| Green Peach Aphid Myzus persicae (Sulzer) is an important pest, whichcould threaten many kinds of plants and vegetables through not only feeding on theplants nutrition but also transmitting plant virus diseases. Pandora neophidis is anobligate aphid pathogen, and it is one of the most wordwide Entomophthoraleanfungi. It can cause epizootics in aphid populations through infecting aphids，whichresulting in the sharp decline in the aphid population. So it is very importantbiological control agents in the aphid control. In this study, we choose M. persicaeand P. neophidis as the research objects. We conducted EPG experiments between P.neophidis-infected M. persicae and normol M. persicae to observe the differentpenetrition and feeding behaviour of the aphid under different infected stage. Also,we conducted the quantitative real-time PCR to choose the steadly expressionreference gene. By measuring the relatively quantify value of P. neophidis in the hostM. persicae bodies using the above chosen reference gene, we developed atheoretical foundation for the further interaction research between P. neophidis and M.persicae and the pathogensis exploration of the process. The experiments results areas follows.(1) Get the waveform P. neophidis-infected M. persicae and the normol M. persicaewaveform using EPG technology. Data analysis are based on the time duration andfrequence of the feeding waveform np、c、pd、E1、E2. Results revealed that thepenetration time waveform c became longer, and the real feeding time waveform Ebecame shorter. And the time duration and frequence of none penetration waveform(np) are influenced greatly. Additaionally, All the parameters compared in the16hbefore the M. persicae death after infected by P. neophidis with the normol M.persicae indicated that the mean time duration and frequence of waveform np and E1are not significantly different. The mean time duration and frequence of waveform cand pd are significantly different. Interestingly, the mean time duration of waveformE2are significantly different, however its mean frequence are not significantlydifferent. Moreover, All the parameters compared in the6h after the P. neophidis-infected M. persicae with the normol M. persicae indicated that the mean time duration of waveform np are not significantly different. The mean frequence ofwaveform np and c are significantly different. The mean time duration and frequenceof waveform pd are significantly different. The mean time duration and frequence ofwaveform E1and E2are not significantly different.(2) Selection of a suitable reference gene is an important prerequisite for precisegene expression analysis by real-time quantitative polymerase chain reaction.Pandora neoaphidis, an obligate aphid pathogenic fungus, which could induce adrastic epizootic to cause the collapse of aphid populations on crops, has no reportson selection of reference genes. To determine potential reference genes fornormalization of real-time PCR data on P. neoaphidis, the transcript levels of3traditional housekeeping genes including18S rRNA(18S),28S rRNA(28S) andelongation factor1alpha-like protein(EF1), were measured in this study. Weinvestigated the expression stability of three candidate reference genes of P.neoaphidis ARSEF5403in different developmental stages including conidia stage,germ tubes stage, early hyphae stage and elongated hyphae stage, as well as underdifferent nutrient conditions including OS-SDB medium, GLEN medium andGrace’s insect cell culture medium. The expression stability of the candidatereference genes was then calculated using three algorithms geNorm, NormFinder andBestKeeper. Results from real-time PCR revealed that designed primers based onGenbank have a good proliferation efficiency and specificity. The analysis withgeNorm algorithms revealed that the stability value (M value) of candidate referencegenes was0.457(18S),0.534(28S) and0.749(EF1) under different developmentalstages,0.389(18S),0.557(28S) and0.607(EF1) under different nutrient conditionsas well. Additionally, the analysis with NormFinder algorithms revealed that thestability value of candidate reference genes was0.084(18S),0.264(28S) and0.509(EF1) under different developmental stages,0.118(18S),0.355(28S) and0.403(EF1) under different nutrient conditions as well.18S was ranked as the mostsuitable reference gene of three candidate reference genes analyzed by geNorm andNormFinder. However, the analysis with BestKeeper algorithms revealed that28Swas the most suitable reference gene under all conditions examined,18S was thenext, EF1was the most unstable reference gene. Comprehensively, the mean rankbased on stability banking analyzed by those three algorithms showed that18S was the most suitable reference gene under all conditions examined.(3) Use the steadily expressed housekeeping gene18S as the reference gene toconduct the real time PCR experiment of the DNA extracted from M. persicaeinfected with P. neoaphidis. Preliminary studies have been carried out on theproliferation process of P. neoaphidis in M. persicae bodies. A rapid DNAextractation methoed based on Chelex-100was developed to extract the genomeDNA of the P. neophidis-infected M. persicae. Then7different infected stage M.persicae were investigated by real-time PCR. Accordig to the gene relativeexpression, a growth model on the proliferation situation of P. neoaphidis in M.persicae bodies was developed and assessed.Conclusively, observation the feeding behaviour differences between P. neoaphidis-infected M. persicae and the normol M. persicae using EPG technology coulddiscribe the process of P. neoaphidis infecting M. persicae in the macroscopy aspect.Meanwhile, analyzed by real-time qPCR using the reference gene18S, proliferationprocess of P. neoaphidis in M. persicae are studied using qPCR in the molecularaspect. Therefore, combination of macroscopy observation outside and microscopyanalysis inside, could provide experiment datas for the P. neoaphidis infecting M.persicae pathogenic process and offer the foundation for the further research relatedto the pathogensis. |
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