玉米与大豆群落捕食者捕食行为的DNA标记检测与控害作用的FQ-PCR量化评价
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摘要
可持续农业与害虫生态管理策略要求以生态系统为调控单位,充分发挥自然控制力,以达到治理有害生物和维护自然平衡的目的。捕食者作为一种重要的自然控制力是调节害虫种群密度、维持自然生态平衡的关键因子。无疑,准确评价捕食者在生态群落中所处的营养地位及精确量化评估其控害能力是应用捕食类群进行生物防治的基础。然而,捕食者尤其是广食性捕食者食物谱复杂且存在一定的选择性,加之研究理念、技术支持等条件的限制,对其所进行的研究与利用工作一直没有突破性进展,成为困扰生态学家与害虫防治学家的研究瓶颈之一。近年来,日趋成熟的DNA分子标记技术为探索群落内营养联系提供了有利的工具,同时荧光定量PCR技术的迅猛发展也为量化研究天敌对害虫的控制作用提供了新的契机。本研究立足玉米田与大豆田害虫与天敌群落,以DNA分子标记与荧光定量PCR技术为工具,准确定性研究群落内天敌对害虫的捕食行为,并拟和各种影响因子,精确定量研究主要天敌对靶标害虫的控制作用,对科学管理害虫、充分利用捕食性天敌、评估引进生物风险和治理有害生物入侵等工作具有重要意义。具体研究内容与结论如下:
1基本明确沈阳地区玉米田与大豆田主要害虫与天敌群落结构及动态
玉米田群落中主要害虫与天敌共50种,其中害虫17种,天敌33种。害虫类群中玉米蚜和禾缢管蚜相对多度最大,分别为46.86%和32.69%;并在7月25日至8月19日期间为优势害虫种群。天敌类群中,蜘蛛类相对多度最大为4.28%,其次为异色瓢虫与大草蛉,相对多度分别占1.25%和1.02%;大部分时间蜘蛛类为优势天敌种群。玉米田群落在6月15日和7月20日两个时期,多样性与均匀度指数同时达到峰值,是维持物种间相互组合抵抗外界干扰和恢复自然平衡的最佳时期,应予以保护。
大豆田群落中主要害虫与天敌共41种,其中害虫15种,天敌26种。害虫类群中,大豆蚜和烟蓟马相对多度最大,分别为55.28%和27.59%;并且在大部分时间为优势害虫种群。天敌类群中,东亚小花蝽相对多度最大为2.97%,其次为蜘蛛类与大草蛉,分别为1.71%和0.54%;大部分时间东亚小花蝽为优势天敌种。大豆田群落多样性与均匀度指数在6月15日至6月25日、7月30日至8月14日两个时期较低,群落最不稳定。其余大部分时期群落多样性与均匀度指数维持在较高水平,是维持物种间相互组合抵抗外界干扰和恢复自然平衡的最佳时期,应予以保护。
2利用DNA分子标记技术研究玉米田与大豆田群落内捕食行为
2.1 COⅠ基因标记检测天敌对大豆蚜的捕食作用
设计大豆蚜COⅠ基因特异片段扩增引物2对(A和B),扩增片段大小分别为197 bp和253 bp。检测田间捕食者对大豆蚜的捕食作用,结果表明,异色瓢虫成虫和幼虫、草蛉成虫、东亚小花蝽成虫和若虫捕食大豆蚜的作用较强,其阳性比率分别为61.90%和80.00%、78.57%、57.14%和62.50%。同时田间检测尚表明,捕食性天敌群体总的阳性比率与大豆蚜的种群密度呈显著正相关。
2.2 COⅡ基因标记检测天敌对玉米蚜的捕食作用
筛选玉米蚜COⅡ基因特异片段扩增引物(ClaCOⅡF/R3),其扩增片段为339bp,检测田间捕食者对玉米蚜的捕食作用,结果表明,大灰食蚜蝇幼虫、草蛉幼虫、异色瓢虫成虫和幼虫、龟纹瓢虫成虫和幼虫捕食玉米蚜的作用较强,其阳性比率分别为66.67%、60.00%、44.83%和46.15%、45.00%和57.14%。
2.3 COⅡ基因标记检测天敌对禾缢管蚜的捕食作用
筛选禾缢管蚜COⅡ基因特异片段扩增引物(BcoaCOⅡF4/R2),其扩增片段为148bp,检测田间捕食者对禾缢管蚜的捕食作用,结果表明,大灰食蚜蝇幼虫、异色瓢虫成虫和幼虫、草蛉成虫和幼虫、龟纹瓢虫幼虫捕食禾缢管蚜的作用较强,其阳性比率分别为55.56%、55.00%和46.15%、40.00%和50.00%、42.86%。
2.4东亚小花蝽捕食朱砂叶螨的DNA分子标记检测
克隆并测序朱砂叶螨COⅠ基因一段大小为480bp的序列,并借此设计朱砂叶螨特异片段扩增引物1对(C),其扩增目的片段为158bp。利用PCR方法鉴定了东亚小花蝽成虫及若虫对朱砂叶螨的捕食作用。
2.5玉米田与大豆田捕食者对异色瓢虫与大草蛉捕食作用的DNA分子标记检测
克隆并测序异色瓢虫COⅠ基因一段大小为500bp的序列,并借此设计异色瓢虫特异片段扩增引物1对(D),其扩增目的片段为293bp。根据草蛉16S ribosomal RNA基因序列(登陆号为AY620151),设计大草蛉特异片段扩增引物1对(E),其扩增片段为193bp。利用所设计引物,分别检测田间捕食者腹内食物成分,结果表明,捕食者类群内部存在捕食行为。大草蛉、步甲和蜘蛛类对异色瓢虫具有捕食能力,其中大草蛉成虫检出率最高,玉米田中为33.33%,大豆田中为40.00%;只有蜘蛛类对大草蛉存在捕食行为,玉米田中检出率为33.33%,大豆田中为25.00%。
3构建荧光定量PCR体系量化研究田间捕食性天敌腹内目标害虫成分的含量
