【作者】 王四宝；

【导师】 黄勇平；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 动物学， 2006， 博士

【摘要】 由松材线虫引起的松树萎焉病(Pine wilt disease)已成为我国南方松林最严重的森林病害,给林业生产和生态环境带来了巨大的损失。该病主要借助羽化后的松褐天牛成虫于补充营养和产卵时进行传播。因此,有效地控制松褐天牛的发生,是防治松树萎焉病的关键。由于松褐天牛危害的隐蔽性,化学防治一般难于奏效。因此,利用生物防治和信号化合物引诱方法成为防治松褐天牛的关键措施。然而,这两种方法在利用过程中存在着各自的局限。虽然球孢白僵菌是松褐天牛最重要的病原真菌,在自然界中对天牛也有着非常高的寄生率,但是防治效果不稳定、控制害虫需要较长的时间、单独使用效果往往不佳。人们对白僵菌在自然界的流行动态知之甚少。具有诱集作用的引诱剂已在松褐天牛种群监测和防治中发挥了重要作用,但目前的引诱剂对补充营养期天牛活性较低。为此,在开发松褐天牛补充营养期引诱剂的基础上,为了充分发挥真菌杀虫剂的持续控制作用和引诱剂的引诱聚集作用,我们开展了应用引诱剂与白僵菌联合使用持续控制松褐天牛的研究。本论文还在筛选鉴定白僵菌新型分子标记的基础上,通过野外调查与室内分子分析相结合,借助于群落生态学和分子生态学的理论和方法,系统科学地评价人为引种白僵菌后的环境宿存能力、安全性和将松褐天牛持续控制在低虫口的生态学效应和作用机制。为了开展这项研究,本论文从以下几个方面入手并获得了相应的结果:第一,应用扫描电镜对松褐天牛的触角感器进行扫描观察表明:松褐天牛触角上共有7种感器,即毛形感器、锥形感器、指形感器、棒形感器、瓶形感器、芽形感器以及刺形感器,其中以毛形和锥形感器的数量最丰富。天牛触角对松树主要挥发物的电生理反应表明:松褐天牛成虫触角端部对α-Pinene (500 mg/ml)的EAG (electroantennographic)反应最强(2.3951±0.26 mV)。不同化合物的EAG反应表明:雌、雄成虫对Carene、(+)-α-Pinene、(-)-β-Pinene、Limonene均有较强的EAG反应,与对照(正已烷或空气)有显著性差异。其中,雄成虫对Carene的EAG反应最为强烈,雌成虫对(+)-α-Pinene的EAG反应最强,与其它化合物差异显著。剂量反应测试表明,当α-Pinene浓度在0.0005 mg/ml以下时,EAG反应值没有明显差异,当剂量超过5.0 mg/ml时,EAG反应值随剂量的增加呈直线快速上升趋势。不同化合物组合对松褐天牛的EAG反应表明:松褐天牛成虫触角对(-)α-Pinene + Carene、(-)α-Pinene + (+)-α-Pinene和(-)-α-Pinene + Limone三种二元混合物的EAG反应强烈,而对其余3种二元化合物和松节油的EAG值相对较低,且与单一组分(-)-α-Pinene的EAG值差异不显著。第二,4年林间诱捕结果表明:根据电生理反应所研制的不同引诱剂对松褐天牛都有一定程度的引诱效果,并对其它鞘翅目和半翅目昆虫也有引诱作用。其中,引诱剂Ma02的引诱活性最强,引诱活性显著高于其它8种引诱剂,Ma02诱捕量达到67.6-77.0头/诱捕器,天牛雌雄比为53.26,捕获到的雌成虫的怀卵率仅为61.43%,而引诱剂Ma99-1、Ma03和Ma04怀卵率分别高达100%、96%和96.67%。此外,Ma02诱捕到的雌天牛携带线虫率高达34.8%,雄成虫携带线虫率为25.0%。有效活性距离试验表明,Ma02的有效引诱活性距离至少达到50m,其有效诱捕范围可达0.78公顷。以上结果表明,Ma02不仅具有较高的引诱活性,而且能诱捕到携带线虫并处于补充营养期的天牛成虫。此外,还筛选出了持续引诱周期长达38天的引诱剂缓释器。不同剂量诱捕效果表明,当诱剂达到120 ml/诱捕器时,引诱效果显著增加。自主设计的宣州诱捕器引诱效果显著高于日式诱捕器,分别为36.4头/诱捕器和9.7头/诱捕器。第三,引诱剂与病原菌的联合使用。在松褐天牛幼虫毒力测定的基础上,通过选用不同来源的球孢白僵菌对成虫进行毒力生物测定,最终筛选出高毒力的白僵菌菌株Bb202-1,其校正死率达到86.9%,侵染率为75%。为了比较引诱剂与无纺布菌条不同联合应用方式的控制效果,共设置了5种不同的处理:不放菌区(对照区)、无纺布菌条区、无纺布菌条+引诱剂缓释器联用区、无纺布菌条+诱木联用区和无纺布菌条+引诱剂缓释器+诱木联用区。天牛种群动态监测表明,后两种联合防治区(诱木+无纺布菌条和引诱剂缓释器+诱木+无纺布菌条)对松褐天牛的种群均具有显著的控制作用,虫口密度下降近50%。死树率统计表明,2004年和2005年后两种联合防治区内死树下降率均显著地高于其它防治区(p<0.01), 2004年死树下降率分别达到65.66%和70.56%,2005年两区均无死树发生,死树下降率达到100%,具有明显的控制效果,但两处理间无显著性差异(p>0.05)。无纺布菌条区和无纺布菌条+引诱剂缓释器联用区的防治效果也显著高于对照区(p<0.05),2005年两处理区的死树下降率达到83.33%和76.67%,但两处理间也无显著性差异(p>0.05)。放菌后不同时间从非防治区引入松褐天牛至不同防治处理区进行罩笼饲养,结果表明:所有放菌区内的天牛感染率均与对照区有显著差异(p<0.01),放菌2天后引入第2、3、4、5处理区的天牛感染率在53.33-63.33%,而对照区仅为3.33%;12天后引入的天牛在4个防治区的感染率在30%-40%,对照区感染率为6.67%;22天后引入的天牛感染率为25-35%,对照区感染率为5%。放菌后不同时间对第1、3、4三种处理区内捕捉到的天牛进行罩笼饲养,放菌2天后从第3、4处理区捕捉到的天牛感染率分别为50%和60%,12天后感染率分别为25%和35%,22天后感染率分别为20%和25%。笼内木段上的侵入孔数和幼虫数统计表明:3、4联合防治区诱集天牛罩笼饲养后,木段内的侵入孔数和幼虫数显著低于对照区,其中侵入孔抑制率达到50.02%-54.41%,木段内幼虫数下降率达到25.9%-41.02%。以上结果表明:诱木和无纺布菌条联合应用在防治松褐天牛过程中表现出明显的优势,充分发挥了引诱剂和白僵菌各自的优良特性,实现了优势互补,为持续控制松褐天牛提供了新方法。第四,白僵菌的分子生态学研究。通过选用33个ISSR引物对39株不同来源的白僵菌的种群结构和遗传多样性研究表明:筛选出的18个ISSR引物扩增结果具有高度的多态性和可重复性,在种间和种内不同菌株间具有较高的遗传多样性。18个ISSR引物在39株白僵菌中共扩增出168条带,平均每个引物扩增出9.3个条带,多态性条带有161条,占总条带的95.8%,其中7条是布氏白僵菌Bbr06所特有,14条为多形白僵菌Ba08特有,18条带为粘孢白僵菌Bv01所特有。在36株球孢白僵菌种群内,共扩增出123条带,其中102条(占82.9%)具有多态性,平均每个引物扩增出8.9 (1-13)条带。供试的球孢白僵菌种内遗传相似性系数为0.651-0.972,3种白僵菌种间遗传相似性系数为0.411-0.720,系统聚类结果表明ISSR分子标记能较好地区分形态上相近的3种白僵菌。运用“磁珠富集法”构建了球孢白僵菌Bb202-1的(CA)n、(CT)n、(AAT)n和(ATG)n 4个微卫星富集文库,共得到429个阳性克隆。其中(CA)12探针富集得到的文库中含有177个克隆,(CT)12文库108个克隆,(AAT)10文库72个克隆,(ATG)10文库72个克隆。其中,(CA)12和(CT)12文库阳性率分别高达98%和92.7%,(AAT)10和(ATG)10文库阳性率较低,分别为29.2%和47.2%。通过对165个阳性克隆进行测序后,共得到65个两侧可设计引物的微卫星DNA非冗余序列,富集效率高达39.39%。其中完全型54个,高达83.07%;不完全型或复合型11个,仅占16.93%。65个微卫星中,含有(CA/GT)n和(CT/GA)n类型的分别为20和22个,以(ATG/TAC)n为重复单位的有4个,没有获得(CGC)n类型的微卫星DNA。