Several pseudomonads, associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic.

Pseudomonas tolaasii, causing brown blotch disease on cultivated mushrooms, and yielding a white line precipitate towards P. "reactans", has been shown to induce lysis of erythrocytes. Some Finnish strains isolated from diseased mushroom fruit bodies, although harboring the typical features of P. tolaasii, proved to be distinct, and have been allocated to a nov. sp. P. costantinii. We examined in these study whether all brown blotch causing agents were hemolytic. The induction of erythrocytes lysis seemed to be a rather common feature of mushroom associated-pseudomonads, especially for strains involved in the production of a white-line-in agar.

Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii, cause of brown blotch disease of the cultivated mushroom Agaricus bisporus.

Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized. Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329. These 16 isolates of P. tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates. Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping. Ribotyping differentiated P. tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes. A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P. tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2). Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T. Sequence determination and analysis of the internally transcribed spacer region ITSI for P. tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars. It is concluded that considerable genotypic differences exist among Finnish isolates of P. tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.

The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans".

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.

Pseudomonas costantinii sp. nov., another causal agent of brown blotch disease, isolated from cultivated mushroom sporophores in Finland.

Pathogenic bacteria are frequently associated with mushroom sporophores exhibiting brown blotch disease symptoms. These bacteria belong mainly to Pseudomonas tolaasii or occasionally to 'Pseudomonas reactans'. Although a group of isolates originating from some Finnish mushroom farms satisfied the two characteristic criteria for diagnosis of infection with P. tolaasii (i.e. yielding a typical brown blotch symptom on Agaricus bisporus sporophores and producing a typical white line in agar when streaked towards the 'P. reactans' LMG 5329 inducing strain), results based on numerical taxonomy, siderotyping, DNA-DNA hybridizations and 16S rDNA phylogenetic analyses supported the view that these isolates constituted a novel species within the genus Pseudomonas, Pseudomonas costantinii. The type strain is PS 3a(T) (= CFBP 5705(T) = HAMBI 2444(T)).