制作玉米蚜、禾缢管蚜绝对定量标准品,并分别构建荧光定量PCR体系。对田间捕食者进行检测表明,大草蛉幼虫腹内含有的玉米蚜COⅡ基因拷贝量最大;其次依次为大草蛉成虫、异色瓢虫幼虫、成虫、大灰食蚜蝇幼虫;再次为龟纹瓢虫成虫、幼虫;蜘蛛类和东亚小花蝽成虫腹内含有的玉米蚜COⅡ基因拷贝数最少;步甲类腹内未检测到玉米蚜COⅡ基因成分。检测田间捕食者对禾缢管蚜的捕食作用,结果表明,大草蛉成虫腹内含有禾缢管蚜COⅡ基因拷贝量最大,其次依次为大草蛉幼虫、异色瓢虫幼虫、成虫;再次为大灰食蚜蝇幼虫、龟纹瓢虫幼虫、成虫;蜘蛛类和东亚小花蝽成虫腹内禾缢管蚜COⅡ基因拷贝数最少;步甲类腹内未检测到禾缢管蚜COⅡ基因成分。
4荧光定量PCR量化研究目标害虫在主要天敌腹内的衰变规律
异色瓢虫成虫喂饲单头玉米蚜,自然消化0,2,4,6h后,可检测到目标片段的拷贝数分别为991.5471、101.1695、20.6750、0个,时间与拷贝数对数的相关关系式为:A=-0.4203B+2.9458(R~2=0.9894);大草蛉成虫喂饲单头玉米蚜,自然消化0,2,4,6h后可检测到目标片段的拷贝数分别为942.9212、565.5247、121.4485、0个,时间与拷贝数对数的相关关系式为:C=-0.2225D+3.0483(R~2=0.9228)。
异色瓢虫成虫喂饲单头禾缢管蚜,自然消化0,2,4,6h后可检测到目标片段的拷贝数分别为2304.2586、125.7857、6.1939、0个,时间与拷贝数对数的相关关系式为:E=-0.6427 F+3.3705(R~2=0.998);大草蛉成虫喂饲单头禾缢管蚜,自然消化0,2,4,6h后可检测到目标片段的拷贝数分别为1605.2458、576.1685、86.0061、0个,时间与拷贝数对数的相关关系式为:G=-0.3177 H+3.2689(R~2=0.970)。
5量化评价主要天敌对目标害虫的控制作用
拟和天敌发生数量、阳性比率、腹内目标害虫成分含量及目标害虫在天敌腹内的衰变速率四个因素评价7月31日至8月14日期间异色瓢虫成虫与大草蛉成虫对玉米蚜的控制能力,结果表明,异色瓢虫成虫对玉米蚜的控制作用(R=6.1429×10~6)强于大草蛉成虫(R=2.7967×10~6);评价异色瓢虫成虫与大草蛉成虫对禾缢管蚜的控制能力,结果表明,异色瓢虫成虫对禾缢管蚜的控制作用(R=15.4896×10~6)亦强于大草蛉成虫(R=8.4966×10~6)。
Considering the requirements of the sustainable agriculture and ecological pest management,we should base ecosystem and full utilize all natural control factors to managing pests and maintaining the natural balance.Without question,as an important natural control factor predators play a key role in pest biological control.The foundation of pest biological control are the evaluating predators' function in the biological community and estimating predators' control effect with quantity accurately.Predators,especial generalist predators prey multiplicate foods and select predation for different foods.The limit of idea and technology made the study of generalist predators' predation maintain a lower level and become a difficult problem to ecologists and experts of pest control.
Recently,the rapid progress of molecular biotechnology of DNA marker and FQ-PCR provide us with valuable opportunities to study complex trophic interactions and evaluate generalist predators' control effect with quantity accurately in the field.In line with the communities structure of insect pests and natural enemies in maize and soybean fields and utilize DNA marker and FQ-PCR techniques,we study predation of predators with quality