Study On Technique For Evaluating Predation On Planthoppers In Rice Ecosystem

The rice planthoppers, including white back planthopper Sogatella fureifera(Horvath), brown planthopper Nilaparvata lugens(St(?)l) and small brown planthopperLaodelphax striatellus(Fall(?)n) (Homoptera: Delphacidae), are the most serious pestsin rice ecosystem in Asia. They can cause serious economic damage to the rice cropdirectly by feeding and indirectly by transmitting virus disease. The resources ofnatural enemies for these planthoppers are abundant, many of the general predator,especially spiders, are the predominant natural enemies and play an important role inplanthopper control. To make full use of the natural enemies, it is necessary andessential to understand the predation on the planthoppers by these predators. In thisdissertation, the COI, ITS1 and ITS2 genes of the three species of planthoppers werecloned and three sets of primers and specific ALLGlo probes for WBPH, BPH andSBPH were designed and developed, respectively. A technique of triplex real timequantitative PCR assay to evaluate predation on the three planthoppers(WBPH, BPHand SBPH) quantitatively was developed and a preliminary investigation wascontacted to study the interactions between the three rice planthopper species andmain predators in rice ecosystem of Jiaxing. The main results are as follows:1. The COI, ITS1 and ITS2 gene of WBPH, BPH and SBPH were cloned,sequenced and analyzed. The comparison of sequences of COI, ITS1 and ITS2 amongthe three species showed that identities of these genes among the three planthopperspecies were 89.02%, 67.98% and 70.11%, respectively.2. Based on the sequences of ITS2 of the three planthoppers, a pair of primerswhich are species specific for WBPH, BPH and SBPH were designed, the lengthes ofthe fragments amplified for WBPH, BPH and SBPH are 192bp, 215bp and 207bp,respectively. A multiplex PCR assay was developed to detect these speciesqualitatively using the multiplex PCR assay.3. A triplex real time quantitative PCR assay for three planthopper species wasdeveloped. The dynamic range of the assay encompasses at least six orders ofmagnitude, with a strong linear relationship between the Ct values and the log₁₀ of theinput number of copies(WBPH, R²=0.99; BPH, R²=0.98; SBPH, R²=0.99). Theslope of each standard curve was -3.825 for WBPH, -3.782 for BPH, and -3.648 forSBPH. The efficiency was 182.57% for WBPH, 183.82% for BPH, and 187.98% forSBPH. The sensitivities for detection of the multiplex assay were 100 copies for WBPH, 1000 copies for BPH, and 100 copies for SBPH respectively. Interference testof multiplex real time PCR assay showed that the multiplex real time PCR assaycould be used to detect each of the three targeted planthopper species, even when oneof the species had very low DNA template concentrations(1:10000). The backgroundDNA of the spider specimen didn't affect amplification efficiency and there are notPCR-inhibitors in the overall DNA content extracted from predator.4. The results from quantitative assays showed that there were 111192839,860074796 and 274680423 target copies in one individual long-winged pregnantfemale adult WBPH, BPH and SBPH, respectively. The average DNA loss rate of theCTAB method is 10.66% by addition recovery experiment. The decay equations anddetection periods of one individual long-winged pregnant female adult in the guts ofPardosa pseudoannulata, are y=0.548x+21.594(R²=0.8744), and 28h for WBPH;y=0.2075x+22.257(R²=0.5047) and 32h for BPH and y=0.5304x+20.588(R²=0.8125),and 24h for SBPH.5. The results from bioassays of the predators collected from rice field duringAugust to October, in Jiaxing showed that many natural enemies could prey all thethree planthopper species, but the positive rates were varied among predators. Thepredators with the highest and lowest positive rate were Coleosoma octomaculatum(73.64%)and Cyrtorrhinus liuidipennis(34.94%) for WBPH, Cyrtorrhinus liuidipennis(72.06%)and Tetragnatha maxillosa(12.71%)for BPH; Pardosa pseudoannulata(79.14%) and Tetragnatha maxillosa(44.92%) for SBPH, respectively.6. The residual copies of rice planthoppers were varied among predators.Throughout the whole rice-growing season, predotors with the highest and lowestresidues were Pardosa pseudoannulata and Tetragnatha maxillosa, respectively. Themean residual copies were 53600 copies, 13587277 copies and 3829821 copies forWBPH, BPH and SBPH in Pardosa pseudoannulata, respectively; and the meanresidual copies were 18327 copies, 716530 copies and 32957 copies for WBPH,BPH and SBPH in Tetragnatha maxillosa, respectively.7. The compositions of prey species for main predators in the different ricegrowing periods were varied. In paddy field with single rice crop transplanted lately,SBPH and WBPH moved in around early July and BPH moved in late July-earlyAugust. At tillering stage on Aug. 14^th, the populations of WBPH and SBPH were stillnot high, Pardosa pseudoannulata, Coleosoma octomaculatum and Ummeliata insecticeps mainly preyed on WBPH and BPH, but the positive rates were low andthere were high proportional predators without any planthoppers in their guts; Atheading and milking stages on Sept.14^th and Oct. 10^th, the BPH population, as well asSBPH population, were higher, Pardosa pseudoannulata, Coleosoma octomaculatum,Ummeliata insecticeps and Cyrtorrhinus liuidipennis mainly preyed on BPH andSBPH, the positive rates were also getting higher, and there were high proportionalpredators with all three speicies of planthoppers in their guts.8. The prey residues of WBPH, BPH and SBPH in different predator during thesame and different periods were compared and the results showed that the averageprey residues of BPH were higher in Pardosa pseudoannulata, Coleosomaoctomaculatum, Ummeliata insecticeps and Cyrtorrhinus liuidipennis from Sept. toOct.; the average prey residues of SBPH were higher in Pardosa pseudoannulata andColeosoma octomaculatum from Aug. to Sept. and higher in Pardosa pseudoannulata,Coleosoma octomaculatum, Ummeliata insecticeps and Cyrtorrhinus liuidipennisfrom Sept. to Oct.; the average prey residues of WBPH were lower in all the predatoras above during the whole rice growing periods. These results suggested that theaverage prey residues were consistent with the population dynamics of BPH, SBPHand WBPH in rice field.