First Report of <i>Pseudomonas extremorientalis</i> Causing Brown Blotch Disease on White Button Mushroom ( <i>Agaricus bisporus</i> ) in China

Agaricus bisporus is one of the most widely cultivated edible fungi globally. From 2020 to 2022, brown blotch lesions (2% incidence) were observed on A. bisporus at a mushroom farm in Guangxi, China. Initially, these brown blotches (0.8-1.5 cm) appeared on the cap. As the fruiting bodies grew, brown blotches expanded, but no putrefaction occurred. To isolate the pathogen, samples (5×5×5 mm) of brown blotches were surface sterilized with 75% ethanol for 30 s, rinsed three times with sterile deionized water (SDW), and then homogenized in sterile 2 mL Eppendorf tubes. After adding 1000 µL of SDW, the suspension was serially diluted from 10⁻¹ to 10⁻⁷. For each dilution, 120 µL was spread onto Luria Bertani (LB) agar plates and incubated at 28°C for 24 h. Morphological observation of the isolates was performed following the method described by Liu et al (2022). Dominant single colonies were round, pale yellow with slight whitish hues, convex, and gram-negative. On King’s medium, yellow fluorescence was observed under UV light. The bacterial cells were rod-shaped (1.0-2.0×0.6-1.2 µm, n=20), possessed a single polar flagellum, and lacked endospores. Seven randomly selected strains were subjected to genomic DNA extraction and amplification of target genes using the method described by Huang et al. (2023). The amplified genes included 16S rRNA (PV670024-PV670030), type I glyceraldehyde 3-phosphate dehydrogenase (gapdh, PV682273-PV682279), citrate synthase (gltA, PV682280-PV682286), DNA topoisomerase subunit B (gyrB, PV682287-PV682293), recombinase A (recA, PV682294-PV682300), and RNA polymerase subunit beta gene (rpoB, PV682301-PV682307). All genes showed over 99% sequence homology with Pseudomonas extremorientalis, and phylogenetic analysis of concatenated multi-gene sequences clustered the isolates with P. extremorientalis LMG 19695. Biochemical tests were performed on three representative isolates using bacterial micro-biochemical reaction tubes. Biochemical tests yielded results consistent with P. extremorientalis (Ivanova et al. 2002): positive for arginine dihydrolase, citrate utilization, and reduction of nitrate; negative for glucose, lactose, and starch. The dominant isolate was identified as P. extremorientalis based on morphological characteristics, biochemical profiles, and phylogenetic analysis. For pathogenicity testing, bacteria were cultured in LB broth at 160 rpm and 28°C for 24 hours, then harvested by centrifugation (4000 rpm, 10 min) and resuspended in SDW to approximately 1×10⁹ CFU/mL. 30 µL of the cell suspension was inoculated onto the cap and tissue of A. bisporus, with equal volumes of SDW as negative controls. All treatments were incubated at 20°C and 80-85% humidity. The experiment consisted of three replicates, with five caps and five tissues in each. At 48 hours post-inoculation, brown blotches developed at the inoculation sites on both the cap and the tissue. Blotches on caps were regular in shape, while those on tissues showed diffusive browning. No lesions appeared in SDW-inoculated controls. Pathogens were successfully re-isolated from inoculated sites based on morphological observations and 16S rRNA sequencing, fulfilling Koch’s postulates. This is the first report of P. extremorientalis causing brown blotch disease in A. bisporus. This research enriches the understanding of A. bisporus brown blotch pathogen diversity and provides support for early disease detection and management.