accurately and evaluate natural enemies' control effect to pests with quantity accurately in the field.This study is significance for ecological pest management,utilizing predatorial natural enemies resource,assessmenting immigrate risk species and managing invasive exotic species. And the main contents and conclusions are stated as following:
1 The research basically finds out the communities structure and their dynamic chang of the insect pests and natural enemies in maize and soybean fields of Shen yang.There were 17 species pest insects and 33 species natural enemies among 50 kinds of species in maize fields. The relative abundance of Rhopalosiphum maidis and Rhopalosiphum padi were higher than the other insect pests,respectively as 46.86%and 32.69%.During the period of time from July 25th to August 19th,they were dominant insect pests.The relative abundance of spiders were 4.28%,higher than the other natural enemies,then followed by Harmonia axyridis and Chrysopa pallens.In the most time spiders were dominant natural enemies.In maize field,on June 15th and July 20th,the community diversity index and the evenness index of the insect pests and natural enemier community become the climax,those periods were the time that community most stable and the best one time to maintain the ability of resisting the outer interferences and returning to the natural balance,so in this period we should avoid the breakage of natural condition.
There were 15 species pest insects and 27 species natural enemies among 41 kinds of species in soybean fields.The relative abundance of Aphis glycines and Thrips tabaci were higher than the other insect pests,respectively as 55.28%and 27.59%.In the most time they were dominant insect pests.The relative abundance of Orius sauteri was 4.28%,higher than the other natural enemies,then followed by spiders and C.pallens.In the most time Orius sauteri was dominant natural enemy.In soybean field,except of two periods of time from June 15th to June 25th and July 30th to August 14th,the community diversity index and the evenness index of the insect pests and natural enemier community maintaining in highly level, we should avoid the breakage of natural condition in this period.
