苏云金杆菌和麦蛾柔茧蜂对印度谷螟的联合控制作用研究
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摘要
许多发展中国家都面临着严重的粮食短缺问题。而这些国家又不能很好的防治在粮食储存过程中发生的微生物、脊椎动物以及昆虫。据估计,在世界范围内每年由于昆虫为害而造成的损失为粮食储藏量的5%。印度谷螟Plodiainterpunctella(Hübner)以幼虫为害各种粮食及其加工品、豆类、油料、干果、干菜、奶粉、蜜饯果品、烟叶、药材等中成药,是极重要的初期性害虫,分布世界各地,其防治仍然依赖化学农药,近年尽管有部分试用苏云金杆菌和天敌昆虫防治,但又有报道印度谷螟同样能对苏云金杆菌产生抗性。目前还没有苏云金杆菌和天敌联合控制印度谷螟的研究报道。为更好利用印度谷螟生物防治因子,我们开展本研究,主要研究内容和结果如下:
本研究通过筛选获得对印度谷螟高毒力苏云金杆菌,并分析了其杀虫晶体蛋白及其基因型组成。首次系统研究了印度谷螟幼虫龄期与食料庇护对麦蛾柔茧蜂(Habrobracon hebetor Say)寄生率的影响,考察了苏云金杆菌和麦蛾柔茧蜂对印度谷螟的联合控制作用。分析了苏云金杆菌对天敌麦蛾柔茧蜂的影响。同时还研究了麦蛾柔茧蜂的搜索行为与利它素。这些研究结果将有利于阐明苏云金杆菌对印度谷螟的控制潜力,不仅为印度谷螟的生物防治提供高毒力苏云金杆菌,而且为制定印度谷螟生物防治方案奠定理论基础。因此,本研究对印度谷螟的生物防治具有重要的科学意义和实践意义。
通过对在封闭环境中,麦蛾柔茧蜂对印度谷螟寄生过程中不同营养水平和寄主所处位置相互作用的研究,评估了食料庇护、寄主龄期及其两者互作对印度谷螟死亡率和麦蛾柔茧蜂羽化率的影响。结果表明,麦蛾柔茧蜂能寄生所有龄期的印度谷螟幼虫(1～4龄),但低龄(1龄和2龄)幼虫被寄生的概率显著低于高龄(3龄和4龄),3龄和4龄幼虫之间被寄生的概率没有显著性差异。在无选择条件下,24和48小时后只有龄期对麦蛾柔茧蜂的寄生率有显著的影响,而食料庇护及其两者互作对麦蛾柔茧蜂的寄生率无显著的影响。但是在选择条件下,24和48小时后龄期和食料庇护对麦蛾柔茧蜂的寄生率都有显著的影响。一般来说,低龄(1龄和2龄)幼虫在无选择条件下的被寄生率显著高于选择条件下的被寄生率。寄生率随着寄生时间的延长而升高,但4龄幼虫的寄生率无论何时都比其他龄期幼虫的寄生率高。在有食料庇护的情况下,印度谷螟幼虫的被寄生率较低。食料庇护和龄期的交互作用没有显著性差异说明,无论有无食料庇护,寄生率都随着虫龄的增长而升高。不同处理间,麦蛾柔茧蜂后代羽化的性比没有显著差异。无论何种处理,在有选择条件下的寄生蜂后代羽化数都比在无选择条件下的高。从上述结果可以看出,麦蛾柔茧蜂具有选择性和专一性的寄生行为,为麦蛾柔茧蜂应用释放提供理论依据。
通过初步的生物测定,我评估了收集到的122株苏云金杆菌(Bt)对印度谷螟的毒性,筛选获得3株比标准株(B.thuringiensis subsp.kurstaki HD-1)对印度谷螟幼虫具有更强毒力的分离株。结果显示全部的Bt分离株能引起印度谷螟58.2%的死亡率,其中,13种分离株能引起90-100%的死亡率。结果,具有最高毒力(引起印度谷螟100%死亡率)的分离株属于H_7,同时最低毒力的分离株属于H_8,相同血清型的不同分离株对印度谷螟的毒力存在差异。在对122种Bt分离株的研究中,IMM-130,IMM-368和IMM-408这三种分离株对防治印度谷螟具高毒力(df=3,f=48.35,P＜0.001),它们的LC_(50)低于标准菌株HD-1。在标准菌株HD-1的LC_(50)是8.48μg/ml的情况下,IMM-408、IMM-130和IMM-368的LC_(50)分别为1.24、2.86和4.62μg/ml。其中以IMM-408的毒力最强。SDS-PAGE分析结果表明,和标准Bt菌株HD-1一样,IMM-130和IMM-368均包含135 kDa和65kDa的杀虫晶体蛋白,而IMM-408包含135kDa晶体蛋白,包括crylAb9,crylCal,crylDal和cry2基因。血清学检测表明IMM-130、IMM-368和IMM-408分别属于H_3、H_4和H_7血清型。
