苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展

马铃薯甲虫是重要的入侵害虫,严重威胁着我国粮食作物马铃薯的生产。苏云金芽胞杆菌是重要的农业害虫生防细菌,对马铃薯甲虫有良好的防治效果。本文围绕苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展与应用进行综述。主要从马铃薯甲虫的入侵与防治手段、苏云金芽胞杆菌的晶体蛋白结构与杀虫机制、对马铃薯甲虫有活性的Bt毒蛋白研究进展、Bt毒蛋白对马铃薯甲虫的作用机制以及马铃薯甲虫对Bt毒蛋白的抗性机制等方面进行了综述。最后,从Bt新基因的挖掘和杀虫机理方面对苏云金芽胞杆菌在马铃薯甲虫防治上的研究进行了展望。     

Detection of organophosphate and pyrethroid resistance alleles in Czech Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) populations by molecular methods

BACKGROUND: Pyrethroids and organophosphates are the most frequently used insecticides for Colorado potato beetle, Leptinotarsa decemlineata (Say), control in the Czech Republic. Based on molecular methods, organophosphate and pyrethroid resistance alleles have been detected in samples from three sites. The accuracy of restriction fragment length polymorphism (RFLP) and bidirectional polymerase chain reaction amplification of specific alleles (Bi‐PASA) for detection of resistance alleles is compared. RESULTS: Leptinotarsa decemlineata from three sites showed higher frequencies of resistance alleles to organophosphates than to pyrethroids. The rates of occurrence of individuals homozygous resistant (RR) to pyrethroids ranged from 20.0 to 22.9%, while the rates of occurrence of individuals RR to organophosphates ranged from 52.9 to 66.7%. The incidences of individuals with resistance alleles to both organophosphates and pyrethroids ranged from 8.6 to 13.6%. No relationship was found between incidence of the pyrethroid resistance allele and site, while incidence of the organophosphate resistance allele differed significantly according to site. CONCLUSION: Both RFLP and Bi‐PASA were suitable for detecting resistance alleles to pyrethroids, and in most cases also for detecting resistance alleles to organophosphates. In contrast to Bi‐PASA, RFLP was also suitable for samples with lower DNA quality when testing for the resistance allele to pyrethroids. On the other hand, RFLP was not as accurate as Bi‐PASA in detection of the organophosphate resistance allele. Copyright © 2010 Society of Chemical Industry     

Control of Colorado beetle (Leptinotarsa decemlineata) in Poland1

Leptinotarsa decemlineata (Colorado beetle) was found in Poland for the first time in 1945 and a great invasion of this pest took place in 1950. It now plays a very important role in Polish plant protection, mainly because the acreage of potato in Poland amounts to about 1.8 million ha. The pest is controlled by repeated insecticide applications to a yearly total of 2 million ha, which is the highest amount of insecticides used in Poland. Research has been carried out in Poland on the following subjects: biology and ecology of Colorado beetle, influence of pest appearance and control on the biocenosis of fields, methods of control, biochemical factors influencing the life cycle, forecasts and warning of appearance, artificial diet, resistance of potato cultivars and reaction of leaf blades of potato to the eggs of Colorado beetle. Research is also carried out on chemical control, modernization of pesticide recommendations and resistance of Colorado beetle to pesticides. The influence of biotic and abiotic factors on the reduction of Colorado beetle numbers has been studied. With a view to possible biological control, predatory bugs (Perillus bioculatus and Podisus maculiventris) were introduced into Poland in 1959 and 1979. Research is also conducted on entomopathogenic fungi, parasitic nematodes, protozoans, spiroplasms and Bacillus thuringiensis.     

Molecular detection of insecticide resistant alleles

The summary discusses techniques used to investigate mechanisms of resistance in the Colorado potato beetle (Leptinotarsa decemlineata) to several classes of pesticides.     