2 Studying the predation using DNA marker in maize and soybean fields
2.1 Identifying the key predators of Aphis glycines using COI gene marker
Two pairs of sequence-characterized primers(A and B) were designed to detect the remains of A.glycines in the gut of various predators in the soybean fields.Primer A amplified single band was about 197 bp and primer B about 253 bp.Using primer A,the percentage of positive responses of A.glycines DNA in the predators collected in fields,such as the larvae of Harmonia axyridis was 80.00%,the nymph of Orius sauteri was 62.50%,the adult of H.axyridis was 61.90%,O.sauteri was 57.14%and Chrysopas was 78.57%,their were all over 50%.Moreover,the tests of samples collected from the field showed that the detection rate of predators by using primer A significantly corresponded to the density of A. glycines in soybean fields.
2.2 Identifying the key predators of Rhopalosiphum maidis using COⅡgene marker
One pair of sequence-characterized primers(ClaCOⅡF/R3) of Rhopalosiphum maidis was selected and it amplified single band was about 339 bp.The positive responses of R. maidis DNA in predators collected in fields,such as the larvae of Syrphus corollae was 66.67%,Chrysopas was 60.00%,H.axyridis was 46.15%,Propylaea japonica was 57.14%, and adult H.axyridis was 44.83%and P.japonica was 45.00%,theirs were all over 40%.
2.3 Identifying the key predators of Rhopalosiphum padi using COⅡgene marker
One pair of sequence-characterized primers(BcoaCOⅡF4/R2) of Rhopalosiphum padi was selected and it amplified single band was about 148 bp.The positive responses of R.padi DNA in predators collected in fields,such as the larvae of S.corollae was 55.56%,Chrysopas was 50.00%,H.axyridis was 46.15%,P.japonica was 42.86%,and adult H.axyridis was 55.00%and Chrysopas was 40.00%,theirs were all over 40%.
2.4 Identifying the predation of Orius sauteri to Tetranychus cinnabarinus using DNA marker
Cloning and sequencing COⅠgene segment of Tetranychus cinnabarinus.The length of the sequence was 480 bp.In line with it,One pair of sequence-characterized primers(C) of T. cinnabarinus was designed and it amplified single band was about 158 bp.We can identifying the adult and nymph of Orius sauteri predation to T.cinnabarinus by it.
2.5 Identifying the key predators of H.axyridis and C.pallens using DNA marker
Cloning and sequencing COⅠgene segment of H.axyridis.The length of the sequence was 500 bp.In line with it,One pair of sequence-characterized primers(D) of T.cinnabarinus was designed and it amplified single band was about 293 bp.One pair of sequence-characterized primers(E) of C.pallens was designed and it amplified single band was about 193 bp.Using primer D and E,the results showed that there were intraguild predation in predacity community.C.pallens、Carabidaes and spiders can consumed H. axyridis in fields.There in to the positive responses of C.pallens was 33.33%in maize fields and 40.00%in soybean fields higher than Carabidaes and spiders.At the same time,the results showed that only spiders consumed C.pallens in fields,the percentage of positive responses was 33.33%in maize fields and 25.00%in soybean fields.
3 FQ-PCR was used in studying the quantity of pest insects gene copes in predators gut contents
The calibration curves for the quantifying analyses of R.maidis and R.padi DNA fragment were set up,and the conditions and system compositions of real-time detection were optimized.Real-time detection for the gut contents of predators collected in fields showed that C.Pallens larva was the most possesser of R.maidis COⅡgene copes,then followed by C.Pallens adult,H.axyridis larva and adult,S.corollae larva,P.japonica adult and larva, spider and O.sauteri adult;C.Pallens adult was the most possesser of R.padi COⅡgene copes,then followed by C.Pallens larva,H.axyridis larva and adult,S.corollae larva, P.japonica larva and adult,spider and O.sauteri adult.
4 FQ-PCR was used in studying the disintegration rate of pest insects gene copes in predators gut contents
When a single R.maidis consumed by adult H.axyridis the relationships between time and delectability copes quantity were evaluated the formula:A=-0.4203B+2.9458 (R~2=0.9894);When a single R.maidis consumed by adult C.Pallens the relationships between time and delectability copes quantity were evaluated the formula: C=-0.2225D+3.0483(R~2=0.9228).When a single R.padi consumed by adult H.axyridis the relationships between time and delectability copes quantity were evaluated the formula: E=-0.6427 F+3.3705(R~2=0.998);When a single R.maidis consumed by adult C.Pallens the relationships between time and delectability copes quantity were evaluated the formula: G=-0.3177 H+3.2689(R~2=0.970).
