【作者】 郭敏；

【导师】 高智谋；

【作者基本信息】 安徽农业大学 ， 植物病理学， 2007， 硕士

【摘要】 本论文首先对安徽铜陵地区牡丹根腐病原菌进行了分离与鉴定,并从生物学特性、生物学性状的遗传稳定性、营养体亲和性、同工酶电泳分析及有效杀菌剂筛选等方面对牡丹根腐病原菌进行了较为系统的研究,主要结果如下:1牡丹根腐病原菌的鉴定从铜陵地区发病的牡丹上分离到12株根腐病原菌,通过对其形态特征与培养性状观察,并结合其生物学特性研究,根据C.Booth、Nelson的镰刀菌分类系统,将该菌确定为茄病镰刀菌[Fusarium solani（Mart.）Sacc.]。2牡丹根腐病菌生物学特性的研究对牡丹根腐病菌（Fusarium solani）的研究结果表明,该菌多数对单糖、双糖、多糖等碳源及有机氮和无机氮均能够利用。其中葡萄糖、麦芽糖、甘油为适宜碳源,产孢量以果糖最高。以有机脲为氮源培养基上产孢量最高。蛋白胨为最适宜的氮源培养基,病原菌在其上生长速率最大。全黑暗条件下气生菌丝较多,产孢量也最高。PSA、淀粉、PDA和茄汁是适合病原菌菌丝生长的培养基。该菌在10-35℃条件下均能生长,最适生长温度为25-30℃,温度低于5℃或高于40℃菌丝不能生长,在25-35℃之间孢子萌发率达到最大;孢子致死温度为55℃。病原菌在pH4.1-11.5之间均能生长,其中在pH5.5-7.0之间病原菌丝生长最好;pH在7.0时分生孢子的萌发率最高。3生物学性状遗传稳定性试验结果表明,牡丹根腐病菌（F solani）的菌落形态和和菌丝生长速率在无性单孢后代可以稳定遗传,单孢后代之间差异不显著。菌株MD-1-5和GH-1-1的各代单分生孢子株在菌落形态上与其对应亲本之间基本相同,其菌落近圆形,边缘规则整齐,质地均匀,气生菌丝较少。菌株MD-1-5和GH-1-1的各代单孢株在生长速率上较为相似。菌株F-3各无性单孢株后代的菌落形态和生长速率与亲本是相似的,F-3各单孢株间与亲本在生长速率上均无显著差异。4营养亲和性测定利用氯酸盐培养基对分离得到12个牡丹根腐病菌（F solani）进行诱变,共获得99个nit突变体,利用不同氮源培养基对突变体进行鉴定,得到nit1型64株,nit3型19株,nitM型16株。来源于同一野生菌株的不同类型nit突变体间可产生互补反应而形成异核体,其中以nitM型突变株互补性最好,将各菌株的nit突变株和测试菌株的nitM配对,结果12个菌株归为3个不同的营养体亲和群（VCGs）。将诱变获得的99个nit突变株分别在PDA斜面上转管培养4次（80 d）后,除4株恢复成野生菌株外,其余多数nit突变菌株表现稳定。5.酯酶同工酶及可溶性蛋白电泳分析采用同工酶电泳技术,对采自安徽铜陵的12株牡丹根腐病菌（Fusarium solani）菌株进行了酯酶同工酶和可溶性蛋白分析。结果表明,供试的12个牡丹根腐病菌菌株的酯酶同工酶在R伪0.78处各有1条酶带,但各个菌株酶带的深浅、浓淡均有差异,根据酶带的强弱可以将其分为3种类型。12个菌株的可溶性蛋白图谱存在较大的差异,根据各菌株的主谱带数可将其分为4种类型:A型具有8条主谱带,共2个菌株,占供试菌株的16.7%;B型具有7条主谱带,共8个菌株,占供试菌株的66.7%;C型和D型分别具有6条和5条主谱带,各有1个菌株,均占供试菌株的8.3%。从谱带迁移的位置可以看出,12个供试菌株的可溶性蛋白图谱主要分布在3个区域内:区域ⅠRf为0.24～0.36;区域ⅡRf为0.42～0.67;区域ⅢRf伪0.70～0.79。在Rf为0.24,Rf为0.36,Rf为0.42处的3条谱带为安徽丹皮根腐病菌的特征性谱带。酯酶同工酶和可溶性蛋白电泳均表明各供试菌株在遗传上具有相似性且种内变异程度较小。6牡丹根腐病菌有效杀菌剂筛选分别以菌丝生长速率法和孢子萌发法测定了6种杀菌剂对牡丹根腐病菌的毒力。菌丝生长速率法测定毒力的结果表明,6种杀菌剂对牡丹根腐病菌的EC₅₀值大小顺序为:嘧菌酯＞百菌清＞恶醚唑＞代森锰锌＞多菌灵＞咪鲜胺;咪鲜胺对牡丹根腐病原菌具有十分显著的抑制效果。孢子萌发法测定毒力的结果表明,百菌清对牡丹根腐病原菌孢子萌发的毒力最强,EC₅₀值为0.343 mg/L;咪鲜胺、恶醚唑、多菌灵、代森锰锌毒力依次减弱;嘧菌酯的毒力最小。更多还原