Resistance to neonicotinoid insecticides in field populations of the Colorado potato beetle (Coleoptera: Chrysomelidae)

BACKGROUND: Neonicotinoid insecticides were first used commercially for Colorado potato beetle [Leptinotarsa decemlineata (Say), Coleoptera: Chrysomelidae] control in the United States in 1995, and since then have been critical for management of this pest. Field populations from the northeastern and midwestern United States were tested from 1998 to 2010 for susceptibility to imidacloprid and thiamethoxam using standard topical dose assays with adults. RESULTS: From 1998 to 2001, imidacloprid resistance was present in only a few locations in the eastern United States. By 2003, imidacloprid resistance was common in the northeastern Unites States. In 2004, imidacloprid resistance in Colorado potato beetle was detected for the first time in the midwestern United States. In 2003, the first case of resistance to thiamethoxam was found in a population from Massachusetts. Neonicotinoid resistance in summer‐generation adults was higher than in overwintered adults from the same locations. By 2009, 95% of the populations tested from the northeastern and midwestern United States had significantly higher LD₅₀ values for imidacloprid than the susceptible population. CONCLUSIONS: The increasing resistance to neonicotinoid insecticides raises concerns for the continued effective management of Colorado potato beetles in potatoes and highlights the need for more rigorous practice of integrated pest management methods. Copyright © 2011 Society of Chemical Industry     

An international campaign coordinated by EPPO to control Colorado Beetle in the Channel Islands

Collaboration between the Plant Protection Services of France and the Channel Islands has helped to keep Jersey and Guernsey free of Colorado Beetle (Leptinotarsa decemlineata) for over 60 years. The Channel Islands lie close to the coast of the Cotentin peninsula where potato fields are still infested with Colorado Beetle. Joint surveys of the coastal potato fields of Cotentin provide valuable information on beetle populations. Beetle flight records together with local weather data provide the necessary information to issue Colorado Beetle alerts to the Channel Islands during the months of May–July. High risk alerts issued by French Plant Protection colleagues are acted on and inspectors are mobilised to search beaches in Jersey and Guernsey for adult beetles. Farmers are not obliged to control Colorado Beetle in France. However, there has been an agreement negotiated in the Cotentin that farmers will treat their fields if beetles are found, with monitoring carried out by staff from a local co‐operative. This co‐operation has proved essential in the Colorado Beetle campaign.     

Insecticide resistance patterns among colorado potato beetle,leptinotarsa decemlineata (Say) (Coleoptera: chrysomelidae), populations in north carolina

A residual bioassay was used to determine insecticide susceptibility in populations of Colarado potato beetle, Leptinotarsa decemlineata (Say), in eastern North Carolina. Mean bioassay mortalities were subjected to correlation analysis to assess patterns of resistance among tested insecticides. Within insecticide classes, cross-resistance patterns were observed between carbaryl and carbofuran (carbamates), fenvalerate and permethrin (pyrethroids), and among azinphosmethyl, methamidophos, and phosmet (organophosphates). Significant associations between insecticides from different insecticide classes were concluded to be due to multiple resistance. Knowledge of multiple and cross-resistance patterns are discussed in relation to the bioassay as a tool for monitoring insecticide resistance.     

Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae (Sulzer)

In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.     

Electrophysiological identification of site-insensitive mechanisms in knockdown-resistant strains (kdr,super-kdr) of the housefly larva (Musca domestica)