还获得了1个(A)45、2个(CGC)6、1个(CTCCT)9和1个(CCTCTT)5等模式的微卫星DNA。通过引物设计,PCR产物电泳检测表明,52对SSR引物能扩增出特异的产物,占80%,其中的44对引物的PCR产物经PAGE检测具有多态性,占67.69%,其中的27对具有高度的多态性,占41.54%。5种不同处理区内的土壤带菌量动态检测表明:4个放菌区的带菌量在放菌后的不同时间内明显高于不放菌的对照区,表明释放菌株在土壤中具有较强的宿存能力。白僵菌寄主昆虫群落和当地虫生真菌群落动态研究表明:白僵菌具有丰富的寄主多样性,寄主的丰度为33种,多度为311株,相对多度高达60.04%。但人工接种式放菌后,寄主昆虫多样性指数和虫生真菌群落多样指数均较为稳定,并没有对寄主昆虫群落和虫生菌群落构成威胁,放菌后的第二年多样性指数反而有上升的趋势。通过对释放菌株与放菌前采集的野生菌的基因型比较分析,从27个SSR标记中获得了2个释放菌株特异的SSR标记。通过对放菌后回收菌株的基因型分析,表明释放菌株在土壤和僵虫中均具有较高的回收率。其中,土壤中释放菌株的回收率在16.67%-66.66%之间波动;僵虫中释放菌株的回收率相对较低,在7.69%-82.14%之间,表明释放菌株已在林间宿存和延续。群体遗传多样性分析表明,释放菌株也没有对土壤和僵虫中的白僵菌种群遗传结构造成不良影响。对释放菌株追踪和寄主谱分析表明,释放菌株虽然能感染林间其它19种非目标寄主昆虫,松茸毒蛾、松褐天牛、马尾松角胫象和黑蚁是释放菌株在林间不同时间内实现寄主转移的关键物种,在食物链的传递中处于重要的地位,为维持林间带菌量和保持遗传稳定性提供了保证。因此,释放菌株通过在土壤中营腐生生活,在不同寄主昆虫间通过寄主转移进行宿存和延续,从而达到持续控制害虫的目的,这也正是真菌杀虫剂发挥生态学效应和持续控制的机制所在。更多还原

【Abstract】 The pine wilt disease caused by the pine wood nematode, Bursaphelenchus xylophylus Nickle has been becoming one of the most serious epidemic diseases of the pine (Pinus spp.) forests in the south of China. The pine sawyer, Monochamus alternatus Hope is the most important vector of the pine wood nematode. Because most of its life time is inside the wood, Monochamus alternatus is difficult to control by spraying chemical insecticides. Biological control, therefore, provides a viable alternative for controlling the vector beetle. The entomopathogenic fungus Beauveria bassiana has been considered to be the most important natural pathogen and the most promising biocontrol agent of the pine sawyer. The application of non-woven fiber bands impregnated with B. bassiana cultures onto infested trees have been demonstrated to be one of the most convenient and effective methods.At the adult stage, the beetle will eclose and look for food and location for oviposition. During this procession, the volatile chemicals play important roles in mediating beetles’behavior. Thus, the attractants have been developed to monitor the population dynamics and suppress the adult populations of pine sawyer. In order to integrate the massive natural epizootic potential of B. bassiana and the merits of synergistic function of attractant, the combined applications of mycoinsecticide and semiochemical attractants were studied for sustainable control of the pine sawyer, Monochanus alternatus. Through the developments of ISSR (Inter-Simple Sequence Repeat) and SSR (Simple Sequence Repeat) molecular markers of Beauvera spp., the studies were conducted to investigate the survival capacity, prevalence and persistence course of the released stain in the field as well as its infective rate on the target insect pest. Based on the theories and methods of community ecology and molecular ecology, further analyses were carried out to evaluate the effects of the released strain on local Beauveria population genetic structures and the nontarget insect pests. From the results of this study, systematical evaluations are made based on the factors of environmental safety of release, ecological effects of the introduced strain, and the mechanisms for sustainable control of the Monochamus alternatus by Beauveria bassiana. Our researches including the following aspects:First, with a scanning electron microscope (SEM), 7 types of sensilla of M. alternatus were observed: Sensilla trichoid, sensilla basiconica, sensilla digit-like, sensilla rod-like, sensilla bottle-like, sensilla bud-like, sensilla chaetica. Overall, Sensilla trichoid and sensilla basiconica are the most abundant sensilla on the antenna surface. The