【作者】 王利军；

【导师】 谭万忠；

【作者基本信息】 西南大学 ， 植物病理学， 2010， 硕士

【摘要】 马铃薯甲虫Leptinotarsa decemlineata (Say);是重要的外来入侵物种和检疫性害虫,国外已开发出多杀菌素等防治马铃薯甲虫的生物杀虫剂,但由于马铃薯甲虫传入我国不久,对其致病微生物的研究国内尚未见报道。因此本研究从新疆马铃薯甲虫发生地采集感病成虫样品,在室内分离昆虫病原菌,并经致病性测定筛选出2个强致病白僵菌菌株,用形态及分子生物学技术鉴定强致病菌,并研究它们的培养特性,最后进行温室盆栽和大田试验测定这2个菌株的防治效果。马铃薯甲虫病原菌的分离：从新疆大量采集感病马铃薯甲虫,采用常规昆虫真菌分离法从虫体上分离纯化后获得47个真菌菌株,初步形态学鉴定分别属于白僵菌(Beauveria)、轮枝菌(Verticillium)、镰刀菌(Fusarium)、青霉(Penicillim)、木霉(Trichoderma)和曲霉(Aspergillus)属等。不同菌株对家蚕的致病性测定：用真菌悬浮液(浓度：1.05×109个／mL)接种家蚕2龄幼虫,结果表明有36个菌株对家蚕有不同程度的致病性,其中白僵菌30株、轮枝菌3株、镰刀菌2株和木霉1株,白僵菌菌株中MJ-07的致病力最强,接种后11d的累积死亡率达到95.6%,致死中时(LT50)为7.5d；轮枝菌MJ-20接种后11d的累积死亡率和LT50分别为61.5%和9.1d；镰刀菌MJ-27和木霉MJ-32的致病力较弱,其接种11d的累积死亡率仅为8.6～11.0%。本试验结果为进一步到新疆进行马铃薯甲虫致病性测定奠定了基础,同时也为今后有关地区在应用病原菌控制马铃薯甲虫时注意保证养蚕业的安全提出了警示。真菌菌株对马铃薯甲虫的致病性：用家蚕初步筛选获得的36个菌株的分生孢子悬浮液(浓度：1.1×109个／mL)分别接种马铃薯甲虫2龄幼虫后观察,它们都具有不同程度的致病力。轮枝菌(Verticillium)、镰刀菌(Fusarium)和木霉(Trichoderma)菌株的致病力比较弱,接菌9d后的虫体累计死亡率(LR9d)只有4.3～11.3%。多数白僵菌(Beauveria)菌株的致病力都较强,其中有5株白僵菌(MJ-01、MJ-03、MJ-07、MJ-08、MJ-09)的致病力非常强,LR9d达到82.3～93.3%,致死中时(LT50)和致死中浓度(LC50)分别为6.71～7.53d和4.53×106～5.89×107mL-1。选择致病力最强的MJ-07和MJ-09菌株作进一步试验,观察它们对成虫和各龄幼虫的致病性。结果表明,MJ-07菌株对1龄、2龄幼虫和成虫的致病性都很强,接菌后5d初见虫体死亡,LR9d分别为100%、93.4%和98.0%；而对3龄和4龄幼虫的致病性相对较弱,LR9d各为70.0%和43.3%；死亡的虫体缩小,后期产生大量分生孢子完全包被虫体。MJ-09对1龄和2龄幼虫的致病性很强,接菌后5d始见虫体死亡,LR9d分别达100%和90.0%；而对3龄和4龄幼虫和成虫的致病性较弱,接种后7d才开始见虫体死亡,LR9d分别只有63.3%、33.0%和13.3%；刚死亡的虫体完全变黑,后期产生的分生孢子量不如MJ-07致死的虫体产孢量大,不能完全包被虫体。本研究结果认为,MJ-07和MJ-09是马铃薯甲虫的强致病力菌株,在马铃薯甲虫的生物控制中将具有潜在的开发应用价值。菌株MJ-07和MJ-09对马铃薯甲虫的控制效果测定：通过前期室内致病性试验,选致病性最强的2株白僵菌(MJ-07和MJ-09)进行室内盆栽和田间防效试验。1.室内盆栽结果表明,2个菌株对1龄和2龄幼虫的致死率显著高于3龄幼虫,喷施MJ-07菌悬液15d后的各龄幼虫的死亡率分别为65.5%、61.8%和30%；而喷施MJ-09菌悬液15d后的死亡率分别为62.3%、57.2%和25%。2.选择1龄和2龄CPB幼虫进行田间试验,喷施MJ-07菌悬液15d后的死虫率分别为45%和41.6%；喷施MJ-09菌悬液15d后的死虫率分别为40.2%和38.3%；作为对照白僵菌制剂的致死率仅有30%,2个菌株的田间致病性高于对照白僵菌制剂,表现出很好的害虫控制效果。白僵菌形态学和分子生物学鉴定：在SDAY培养基上培养菌落呈茸毛状,MJ-07培养初期多呈白色且菌落较厚,MJ-09菌落淡黄色,背面无色,或淡黄色。气生菌丝透明、光滑、薄壁,分枝较少且较纤细,宽1.8～2.8μm；分生孢子梗着生在菌丝上,粗1-2μm,产孢细胞常着生于短的并稍有膨大的柄细胞上,常呈浓密的菌丛,基部多为球形、近球形至梭形膨大；产孢轴较长,呈“之”字形弯曲。分生孢子单孢、壁薄、透明、光滑,多为球形、近球形,直径2～3×2～2.5μm。采用真菌通用引物对(ITS4和ITS5),通过聚合酶链式反应(PCR)分别对菌株MJ-07和MJ-09的核糖体DNA内转录间隔区(rDNA ITS)的特异性扩增,得到了长度分别为581bp和537bp的特异性片段(GenBank登录号分别为GU233851和GU989641),Blast比对后结合形态学特征鉴定它们的无性时期均为球孢白僵菌(Beauveria bassiana),有性时期为球孢虫草菌(Cordyceps bassiana)。白僵菌菌株主要培养特性：在供试的8种培养基中,SDAY培养基最适MJ-07和MJ-09菌株生长,其产孢强度也最大；6个温度处理中最适生长温度为25℃,孢子萌发最适温度25～28℃；菌株在供试的9种碳源和9种氮源中,对葡萄糖和蔗糖2种碳源及胰蛋白胨和酵母浸膏2种氮源利用最好；菌株在全光照条件下菌丝生长最好及产孢量最大；分生孢子的致死条件为55℃处理10min；紫外光照射对分生孢子萌发有显著影响,照射60min时,24h内没有孢子萌发；分生孢子在2%葡萄糖+1%蛋白胨培养基中萌发最早。5种常用马铃薯甲虫杀虫剂对MJ-07和MJ-09生长产孢的试验结果显示,5种供试杀虫剂对2个菌株的菌落生长和孢子萌发均有不同程度的抑制作用。其中高效氯氟氰菊酯和吡虫啉对2个菌株的抑制作用较小,对MJ-07的孢子萌发抑制率分别为9.5%和13.5%,对MJ-09孢子萌发率分别为10.9%和13.3％；而灭多威、啶虫脒、甲氨基阿维菌素苯甲酸盐对2个菌株均有较强的抑制效果。因此在田间应用时应注意杀虫剂对白僵菌的抑制作用。虫生真菌(entomogenous fungi)是自然界广泛分布的一类珍贵的生物资源。随着科学技术的发展和绿色植保方针的提出,生物防治在马铃薯甲虫的综合治理中发挥重要的作用,本研究中筛选鉴定的2个白僵菌菌株具有明显的应用潜力,安全有效的马铃薯甲虫真菌杀虫剂将来会被研制出来并广泛应用于马铃薯甲虫的控制实践。更多还原