5 FQ-PCR was used in quantitive evaluation control effect of predators
For the purpose of evaluating the control effect of H.axyridis and C.Pallens on R. maidis and R.padi by comprehensively analyzing the number,positive rate,possess pest insects gene copes and digestive speed of natural enemies,the results showed that the control effect of H.axyridis on R.maidis(R=6.1429x10~6) and R.padi(R=15.4896x10~6 ) is stronger than that of C.pallens.(R=2.7967x 10~6 and R=8.4966x 10~6).
1基本明确沈阳地区玉米田与大豆田主要害虫与天敌群落结构及动态
玉米田群落中主要害虫与天敌共50种,其中害虫17种,天敌33种。害虫类群中玉米蚜和禾缢管蚜相对多度最大,分别为46.86%和32.69%;并在7月25日至8月19日期间为优势害虫种群。天敌类群中,蜘蛛类相对多度最大为4.28%,其次为异色瓢虫与大草蛉,相对多度分别占1.25%和1.02%;大部分时间蜘蛛类为优势天敌种群。玉米田群落在6月15日和7月20日两个时期,多样性与均匀度指数同时达到峰值,是维持物种间相互组合抵抗外界干扰和恢复自然平衡的最佳时期,应予以保护。
大豆田群落中主要害虫与天敌共41种,其中害虫15种,天敌26种。害虫类群中,大豆蚜和烟蓟马相对多度最大,分别为55.28%和27.59%;并且在大部分时间为优势害虫种群。天敌类群中,东亚小花蝽相对多度最大为2.97%,其次为蜘蛛类与大草蛉,分别为1.71%和0.54%;大部分时间东亚小花蝽为优势天敌种。大豆田群落多样性与均匀度指数在6月15日至6月25日、7月30日至8月14日两个时期较低,群落最不稳定。其余大部分时期群落多样性与均匀度指数维持在较高水平,是维持物种间相互组合抵抗外界干扰和恢复自然平衡的最佳时期,应予以保护。
2利用DNA分子标记技术研究玉米田与大豆田群落内捕食行为
2.1 COⅠ基因标记检测天敌对大豆蚜的捕食作用
设计大豆蚜COⅠ基因特异片段扩增引物2对(A和B),扩增片段大小分别为197 bp和253 bp。检测田间捕食者对大豆蚜的捕食作用,结果表明,异色瓢虫成虫和幼虫、草蛉成虫、东亚小花蝽成虫和若虫捕食大豆蚜的作用较强,其阳性比率分别为61.90%和80.00%、78.57%、57.14%和62.50%。同时田间检测尚表明,捕食性天敌群体总的阳性比率与大豆蚜的种群密度呈显著正相关。
2.2 COⅡ基因标记检测天敌对玉米蚜的捕食作用
筛选玉米蚜COⅡ基因特异片段扩增引物(ClaCOⅡF/R3),其扩增片段为339bp,检测田间捕食者对玉米蚜的捕食作用,结果表明,大灰食蚜蝇幼虫、草蛉幼虫、异色瓢虫成虫和幼虫、龟纹瓢虫成虫和幼虫捕食玉米蚜的作用较强,其阳性比率分别为66.67%、60.00%、44.83%和46.15%、45.00%和57.14%。
2.3 COⅡ基因标记检测天敌对禾缢管蚜的捕食作用
筛选禾缢管蚜COⅡ基因特异片段扩增引物(BcoaCOⅡF4/R2),其扩增片段为148bp,检测田间捕食者对禾缢管蚜的捕食作用,结果表明,大灰食蚜蝇幼虫、异色瓢虫成虫和幼虫、草蛉成虫和幼虫、龟纹瓢虫幼虫捕食禾缢管蚜的作用较强,其阳性比率分别为55.56%、55.00%和46.15%、40.00%和50.00%、42.86%。
2.4东亚小花蝽捕食朱砂叶螨的DNA分子标记检测
克隆并测序朱砂叶螨COⅠ基因一段大小为480bp的序列,并借此设计朱砂叶螨特异片段扩增引物1对(C),其扩增目的片段为158bp。利用PCR方法鉴定了东亚小花蝽成虫及若虫对朱砂叶螨的捕食作用。
2.5玉米田与大豆田捕食者对异色瓢虫与大草蛉捕食作用的DNA分子标记检测
克隆并测序异色瓢虫COⅠ基因一段大小为500bp的序列,并借此设计异色瓢虫特异片段扩增引物1对(D),其扩增目的片段为293bp。根据草蛉16S ribosomal RNA基因序列(登陆号为AY620151),设计大草蛉特异片段扩增引物1对(E),其扩增片段为193bp。利用所设计引物,分别检测田间捕食者腹内食物成分,结果表明,捕食者类群内部存在捕食行为。大草蛉、步甲和蜘蛛类对异色瓢虫具有捕食能力,其中大草蛉成虫检出率最高,玉米田中为33.33%,大豆田中为40.00%;只有蜘蛛类对大草蛉存在捕食行为,玉米田中检出率为33.33%,大豆田中为25.00%。