data of electroantennographic (EAG) responses of different parts of the antennae toα-Pinene indicated that in contrast to the basal part, the apical part had the strongest response (2.3951±0.26mV) EAG value was significantly increased at the dose of more than 5 mg/ml. Carene could provoke the most significant EAG response by the male beetles while the females have the strongest response to Rα-Pinene. In general, the findings showed that the mixture of compounds Rα-Pinene and carene elicited the strongest EAG responses by both the male and female beetles.Second, the field trapping in four years showed that different attractant formulae had varied degree of trapping ability to pine sawyer, and some of them could also attract other species of Coleopteran or Hemipteran insects. Among them, the formula Ma02 showed the significantly higher attractancy than other 8 tested attractants with the mean capturing effect was 67.6-77.0 individuals per trap. The percentage of female beetles with pregnant eggs captured by Ma02 was 61.43%, and was significantly lower than those of Ma99-1(100%), Ma03(96.0%) and Ma04(96.67%). The percentage of the beetles carring nematode captured by Ma02 was 25.0% for the male, and 34.8% for the female. The observation of trapping capacity indicated that the effective distance ranged from 20-50m by Ma02 but significantly reduced when beyond 70m. The effectively attractive area could be up to 0.78 ha. The above results showed that the attractant Ma02 could attract not only the ovipositing stage of female adults, but also the nutrition replenishing stage beetles. The attractiveness of three different attractant dispensers to Monochamus alternatus adults was also investigated. The attractant dispenser B (specified control dispensing plastic bottle) had comparatively stronger effect, could be used for up to 38 days. The attractiveness of different dose of attractant showed that the efficiency enhanced with the increase of attractant volume. The effect was not significantly enhanced when the dosage of attractant was increased from 20 ml to 80 ml per bottle, but was significantly improved when the dosage reached up to 120 ml. As for trap, Xuanzhou trap designed by us was superior to the imitated Japanese trap, the trapping efficiency was 36.4 individuals/trap, 9.7 individuals/trap, respectively.Third, combined application of attractants and fungi. The virulence of different entomogenous fungi on adult pine sawyers, Monochamus alternatus was tested. Beauveria bassiana strain Bb202-1 was the highest virulent, with sawyer mortality up to 86.9%, infection rate to 75%, and LT50 at 6.77d. In order to compare the efficacy of different combined applications with control, Five treatments were conducted: (1) control (CK); (2) treated with non-woven fiber bands impregnated with Bb202-1 alone (NFB); (3) fungal bands combined with attractant (NFB+Attractant); (4) fungal bands combined with bait tree (NFB+bait tree); and (5) fungal bands combined with attractant and bait tree (NFB+Attractant+bait tree). Relative population dynamics of Monochamus alternatus captured by attractant-baited trap were monitored for five treatments every four days. The results showed that the treatments of NFB+bait tree and NFB+Attractant+bait tree had obviously increased controlling effects, the relative beetle population densities all declined by nearly 50%. The pine tree mortality in plots using five different treatments against the Monochamus alternatus were investigated