本研究评估了Bt和麦蛾柔茧蜂对印度谷螟的联合控制作用。Bt和麦蛾柔茧蜂引起印度谷螟幼虫的死亡率分别为41.67%和35.35%。Bt和麦蛾柔茧蜂联合使用时能引起86%的校正死亡率,显著高于其它的处理。Bt感染影响麦蛾柔茧蜂产卵和后代的发育,它在正常寄主上产的卵显著多于在被Bt感染的寄主上产的卵,用Bt和天敌联合防治后寄主产生的寄生蜂数少于不用Bt处理寄主产生的寄生蜂数。联合防治试验中麦蛾柔茧蜂幼虫的死亡率显著高于对照,产生的麦蛾柔茧蜂后代个体显著小于对照,但用联合防治和对照的麦蛾柔茧蜂后代发育历期没有显著差异,后代成虫的寿命也没有显著差异。对照处理的后代性比1:2.27(雄性:雌性),显示雌性较多,而联合防治处理的后代性比也有相同的趋势。研究结果显示用Bt和寄生蜂联合防治印度谷螟比任何一种单一防治效果要好。
最后,研究了麦蛾柔茧蜂雌虫对干净食料、幼虫取食过的食料、幼虫以及幼虫排泄物提取物的反应。结果显示,正己烷提取物能引起麦蛾柔茧蜂的寄生行为。幼虫排泄物提取物比其它提取物能显著刺激更多的寄生行为。寄生经验在寄生蜂寻觅寄主的过程中发挥了巨大作用,也就是和寄主接触过的寄生蜂雌虫对滤纸片的反映会比没有寄生经验的雌虫强烈。正己烷比丙酮、乙醚、甲醇更易溶解与印度谷螟相关物质的活性组分。用硅胶柱层析法分馏幼虫和幼虫排泄物的粗提物,各馏分再用麦蛾柔茧蜂雌虫检测,最能引起麦蛾柔茧蜂的寄生行为的馏分用气质联用(GC-MS)分析,两种馏分都有三个主要的波峰,结果显示提取物主要含有碳氢化合物。两种提取物的碳氢化合物都主要是饱和的长烷烃链(1,3二甲基苯、2-acyl-1,3-cyclohexanediones)。起作用的馏分中还包括其他的化学物质:甲苯、异三十烷、乙基环己烷、二十九烷和十六烷。这些结果显示,这些成分包含能引起麦蛾柔茧蜂搜寻和寄生行为的利他素类化学物质,而且这些混合物(利它素)将来可能被合成,并用于储藏物表面以提高麦蛾柔茧蜂的寄生率等印度谷螟生物防治实践。
Many developing countries are confronted with acute food shortage,and this could probably be linked to their inability to protect and preserve their food produce from quality and quantity deterioration that result from microbial,vertebrate and insect pest infestation.The annual losses incurred in storage due to insect attack are estimated as 5%worldwide.Plodia interpunctella Hübner(Lepidoptera:Pyralidae) is a cosmopolitan insect infesting a broad range of commodities,including raw or processed cereal.
The key objectives of this research were to:a.screen and evaluate some Bacillus thuringiensis isolates against P.interpunctella and determine the LC_(50) of highly toxic strains,b.to evaluate the role of host-instar and refuge on the parasitization behavior of Habrobracon hebetor Say(Hymenoptera:Braconidae),c. Investigate the effect of combining B.thuringiensis with H.hebetor for management of P.interpunctella infestation and to assess the influence of B.thuringiensis on H. hebetor during this combination treatment,d.investigate the host-finding behavior in H.hebetor,by extracting and identifying kairomone from P.interpunctella responsible for eliciting parasitization behavior in the parasitoid.
