First Report of Brown Blotch Disease Caused by <i>Pseudomonas tolaasii</i> in Cultivated <i>Pleurotus tuoliensis</i> in China

Pleurotus tuoliensis (Bailinggu) is a precious edible mushroom native to Iran and the northwest of China (Zhao M et al. 2016). In 2019, symptoms of brown blotch disease were observed on cultivated P. tuoliensis in Urumqi (43.46° N, 87.40° E), Xinjiang, China. Subsequently, a disease survey conducted across different mushroom houses from 2019 to 2024 revealed disease incidence rates ranging from 35% to 80%. The symptoms were initially characterized by brown-to-yellowish brown on the surface of P. tuoliensis caps. Later, symptoms appeared as small and water-soaked lesions that gradually expanded to cover the entire mushroom (Fig. 1). The infected tissues decayed and emitted a pronounced odor, significantly reducing the quality and yield of P. tuoliensis. Ten fruiting bodies of Bailinggu at different stages of disease were collected from five mushroom houses. Tissue (5 × 5 mm) from lesion margins was surface-sterilized in 75% ethanol for 30 s, rinsed three times in sterile water, and macerated (Liu et al. 2022). Five bacterial isolates were obtained. After incubation at 28℃ for 48 h, colonies were round, convex, smooth, with entire margins, and milky white on Luria-Bertani (LB) medium (Fig. 2). Gram staining revealed that the isolated strain was Gram-negative (Fig. 3). Electron microscopy images showed that the cells were short rod-shaped (0.5 to 0.9 × 1.5 to 2.3 μm) and motile, with one to two polar inserted flagella (Fig. 4). The 16S rRNA gene of five isolates (XJ2-3, XJ2-10, XJ4-6, XJ4-11, and XJ7-2) was amplified using universal primers 27F/1492R (Heuer et al. 1997) and sequenced (GenBank accessions MW532693 to MW532697). BLAST analysis revealed 99.44% to 99.72% identity with the Pseudomonas tolaasii type strain (AF255336). Maximum-likelihood phylogenetic analysis clustered the isolates in a well-supported clade with the P. tolaasii type strain, distinct from other Pseudomonas species (Fig. 5). This analysis included key references for context: the P. tolaasii type strain CGMCC 1.2960 (= ATCC 33618 = LMG 2342)(Osdaghi et al. 2019), our pathogenic Koch's postulate-fulfilling re-isolate R36, and two non-pathogenic isolates (24c, 62b) from symptomatic tissue. Furthermore, the identity was confirmed as P. tolaasii by a nested PCR assay targeting the tolaasin biosynthetic gene. The first-round amplification with primers Pt-1A/Pt-1D1 yielded a 449 bp fragment, followed by a second round with the inner primer pair Pt-PM/Pt-QM, which produced a specific 249 bp amplicon (Lee H I et al. 2002) (Fig. 6). Finally, pathogenicity was confirmed via Koch's postulates. A bacterial suspension in sterile water (108 cfu/mL) was applied on P. tuoliensis caps by needling and spraying. Sterile water was used as control. All inoculated caps developed symptoms similar to those observed in the field, while controls remained asymptomatic. The pathogen was re-isolated from symptomatic tissue (Fig. 7). P. tolaasii causes brown blotch on many mushrooms, including Agaricus bisporus, Pleurotus ostreatus, Flammulina filiformis, and Cordyceps militaris, and so on (Azu Okorley et al. 2019; Han et al. 2012; Liu et al. 2022). To our knowledge, this is the first report of P. tolaasii infecting P. tuoliensis in China. This finding extends the known host range of P. tolaasii and highlights the pathogen's adaptability, providing an essential basis for developing targeted management strategies.