Pathogen identification of pear canker and screening of biocontrol bacterial strains

【Objective】Pear diseases pose a substantial threat to the pear industry in China. The use of traditional chemical pesticides has led to increasingly prominent environmental pollution and resistance problems. Therefore, there is an urgent need to develop environmentally friendly disease control methods. In 2024, a newly emerged bark canker disease was observed on pear trees in Harbin City. Infected trees developed water-soaked and swollen lesions on the bark of 1-3-year-old branches, typically surrounded by a halo. As the disease progressed, multiple lesions formed on a single branch, eventually darkening, cracking open, and producing distinct black pycnidia. The bark desiccated, and in severe cases, branches fractured, severely impairing tree growth and productivity. This study aimed to identify the causal pathogen of this emerging disease and to screen for effective biocontrol bacterial strains, thereby providing a scientific basis for its prevention and management.【Methods】Symptomatic branches were surface-sterilized, and single-spore isolation was used to obtain pure fungal cultures. The pathogenicity of the isolate was tested using Koch’s postulates on healthy pear twigs cultivated hydroponically, applying non-wounded, wounded, and heat-injured inoculation methods. Pathogen identification was based on both morphological characteristics and multi-locus phylogenetic analysis using ITS, LSU, and Tef-1α gene sequences. Endophytic bacteria were isolated from healthy pear branches using tissue separation. Candidate biocontrol strains were screened through dual culture and co-culture assays with the patho-genic isolate. The most effective antagonistic strain was further characterized based on morphological, physiological, and biochemical properties, and identified using phylogenetic analysis of 16S rRNA and gyrB gene sequences.【Results】(1) A fungal isolate designated LA-1 was obtained and found to reproduce field symptoms under wounded and heat-injured inoculations. These symptoms included water-soaked swelling and black pycnidia formation. The same fungus was re-isolated from symptomatic tissues and showed identical morphological and molecular characteristics to the original LA-1 isolate. ITS sequencing confirmed 100% identity with LA- 1. (2) Morphological observations revealed that LA-1 formed immersed to semi-immersed, discoid, and dark brown to black pycnidia, measuring 650 to 950 μm in diameter (mean: 740 μm, n=30). Pycnidia were multi-locular with shared walls, and individual locules measured 40 to 120 μm (mean: 75 μm, n=30). Conidiophores were hyaline, clavate, occasionally swollen, and 0.75 to 2.25 μm wide (mean: 1.5 μm, n=30). Conidia were unicellular and oval to cylindrical, containing oil droplets, and displayed a frontal central depression. They transitioned from hyaline to dark brown upon maturation and measured (15.5-) 17.0 to 20.5 (-22.5) μm × (6.0-) 6.5 to 8.0 (-8.5) μm, averaging 18.5 μm × 7.0 μm (n=50). Colonies on PDA were initially white and floccose, later producing olive pigment and forming pycnidia after 13 days of incubation in the dark. These features were consistent with descriptions of Aplosporella ginkgonis. (3) Gene sequences obtained included 603 bp (ITS), 1341 bp (LSU), and 170 bp (Tef-1α). BLAST analysis revealed that the ITS and Tef-1α sequences of the isolate exhibited ＞99% similarity to those of A. ginkgonis and A. longipes in the NCBI database. However, LSU comparisons were inconclusive due to length variation. To resolve species identity, maximum likelihood and Bayesian phylogenetic trees were constructed using the K80+R2+FO and K80+I models via IQ-TREE and MrBayes 3.2.6, respectively. Both analyses yielded identical topologies, with LA-1 clustering with A. ginkgonis with full statistical support (BS/PP = 100/1). Notably, several strains formerly designated as A. longipes were also nested within the A. ginkgonis clade. Based on morphological traits, multi- gene phylogeny, and ecological context, LA-1 was conclusively identified as A. ginkgonis. (4) Furthermore, eleven bacterial strains were isolated from healthy pear branches. Dual culture and co-culture screening identified strain B10 as exhibiting the strongest antagonistic activity against LA-1. Colonies of B10 were milky white and opaque, with wrinkled surfaces and irregular edges on LB agar. Biochemical tests confirmed it was a Gram- positive bacterium. Phylogenetic analysis based on 16S rRNA and gyrB sequences identified B10 as Bacillus velezensis. Importantly, B10 produced heat-stable antimicrobial metabolites; even after autoclaving at 121 ℃, its fermentation broth retained strong inhibitory effects on LA-1. Additionally, B10 suppressed the growth of other wood-infecting fungi, including Cytospora, Phaeobotryon, Diaporthe, and Phomopsis species.【Conclusion】A. ginkgonis was identified as the causal agent of a newly emerged bark canker disease on pear trees in Harbin, marking the first report of this pathogen affecting pear in China. Moreover, the identification of Bacillus velezensis B10 as an effective biocontrol agent offers a promising alternative to chemical fungicides. Its broad- spectrum activity and thermal stability highlight its potential for integrated management of wood-infecting pathogens in pear orchards.