in years 2004 and 2005. The results showed that the decrease rates of dead pine trees in the treatment plots of NFB+bait tree and NFB+Attractant+bait tree was 65.66 and 70.56% in 2004, respectively and no dead pine tree was found in 2005. The tree mortality was decreased by 100%, which were significantly higher than the other treatments. But there was no significant difference between the NFB+bait tree and NFB+Attractant+bait tree plots. The efficacy of the NFB and NFB+Attractant was also significantly higher than that of control, the tree mortalities were decreased by 83.33% and 76.67% in 2005, respectively. The cage experiment by importing the adult beetles and rearing inside the treatment plots showed that the mean percentage infection rates of the imported beetles in 4 fungal bands treated plots were significantly different from the control, but the infection declined with time. The infection rates in 4 fungal bands treated plots were ranged at 53.33-63.33% 2 days after bands application, 30-40% for 12th day, 25-35% for 22th day, but the highest infection of untreated control was only 6.67%. The cage experiment by exporting the adult beetle and rearing outside the treatment plots showed that the infections of the exported beetles from NFB+Attractant and NFB+bait tree were significantly higher than those of the control at 2, 12, and 22 days after band application. The mean percentage of infection of the beetles exported from NFB+Attractant and NFB+bait tree was 50% and 60% at 2th day, 25% and 35% at 12th day, 20% and 25% at 22th day, respectively. The anatomization of log section in the cage showed that the number of invading holes and larvae of the treatment of NFB+Attractant and NFB+bait tree were decreased by 50.02-54.41% and 25.9-41.02%, respectively, which were significantly lower than that of the control. The above results showed that the combined applications of fungal bands and semiochemical attractant evidently demonstrated synergistic effects by complementing each other’s disadvantages to offer the great potential for controlling Monochamus alternatus.Fourth, the molecular ecology study using molecular markers. ISSR markers were used to investigate genetic diversity among 39 isolates of Beauveria spp. (36 B. bassiana, one B. brongniartii, one B. amorpha, and one B. velata) isolated from different insect hosts and geographical origins. Eighteen of 33 primer pairs harboring different SSR were chosen for their good reproducibility and the generation of high polymorphisms. In total, one hundred and sixty-eight highly reproducible fragments were amplified from all 39 isolates with an average yield of 9.3 markers per primer pair. Among these, 161 (95.8%) were polymorphic among strains. For 36 B. bassiana isolates, 8.9 (1-13) markers per primer were acquired, and the total of 123 fragments were amplified, in which 102 (82.9%) were polymorphic. Among the 168 polymorphic bands, 7 bands were considered to be specific for B. brongniartii isolate Bbr06, 14 bands for B. amorpha isolate Ba08, and 18 bands for B. velata isolate Bv01. Within 36 B. bassiana isolates, genetic similarity ranged from 0.651 to 0.972. However, the genetic similarity values among different Beauveria species ranged from 0.411 to 0.720, suggesting that ISSR technique was successful in differentiating the 3 closely related species from B. bassiana. The results also indicated that there was a positive association between the genotypes of B. bassiana isolates and their