The effects of pyrethroids were studied upon isolated segmental nerves and neuromuscular junctions in both susceptible (Cooper) and knockdown-resistant (kdr; super-kdr) strains of housefly larvae (Musca domestica L.). Isolated segmental nerves contained neither cell bodies nor synaptic contacts; thus, any effects of pyrethroids were attributed solely to their actions upon voltage-dependent Na⁺ channels. Threshold concentrations of the type II pyrethroid, deltamethrin, required to elevate the spontaneous firing rate of these nerves were determined. Both resistant strains were about ten times less sensitive to deltamethrin than the susceptible strain, but insensitivity of super-kdr nerves was no greater than in the less resistant kdr strain. At neuromuscular junctions, the minimum concentrations of pyrethroids needed to trigger massive increases in the frequency of miniature excitatory postsynaptic potentials (mEPSPs) were determined for deltamethrin and the type I pyrethroid, fenfluthrin. With fenfluthrin there was no detectable difference between the junctions of kdr and super-kdr strains, which were both about ten-fold less sensitive than Cooper junctions. With deltamethrin, kdr junctions were about 30 times less sensitive than those of Cooper; super-kdr junctions were dramatically insensitive to deltamethrin, being some 10000- and 300-fold less sensitive than those of Cooper and kdr respectively. Thus, in the synaptic assay, super-kdr conferred an extension in resistance over kdr only against the type II pyrethroid, it being ineffective against fenfluthrin. We suggest that kdr resistance comprises at least two site-insensitive areas within the nervous system. One involves insensitivity of the Na⁺ channel and has similar efficacy in both kdr and super-kdr strains against type I and II pyrethroids; the other is associated with the presynaptic terminal and is particularly effective in super-kdr resistance against type II pyrethroids. The latter could be associated with Ca²⁺-activated phosphorylation of proteins involved with neurotransmitter release. Such phosphorylation reactions are known to be perturbed by pyrethroids, especially by type II compounds.     

Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.

Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed.     

High-throughput approach to detection of knockdown resistance (kdr) mutation in mosquitoes, Culex quinquefasciatus, based on real-time PCR using single-labelled hybridisation probe/melting curve analysis

BACKGROUND: Knockdown resistance (kdr) mutation (L1014F) is a well‐defined mechanism of resistance to pyrethroids and DDT in many insect species. Sensitive detection of the mutations associated with resistance is a prerequisite for resistance management strategies. The authors have developed a new real‐time molecular diagnostic assay based on SimpleProbe^®/melting curve analysis for large‐scale kdr genotyping in the wild population of Culex quinquefasciatus Say, the principal vector of bancroftian filariasis. Melting curve analysis is based on the thermal stability difference between matched and mismatched DNA duplexes. The application of SimpleProbe^® chemistry in insects described here is novel in entomology research. RESULTS: The mosquitoes homozygous for knockdown‐resistant and knockdown‐susceptible allele showed melting peaks at 60.45 °C ( ± 0.25) and 64.09 °C ( ± 0.24) respectively. The heterozygous mosquitoes yielded both peaks at approximately 60.5 °C ( ± 0.2) and 64.20 °C ( ± 0.23). Among the 92 samples genotyped, 16 were found to be homozygous resistant, 44 homozygous susceptible and 32 heterozygous. Comparative assessments were made of all the reported methods for kdr genotyping. CONCLUSION: The present method is cheaper, faster, more reliable and versatile than other alternatives proposed in detecting correct kdr genotypes in mosquitoes. This is the first report using a single‐labelled hybridisation probe to detect point mutations in insect populations. Copyright © 2010 Society of Chemical Industry     

Use of biochemical and DNA diagnostics for characterising multiple mechanisms of insecticide resistance in the peach-potato aphid,Myzus persicae (Sulzer)

The peach-potato aphid Myzus persicae (Sulzer) can resist a range of insecticides by over-producing detoxifying esterase and having mutant-insensitive forms of the target proteins, acetylcholinesterase (AChE), and the sodium channel. Using a combination of bioassays, biochemical and DNA diagnostics, it is now possible to diagnose all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. A survey of 58 samples of wide geographic origin showed that all 46 resistant clones had amplified esterase genes (E4 or FE4) conferring broad-spectrum resistance to pyrethroids, organophosphates and carbamates. These occurred in combination with insensitive AChE (11 clones), conferring resistance to pirimicarb and triazamate, and/or mutant sodium channel genes (25 clones), conferring knockdown (kdr) resistance to pyrethroids and DDT. Amplified esterase genes were in linkage disequilibrium with both insensitive AChE and the kdr mutation, reflecting tight physical linkage, heavy selection favouring aphids with multiple mechanisms, and/or the prominence of parthenogenesis in many M. persicae populations. An ability to monitor individual mechanisms with contrasting cross-resistance profiles has important implications for the development of resistance management recommendations. ©1997 SCI     

