In 2006, symptoms of brown blotch were observed on cultivated Pleurotus eryngii (king oyster mushroom) in Castilla-La Mancha (Spain). Subsequently, between January and May of 2008, brown blotch affected 39.75% of the blocks of substrate cultivated, resulting in a considerable loss of production. Symptoms observed were principally characterized by a yellowish brown-to-orangish color, first of the cap and then of the stalk. Some samples also showed a slightly concave cap. From samples collected from four different king oyster mushroom farms, a fluorescent gram-negative bacterium was recovered on King's B medium and identified as Pseudomonas tolaasii by the LOPAT scheme and other tests (2). The bacterium isolated had the following characteristics: oxidative, positive for oxidase and arginine dihydrolase, negative for levan production, pectinolitic activity on potato slices, and tobacco hypersensitivity. Results from other tests were as follows: negative for esculin hydrolysis and positive for gelatine, casein, and Tween 80 hydrolysis; mannitol, erythritol, sorbitol, m-inositol, and adonitol were used as a sole carbon source, but not sucrose, d-tartrate, or trigonelline. The white line test was performed (4) using P. reactans LPPA 540 and the presumptive isolates of P. tolaasii were positive. The gene encoding the 16S rRNA from two isolates (LPPA532 and LPPA533) was sequenced after PCR amplification (2) and their nucleotide sequences (1,400 bp; EMBL Accession No. FM864215 for LPPA 532) proved to be identical. The amplified sequences were compared with DNA sequences available in databases (GenBank, EMBL, DDBJ, and PDB) by using BLAST. An identity of 99% was obtained with 16S rDNA of three P. tolaasii strains (GenBank Accession Nos. AF320990, AF094750, and AF255336). Four isolates were selected for pathogenicity tests. Bacterial suspensions were grown for 16 h in yeast peptone glucose broth (approximately 108 CFU/ml) and were inoculated by puncture into 10 mushroom caps using sterilized wooden toothpicks (4). Sterilized distilled water was used as a control. These were then incubated at room temperature in glass dishes. Assays were conducted twice and the results were recorded after 10 days. The symptoms that developed after infection were similar to those observed in the crop, while the control mushrooms remained symptomless. Bacteria sharing the characteristics of the inoculated isolates were recovered from symptomatic caps. P. tolaasii has been described as causing brown blotch on Pleurotus eryngii (1,3), but to our knowledge, this is the first report of P. tolaasii causing brown blotch on Pleurotus eryngii in Spain.
References: (1) J. F. Bradbury. No. 891 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1987. (2) A. J. González et al. Appl. Environ. Microbiol. 69:2936, 2003. (3) A. Russo et al. Microbiol. Res. 158:265, 2003. (4) J. M. Wells et al. Phytopathology 86:1098, 1996.