| The rice stem borer, Chilo suppressalis, is one of the most important pest insects on rice. Chemical control is an important way to control its damage. Because it must be taken into account the security of insecticide application in paddy field, effectiveness of controlling borers and insecticide cost endured in food crop, few insecticides could be practically used so that normally only single insecticide was used extensively and insecticide resistance developed rapidly in this pest. The rice stem borers from Jiangsu and Zhejiang were found developed high level resistance to most insecticides controling borers, such as BHC, monosultap and triazophos, in succession. Then fipronil was introduced to meet the urgent need to control borers in these resistant areas and used subsequently to control many others rice pest insects as an important substitute of highly toxic pesticides due to its different action mechanism. But fipronil was banned on rice pests control because of its ecotoxicity problem after it was used some time. Thus, monitoring and study on the resistance dynamic changes of C. suppressalis to fipronil has important significance in understanding resistance development trend of novel insecticides and reasonable use of insecticides. In addition, for resistance management of C. suppressalis, exploiting efficient agricultural control technology would be very important to reduce borer population density and decrease pesticides application to delay development of resistance, because there were not enough insecticides for alternative use.Based on practice, this work made continuous resistance monitoring of C. suppressalis field population, selection with fipronil in laboratory, and test resistance cost and fitness changes to reveal the development pattern and mechanisms of resistance in rice stem borer for reasonable chemical control. Efficient cultivation techniques were also developed for control of the overwintering C. suppressalis in mechanized agriculture taking super rice as sample because of the borer occuring seriously on it. And separated rice seedling nurse was proved to decrease C. suppressalis population density by survey on efficient host plants of borers in spring. In this study, it was tried to explore integrated management technology for resistant C. suppressalis on two aspects of chemical and agricultural control.1 Field survey on insecticide resistance in C. suppressalisBy means of topical application, the resistance to conventional insecticides was surveyed in rice stem borer, Chilo suppressalis, from Cangnan, Zhejiang, aiming to provid useful information for rational insecticides use in controlling rice stem borer. The results showed that Cangnan population developed high level of resistance to triazophos (RR=297), medium level of resistance to trichlorphon (RR= 12.8) and fipronil (RR=16), and also had low level resistance to pyridaphenthion and diazinon (RR=5.9 and 5.6 respectively), but had no resistance to deltamethrin, acephate and avermectin. It was found by the discussion that the rice stem borer only exhibit distinct resistance to the insecticides which were used intensively for long time. The resistance to fipronil was further monitored in different year and areas. It was found that most borer populations were susceptible to fipronil and the resistance was detected only in Cangnan and few other cities nearby where fipronil was used intensively. The level of resitance to fipronil kept on decreasing following its reduced using and forbidden.The results revealed not only that the stem borer had risk to develop resistance to fipronil but also that the resistance to any new insecticides could develop rapidly if used intensively. Thus, it is concluded that on the one hand, multiple use of different kinds of insecticides should be put into practice as far as possible for the resistance management of the rice stem borer, on the other hand, efficient agricultural measures and other integrated management measures should be developed to depress population density of borers and reduce insecticides application. In addition, based on toxicity test results, reasonable suggestions were proposed on use of insecticides for control of the borer.2 Sublethal effect of fipronil against resistant and susceptible rice stem borerStudy on sublethal effects of insecticides to resistant and susceptible insects is helpful to understand their sustained efficiency and effect on resistance development. The rice stem borers from both resistant (Cangnan, Zhejiang) and susceptible (Taihu, Anhui) populations were treated with their own sublethal doses (LD10 and LD25) of fipronil to examine if there were any sublethal effects. The results showed that the sublethal doses obviously depressed survivorship and fecundity of the borers, and prolonged the larval duration of C.suppressalis. The effect of fipronil on survivorship of the resistant population was stronger than that of the susceptible one. In addition, specific activity of CarE in susceptible population increased significantly after the