A study was conducted to examine the interactions between trophic levels during parasitism and host location by H.hebetor within a closed environment,by carrying out multiple tests to evaluate the role of refuge and host-instar on the mortality of P.interpunctella and on the emergence of H.hebetor.Results showed that H.hebetor was able to parasitize all(first-fourth) instars of P.interpunctella,but significantly fewer early(first and second) instars were parasitized.Interaction among instar x refuge had no significant effect on parasitism.In no-choice condition,only instar factor was observed to have any significant effect on parasitism at both 24 h and 48 h post treatment.While under choice condition:instar,refuge and not interaction abetween them,significantly affected parasitism by the wasp at both 24 h and 48 h post treatment.Generally,the percentage of early(first and second) instars parasitized was higher under no-choice condition than parasitism observed for the same larvae stages in choice condition.Parasitism appeared to increase in conjunction with the increase in exposure time.Significantly more fourth instar larvae were parasitized than other larvae from the other stages irrespective of the duration of exposure.Fewer hosts were parasitized in the presence of refuge as opposed to the higher parasitism observed in the absence of refuge.The non significant interaction between instar x refuge explained that,irrespective of the presence of refuge or not,percentage of host parasitized by H.hebetor also increased with increase in larval stage.Differences between male and female emergences appeared non significant.Results also revealed that only host-instar significantly affected parasitoid yield.It was however noted that parasitoid emergence was higher under no-choice condition than in choice.There was no significant difference between the parasitoid yield of third and fourth instar larvae. Based on the results from this investigation,H.hebetor could therefore be described as a weak competitor with an opportunistic and specialized behavior,thereby giving more insight to its ecology and biology.
Through preliminary bioassays,the toxicity of a collection of 122 isolates of B. thuringiensis were evaluated against P.interpunctella.Afterwards,3 strains that demonstrated significantly greater potency than the standard strain(B.thuringiensis serovar kurstaki HD-1) against Lepidoptera were selected and analyzed in detail. Results indicated that 58.2%of the entire B.thuringiensis isolates evoked≥60% mortality to P.interpunctella and were thus considered toxic.Among these,13 isolates were distributed within the 90%to 100%mortality groups.The results also showed that different isolates belonging to the same serotype had different toxic level to P.interpunctella.The highest frequency of toxicity(causing 100%mortality of Indianmeal moth) was observed in isolates belonging to H_7,while the lowest frequency of toxicity was recorded in isolates belonging to H_8.Among the 122 B. thuringiensis isolates evaluated,3 three isolates exhibited a significantly(df=3,f= 48.35,P＜0.001) greater potency against P.interpunctella,with their LC_(50) lower than the standard strain HD-1.Treatments with spore-crystal mixture caused higher larval mortality than soluble protein.The LC_(50) s for these 3 isolates IMM-408,IMM-130 and IMM-368 are 1.24,2.86 and 4.62μg/ml respectively while that of isolate HD-1 was 8.48μg/ml.Of the 3 highly toxic strains,IMM-408 was found to be most toxic. SDS-PAGE of 3 isolates that yielded 100%mortality against P.interpunctella indicated that both isolates IMM-130 and IMM-368 contained～135 kDa and 65 kDa crystal proteins,similar to standard B.thuringiensis strain HD-1,while isolate IMM-408 contained～135 kDa crystal protein,and contained cry1Ab9,cry1Ca1,cry1Da1 and cry2 gene.The serological tests conducted showed that isolates IMM-130,IMM-368 and IMM-408 belong to serotypes H_3,H_4 and H_7 respectively
Furthermore,the suitability of combining microbial pesticides and natural enemies for integrated pest management(IPM) of stored cereal in China was evaluated using laboratory assays.This was achieved by studying the interactions between B.thuringiensis,B.thuringiensis-intoxicated host larvae and H.hebetor during control of P.interpunctella.B.thuringiensis or H.hebetor alone caused 41.67%and 35.35%P.interpunctella larval mortality respectively.The B. thuringiensis-parasitoid combination treatment significantly evoked more P. interpunctella mortality(86%) than other treatments.Progeny development of H. hebetor was dependent upon its susceptibility to B.thuringiensis contaminated host. However,more eggs were significantly laid by H.hebetor on hosts placed in control (257 eggs) than on hosts in B.thuringiensis contaminated diets(93 eggs).Hatchability of H.hebetor eggs were not affected in all treatments,data between treatment and control did not vary significantly.Fewer wasps emerged from B.thuringiensis-parasitoid combine treatment than in none B.thuringiensis treatments.H.hebetor larval mortality was significantly higher in combine treatments than in control. Although the size of adult parasitoids that emerged from combine treatments appeared smaller than those in the control,their developmental period was not significantly different.Similarly,no significant difference was observed in the longevity of adult wasps emerging from both treatments.The sex ratio of H.hebetor progeny in control experiment was female biased in the ratio of 1:2.27 male:female and this trend is similar to the observations in combine treatment Therefore,since B.thuringiensis did not prevent parasitoid development,this investigations is suggestive that a combine treatment with B.thuringiensis and parasitoid release,would produce better protection against P.interpunctella.
