Pore-Forming Peptide Toxins Obtained from Various Pseudomonas Sp. Causing Brown Blotch Disease

Abstract

Brown blotch disease is one of the most serious diseases on many cultivated mushrooms, such as oyster mushrooms, Agaricus bisporus, and Flammulina velutipe. The pathogen, identified as Pseudomonas tolaasii, secretes a lipodepsipeptide toxin, tolaasin. It is a pore-forming peptide toxin and causes the disease by making pores on the membranes and disrupting the cellular and fruiting body structure of mushrooms. Forty-three bacteria were isolated from the cultivated mushrooms tissues of the farms reported the outbreaks of brown blotch disease and identified by the sequence comparison of 16S rRNA genes. Five different species of Pseudomonas, P1-P5 subgroups, were identified. The P1 group bacteria, including 23 strains, have been identified as the main pathogen secreting tolaasin peptide. P2-P5 groups are other than P. tolaasii and they also cause brown blotches in pitting and cultivation tests of oyster mushroom. In the pitting test using mushroom caps, all five subtypes were able to form brown blotches. These results suggest that subgroups P2-P5 also secrete tolaasin-like peptides causing brown blotches. In order to characterize various pore-forming properties of these peptides, the effects of temperature on the toxicities of these peptide toxins were measured. The hemolytic activities of toxins from P1 to P5 subgroups were decreased by increasing temperature from 40 to 100°C. The toxicity of toxins was decreased by increasing incubation time for heat treatment. The tolaasin of P. tolaasii, P1 group, is an 18 amino acid-peptide, its molecular mass is 1985 Da, and it forms a left-handed α-helix. Characteristics of the peptide toxins of P2-P5 subgroups are not known. We have isolated these toxins and compared them with tolaasin by gel permeation chromatography, ion-exchange column chromatography, HPLC analysis, and mass spectroscopy.