【作者】 章战华；

【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】 本论文研究了温度、pH和营养条件对来源于棉花、葡萄、苹果和甜瓜的粉红单端孢(Trichothecium roseum Link)菌丝生长影响及其分生孢子的致死温度;并在人工接种的条件下,测定了不同寄主来源的粉红单端孢的孢子悬浮液和菌丝块接种时对苹果的致病力;首次较为系统地研究了粉红单端孢对植物病原真菌的拮抗活性及其机制。主要研究结果如下:1.对粉红单端孢生物学特性的研究结果表明,该菌在10～35℃条件下均能生长,适宜生长温度为15℃至30℃,10℃和35℃时生长缓慢,在25℃时生长最好,温度低于5℃或高于40℃菌丝不能生长。方差分析表明:不同菌株之间,S3与S1、S2、S4间存在极显著差异。粉红单端孢在pH值3～11.5之间均能够生长,菌丝生长呈单峰变化。在pH值4.5～7.5之间生长最好,即在中性偏酸环境条件下比中性偏碱的条件下菌丝生长好;不同寄主间比较,来自葡萄的菌株S2生长最好,来自苹果的菌株S3生长较差。不同来源的菌株之间分生孢子致死温度有差异,S1(棉花)、S3、S4(甜瓜)的分生孢子致死温度分别为54℃,S2的分生孢子致死温度分别为52℃。2.研究了碳氮源营养对粉红单端孢的影响,结果表明,该菌对多种单糖、双糖和多糖等碳源及有机氮和无机氮均能够利用。就碳源而言,菌丝线性生长各菌株以在葡萄糖为碳源的培养基上最快,其次是麦芽糖、甘油、可溶性淀粉、蔗糖,数据显示不同的菌株对不同碳源的利用稍有差异。方差分析表明,葡萄糖与其他几种碳源间差异显著(5%水平),其他碳源之间差异不显著。菌株之间的比较,S2与S1、S3、S4间差异极显著(1%水平);S3与S1、S2、S4间差异极显著(1%水平)。液体培养时,以可溶性淀粉为碳源时菌丝干重值最大,其次是葡萄糖、甘油、蔗糖、麦芽糖。方差分析表明,可溶性淀粉与葡萄糖、甘油、蔗糖、麦芽糖差异极显著(1%水平)。就氮源而言,蛋白胨最适合菌丝线性生长,其次是硝酸铵、硝酸钠、脲、硫酸铵;方差分析表明,蛋白胨与其他氮源间差异极显著(1%水平),其他氮源之间差异不显著。菌株之间的比较,S1、S2与S3与S4间差异显著(5%水平)。液体培养时以蛋白胨为氮源的培养基最适合病菌生长,菌丝干重值最大,其次是脲、硝酸铵、硝酸钠、硫酸铵。方差分析表明,蛋白胨、脲、硝酸铵和硝酸钠、硫酸铵、CK之间差异极显著(1%水平);硝酸铵、硝酸钠之间差异不显著;菌株之间比较,S3与其他菌株间差异极显著(1%水平)。3.采用聚丙烯酰胺凝胶电泳方法,分析了6个不同寄主来源的粉红单端孢菌株的酯酶同工酶和可溶性蛋白的电泳图谱。结果表明,6个不同来源的菌株的酯酶电泳图谱有2条共同的酶带,即Rf0.48和Rf0.64,但不同来源的菌株间谱带的数量和强弱有差异;可溶性蛋白电泳图谱在Rf0.52和Rf0.60有特征谱带,不同来源的菌株间谱带数量和强弱也有差异。本实验结果还表明,同工酶电泳方法简单易行,能反映粉红单端孢种间共同的特征谱带以及种内不同寄主菌株之间的差异,是粉红单端孢种及种下分类的一个可行的辅助手段。4.在人工接种条件下,以苹果为供试植物材料,测定了不同来源的粉红单端孢菌株致病力强弱。用孢子悬浮液创伤接种苹果,来自棉花的S1菌株致病力明显强于其它菌株,达到极显著水平(1%水平),其他菌株间差异不显著;用菌丝块创伤接种苹果,来自苹果的S3菌株造成的病斑明显大于其它菌株,差异达到显著水平(5%水平)。上述结果表明,不同来源的粉红单端孢菌株对同一寄主植物的致病力有显著差异,也即粉红单端孢菌株间存在致病力分化现象。菌丝块创伤接种所致病斑面积比孢子悬浮液创伤接种所致病斑面积大。5.研究了粉红单端孢菌对植物病害的抗菌活性。在PSA平板对峙培养试验中,粉红单端孢菌的菌丝生长速度比棉花黄萎病菌、棉花枯萎病菌、茄褐纹病菌、百合炭疽病菌、水稻恶苗病菌、油菜菌核病菌、草莓灰霉病菌、辣椒灰霉病菌、牡丹根腐病菌等植物病原菌慢,两者的边缘很快接触,没有抑菌带出现;有些病原真菌,例如油菜菌核病菌,其生长速度较快,很快侵占到粉红单端孢菌菌落周围,甚至将粉红单端孢菌落覆盖。结果表明,在对峙培养中,粉红单端孢菌不具有营养和空间竞争优势,甚至可以说是处于劣势。在滤液拮抗试验中,粉红单端孢菌的发酵滤液和发酵灭活滤液均对供试的植物病原菌表现出强烈的抑制作用:滤液浓度分别在6.25%、12.5%、25%时,对油菜菌核病菌的相对抑菌率分别为40.64%、58.98%、100%;灭活滤液的浓度在6.25%、12.5%、25%时,对油菜菌核病菌的相对抑菌率达到25.00%、48.75%、100%。滤液和灭活滤液的浓度在6.25%、12.5%、25%时对棉花黄萎病菌的相对抑制率分别达到14.18%、53.15%、69.31%和4.61%、27.80%、66.28%。粉红单端孢菌的抗菌活性很大程度上在于其代谢产物,包含有热稳定性物质和热不稳定物质,是一种极有潜力的,可用来开发成农药的生防菌。更多还原

【Abstract】 The effects of temperature,pH and nutriment on the mycelial growth,dry mycelium weight and thermal death point of their conidium of Trichothecium roseum from different hosts including cotton,grape,apple and melon were studied,and pathogenicity of conidium and mycelium of T.roseum to apple fruit was tested on condition of artificial inoculation in laboratory respectively.Moreover,the antagOnism to pathogenic fungus was analysed systematically for the first time and the cultural filtrate of the T.roseum in the liquid culture was regarded as the one of most inhibitor agent.The main results were as follows.1.The research of biological characteristics of T.roseum showed that the mycelium could grow at 10-35℃,with the optimum temperature being in the range of 15-30°.It couldn’t grow below 5℃or above 40℃.The lethal temperature of conidial of S1,S3 and S4 strain was 54℃、10 min.and the lethal temperature of conidial of S2 was 52℃,10min. The