【作者】 徐岩岩；

【导师】 李宝聚；

【作者基本信息】 中国农业科学院 ， 农业微生物学， 2013， 博士

【摘要】 托拉斯假单胞杆菌（Pseudomonastolaasii）是平菇（Pleurotusostreatus）细菌性褐斑病的病原菌，还可引起双孢菇（Agaricusbisporus）、香菇（Lentinulaedodes）、金针菇（Flammulinavelutipes）和杏鲍菇（Pleurotuseryngi）等的褐斑病，由该病原菌引起的细菌性褐斑病在世界大面积发生，造成了巨大的经济损失。本论文以托拉斯假单胞杆菌为研究对象，从病原菌的侵染来源、传播途径、弱毒灭活菌诱导平菇抗病性及其机理等方面进行研究，为安全高效防治平菇细菌性褐斑病提供理论基础。1．建立了托拉斯假单胞杆菌选择培养基与实时荧光定量PCR检测技术。一种为选择性培养基检测技术，对托拉斯假单胞杆菌有良好的特异性，分离模拟带菌土样的托拉斯假单胞杆菌回收率可以达到70%，可用于病原菌侵染来源及传播途径的追踪检测；一种为实时荧光定量PCR（Real-timePCR）检测技术，采用托拉斯假单胞杆菌毒素基因的特异性引物对Pt-1A/Pt-1D1，建立了良好的检测技术体系，对托拉斯假单胞杆菌的检测范围为102~109cfu/mL，经过选择性培养基富集后，灵敏度进一步提高了100倍。2．确定土壤和培养料是托拉斯假单胞杆菌的侵染来源，水、平菇孢子和菇蝇为其传播途径。利用选择性培养基技术检测确定托拉斯假单胞杆菌可在土壤中存活及越冬，多年连作栽培棚内0-10cm土层中带菌量可达900cfu/g；培养料带菌引发平菇细菌性褐斑病病指可达74.07；保湿过程中飞溅的水经过发病子实体后带菌量可达2×103cfu/mL；平菇孢子携带托拉斯假单胞杆菌在水平距离可弹射300cm；每只菇蝇携带的托拉斯假单胞杆菌可达3×103cfu。3．首次研究发现托拉斯假单胞杆菌弱毒菌株可诱导平菇产生系统抗病性。从363株细菌菌株中分离筛选得到一株弱毒无致病力菌株，鉴定为托拉斯假单胞杆菌（Pseudomonastolaasii）。经多次验证，托拉斯假单胞杆菌弱毒灭活菌对平菇细菌性褐斑病具有良好的诱抗效果，最佳诱导浓度为3×108cfu/mL，最佳诱导间隔期为24h，最佳诱导次数为3次，诱导抗病效果最高可达80.10%。4．研究发现托拉斯假单胞杆菌弱毒灭活菌诱导平菇后可抑制致病菌的繁殖，产生生理抗性及抗病基因的过量表达，使平菇产生系统抗病性。第一，托拉斯假单胞杆菌弱毒灭活菌诱导平菇后可抑制致病菌的繁殖。利用荧光显微观察与荧光定量PCR检测技术发现，托拉斯假单胞杆菌强致病菌在未诱导的平菇表层上，接种至48h可达107cfu/mm~2，而托拉斯假单胞杆菌弱毒灭活菌株诱导平菇后，表层致病菌数量先下降后上升，60h最终数量未超过103cfu/mm~2。第二，托拉斯假单胞杆菌弱毒灭活菌可诱导平菇产生生理抗性。平菇在托拉斯假单胞杆菌弱毒灭活菌诱导作用下，酪氨酸酶、漆酶和β-N-乙酰胞壁质酶活性0-24h之间显著升高，总酚和醌类物质含量明显增加，使得平菇达到一种“应激过敏状态”，为托拉斯假单胞杆菌强致病菌的侵染提前达到防御状态，阻止致病菌的入侵。第三，托拉斯假单胞杆菌弱毒灭活菌可诱导平菇抗病基因的表达。利用同源克隆技术获得平菇HOG（Highosmolarityglycerol）基因cDNA序列，荧光定量PCR技术研究其表达量变化，结果发现平菇在托拉斯假单胞杆菌弱毒灭活菌诱导作用下，HOG基因0-48h之间过量表达，24h达到最大值（为对照的1.2倍）。HOG基因过量表达与抗病性增加表现趋势一致，说明HOG基因与平菇诱导抗病性相关。本研究首次对弱致病菌诱导平菇抗病性及其机理进行了研究，发现托拉斯假单胞杆菌弱毒灭活菌株作为生物激发子可以诱导平菇产生系统抗病性，抑制病原菌的增殖，产生生理抗性及防卫基因表达。该研究结果，一方面在理论上初步探索了弱致病菌诱导食用菌抗病的机理，另一方面，为食用菌病害生物防治提供了新思路，为食用菌生防制剂的开发提供了新依据。更多还原

【Abstract】 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.更多还原