3构建荧光定量PCR体系量化研究田间捕食性天敌腹内目标害虫成分的含量
制作玉米蚜、禾缢管蚜绝对定量标准品,并分别构建荧光定量PCR体系。对田间捕食者进行检测表明,大草蛉幼虫腹内含有的玉米蚜COⅡ基因拷贝量最大;其次依次为大草蛉成虫、异色瓢虫幼虫、成虫、大灰食蚜蝇幼虫;再次为龟纹瓢虫成虫、幼虫;蜘蛛类和东亚小花蝽成虫腹内含有的玉米蚜COⅡ基因拷贝数最少;步甲类腹内未检测到玉米蚜COⅡ基因成分。检测田间捕食者对禾缢管蚜的捕食作用,结果表明,大草蛉成虫腹内含有禾缢管蚜COⅡ基因拷贝量最大,其次依次为大草蛉幼虫、异色瓢虫幼虫、成虫;再次为大灰食蚜蝇幼虫、龟纹瓢虫幼虫、成虫;蜘蛛类和东亚小花蝽成虫腹内禾缢管蚜COⅡ基因拷贝数最少;步甲类腹内未检测到禾缢管蚜COⅡ基因成分。
4荧光定量PCR量化研究目标害虫在主要天敌腹内的衰变规律
异色瓢虫成虫喂饲单头玉米蚜,自然消化0,2,4,6h后,可检测到目标片段的拷贝数分别为991.5471、101.1695、20.6750、0个,时间与拷贝数对数的相关关系式为:A=-0.4203B+2.9458(R~2=0.9894);大草蛉成虫喂饲单头玉米蚜,自然消化0,2,4,6h后可检测到目标片段的拷贝数分别为942.9212、565.5247、121.4485、0个,时间与拷贝数对数的相关关系式为:C=-0.2225D+3.0483(R~2=0.9228)。
异色瓢虫成虫喂饲单头禾缢管蚜,自然消化0,2,4,6h后可检测到目标片段的拷贝数分别为2304.2586、125.7857、6.1939、0个,时间与拷贝数对数的相关关系式为:E=-0.6427 F+3.3705(R~2=0.998);大草蛉成虫喂饲单头禾缢管蚜,自然消化0,2,4,6h后可检测到目标片段的拷贝数分别为1605.2458、576.1685、86.0061、0个,时间与拷贝数对数的相关关系式为:G=-0.3177 H+3.2689(R~2=0.970)。
5量化评价主要天敌对目标害虫的控制作用
拟和天敌发生数量、阳性比率、腹内目标害虫成分含量及目标害虫在天敌腹内的衰变速率四个因素评价7月31日至8月14日期间异色瓢虫成虫与大草蛉成虫对玉米蚜的控制能力,结果表明,异色瓢虫成虫对玉米蚜的控制作用(R=6.1429×10~6)强于大草蛉成虫(R=2.7967×10~6);评价异色瓢虫成虫与大草蛉成虫对禾缢管蚜的控制能力,结果表明,异色瓢虫成虫对禾缢管蚜的控制作用(R=15.4896×10~6)亦强于大草蛉成虫(R=8.4966×10~6)。
Considering the requirements of the sustainable agriculture and ecological pest management,we should base ecosystem and full utilize all natural control factors to managing pests and maintaining the natural balance.Without question,as an important natural control factor predators play a key role in pest biological control.The foundation of pest biological control are the evaluating predators' function in the biological community and estimating predators' control effect with quantity accurately.Predators,especial generalist predators prey multiplicate foods and select predation for different foods.The limit of idea and technology made the study of generalist predators' predation maintain a lower level and become a difficult problem to ecologists and experts of pest control.