Lastly,the response of gravid female H.hebetor to extracts from clean diet, infested diet,larvae and frass of P.interpunctella was investigated.Result revealed that acetone and hexane extracts elicited searching behavior in H.hebetor.Extracts of frass significantly stimulated more parasitization behavior in the parasitoid than extracts of other P.interpunctella-related materials tested.It was also noted that experience plays a huge role in parasitoid foraging efficiency.Females that had previous contact with host produced stronger responses towards treated patch,than their inexperienced counterparts.The active components in P.interpunctella-related materials were more soluble in hexane,than in acetone,ethyl ether or methanol. Using silica gel flash column chromatography,crude extracts of larva and frass were fractionated,then the response of H.hebetor to each fraction was evaluated and most active fractions were analyzed by gas chromatography-mass spectrometry(GC-MS). Three main peaks were observed on the GC of both fractions.Result showed that extracts contained hydrocarbons.The hydrocarbons which are mainly saturated long chain alkanes(1,3-dimethyl benzene,2-acyl-1,3-cyclohexanediones) are present in both extracts.Other chemicals extracted from the active fractions are toluene, squalane,ethyl cyclohexane,nonacosane and hexadecane.These results showed that P.interpunctella-related materials that were tested contained infochemicals which elicits searching/ parasitization behavior in the parasitoid H.hebetor.And these compounds(kairomone),may be synthesized and applied to surface of stored produce in other to enhance H.hebetor parasitization performance.
本研究通过筛选获得对印度谷螟高毒力苏云金杆菌,并分析了其杀虫晶体蛋白及其基因型组成。首次系统研究了印度谷螟幼虫龄期与食料庇护对麦蛾柔茧蜂(Habrobracon hebetor Say)寄生率的影响,考察了苏云金杆菌和麦蛾柔茧蜂对印度谷螟的联合控制作用。分析了苏云金杆菌对天敌麦蛾柔茧蜂的影响。同时还研究了麦蛾柔茧蜂的搜索行为与利它素。这些研究结果将有利于阐明苏云金杆菌对印度谷螟的控制潜力,不仅为印度谷螟的生物防治提供高毒力苏云金杆菌,而且为制定印度谷螟生物防治方案奠定理论基础。因此,本研究对印度谷螟的生物防治具有重要的科学意义和实践意义。