treatment with sublethal doses of fipronil. The Km value of CarE increased in both resistant and susceptible populations when treated, indicating that CarE was induced and isoenzyme compositions changed. Thus, it was thought that sublethal doses of fipronil had strong sustained effect on the borers, which had greater negative impact to resistant population than to susceptible one and was unfavorable for its rapid resistance development. CarE played apparent role on detoxification of fipronil and resistance development in C. suppressalis. Its isoenzymes involved need further studies.3 Resistance selecting with fipronil and fitness change of the resistant strainTo ascertain the development pattern of resistance to fipronil of the rice stem borer and; offer the theoretical basis for rational use of the insecticide, the borers collected from Cangnan County were continuously selected with fipronil for 15 generations in the laboratory, and its level of resistance to fipronil reached almost 20-fold. The realized heritability (h2) estimated was 0.3532, indicating that the rice stem borer had definite resistance risk to fipronil. In addition, life table was used to compare the fitness of the resistant and susceptible strains of the rice stem borer. The results revealed that the rice stem borer experienced biological fitness loss when it evolved median level resistance to fipronil. The decreased pupation rate and number of effective eggs per female were observed in resistant strain. The calculated fitness was only 0.78 for the Cn-R resistant strain as compared with Cangnan susceptible strain. This showed that the resitant strain had significant survival disadvantages in reproduction. Obviously, though the rice stem borer had resistance risk to fipronil, resistant strain had low fitness and resistance level decreased siginificantly when the seclection was suspended. All of these indicated that fipronil resistance development could be delayed by limited using and rotational use with other insecticides.4 Biochemical mechanisms for resistance to fipronil in the rice stem borerSynergism experiments in vivo with TPP, PBO and DEM were performed to make clear the roles of detoxification enzymes in fipronil resistance with selected resistant and susceptible strains by topical application. The results revealed that PBO had significant synergism on fipronil in both resistant (SR=2.8) and susceptible borers (SR=2.1), and DEM also had obvious synergistic effect (SR=2.4) in resistant borers but not in susceptible ones (SR=0.7). TPP had slight effect in resistant strain (SR=1.1) and no effect in susceptible one (SR=0.9). Enzyme activity assays demonstrated that the resistant strain had higher activity of microsomal O-demethylase and esterase than the susceptible strain (1.29- and 1.34-fold, respectively), and glutathione S-transferase activity was obviously higher in the resistant strain than in the susceptible one when CDNB was used as substrate.So, these results confirmed that increased activity of esterase, microsomal-O-demethylase, and particularly glutathione S-transferase should be involved in the resistance mechanism of rice stem borer.After continuously selected with fipronil for 6 generations, the borers were tested for cross resistance. It was found that its resistance to fipronil rise by 4.2-folds (RR from 3.8 to 15.7). At the same time, its resistance to monosultap, cypermethrin and endosulfan increased by 3.3,2.2 and 3.2 times, respectively. So, there might be some cross resistance between fipronil and these pesticides. But its resistance to triazophos, abamectin, chlorpyrifos and chlorantraniliprole changed slightly, suggesting that there was no cross resistance among the insecticides and fipronil. It was deduced that the cross resistance was induced due to the same target-site or metabolic detoxification mechanisms.5 Cloning and sequence analysis on a subunit gene of target GABARTo inspect if there is molecular differentiation relation to fipronil resistance in GABA receptor gene, we cloned the full sequence of a GABA receptor gene with full length 1957 bp,1491 bp ORF,71 bp 5’UTR and 395 bp 3’UTR in the susceptible rice stem borer with RT-PCR and RACE techniques. The deduced protein sequence includes 496 amino acids. Molecular weight was calculated as 55.1 Kda. The cloned gene from the rice stem borer had the typical characteristics of the GABARα-subunits. Homology analysis indicated that the cloned gene shared high similarity (64-94%) with those of other insects. So, the cloned gene was thought to be GABAR gene of the rice stem borer. GABAR genes from different individuals of the resistant and the susceptible strains of the rice stem borer were cloned and their full sequences were compared. But no difference was found between the genes from resistant and susceptible strains of the rice stem borer. So it may exclude that the selected resistance relate to the mutations in the cloned target gene. If there were some other mutations in other target subunits relation to fipronil resistance need further