First report of Burkholderia gladioli causing yellow spot disease on Pleurotus pulmonarius in China

Pleurotus pulmonarius is a commercially important edible mushroom widely cultivated in China. In July 2021, fruiting bodies of P. pulmonarius exhibiting yellow spot disease (with an incidence of 5%) were observed in a cultivation facility located in Xixiangtang District, Nanning, China. The initial symptoms appeared as yellowing at infection sites, then developed into sunken lesions with a diameter ranging from 1–3 mm, while fruiting bodies continued to grow without rotting. Infected cap tissues were surface-sterilized, homogenized in sterile deionized water (SDW), serially diluted, and plated on LB medium. After 24 h at 30°C, dominant ivory, round, convex, opaque colonies were purified, yielding five isolates. To identify the pathogen, three genes were amplified and sequenced: 16S rRNA gene (GenBank PV368322–PV368326) with 27F/1492R (Galkiewicz and Kellogg, 2008), the gyrB (OR756290, PV387103–PV387106) with UP-1E/APrU (Yamamoto et al. 2000), and the rpoB (OR756291, PV387108–PV 387111) with VIC4/VIC6 (Tayeb et al. 2008). BLASTn comparison indicated that isolate GXU5-1 shared 99.71% (1,385/1,389 bp), 98.75% (873/884 bp), and 99.39% (983/989 bp) similarity with the B. gladioli BBB-01 (CP068049.1), while detailed results for all five isolates are provided in Supplementary Table. A multi-locus phylogenetic analysis (MLSA) based on the concatenated sequences of the three genes (16S rRNA-gyrB-rpoB) grouped the isolates within B. gladioli clade. The isolates were Gram-negative rods without fluorescent pigment production on King’s B medium and were positive for catalase, oxidase, gelatinase, and urease activities, utilized glucose, sucrose, and arabinose; and were negative for salicin, rhamnose, lactose, arginine dihydrolase, and nitrate reductase. These characteristics are consistent with genus Burkholderia, including B. gladioli (Coenye et al., 1999). Combined with morphological and molecular analysis, the isolates were identified as B. gladioli. For pathogenicity test, P. pulmonarius was pre-cultivated in plastic bags until young fruiting bodies formed. A 2 μL aliquot of 2 McFarland suspension (approximately 6x10 8 CFU/mL) of B. gladioli was directly dropped onto the caps surface of three cultivation bags, with three additional bags treated with SDW as control. All treatments were incubated at 25°C and 80–85% relative humidity, and the experiment was repeated three times. No symptoms developed in SDW-treated controls; after 24 h, the pathogen was re-isolated from symptomatic inoculated caps and molecularly confirmed, fulfilling Koch’s postulates. B. gladioli was formally reported as a plant pathogen (Lu et al. 2007). In the mushroom industry, it has been confirmed to cause soft rot disease on edible mushrooms of Agaricus bisporus and P. ostreatus (Gill et al. 1997; Lee et al. 2010). However, the symptoms we observed differ markedly from these previously reported cases, possibly due to differences in host species. To our knowledge, this is the first report of B. gladioli causing yellow spot disease on P. pulmonarius in China, posing a threat to the mushroom’s commercial cultivation and providing a basis for disease management strategies.