【作者】 王达；

【导师】 黄大庄；

【作者基本信息】 河北农业大学 ， 森林培育， 2009， 博士

【摘要】 应用绿僵菌防治光肩星天牛成虫及幼虫是光肩星天牛综合治理的一条重要途径。为了揭示绿僵菌对光肩星天牛幼虫的致病机理及控制潜能,明确绿僵菌与光肩星天牛幼虫的互作关系,本文以黄粉虫诱集法从土壤中诱集并筛选到对光肩星天牛幼虫具有较高致病力的绿僵菌MS01菌株,并系统研究了其生物学特性、对光肩星天牛的致病性及其影响因子(温度、湿度、传代方式及传代次数)、绿僵菌对光肩星天牛幼虫的入侵和致病过程、光肩星天牛幼虫对绿僵菌侵染的防御反应、光肩星天牛感染绿僵菌后的病理学变化。研究结果如下:以黄粉虫为饵虫从采集的不同土壤样品中诱集分离到8株绿僵菌,并对光肩星天牛幼虫进行生物测定。结果表明,不同菌株对50 d龄光肩星天牛幼虫的致病力存在差异,其中以MS01菌株的致病力最高,校正死亡率和感染率分别为100%和83.33%,致死中时LT50为2.69 d。MS01菌株与市售菌粉经虫体复壮后对95 d龄光肩星天牛幼虫的生物测定结果表明,相对于市售菌株,MS01的复壮菌株致病力更高,至第8天校正死亡率达100%,感染率为90.4%,LT50为5.25 d。MS01菌株对95 d龄光肩星天牛幼虫的致死中浓度LC50为1.68×106孢子·mL-1。因此确认绿僵菌MS01菌株对光肩星天牛幼虫具有较高致病力。通过生物学研究确定了MS01菌株营养生长和产孢的最适培养基为PPDA。该菌株在26℃下营养生长最好,28℃下产孢量最高、孢子萌发速度最快、萌发率最高。该菌株营养生长和产孢的最适相对湿度范围为95%～100%,相对湿度与孢子萌发速度、萌发率呈正相关,相对湿度100%时达到最高。光肩星天牛成虫接种低、中、高三种浓度的绿僵菌MS01菌株后,其产卵量,卵的孵化率和孵化幼虫存活率均明显降低,且接种浓度与产卵量和孵化幼虫存活率呈负相关。MS01菌株对光肩星天牛幼虫产生致病作用的最适温度为26℃,最适湿度为100%。通过PPDA培养基传代2次后对菌株致病力无明显影响,传代4次后会导致菌株致病力的降低;而通过光肩星天牛幼虫传代培养会不同程度提高菌株的致病力。扫描电镜和透射电镜观察表明,绿僵菌侵染光肩星天牛幼虫的主要部位是腹部节间膜,附着分生孢子数量较多、萌发较快、萌发率和侵入率较高;其次是气门附近区域。这两处是利于绿僵菌侵入的薄弱环节。接菌后12 h,分生孢子在光肩星天牛幼虫体表开始萌发并出现芽状突起,随后产生芽管和各种附着结构,接菌后16 h,寄主表皮层中观察到少量菌丝段,至24 h菌丝已普遍侵入,在入侵过程中芽管依靠机械压力和酶的共同作用进行穿透。36 h～48 h分生孢子即可穿透体壁进入血腔。菌丝和菌丝段经血淋巴循环侵入各器官组织。48 h～72 h,随着菌丝和菌丝段在肠壁细胞中迅速大量增殖,肠壁组织逐渐被破坏,中肠微绒毛脱落,肠壁细胞形成空泡,围食膜被分解消失,最终中肠组织崩溃、解体。当幼虫被绿僵菌不同传代菌株接种后,其血淋巴酚氧化酶活性虽起伏变化但全部出现一个高峰值,表明寄主因受到绿僵菌入侵的刺激,其免疫互作使酚氧化酶活性上升至峰值,但随着菌株适应性的逐渐增强而使酚氧化酶活性呈现波动式下降。对接种不同菌株的幼虫血淋巴中酚氧化酶活性的差异显著性分析表明,相对于寄主传代的菌株,经培养基传代的菌株接种光肩星天牛幼虫后引起的血淋巴酚氧化酶活性较高。光肩星天牛幼虫的血细胞对绿僵菌的侵入具有一定的防御能力,参与防御反应的血细胞主要是粒血细胞和浆血细胞,在防卫反应高峰期(48 h～72 h),血细胞总数急剧上升,粒血细胞和浆血细胞比例发生改变,同时血细胞出现粘附、聚集、吞噬、包被、形成结节或囊块等一系列的防御反应。但由于菌丝段在血液中的迅速增殖,血细胞防御机制对绿僵菌只起暂时的阻碍和抑制作用,最终反被其瓦解。光肩星天牛成虫接种低、中、高三种浓度的绿僵菌MS01菌株后,其取食量明显降低,接种浓度与取食量呈负相关。光肩星天牛幼虫感染绿僵菌后,其取食量和体重均低于对照组;其血淋巴蛋白浓度虽波动较剧烈但总体呈不断下降趋势;光肩星天牛幼虫血淋巴中共检测出17种游离氨基酸,感染绿僵菌后不同时段,游离氨基酸总量呈现先下降后上升的变化趋势,不同种类的氨基酸含量变化情况不一致。绿僵菌的侵染引发了光肩星天牛幼虫体内主要保护酶和解毒酶的活力发生了不同程度的改变。在染菌初期超氧化物歧化酶(SOD),过氧化物酶(POD)和过氧化氢酶(CAT)活力迅速提高,在染菌后期SOD和POD活力有不同程度的下降,而CAT活力却上升到较高的水平。羧酸酯酶(CarE)活性变化表现为先上升而后又逐渐下降;谷胱甘肽-S-转移酶(GSTs)活性变化总体表现为激活→抑制→激活→抑制;对乙酰胆碱酯酶(AchE)活力的影响,可归纳为抑制→激活→抑制→激活→抑制。明确了光肩星天牛幼虫感染后组织病理变化及绿僵菌在寄主体内的发展过程。绿僵菌侵入后,首先使寄主表皮层和皮细胞层分离,进入寄主血腔后,随着菌丝在体腔内大量繁殖,受侵染的各器官和组织均发生明显的病变,如脂肪体松散、肌肉组织出现裂缝、气管组织解体破坏、马氏管变形、消化道解体等。更多还原

【Abstract】 Using Metarhizium anisopliae offers an important approach in the integrated management of Anoplophora glabripennis adults and larvae. In order to reveal the pathogenic mechanism of M. anisopliae on A. glabripennis and its application potentials, and also to clarify the interaction between M. anisopliae and A. glabripennis larvae, a M. anisopliae isolate MS01 highly virulent to A. glabripennis larvae was screened from the isolates baited from the soil using“Tenebrio molitor Bait Method”. The biological characteristics, the pathogenicity against A. glabripennis and impact factors to the pathogenicity (temperature, humidity, passage manners and passage times), as well as infection and pathogenicity process of M. anisopliae on A. glabripennis larvae, defense response of the host against M. anisopliae and the pathological changes of A. glabripennis infected by M. anisopliae were studied. The results of this study indicated that:Eight isolates of M. anisopliae were baited and isolated from different soil samples using Tenebrio molitor as bait. Bioassays of the isolates were conducted on A. glabripennis larvae. The results showed that the pathogenicity of the isolates to 50 d A. glabripennis larvae differed from each other and the isolate MS01 was the most virulent one. The