5-7 days. The emerging fungal colonies were subcultured and purified through hyphal tip transfer. The purified isolates were stored at 4 ℃ for further use and were also maintained on PDA medium supplemented with streptomycin to suppress bacterial contamination. All fungal cultures were incubated at 25 ℃ in the dark until colonies were fully developed. Pathogenicity was confirmed through wound inoculation assays conducted under aseptic laboratory conditions. The purified fungal isolates were cultured on PDA plates at 26 ℃ in darkness for 7 days prior to inoculation. The healthy leaves were surface-cleaned with ultrapure water, wiped with 75% ethanol, and rinsed three times with sterile water before being dried using sterile tissue paper. Wounding was performed with sterile insect pins under a laminar flow hood. The mycelial plugs (8 mm in diameter) were excised from the colony margin using a sterile cork borer and placed on the wounded sites. The sterile PDA plugs served as negative controls. The inoculated leaves were incubated at 25 ℃ under dark conditions for 5 days. Each treatment was conducted with nine replicates, and symptom development was monitored and recorded throughout the incubation period. Preliminary identification of the isolated pathogen was conducted by observing the morphological characteristics of reproductive structures under a light microscope. For molecular identification, the genomic DNA was extracted from the purified isolates, and the internal transcribed spacer (ITS), actin (ACT), and large subunit (LSU) gene regions were amplified by polymerase chain reaction (PCR). PCR products were purified and sequenced by Shanghai Sangon Biotech Co., Ltd. The obtained sequences were analyzed and aligned using BioEdit software. A multi-locus phylogenetic tree was constructed using the maximum likelihood (ML) method implemented in RAxML software to confirm species identity. An in vitro mycelial growth inhibition assay was employed to evaluate the sensitivity of the isolated pathogen to various fungicides. Eleven commercial fungicides were tested, formulated as suspension concentrates, aqueous solutions, emulsifiable concentrates, wettable powders, microemulsions, and water-dispersible granules. The mycelial discs were inoculated onto PDA plates amended with the respective fungicides at designated concentrations and incubated at 25 ℃ in darkness. The colony diameters were measured after incubation to assess fungal growth inhibition.【Results】The ITS, ACT, and LSU gene fragments of strain SH0019 were amplified and sequenced, yielding fragments of 542 bp, 218 bp, and 1291 bp in length, respectively. BLAST analysis against the GenBank database revealed that the nucleotide sequence identities of the rDNA ITS, ACT, and SSU regions of strain SH0019 with Alternaria alternata were 99.81%, 99.04%, and 100%, respectively. A concatenated multi-locus phylogenetic tree was constructed using the maximum likelihood (ML) method implemented in RAxML, based on the GTR+ nucleotide substitution model. The phylogenetic analysis showed that strain SH0019 clustered together with A. alternata. Combined with the morphological characteristics and molecular data, the strain SH0019 was identified as A. alternata. The artificial inoculation experiments were conducted on seven test plant species. The pathogenicity determination results of this pathogen on the following seven Malus plants (Malus‘Indian Magic’, Malus‘Radiant’, Malus‘Flame’, Malus micromalus, Malus halliana, Malus baccata, Malus mandshurica) showed that A. alternata could infect five host plants. It had the strongest pathogenicity to Malus‘Donald Wyman’and the weakest effect to Malus halliana, while it did not obviously infected Malus baccata and Malus mandshurica. Although strain the SH0019 exhibited pathogenicity across different Malus species, the lesion size varied among the species, with the smallest average lesion diameter (4.3 mm) observed on Malus halliana, indicating differences in host resistance levels. The fungicide sensitivity tests were conducted using 11 fungicides. The mycelial growth inhibition assay showed that among the single agents tested, iprodione and prochloraz exhibited the strongest inhibitory effects against the strain SH0019, with EC50 values of 1.735 mg · L- 1 and 5.495 mg·L-1 , respectively, which were significantly lower than those of the other fungicides. In contrast, mancozeb demonstrated the weakest activity, with an EC50 of 222.923 mg · L- 1 . Further screening of fungicide combinations revealed that the 50% benzyl·difenoconazole formulation exhibited the highest inhibitory activity, with an inhibition rate of 71.65% and an EC50 value of 0.179 mg·L-1 . The inhibitory effect of 50% benzyl· difenoconazole was superior to that of 43% tebuconazole and 25% difenoconazole single formulations.【Conclusion】The causal agent for the leaf spot of Malus‘Donald Wyman’was identified as Alternaria alternata based on Koch’s postulates, morphological characteristics, and multi-locus sequence analysis of ITS, ACT, and SSU genes. Among the tested fungicides, benzyl · difenoconazole demonstrated the highest antifungal activity and may serve as an effective candidate for controlling the disease although the field trials should be conducted to further testify the efficiency of the chemicals before practical application.