通过对在封闭环境中,麦蛾柔茧蜂对印度谷螟寄生过程中不同营养水平和寄主所处位置相互作用的研究,评估了食料庇护、寄主龄期及其两者互作对印度谷螟死亡率和麦蛾柔茧蜂羽化率的影响。结果表明,麦蛾柔茧蜂能寄生所有龄期的印度谷螟幼虫(1～4龄),但低龄(1龄和2龄)幼虫被寄生的概率显著低于高龄(3龄和4龄),3龄和4龄幼虫之间被寄生的概率没有显著性差异。在无选择条件下,24和48小时后只有龄期对麦蛾柔茧蜂的寄生率有显著的影响,而食料庇护及其两者互作对麦蛾柔茧蜂的寄生率无显著的影响。但是在选择条件下,24和48小时后龄期和食料庇护对麦蛾柔茧蜂的寄生率都有显著的影响。一般来说,低龄(1龄和2龄)幼虫在无选择条件下的被寄生率显著高于选择条件下的被寄生率。寄生率随着寄生时间的延长而升高,但4龄幼虫的寄生率无论何时都比其他龄期幼虫的寄生率高。在有食料庇护的情况下,印度谷螟幼虫的被寄生率较低。食料庇护和龄期的交互作用没有显著性差异说明,无论有无食料庇护,寄生率都随着虫龄的增长而升高。不同处理间,麦蛾柔茧蜂后代羽化的性比没有显著差异。无论何种处理,在有选择条件下的寄生蜂后代羽化数都比在无选择条件下的高。从上述结果可以看出,麦蛾柔茧蜂具有选择性和专一性的寄生行为,为麦蛾柔茧蜂应用释放提供理论依据。
通过初步的生物测定,我评估了收集到的122株苏云金杆菌(Bt)对印度谷螟的毒性,筛选获得3株比标准株(B.thuringiensis subsp.kurstaki HD-1)对印度谷螟幼虫具有更强毒力的分离株。结果显示全部的Bt分离株能引起印度谷螟58.2%的死亡率,其中,13种分离株能引起90-100%的死亡率。结果,具有最高毒力(引起印度谷螟100%死亡率)的分离株属于H_7,同时最低毒力的分离株属于H_8,相同血清型的不同分离株对印度谷螟的毒力存在差异。在对122种Bt分离株的研究中,IMM-130,IMM-368和IMM-408这三种分离株对防治印度谷螟具高毒力(df=3,f=48.35,P＜0.001),它们的LC_(50)低于标准菌株HD-1。在标准菌株HD-1的LC_(50)是8.48μg/ml的情况下,IMM-408、IMM-130和IMM-368的LC_(50)分别为1.24、2.86和4.62μg/ml。其中以IMM-408的毒力最强。SDS-PAGE分析结果表明,和标准Bt菌株HD-1一样,IMM-130和IMM-368均包含135 kDa和65kDa的杀虫晶体蛋白,而IMM-408包含135kDa晶体蛋白,包括crylAb9,crylCal,crylDal和cry2基因。血清学检测表明IMM-130、IMM-368和IMM-408分别属于H_3、H_4和H_7血清型。
本研究评估了Bt和麦蛾柔茧蜂对印度谷螟的联合控制作用。Bt和麦蛾柔茧蜂引起印度谷螟幼虫的死亡率分别为41.67%和35.35%。Bt和麦蛾柔茧蜂联合使用时能引起86%的校正死亡率,显著高于其它的处理。Bt感染影响麦蛾柔茧蜂产卵和后代的发育,它在正常寄主上产的卵显著多于在被Bt感染的寄主上产的卵,用Bt和天敌联合防治后寄主产生的寄生蜂数少于不用Bt处理寄主产生的寄生蜂数。联合防治试验中麦蛾柔茧蜂幼虫的死亡率显著高于对照,产生的麦蛾柔茧蜂后代个体显著小于对照,但用联合防治和对照的麦蛾柔茧蜂后代发育历期没有显著差异,后代成虫的寿命也没有显著差异。对照处理的后代性比1:2.27(雄性:雌性),显示雌性较多,而联合防治处理的后代性比也有相同的趋势。研究结果显示用Bt和寄生蜂联合防治印度谷螟比任何一种单一防治效果要好。
最后,研究了麦蛾柔茧蜂雌虫对干净食料、幼虫取食过的食料、幼虫以及幼虫排泄物提取物的反应。结果显示,正己烷提取物能引起麦蛾柔茧蜂的寄生行为。幼虫排泄物提取物比其它提取物能显著刺激更多的寄生行为。寄生经验在寄生蜂寻觅寄主的过程中发挥了巨大作用,也就是和寄主接触过的寄生蜂雌虫对滤纸片的反映会比没有寄生经验的雌虫强烈。正己烷比丙酮、乙醚、甲醇更易溶解与印度谷螟相关物质的活性组分。用硅胶柱层析法分馏幼虫和幼虫排泄物的粗提物,各馏分再用麦蛾柔茧蜂雌虫检测,最能引起麦蛾柔茧蜂的寄生行为的馏分用气质联用(GC-MS)分析,两种馏分都有三个主要的波峰,结果显示提取物主要含有碳氢化合物。两种提取物的碳氢化合物都主要是饱和的长烷烃链(1,3二甲基苯、2-acyl-1,3-cyclohexanediones)。起作用的馏分中还包括其他的化学物质:甲苯、异三十烷、乙基环己烷、二十九烷和十六烷。这些结果显示,这些成分包含能引起麦蛾柔茧蜂搜寻和寄生行为的利他素类化学物质,而且这些混合物(利它素)将来可能被合成,并用于储藏物表面以提高麦蛾柔茧蜂的寄生率等印度谷螟生物防治实践。
Many developing countries are confronted with acute food shortage,and this could probably be linked to their inability to protect and preserve their food produce from quality and quantity deterioration that result from microbial,vertebrate and insect pest infestation.The annual losses incurred in storage due to insect attack are estimated as 5%worldwide.Plodia interpunctella Hübner(Lepidoptera:Pyralidae) is a cosmopolitan insect infesting a broad range of commodities,including raw or processed cereal.
