Developing DNA Markers And FQ-PCR To Study Predation In Maize And Soybean Communities

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 fields2.1 Identifying the key predators of Aphis glycines using COI gene markerTwo 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 COâ…¡gene markerOne pair of sequence-characterized primers(ClaCOâ…¡F/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 COâ…¡gene markerOne pair of sequence-characterized primers(BcoaCOâ…¡F4/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 markerCloning and sequencing COâ… gene 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 markerCloning and sequencing COâ… gene 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 contentsThe 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 COâ…¡gene 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 COâ…¡gene 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 contentsWhen 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 predatorsFor 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).