corrected mortality, the infection rate and LT50 were 100%, 83.33% and 2.69 d respectively. Furthermore, bioassays of MS01 and commercially available fungus powder were conducted on 95 d Anoplophora glabripennis larvae after insect rejuvenation. The results indicated that the pathogenicity of the rejuvenated strain of MS01 was higher than that of the commercially available strain. Up to the 8th day the corrected mortality, the infection rate and LT50 were 100%, 90.4% and 5.25 d respectively. The LC50 of the MS01 isolate to 95 d Anoplophora glabripennis larvae was defined to be 1.68×106conidia·mL-1. Therefore, M. anisopliae isolate MS01 was confirmed to be more pathogenical to A. glabripennis larvae.The most proper culture media for the mycelia growth and conidia production of isolate MS01 was determined to be PPDA. The strain presented the best nutritional growth at 26℃. Furthermore, the strain showed highest conidia production, fastest conidia germination and highest germination rate at 28℃. The optimal relative humidity for the nutritional growth and conidia production of the isolate was ranged from 95% to 100%. The conidia germinating speed and germination rate were positively correlated to the relative humidity, and reached maximum when relative humidity was 100%.The oviposition amount, the incubation rate and the survival rate of incubated larvae were significantly reduced after A. glabripennis adults had been inoculated by M. anisopliae isolate MS01 in low, medium and high concentrations. The oviposition amount and the survival rate of incubated larvae were negatively correlated to inoculation concentration. The optimal temperature and humidity for the MS01 isolate to be pathogenical to A. glabripennis larvae were 26℃and 100% respectively. There was no significant impact to the pothogenicity of the strain after 2 times passage on PPDA culture media, however, 4 times passage resulted in lower pothogenicity. On the other hand, the pathogenicity of the strains was enhanced at different degrees when the passage had been performed via A. glabripennis larvae.Examination of the invasion process by using scanning and transmission electron microscope showed that M. anisopliae attacked A. glabripennis larvae mainly through abdominal intersegmental membrane, at where more attached conidia, faster germination, higher germination and penetration rates were observed. The next actively intruded area was the area around the valves. The two places were vulnerable to M. anisopliae invasion. Conidia germinated and grew gemmiform protuberance on the cuticle of A. glabripennis larvae within 12 h post-inoculation, then germ tube and various attaching structure were produced. The host cuticle was invaded by a few hyphal bodies 16 h post-inoculation. Up to 24 h hyphae have widely penetrated into host cuticle. The penetration was successful as a result of joint efforts by mechanical pressure and enzymatic activity produced by the germ tube. Conidia penetrated the integument into the haemocoele of A. glabripennis larvae from 36 h to 48 h post-inoculation. The hyphae and hyphal bodies invaded all organs and tissues via hemolymph circulation. Along with the quick and vast reproduction of the hyphae and hyphal bodies in the midgut epithelium, the midgut tissues were gradually destroyed, the midgut microvilli exfoliated, vacuole formed in the midgut epithelium, the peritrophic membrane was decomposed and disappeared, eventually