【Abstract】 Colorado potato beetle [CPB, Leptinotarsa decemlineata (Say)] is an important invasive alien species and quarantine pest and it is now distributed in northern Xinjiang Autonomous Region of China.The objectives of the present work were to isolate entomogenous fungi from diseased samples of CPB adults and to screen highly pathogenic isolates for future applications in biological congtrol of the CPB pest.Isolation of CPB pathogens:Diseased samples of CPB adults were collected from different venues of northern Xinjiang and 46 fungal isolates were obtained using the conventional tissue culture techniques in the laboratory.Initial morphological identification showed that these fungi belonged, respectively, to Beauveria, Verticillium, Fusarium, Penicillim, Trichoderma and Aspergillus genera.Pathogenicity tests on silkworms:2nd-instar larvae of Bombyx mori Linnaeus were inoculated with fungal suspension. The results showed that the 36 isolates were pathogenic to silkworms including 30 isolates of Beauveria sp.,3 isolates of Verticillium sp.,2 isolates of Fusarium sp. and 1 isolate of Trichoderma sp.The pathogenicity of Beauveria isolate MJ-07 was the strongest; the cumulative mortality rate reached 95.6% 11d after inocultion and the mid lethal time (LT50) was 7.5d; the inoculation with Verticillium MJ-20 cumulative mortality and LT50, respectively 61.5% and 9.1d; Fusarium MJ-27 and Trichoderma MJ-32 would be weak pathogenicity, after the 11d of the cumulative mortality rate was only 8.6-11.0%. The test results to determine the pathogenicity of CPB had a foundation for the areas concerned but also for future control of CPB in the application of fungus when taken to ensure the safety.Pathogenicity tests on CPB: A number of 36 fungal isolates were obtained from diseased adults of CPB, collected from Kashi, Akeshu and Artai of Xinjiang Autonomous Region of China, and they were morphologically identified as the species of Beauveria (30 isolates), Verticillium (3 isolates), Fusarium (2 isolates) and Trichoderma (1 isolate), respectively. Results from inoculation of CPB 2nd-instar larvae with spore suspension of 1.1×109 mL-1 showed that all the Verticillium, Fusarium and Trichoderma isolates had only relatively low pathogenicity to CPB. The cumulative lethal rates of these isolates after 9 days of inoculation (LR9d) were only 4.3%-11.3%. Most of the Beauveria isolates were shown much higher pathogenic than other tested fungi. Amongst Beaveria isolates,5 (MJ-01, MJ-03, MJ-07, MJ-08 and MJ-09) were highly pathogenic to CPB larvae and the death of larvae was first observed only 5 days after inoculation whilst the LR9d were 82.3%-93.3% and the mid lethal time (LT50)and mid lethal contrations(LC50) were 6.71-7.53d and 4.53x106-5.89x107 mL-1, respectively. The most virulent two isolates of Bearveria (MJ-07 and MJ-09) were further tested agianst CPB adult and 1st-4th instar larvae. MJ-07 was shown very pathogenic to 1st-,2nd instar larvae and adults with LR9d of 100%,93.3% and 98.0%, but much less virulent to 3rd- and 4th-instar larvae with LR9d of 70.0%, and 43.3%. The bodies of MJ-07 killed CPB became contracted and were completely wraped by the properously produced mycelia and conidiospores by the fungus. MJ-09 was demonstrated highy pathogenic to 1st- and 2nd instar larvae with LR9d of 100% and 90.0%, but significanly less virulent to 3rd-,4th-instar larvae and adults with LR9d of only 63.3%,33.0% and 13.3%. The bodies of MJ-09-killed CPB became dark-brown and were partially covered by the less properous mycelia and conidia of the fungus. It can be concluded