The key objectives of this research were to:a.screen and evaluate some Bacillus thuringiensis isolates against P.interpunctella and determine the LC_(50) of highly toxic strains,b.to evaluate the role of host-instar and refuge on the parasitization behavior of Habrobracon hebetor Say(Hymenoptera:Braconidae),c. Investigate the effect of combining B.thuringiensis with H.hebetor for management of P.interpunctella infestation and to assess the influence of B.thuringiensis on H. hebetor during this combination treatment,d.investigate the host-finding behavior in H.hebetor,by extracting and identifying kairomone from P.interpunctella responsible for eliciting parasitization behavior in the parasitoid.
A study was conducted to examine the interactions between trophic levels during parasitism and host location by H.hebetor within a closed environment,by carrying out multiple tests to evaluate the role of refuge and host-instar on the mortality of P.interpunctella and on the emergence of H.hebetor.Results showed that H.hebetor was able to parasitize all(first-fourth) instars of P.interpunctella,but significantly fewer early(first and second) instars were parasitized.Interaction among instar x refuge had no significant effect on parasitism.In no-choice condition,only instar factor was observed to have any significant effect on parasitism at both 24 h and 48 h post treatment.While under choice condition:instar,refuge and not interaction abetween them,significantly affected parasitism by the wasp at both 24 h and 48 h post treatment.Generally,the percentage of early(first and second) instars parasitized was higher under no-choice condition than parasitism observed for the same larvae stages in choice condition.Parasitism appeared to increase in conjunction with the increase in exposure time.Significantly more fourth instar larvae were parasitized than other larvae from the other stages irrespective of the duration of exposure.Fewer hosts were parasitized in the presence of refuge as opposed to the higher parasitism observed in the absence of refuge.The non significant interaction between instar x refuge explained that,irrespective of the presence of refuge or not,percentage of host parasitized by H.hebetor also increased with increase in larval stage.Differences between male and female emergences appeared non significant.Results also revealed that only host-instar significantly affected parasitoid yield.It was however noted that parasitoid emergence was higher under no-choice condition than in choice.There was no significant difference between the parasitoid yield of third and fourth instar larvae. Based on the results from this investigation,H.hebetor could therefore be described as a weak competitor with an opportunistic and specialized behavior,thereby giving more insight to its ecology and biology.