geographical origins, but no clear correlation with their insect hosts.Four enriched microsatellite libraries, (CA)n, (CT)n, (AAT)n and (ATG)n were constructed for Beauveria bassiana strain Bb202-1 by using magnetic beads. In total, 429 positive clones were obtained. Of which, 177 clones were for the library constructed by probing with (CA)12 , 108 clones for (CT)12 probe, 72 clones for (AAT)10 probe, and 72 clones for (ATG)10 probe. PCR detection showed that the percentages of positive clones in library (CA)12 (98%) and (CT)12 (92.7%) were higher than those of other two libraries. The percentages of positive clones in library (AAT)10 and (ATG)10 were 29.2% and 47.2%, respectively. Of 165 sequenced positive clones, 65 could successfully provide working primer pairs, accounting for 30% of the total number. Of 65 loci isolated, 54 were perfect (83.07%) and the rest were imperfect (16.93%). Among those, 52 loci could yield amplified products with expected sizes, and of these, 44 amplified SSR products were polymorphic by PAGE analysis, and 27 of them were highly polymorphic, accounting for 41.54%. Investigation of the population dynamics of B. bassiana surviving in soil with a selective dodine-oat medium showed that the inoculum density of B. bassiana in soil of the 4 fungal treatments plots was higher than that of untreated control plot, which suggested that the released strain had higher ability of colonizing in the soil of pine ecosystem. The estimates of community biodiversities of insect hosts and entomopathogenic fungi for different periods showed that B. bassiana had abundant diversity of the insect hosts where the species richness of insect hosts was up to 33, the abundance was 311 and the relative abundance was 60.14%. After fungal releases with an inoculative application strategy, the community diversity index of both insect hosts and entomopathogenic fungi were relatively stable, and did not pose any threat to community diversity of both insect hosts and entomopathogenic fungi. On the contrary, the diversity indexes were increased at the second year after application.Genotyping analysis of B. bassiana strains collected before and after fungal release showed that 2 out of 27 polymorphic SSR markers were specific to the released strain. The detection of recovery showed that the released strain had the higher recovery rates from both the soil and cadaver samples. The recovery rates ranged from 16.67%-66.66% for soil samples and 7.69%-82.14% for dead insects, indicating that the released strain had successfully colonized and dispersed in the pine ecosystem. Analysis of population genetic structure revealed that the artificial releasing strain in an inoculative way also did not impact the genetic diversity of indigenous B. bassiana populations. Analysis of host species infected by the released strain showed that the released strain could also invade 19 species of non-target insect hosts at different seasons. Of those, the species of Dasychira axutha, Monochamus alternatus, Shirahoshizo patruelis, and Lasius niger played crucial roles for the released strain to transfer among different food chains and provided an assurance for maintaining the stability of inoculum density and genetic diversity in the pine ecosystem. In short, the released strain could colonize and persist in the pine ecosystem by surviving as a saprophyte in soil and jumping among different insect hosts to maintain the sustainable control effect against the beetles in the field.更多还原