【作者】 邓彩萍；

【导师】 骆有庆； 刘红霞；

【作者基本信息】 北京林业大学 ， 森林保护， 2009， 博士

【摘要】 光肩星天牛Anoplophora glabripennis是我国林木的重要蛀干害虫。通过对光肩星天牛及其它天牛自然种群生命表的研究,发现卵期和初龄幼虫期的死亡率很高,对种群动态有很大影响,但死亡原因和机制不清。本论文以光肩星天牛刻槽为研究对象,分析比较了北京和宁夏两个不同气候区的刻槽含水量、pH值及微生物多样性。同时在所分离的微生物中筛选了粘质沙雷氏菌Serratia marcescens以及来源于刻槽的球孢白僵菌Beauveriabassiana作为研究菌株,研究了其对光肩星天牛低龄幼虫的致病性以及侵染过程,同时对与致病性相关的酶活性进行了研究,主要结果如下:1.北京地区和宁夏地区杨树和柳树的刻槽含水量规律基本一致,人工刻槽一般中晚期含水量较高,与健康树皮相比差别不大。真实刻槽一般也是中晚期含水量较高,和健康树皮相比,一般高于健康树皮。从整体平均值来看,真实刻槽＞健康树皮＞人工刻槽。2.两个地区树种pH均以真实刻槽最高,平均值范围在7.88～9.97。健康树皮的pH为6.06～7.01,中性或略偏酸性;人工刻槽pH为6.20～8.20。总体来看,pH表现为:真实刻槽＞人工刻槽＞健康树皮。3.对北京、宁夏两个地区5个树种上的真实刻槽、人工刻槽及健康树皮的真菌分离,共得到1449株菌株,经鉴定隶属于18属,其中接合菌亚门2属;半知菌亚门真菌有16属。两个地区不同类型的刻槽,均以Fusarium和Alternaria真菌占优势菌,而且真实刻槽中以Fusarium为优势属,在人工刻槽中则以Alternaria为优势属。从多样性指数和均匀度指数来看,北京地区的真实刻槽和人工刻槽均高于宁夏地区的同类型刻槽。而健康树皮的两个指数则是宁夏地区高于北京地区。细菌主要为黄单胞杆菌属Xanthomonas Dowson和一种未定名细菌。在旱柳所有类型的刻槽中均发现有粘质沙雷氏菌Serratia marcescens。放线菌种类则只分离到一种,初步鉴定为链霉菌属Streptomyces spp.。在无菌条件下,用天牛刻槽饲养无菌条件下孵化出的小幼虫时,发现幼虫感病死亡,表明此致病菌来源于刻槽。病原菌经鉴定为球孢白僵菌Beauveriabassiana。在刻槽外,有白星花金龟成虫、蛱蝶类、蚂蚁、蜜蜂吸食刻槽外部流出的汁液。在刻槽内有麻蝇科幼虫、食蚜蝇科幼虫、露尾甲科幼虫等腐生性昆虫存在。4.对球孢白僵菌和粘质沙雷氏菌进行了低龄幼虫室内致病力测定,表明两种致病菌对光肩星天牛低龄幼虫都有一定的致死率。木段接菌实验中球孢白僵菌效果可达到76.8%,但粘质沙雷氏菌的效果不理想。5.采用石蜡切片技术,对受两种致病菌侵染的幼虫体进行观察,结果显示:被球孢白僵菌感染的虫体局部上皮组织崩解,皮下脂肪与上皮组织分离。脂肪体组织细胞萎缩、死亡。脂球也变得不明显。肌肉发生解离,产生很多大型液泡。在中肠中,环肌轻则向内皱褶,重则与消化道肌肉分离,导致组织解离。菌丝侵入血细胞出现空泡并变形。6.初感染细菌的虫体,表皮细胞部分区域已发生降解,遭到破坏;肌肉部分解离,有些区域被空泡状物充盈;中肠肌肉细胞轮廓不清晰。体表及中肠环肌仅剩薄层。待感染一天左右时,整个体腔内部一片混沌,各组织辨认不清。7.通过扫描电镜观察,本白僵菌很少产生附着胞结构,通过孢子萌发形成芽管直接侵入幼虫体表。芽管平均长度在3.08μm时即可侵入幼虫体表,同时在侵入后期,新发现菌丝之间或者孢子之间有丝状结构,以及菌丝之间形成网状结构。8.研究了两种致病菌的产酶情况,结果显示,球孢白僵菌可产生两种酶:胞外蛋白酶和胞外几丁质酶。明胶培养基和虫尸粉培养基的胞外蛋白酶活相对于酵母培养基都比较高。26～28℃明胶培养基蛋白酶活性较高,35℃时虫尸粉酶活性较高。三种培养基诱导胞外几丁质酶都在26～30℃酶活较高,在33～35℃活性都很低。26℃时胶体几丁质的诱导酶活最高,30℃时则以虫尸粉培养基诱导较好。粘质沙雷氏菌在30～37℃下均以胶体几丁质诱导效果最好,33℃下酶活最高,虫尸粉培养基的诱导效果次之,但两者相比,差异达到极显著。细粉几丁质在每个温度下诱导效果都很差。40℃下三种培养基诱导酶活均很低。更多还原