the midgut tissues collapsed and disintegrated from 48 h to 72 h.When the larvae had been inoculated by M. anisopliae strains descended with different passage manners, though the phenoloxidase activity in the haemolymph fluctuated, all tested samples showed a maximum value. It indicated that, when the host was stimulated by M. anisopliae invasion, the immune interaction pushed the phenoloxidase activity to the maximum. However, the phenoloxidase activity decreased in a fluctuated manner along with the gradual enhancement of the adaptability of the invading strains. The analysis of significance of difference was performed on the phenoloxidase activity in the haemolymph of larvae inoculated with different strains. The result showed that, compared to those strains descended through hosts, the strains descended through culture media would trigger higher phenoloxidase activity after inoculated on A. glabripennis larvae.The hemocytes of A. glabripennis larvae were able to resist the invasion of M. anisopliae to some extent. Granulocytes and plasmatocytes were the main hemocytes involved in the defense. During peak defense period (48 h～72 h), the total amount of hemocytes increased sharply, while the ratio of granulocytes and plasmatocytes changed. The defense activities of the hemocytes mainly included adhesion, aggregation, phagocytosis, encapsulation and nodule formation. Due to fast reproduction of the hyphal bodies in the blood, the defense mechanism of the hemocytes inhibited and suppressed the invasion only temporarily, and was eventually overrun by M. anisopliae.The food consumption of A. glabripennis adults remarkably decreased after inoculation with M. anisopliae strain MS01 of low, medium and high concentrations. The food consumption was negatively correlated with inoculation concentration. The food consumption and weight of A. glabripennis larvae were all lower than those of the control after infected by M. anisopliae. The content of the proteins in the hemolymph showed a generally decreasing trend although fluctuated significantly. Total 17 free amino acids were found in the hemolymph of A. glabripennis larvae. During different post-infection periods, the total contents of free amino acids fell first and rose later. The contents of different amino acids fluctuated differently.The activities of the main protective enzymes and detoxifying enzymes changed at different degrees due to invasion of M. anisopliae. During the initial infection stage, the activities of SOD, POD and CAT increased quickly, while during the later stage, the activities of SOD and POD decreased at different degrees but that of CAT rose to a high level. The CarE activity rose first and then gradually decreased. The trend of changes of GSTs activity could be summarized as activated→suppressed→activated→suppressed. The impact on AchE activity could be summarized as suppressed→activated→suppressed→activated→suppressed.The histopathological changes of A. glabripennis larvae after infection and the development process of M. anisopliae in the host body were clarified. The invading M. anisopliae first separated the host cuticle and the epithelium. Along with the vast proliferation of hyphal bodies in the host haemocoel, apparent pathological changes occured at all infected organs and tissues, e.g. fat bodies loosened, cracks appeared in musles, trachea tissue disintegrated, malpighian tubes deformed and digestive tract disintegrated.更多还原