Recently,the rapid progress of molecular biotechnology of DNA marker and FQ-PCR provide us with valuable opportunities to study complex trophic interactions and evaluate generalist predators' control effect with quantity accurately in the field.In line with the communities structure of insect pests and natural enemies in maize and soybean fields and utilize DNA marker and FQ-PCR techniques,we study predation of predators with quality accurately and evaluate natural enemies' control effect to pests with quantity accurately in the field.This study is significance for ecological pest management,utilizing predatorial natural enemies resource,assessmenting immigrate risk species and managing invasive exotic species. And the main contents and conclusions are stated as following:
1 The research basically finds out the communities structure and their dynamic chang of the insect pests and natural enemies in maize and soybean fields of Shen yang.There were 17 species pest insects and 33 species natural enemies among 50 kinds of species in maize fields. The relative abundance of Rhopalosiphum maidis and Rhopalosiphum padi were higher than the other insect pests,respectively as 46.86%and 32.69%.During the period of time from July 25th to August 19th,they were dominant insect pests.The relative abundance of spiders were 4.28%,higher than the other natural enemies,then followed by Harmonia axyridis and Chrysopa pallens.In the most time spiders were dominant natural enemies.In maize field,on June 15th and July 20th,the community diversity index and the evenness index of the insect pests and natural enemier community become the climax,those periods were the time that community most stable and the best one time to maintain the ability of resisting the outer interferences and returning to the natural balance,so in this period we should avoid the breakage of natural condition.
There were 15 species pest insects and 27 species natural enemies among 41 kinds of species in soybean fields.The relative abundance of Aphis glycines and Thrips tabaci were higher than the other insect pests,respectively as 55.28%and 27.59%.In the most time they were dominant insect pests.The relative abundance of Orius sauteri was 4.28%,higher than the other natural enemies,then followed by spiders and C.pallens.In the most time Orius sauteri was dominant natural enemy.In soybean field,except of two periods of time from June 15th to June 25th and July 30th to August 14th,the community diversity index and the evenness index of the insect pests and natural enemier community maintaining in highly level, we should avoid the breakage of natural condition in this period.
2 Studying the predation using DNA marker in maize and soybean fields
2.1 Identifying the key predators of Aphis glycines using COI gene marker
Two pairs of sequence-characterized primers(A and B) were designed to detect the remains of A.glycines in the gut of various predators in the soybean fields.Primer A amplified single band was about 197 bp and primer B about 253 bp.Using primer A,the percentage of positive responses of A.glycines DNA in the predators collected in fields,such as the larvae of Harmonia axyridis was 80.00%,the nymph of Orius sauteri was 62.50%,the adult of H.axyridis was 61.90%,O.sauteri was 57.14%and Chrysopas was 78.57%,their were all over 50%.Moreover,the tests of samples collected from the field showed that the detection rate of predators by using primer A significantly corresponded to the density of A. glycines in soybean fields.
2.2 Identifying the key predators of Rhopalosiphum maidis using COⅡgene marker
One pair of sequence-characterized primers(ClaCOⅡF/R3) of Rhopalosiphum maidis was selected and it amplified single band was about 339 bp.The positive responses of R. maidis DNA in predators collected in fields,such as the larvae of Syrphus corollae was 66.67%,Chrysopas was 60.00%,H.axyridis was 46.15%,Propylaea japonica was 57.14%, and adult H.axyridis was 44.83%and P.japonica was 45.00%,theirs were all over 40%.