that the Beauveria isolates MJ-07 and MJ-09 are pathogens of CPB with high virulence and may be potential candidates for using to develop mycopesticide against CPB.Greenhouse and field tests of MJ-07 and MJ-09 against CPB: The two most pathogenic isolates of Beauveria (MJ-07 and MJ-09) were applied to control CPB and field the results indicated that CPB was effectively controlled by spraying spore suspensions of both MJ-07 and MJ-09. In the greenhouse tests, the lethal rates of 1st and 2nd instar larvae reached more than 62% at 15d after treatment. In the field, CPB lethal rates of about 40% were recorded 15d after separate sprays of the two isolates on the 1st and 2nd-instar larvae. The control efficacy was significantly higher than that of a commonly used Beaveria bassiana pesticide (control,30%).Morphological and molecular identification of MJ-07 and MJ-09:On SDAY medium plates, the colonies were downy, whiteor yellowish wite. Aerial hyphae transparent, thin-walled, branches were thin and slender, width 1.8-2.8μm; conidia were produced on the conidiophores (sized 1-2μm in diameter). They were single-spored with thin walls, transparent, smooth, mostly spherical, sized 2-3 x 2-2.5μm (in diameter).The rDNA ITS sequences were obtained from the genomic DNAs of MJ-07 and MJ09 through polymerased chain reaction (PCR) with the general fungal primer pair ITS4 and ITS5. These sequences were registered in Genbank with accesion numbers of GU233851 and GU989641, respectively, and they shared maximum identities of 98%-100% with those from isolates of Cordyceps bassiana existed in Genbank.Therefore, MJ-07 and MJ-09 were identified as Cordyceps bassiana based on the about morphological and molecular descriptions and the asexual stage of the fungus was Beaveria bassiana.Cultural conditions of Beauveria isolates:For the two B.bassiana, MJ-07 and MJ-09, the best medium was SDAY. The most suitable temperature for its colony growth was 25℃.The optimum germination temperature of 25-28℃. The fungual isolates grew normally on media with glucose, sucrose, tryptone and yeast extract sources. Light treatment had positive effect on fungal sporulation. Conidial lethal conditions for 55℃treatment 10min. UV radiation had negative effect on spore germination and after treatment of 60min-UV irradiation for 24h there was no spore germination.The conidia germinated best on medium plate with 2% glucose+1% peptone medium.The five insecticides commonly used for CPB control were tested to show their effect on MJ-07 and MJ-09. Results showed that lambda-cyhalothrin and imidacloprid had minimum reverse effect on the mycelial growth and spore germination of the isolates while the other three insecticides (methomyl, emamectin benzoate and acetamiprid) significantly suppressed mycelial growth and spore germination of the two isolates.Entomogenous fungi as a class of valuable biological resources are widely distributed in natural fields or environment. With the rapid development of science and technology entomogenous fungi will be given greater attention in crop pest management. The two isolates of Beaveria bassiana had been shown potentially valuable in producing mycoinsecticides and in future CPB control.更多还原