mycelium could grow under the condition of pH 3 to 11.5,showing single peak curve, with the optimum pH range being between pH 4.5 to pH7.5.2.The pathogen could use many carbohydrates and nitrogen sources.For mycelial growth on solid media,the optimum carbon source was glucose,next being maltose and glycerol.Analysis of variance showed that there were significant difference（P＜0.05） between glucose and the others carbohydrates sources.The more suitable carbon sources for mycelial growth in liquid medium（mycelial dry weight）were soluble starch and glucose.Analysis of variance showed that There were very significant difference（P＜0.01） between soluble starch and the others carbohydrates sources.And for mycelial linear growth,the optimum nitrogen sources were peptone,next being NH₄NO₃ and NaNO₃.The growth of T.roseum in liquid medium showed that peptone,as a sources of nitrogen,was optimum for mycelial growth.Analysis of variance showed that there were very significant difference（P＜0.01）between peptone and the others nitrogen sources.There were not different among the others nitrogen sources.3.The polyacrylamicide gel electrophoresis analysis of isozymes and soluble proteins of 6 isolates of T.roseum from different hosts were studied to compare their differences in isoenzyme zymogram and electrophoresis map.The results showed that there were clear differences in soluble protein electrophoresis map between different isolates in T.roseum, and that there were common two zymograms in 6 strains.It is suggested that electrophoresis technique was a useful tool in the classification of T.roseum could be used to classify T.roseum on both specific and infraspecific ranks.4.Pathogenicity of conidium of S1 strain had obviously strong than the others strain on condition of artificial inoculation in laboratory,reaching a extreme significant difference（P＜0.01）and pathogenicity of mycelial of S3 strain had obviously strong than the others strains on condition of artificial inoculation while there had no difference in the others strains.5.The inhibition activity of T.roseum had not been manifested in the dual-culture of itself and the tested pathogenic fungus,which indicated that the mycelial growth on solid media had no advantage than pathogenic fungus.However,the cultural filtrate of the T. roseum in the liquid culture showed powerful anti-fungal activity and broad inhibitory spectrum.The relative inhibitory effect on Sclerotinia sclerotiorum were 40.64%,58.98%, 100%When the cultural filtrate concentration were 6.25%,12.5%,25%respectively and were 25.00%,48.75%,100%when the disinfected cultural filtrate concentration were same.The relative inhibitory effect on Verticillium dahliae were 14.18%,53.15%,69.31% When the cultural filtrate concentration were 6.25%,12.5%,25%respectively and were 4.61%,27.80%,66.28%when the disinfected cultural filtrate concentration were same.It is tested that the inhibition activity of T.roseum mainly attributed to ferment substance. Therefore,T.roseum may be regarded as a very potential biocontrol agent.更多还原