2.3 Identifying the key predators of Rhopalosiphum padi using COⅡgene marker
One pair of sequence-characterized primers(BcoaCOⅡF4/R2) of Rhopalosiphum padi was selected and it amplified single band was about 148 bp.The positive responses of R.padi DNA in predators collected in fields,such as the larvae of S.corollae was 55.56%,Chrysopas was 50.00%,H.axyridis was 46.15%,P.japonica was 42.86%,and adult H.axyridis was 55.00%and Chrysopas was 40.00%,theirs were all over 40%.
2.4 Identifying the predation of Orius sauteri to Tetranychus cinnabarinus using DNA marker
Cloning and sequencing COⅠgene segment of Tetranychus cinnabarinus.The length of the sequence was 480 bp.In line with it,One pair of sequence-characterized primers(C) of T. cinnabarinus was designed and it amplified single band was about 158 bp.We can identifying the adult and nymph of Orius sauteri predation to T.cinnabarinus by it.
2.5 Identifying the key predators of H.axyridis and C.pallens using DNA marker
Cloning and sequencing COⅠgene segment of H.axyridis.The length of the sequence was 500 bp.In line with it,One pair of sequence-characterized primers(D) of T.cinnabarinus was designed and it amplified single band was about 293 bp.One pair of sequence-characterized primers(E) of C.pallens was designed and it amplified single band was about 193 bp.Using primer D and E,the results showed that there were intraguild predation in predacity community.C.pallens、Carabidaes and spiders can consumed H. axyridis in fields.There in to the positive responses of C.pallens was 33.33%in maize fields and 40.00%in soybean fields higher than Carabidaes and spiders.At the same time,the results showed that only spiders consumed C.pallens in fields,the percentage of positive responses was 33.33%in maize fields and 25.00%in soybean fields.
3 FQ-PCR was used in studying the quantity of pest insects gene copes in predators gut contents
The calibration curves for the quantifying analyses of R.maidis and R.padi DNA fragment were set up,and the conditions and system compositions of real-time detection were optimized.Real-time detection for the gut contents of predators collected in fields showed that C.Pallens larva was the most possesser of R.maidis COⅡgene copes,then followed by C.Pallens adult,H.axyridis larva and adult,S.corollae larva,P.japonica adult and larva, spider and O.sauteri adult;C.Pallens adult was the most possesser of R.padi COⅡgene copes,then followed by C.Pallens larva,H.axyridis larva and adult,S.corollae larva, P.japonica larva and adult,spider and O.sauteri adult.
4 FQ-PCR was used in studying the disintegration rate of pest insects gene copes in predators gut contents
When a single R.maidis consumed by adult H.axyridis the relationships between time and delectability copes quantity were evaluated the formula:A=-0.4203B+2.9458 (R~2=0.9894);When a single R.maidis consumed by adult C.Pallens the relationships between time and delectability copes quantity were evaluated the formula: C=-0.2225D+3.0483(R~2=0.9228).When a single R.padi consumed by adult H.axyridis the relationships between time and delectability copes quantity were evaluated the formula: E=-0.6427 F+3.3705(R~2=0.998);When a single R.maidis consumed by adult C.Pallens the relationships between time and delectability copes quantity were evaluated the formula: G=-0.3177 H+3.2689(R~2=0.970).
5 FQ-PCR was used in quantitive evaluation control effect of predators
For the purpose of evaluating the control effect of H.axyridis and C.Pallens on R. maidis and R.padi by comprehensively analyzing the number,positive rate,possess pest insects gene copes and digestive speed of natural enemies,the results showed that the control effect of H.axyridis on R.maidis(R=6.1429x10~6) and R.padi(R=15.4896x10~6 ) is stronger than that of C.pallens.(R=2.7967x 10~6 and R=8.4966x 10~6).
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