【Abstract】 The causal organism of tree peony root rot was identified and a systematic research was done on biological characteristics, inheritance stability of biological characters, vegetative compatibility, electrophoresis analysis on esterase isozymes and soluble protein of Fusarium solani and on screening of effective fungicides controlling causal organism of tree peony root rot. The test results were as follows:1. Identification of the Causal Organism of Tree Peony Root RotAccording to the classification system of C. Booth and Nelson, 12 strains isolated from tree peony root rot in Tongling, Anhui were identified as Fusarium solani through its morphology and culture characteristics and biological characteristics.2. Biological characteristics of Fusarium solani caused tree peony root rotThe studies on the biological characteristics of Fusariun solani showed that the pathogen could use many carbohydrate and nitrogen sources, but the optimum carbon sources were glucose, maltose and mannose. The optimum medium for conidial production was sucrose. Fructose and maltose, as sources of carbon, were more optimum for mycelial growth of Fusariun solani inTiquid medium. The optimum nitrogen sources were （NH₄）₂HPO₄, KNO₃ and NH₄NO₃ respectively. The optimum medium for conidial production was NH₄NO₃, and the growth of Fusariun solani in liquid medium showed that NaNO₃, as a source of nitrogen, was optimum for mycelial growth. The optimum media for mycelial growth of Fusariun solani were potato I and potato II, and the conidial production in the soluble starch medium was the highest one. The hyphae could grow in 10-35℃, the optimum temperature was in the range of 25-30℃, and the temperature lower than 5℃or higher than 40℃was not fit for the mycelial growth, the lethal temperature of conidial was 52℃for ten minutes. The mycylia could grow under the condition of pH 4.1-12.5, and the optimum were pH 5.5 and pH 10.3, the optimum pH for conidia spore germination was between 3.99 and 7.0.3. Inheritance stability of biological character of Fusarium solani isolates inconidium progenyThe test result showed that the colony morphology and mycelial growth rate could be stably inherited in macroconidia progeny, no significant difference on morphology and mycelial growth rate among spores in later generation. The colony morphology of MD-1-5 and GH-1-1, with nearly circular and neat colony, was identical with its parental strains in each generation. The mycelial growth rates of strains MD-1-5 and GH-1-1 were nearly the same in each macroconidia generation. The strains of asexual spores of F-3 were nearly the same to its parental strain on colony morphology and mycelial growth rates, and no significant differences were existed in strains from F-3 on mycelial growth rate.4. Studies on the vegetative compatibility of Fusarium solani causing treepeony root rotAmong 12 Fusarium solani isolates, we totally got 99 nit mutants by using chlorate medium. All of the nit mutants were then transferred into the media containing different nitrate as single nitrogen source to find which nit type they belonged to. Among the 99 nit mutants, 64 nit mutants were nit1,19 nit mutants were nit3, and 16 nit mutants were nitM. Complementation occurred between the different nit mutantphenotypes derived from the same parental strain. Complementation occurred readily and reliably in nitM which was used to identify the vegetative compatibility of other nit mutants, and the test results showed 12 isolates belongs to 3 vegetative compatibility groups. The nit mutants were all transferred on the PDA medium, and the most majority of nit mutants were stable except 4 nit mutants.5. Analysis on esterase isozymes and soluble protein electrophoresisThe soluble protein and esterase isozyme of 12 isolates of Fusarium solani, the causal organism of tree peony root rot in Tongling, were analyzed by the technique of electrophoresis. The results showed that there was only one main band at Rf 0.78 in esterase isozyme electrophoresis of the 12 isolates. The soluble protein patterns were obviously different among the 12 isolates tested, and they can be classified into 4 types. Of 12 isolates, 2 isolates belonged to Type A having 8 main bands, occupying 16.7% of the total. Eight isolates belonged to Type B having 7 main bands, occupying 66.7%. Type C and Type D had 6 and 5 main bands respectively, including 1 isolate （occupying 8.3%） each. The soluble protein bands were mainly distributed in 3 areas: the Rf in Area I was from 0.24 to 0.36, Rf in Area II was from 0.42 to 0.67, and Rf in Area IIIwas from 0.70 to 0.79. The three bands at Rf 0.24, Rf 0.36 and Rf 0.42 were the specific bands of the pathogenic fungus. Both esterase isozyme and soluble protein electrophoresis showed similarity among the isolates tested and low variation in species.6. Screening tests of the effective fungicides controlling tree peony root rotToxicity of 6 fungicides to Fusarium solani which caused tree peony root rot were tested by the means of mycelial growth method and spore germination method under the condition of laboratory, respectively. The toxicity result by the way of mycelial growth method showed that the order of EC50 values of 6 fungicides was as following: Azoxystrobin>Chlorothalonil>Difenoconazole>Mancozeb> Carbendazin> Prochloraz; the inhibition effect of Prochloraz against the pathogen was significant among those fungicides being tested. The toxicity result by mean of spore germination method showed that Chlorothalonil, with EC₅₀ value as 0.343 mg·L^-1, has the highest toxicity on the fungus. Toxicity of Prochloraz, Difenoconazole, Carbendazin, Mancozeb were gradually weakened in turn, with the toxicity of Azoxystrobin being the lowest one.更多还原