Through preliminary bioassays,the toxicity of a collection of 122 isolates of B. thuringiensis were evaluated against P.interpunctella.Afterwards,3 strains that demonstrated significantly greater potency than the standard strain(B.thuringiensis serovar kurstaki HD-1) against Lepidoptera were selected and analyzed in detail. Results indicated that 58.2%of the entire B.thuringiensis isolates evoked≥60% mortality to P.interpunctella and were thus considered toxic.Among these,13 isolates were distributed within the 90%to 100%mortality groups.The results also showed that different isolates belonging to the same serotype had different toxic level to P.interpunctella.The highest frequency of toxicity(causing 100%mortality of Indianmeal moth) was observed in isolates belonging to H_7,while the lowest frequency of toxicity was recorded in isolates belonging to H_8.Among the 122 B. thuringiensis isolates evaluated,3 three isolates exhibited a significantly(df=3,f= 48.35,P＜0.001) greater potency against P.interpunctella,with their LC_(50) lower than the standard strain HD-1.Treatments with spore-crystal mixture caused higher larval mortality than soluble protein.The LC_(50) s for these 3 isolates IMM-408,IMM-130 and IMM-368 are 1.24,2.86 and 4.62μg/ml respectively while that of isolate HD-1 was 8.48μg/ml.Of the 3 highly toxic strains,IMM-408 was found to be most toxic. SDS-PAGE of 3 isolates that yielded 100%mortality against P.interpunctella indicated that both isolates IMM-130 and IMM-368 contained～135 kDa and 65 kDa crystal proteins,similar to standard B.thuringiensis strain HD-1,while isolate IMM-408 contained～135 kDa crystal protein,and contained cry1Ab9,cry1Ca1,cry1Da1 and cry2 gene.The serological tests conducted showed that isolates IMM-130,IMM-368 and IMM-408 belong to serotypes H_3,H_4 and H_7 respectively
Furthermore,the suitability of combining microbial pesticides and natural enemies for integrated pest management(IPM) of stored cereal in China was evaluated using laboratory assays.This was achieved by studying the interactions between B.thuringiensis,B.thuringiensis-intoxicated host larvae and H.hebetor during control of P.interpunctella.B.thuringiensis or H.hebetor alone caused 41.67%and 35.35%P.interpunctella larval mortality respectively.The B. thuringiensis-parasitoid combination treatment significantly evoked more P. interpunctella mortality(86%) than other treatments.Progeny development of H. hebetor was dependent upon its susceptibility to B.thuringiensis contaminated host. However,more eggs were significantly laid by H.hebetor on hosts placed in control (257 eggs) than on hosts in B.thuringiensis contaminated diets(93 eggs).Hatchability of H.hebetor eggs were not affected in all treatments,data between treatment and control did not vary significantly.Fewer wasps emerged from B.thuringiensis-parasitoid combine treatment than in none B.thuringiensis treatments.H.hebetor larval mortality was significantly higher in combine treatments than in control. Although the size of adult parasitoids that emerged from combine treatments appeared smaller than those in the control,their developmental period was not significantly different.Similarly,no significant difference was observed in the longevity of adult wasps emerging from both treatments.The sex ratio of H.hebetor progeny in control experiment was female biased in the ratio of 1:2.27 male:female and this trend is similar to the observations in combine treatment Therefore,since B.thuringiensis did not prevent parasitoid development,this investigations is suggestive that a combine treatment with B.thuringiensis and parasitoid release,would produce better protection against P.interpunctella.
Lastly,the response of gravid female H.hebetor to extracts from clean diet, infested diet,larvae and frass of P.interpunctella was investigated.Result revealed that acetone and hexane extracts elicited searching behavior in H.hebetor.Extracts of frass significantly stimulated more parasitization behavior in the parasitoid than extracts of other P.interpunctella-related materials tested.It was also noted that experience plays a huge role in parasitoid foraging efficiency.Females that had previous contact with host produced stronger responses towards treated patch,than their inexperienced counterparts.The active components in P.interpunctella-related materials were more soluble in hexane,than in acetone,ethyl ether or methanol. Using silica gel flash column chromatography,crude extracts of larva and frass were fractionated,then the response of H.hebetor to each fraction was evaluated and most active fractions were analyzed by gas chromatography-mass spectrometry(GC-MS). Three main peaks were observed on the GC of both fractions.Result showed that extracts contained hydrocarbons.The hydrocarbons which are mainly saturated long chain alkanes(1,3-dimethyl benzene,2-acyl-1,3-cyclohexanediones) are present in both extracts.Other chemicals extracted from the active fractions are toluene, squalane,ethyl cyclohexane,nonacosane and hexadecane.These results showed that P.interpunctella-related materials that were tested contained infochemicals which elicits searching/ parasitization behavior in the parasitoid H.hebetor.And these compounds(kairomone),may be synthesized and applied to surface of stored produce in other to enhance H.hebetor parasitization performance.
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