First Report of Pantoea beijingensis Induced Soft Rot Disease of Pleurotus nebrodensis in China.

Abstract

Pleurotus nebrodensis is a popular edible fungus in China. In December 2009, soft rot disease appeared on fruiting bodies of P. neberodensis in greenhouses located at Haidian District of Beijing, China. Early symptoms were water-soaked lesions on the pilei of the fruiting bodies. Lesions then spread and purulent tissues were formed. Severe soft rot induced production of deformed fruiting bodies and offensive odor. Diseased fruiting bodies stopped growth and commercial losses were estimated at 5 to 10%. Internal sections of the diseased pilei about 0.5 × 0.5 cm were suspended in 0.85% NaCl and the suspension was spread on trypticase soy agar (TSA) media. After incubation at 30°C for 2 days, dominant bacterial colonies were yellow, smooth, round, and convex. Cells were gram-negative, short rods, non-capsulated, motile, and non-spore forming. The 16S rRNA gene (1,408 bp, GenBank Accession No. KF849293) was amplified by using the universal forward primer P1 (5'-AGAGTTTGATCCTGGTCAGAACGCT-3') and the universal reverse primer P6 (5'-TACGGCTACCTTGTTACGACTTCACCCC-3'). Neighbor-joining tree analysis based on 16S rRNA gene sequences demonstrated the isolates belonged to the genus Pantoea and showed highest similarity with Pantoea beijingensis LMG 27579T (99.9% similarity, 2 base pair differences), which was isolated from the diseased fruiting bodies of P. eryngii in China. Pantoea sp. PA4, reported to be the pathogen of soft rot disease of P. eryngii in Korea (1), showed 99.8% similarity with P. ananatis ATCC 33244T, which suggested our isolates belonged to a different species with Pantoea sp. PA4 (supported by 97.9% similarity between the present isolates and Pantoea sp. PA4) (2). Results of physiological properties and enzyme activities determined by the API 20E, API 20NE, API 50CH, API ZYM (bioMérieux), and GN2 MicroPlate (Biolog) system showed that there was no difference between the present isolates and P. beijingensis LMG 27579T. Pathogenicity tests for these isolates were performed with bacterial suspensions (approximately 1 × 106 CFU/ml) that were grown for 24 h in trypticase soy broth (TSB). Fruiting bodies of P. nebrodensis were induced after spawn was run for 30 days in plastic bags. When the young fruiting bodies were forming, the prepared bacterial suspension was sprayed onto the surface of the pilei. Sterilized TSB media was used as a negative control. All inoculated fruiting bodies were then incubated at 16°C with 80 to 95% relative humidity. Assays were conducted twice and five fruiting bodies were used each time. Results were observed after 5 to 10 days of incubation. The symptoms that developed were similar to those observed in the original samples. The negative control remained symptomless. Koch's postulates were fulfilled by re-isolating bacteria, which were identical to the original isolates based on 16S rRNA gene sequence analysis, physiological properties and enzyme activities. Pantoea ananatis was first reported as a pathogen of Pleurotus eryngii, causing soft rot disease (1), but to the best of our knowledge, this is the first report of P. beijingensis- induced soft rot disease of P. nebrodensis in China. This disease may be an emerging disease problem in the near future and information on this pathogen will be useful in the development of management practices.