proved.6 Study on agricultural control of C. suppressalisIt is an important parthway for insect resistace management to decrease the insecticides usage by integrated control methods. Based on occurring characteristics of the rice stem borer and farm operation habits, it tried to exploit efficient agricultural control techniques through investigations. Firstly, surveys on vertical distribution and development of C.suppressalis overwintering populations in common and super rice plants at harvesting time, and field trials were performed to seek efficient ways to lower the borer’s population density. The results showed that the borer larvae were distributed to a greater height in the plants of super rice than in common rice, making control through agricultural operations more feasible. For common rice, over 40% of overwinting borer larvae were distributed whthin 10 cm plant, and 80% of the larvae whthin 20 cm plant above soil surface. In comparison, for super rice, only 14.3% of borer larvae distributed within 10 cm plant, 39.2% of the larvae within 20 cm plant above soil surface. Over 60% of the borers larvae developed to 5th instar in common rice, but 63.9% of larvae developed to 6th instar in super rice at harvesting time. Leaving stubble height had a great influence on density of rice stem borer overwinting in rice stub. Pre-winter rotary tillage only resulted in less than 30% decrease rate of overwintering borers but could enhance the population reduction effect of irrigation the following spring. Spring irrigation after the pre-pupation dispersal movements of borers killed 60-77% of the overwintering population in super rice. In addition, we found that burning fields after harvest, which had little effect on borers in common rice but killed 84.5% of borers in super rice, seemed the best remedy, though this practice is forbidden in China for ecological reasons. Based on the results obtained, efficient control of borers on rice can be achieved by harvesting leaving short stubbles, followed by rotary tillage and spring irrigation. This regime can reduce the borer population in super rice by more than 98%. Secondly, the host plant selection of rice stem borer adults for oviposition in spring and survival of neonate larvae in different hosts were investigated in fields. It was found that the borer adults much more prefer to oviposit on rice and water-oats plants than on other plants under natural condition. The borers oviposited more eggs on rice plants than on water-oats plants, with significant difference. But no eggs were examined on wheat, corn, sugar cane and weed in fallow field; The results indicated that survival rate of the neonate larvae was highest in rice plant, second in wheat, weed in fallow field and water-oats plants, and lowest in corn and sugar cane. It can be primarily concluded from above results that the reproduction host plants of the rice stem borer adults were most from rice and few from water-oats plant. There was little chance that borer adults oviposited on wheat and fallow field in spring, and com and sugar cane fields were difficult to be efficient reproduction sites for the rice stem borers. Thus, it was thought that separated rice seedling nurse to keep back C. suppressalis oviposit on rice may decrease borer population density efficiently in one year.It was found in this paper that C. suppressalis had developed different level of resistance to many conventional insecticides and also developed rapidly median level of resistance to fipronil in the areas where it was used largely. But the results on selection with fipronil in laboratory, test resistance cost fitness and sublethal effect of fipronil all indicated that C. suppressalis was difficult to develop further resistance to fipronil. It demonstrated that resistance of C. suppressalis was mainly due to intensive application of single insecticide in stages. Hence it must be taken measures of rational use and decreasing application of insecticides for resistance management. On the aspect of decreasing insecticides application, it was testified several efficient agricultural operations and a set of cultivation techniques was provided to control overwintering C. suppressalis population. Simultaneously, through survey in the fields it can be primarily concluded main reproduction host plants of C. suppressalis in spring and testified that separated rice seedling nurse to control 1st generation of C.suppressalis may decrease borer population density in one year.Above all of these results have important practical value for efficient integrated resistance management of the C.suppressalis.In addition,the results of study on cross-resistance and resistance mechanism reveled that resistance of C.suppressalis to fipronil was mainly due to enhanced detoxifying enzyme activity.Andαsubunit gene of GABA receptor from C.suppressalis was first cloned. There results will found the bases for resitance molecular mechanism of C. suppressalis and further study on GABA receptor. |
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