【Abstract】 Anoplophora glabripennis,Asian longhorned beetle(ALB),is one of most important boring pest in China.According to natural population life table of ALB and other longhorned beetles, the mortalities of eggs and low instar larvae are high,and have significant influence on population dynamics.But the reasons remain unknown.This study reports the water content and pH of ALB oviposition slits in two climatic regions, Beijing and Ningxia,and microorganism community structure and diversity in oviposition slits. Two microorganism strains,Serratia marcescens and Beauveria bassiana,were isolated from oviposition slit directly or indirectly.Pathogenicity,invasion behavior and correlative enzyme activity of these two strains were studied.The main research conclusions were listed as follows:1.The water content of oviposition slits or imitated slits of poplar and willow in Beijing was similar to that in Ningxia.As for imitated slits,water contents of middle stage and late stage were high.The difference in water content between imitated slits and healthy bark was not apparent.Similarly,the water contents of middle stage and late stage of oviposition slits were high,which were higher than healthy barks.The trend of average water content from high to low was oviposition slits,healthy bark,imitated slits.2.pH of oviposition slits was the highest than that of other bark types.The average pH was 7.88～9.97.pH of healthy bark was neutral or little acidic(6.06～7.01),while pH of imitated slits(6.20～8.20) was higher than that of healthy bark.Therefore,the trend of average pH was oviposition slits＞imitated slits＞healthy bark.3.There were 1449 fungi strains isolated from oviposition slits,imitated slits and healthy bark from 5 tree species in Beijing and Ningxia.The isolated fungi were designated as 18 genera. Among them,2 genera were identified as Zygomycotina,16 genera as Deuteromycotina. Fusarium and Alternaria were dominated genera in three bark types of two areas.Fusarium was dominate genus in oviposition slits,whereas Alternaria was dominate genus in imitated slits.As for diversity index and species evenness,fungi population of oviposition slits and imitated slits in Beijing were higher than that in Ningxia.Fungi population of healthy bark in Beijing was lower than that in Ningxia.Xanthomonas Dowson and another unidentified strain were the main isolates in three types of bark.Serratia marcescens was found in three types of bark of Salix matsudana.Only one kind of actinomycete was isolated from three types of bark,and identified as Streptomyces spp.ALB larvae were found to dead when incubated with oviposition slit in aseptic condition. The pathogen was identified as Beauveria bassiana,suggesting that originated from oviposition slit.In addition,there were some insects living inside or outside of oviposition slits,such as potosia brevitarsis(Lewis),and insect adults of Nymphalidae,Formicidae,Apidae,attracted by large amount of sap flowing out of slits.Inside oviposition slits,saprophytic larvae of Nitidulidae, Sarcophagidae and Syrphidae were found.4.Laboratory virulence of B.bassiana and S.marcescens on low instar ALB larvae were tested.The results showed that the two pathogenic strains were pathogenic to ALB larvae.Trunk tests showed that B.bassiana had a good control effect(76.8%),but no control effect on S. marcescens.5.The infected larvae of ALB by B.bassiana showed that the part of epithelial tissue was disintegrated and subcutaneous fat was separated from epithelial tissue.Fat cell became atrophic and dead.Fat globule also became unclear.Muscle was disintegrated and some large vacuoles appeared with low coloration effect.In midgut,circular muscle was disintegrated,arranged in disorder and rumpled inward.Vacuoles appeared in blood corpuscle and blood corpuscle became distorted.6.Larvae infected by S.marcescens were found that part of cuticle was destroyed and degraded.Part of muscle was separated and filled with some vacuoles.Profile of midgut muscle became unclear.Circular and midgut circle muscle became a piece of lamina.One day later,all viscera became chaotic and tissues were indistinguishable.7.B.bassiana could invade circular directly by germ-tube.The strain produced few appresoriums.Germ-tube could invade circular with mean length 3.08μm.Silk in between hypha,silk between spores and hypha,and net among hypha were found in late infection.These phenomena have not been reported to date.8.B.bassiana could produce two main extracellular enzymes:extracellular protease and extracellular chitinase.The activity of extracellular protease induced in gelatin medium and larva powder medium was higher than that in yeast medium.High enzyme activity in gelatin medium appeared at 26～28℃,and in larva powder medium appeared at 35℃.Three mediums induced extracellular chitinase having higher enzyme activity at 26～30℃.Enzyme activity was low at 33～35℃.Colloidal chitin medium induced highest enzyme activity at 26℃,and highest enzyme activity at 30℃was larva powder medium.Extracellular chitinase of S.marcescens had a good induced effect in colloidal chitin medium at 30～37℃,and highest enzyme activity appeared at 33℃.Larva powder medium induced effect was lower than colloidal chitin.The difference in activity of enzymes induced by the two mediums was very significant.Chitin powder medium induced very low enzyme activity at 5 temperatures.Induced enzyme activities in three mediums were all low at 40℃.更多还原