Study On Transmission Routines Of Pseudomonas Tolaasii And Mechanism Of Induced Resistance By Its Attenuated Strain In Pleurotus Ostreatus

Pseudomonas tolaasii is the pathogen of brown blotch disease of Pleurotus ostreatus, which can also cause brown blotch disease of Agaricus bisporus, Lentinula edodes, Flammulina velutipes, and Pleurotus eryngi. Brown blotch disease of Pleurotus ostreatus occurs in a large area of the world, resulting in huge economic losses. In this paper P. tolaasii is chosen as the research object, to investigate pathogen infection source, transmission, and induced resistant effect and mechanism of P. ostreatus by inactivated attenuated P. tolaasi. This research can offer a theoretical basis for safe and efficient prevention of brown blotch disease on P. ostreatus.1. The selective medium and Real-time fluorescence quantitative Polymerase Chain Reaction (RT-PCR) of P. tolaasii were established. The selective medium had the best specificity for P. tolaasii. The recovery rate can reach to70%, isolating P. tolaasii from simulated contaminated soil samples. Thus, the selective medium can be used for detection of infection source, transmission of P. tolaasii in mushroom farms. Meanwhile, a good RT-PCR detection system for P. tolaasii was established by using Tolaasin gene-specific primers (Pt-lA/Pt-1D1). The RT-PCR detection range of P. tolaasii was102～109cfu/mL, and the sensitivity was100times higher after selective medium enrichment.2. The results of sources and transmission of P. tolaasii showed that soil and compost were the main pathogens sources, and the pathogens can be transmitted via water, Pleurotus ostreatus spore, and Phorid Flies. The detection result showed that P. tolaasii can survive many years in soil by using selective medium, and the number of P. tolaasii can reach to900cfu/g in0-10cm soil of monocropping mushroom farms; The disease index of compost with P. tolaasii resulting in brown blotch disease reached to74.07; The number of P. tolaasii in the water after passing diseased sporocarp to adjacent sporocarp in the moisturizing process can reach to2×103cfu/mL, which could result in brown blotch disease; Pleurotus ostreatus spore was one of the routes of transmission, and catapulted300cm carrying P. tolaasii spreading to neighboring sporocarp only in the horizontal distance; The number of P. tolaasii of a Phorid Flies carrying was up to3×103cfu in mushroom farm.3. Systemic acquired resistance of P. ostreatus induced by attenuated P. tolaasii was reported for the first time in our paper. An attenuated bacterial strain without pathogenicity was discovered from363bacteria strains on the sporocarp, which was then identified as Pseudomonas tolaasii. The inactivated attenuated P. tolaasii have a good acquired resistance for P. ostreatus in several tests, and the best induction concentration, induction interval, and induction times for resistance of P. ostreatus by inactivated attenuated P. tolaasii were3×108cfu/mL,24h, and3times, respectively, and control efficiency was up to80.10%under this condition.4. Pleurotus ostreatus after induced by inactivated attenuated P. tolaasii, can suppress pathogen reproduction, produce physiological resistance and overexpress resistance gene, which resulting in systemic acquired resistance in P. ostreatus. First, P. ostreatus induced by inactivated attenuated P. tolaasii can suppress pathogen reproduction. Using both fluorescence microscopy and RT-PCR technology, we found the density of P. tolaasii pathogen on control P. ostreatus surface was up to107cfu/mm2after48hours of inoculation, while the pathogen density on induced P. ostreatu firstly decreased, and then increased between0-36h with the final less than103cfu/mm2until60h.Second, P. ostreatus induced by inactivated attenuated P. tolaasii can produce physiological resistance. After induced by inactivated attenuated P. tolaasii, the activity of P. ostreatus tyrosinase, laccase and β-N-acetylmuramidase were significantly higher within24h, and total phenols and quinones content increased dramatically, which result in producing a "stress allergic state", suppressing P. tolaasii in the earlier infection stage.Third, P. ostreatus induced by inactivated attenuated P. tolaasii can induce overexpression of defense genes. cDNA sequence of Pleurotus ostreatus HOG (High osmolarity glycerol) gene was obtained using the homology cloning technology. RT-PCR analysis of the P. ostreatus HOG gene after induced by inactivated attenuated P. tolaasii showed that, HOG gene was overexpressed within48h, and reached its maximum value (1.2times control) at24h. HOG gene overexpression and resistance increasing showed the same trend, which illustrated the relationship between HOG gene and Pleurotus ostreatus acquired resistance.This study investigated the attenuated pathogen inducing resistance effect and mechanism of P. ostreatus acquired resistance for the first time in the paper. The inactivated attenuated P. tolaasii as the biological elicitor induced P. ostreatus to produce systemic acquired resistance, to suppress pathogen reproduction, to produce physiological resistance and to overexpress resistance gene. The study performed a preliminary exploration on the disease-resistant mechanism of edible mushroom induced by attenuated pathogen in theory, on the other hand, provided a new idea on edible mushroom biological control, and an evidence for development of edible mushroom biocontrol agent.