Synthesis and insecticidal activity of 3-(haloalkyl-1,3-dienyl)-2,2-dimethylcyclopropanecarboxylates

New 2,2-dimethylcyclopropanecarboxylic acids bearing halogenated 1,3-alkadienyl substituents at position 3 of the cyclopropane ring were synthesised and the insecticidial activities of their 3-phenoxybenzyl and α-cyano-3-phenoxybenzyl esters against housefly (Musca domestica), Colorado potato beetle (Leptinotarsa decemlineata) and Egyptian cotton leafworm (Spodoptera littoralis) were determined. The activity of these compounds varied according to the relative position of the halogen substituents on the diene chain. The observed changes of activity with the structure of the side chain are discussed in terms of the coplanarity of the conjugated double bonds.     

Density—yield relationships for Colorado potato beetle adults on potatoes

A model composed of two straight lines describes the relation between overwintering spring adults of Colorado potato beetle [Leptinotarsa decemlineata (Say)] and potato yield. Potato vines (Solarium tuberosum (L.), cv. ‘Superior’) had a damage boundary of around 5.8 spring adults per plant. However, under conditions favorable for reproduction, overwintering adults of 0.07 beetles per plant would produce a large population of first-generation larvae that would reduce considerably the potato yield if not controlled. There is no appreciable seasonal overlapping between adults and young larvae in the field. Monitoring activities should therefore be directed at the beginning of the season against overwintering adults and later against first-generation larvae. For summer adults, an action threshold of 10 per plant is proposed.     

Control of Colorado Potato Beetle with the SIMLEP Decision Support System*

The Colorado Potato Beetle (Leptinotarsa decemlineata) is a serious threat in potato‐growing areas in Europe. Control requires a high insecticide input and meanwhile L. decemlineata has developed resistance to all commonly used insecticides. In order to establish an integrated pest management system a temperature‐driven decision support system (SIMLEP DSS) has been elaborated and consists of two modules. SIMLEP 1 is a regional forecasting model for the first occurrence of hibernating beetles and the start of egg laying. SIMLEP 3 is a field–specific model which forecasts the occurrence of the developmental stages of L. decemlineata. From 1999 to 2004 SIMLEP 3 was validated in Germany, Austria, Italy and Poland. In about 90% of cases SIMLEP 3 correctly predicted the periods of maximum egg laying and young larval occurrence, which are the optimal periods for field assessments and treatments with conventional and biological insecticides. SIMLEP 3 is used in practice in Germany and Austria on a large scale and in the western part of Poland. The SIMLEP DSS contributed a lot to the improvement of farmers’L. decemlineata control measures.     

Evaluation of the resistance of transgenic potato plants expressing various levels of Cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say) in the laboratory and field

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, is a destructive pest. The CPB is a quarantine pest in China, but has now invaded the Xinjiang Uygur Autonomous Region and is continuing to spread eastwards. To control the damage and overspreading, transgenic potato plants expressing Cry3A toxin were developed, and their resistance to CPB was evaluated by bioassays in the laboratory and field in 2009, 2010 and 2011. RESULTS: The insect resistance of the high‐dose (HD) transgenic lines was significantly greater than the middle‐dose (MD) and low‐dose (LD) transgenic lines regarding leaf consumption, biomass accumulation and mortality. The HD and MD transgenic lines showed 100% mortality when inoculated with first‐ and second‐instar larvae; however, the LD transgenic lines showed about 50% mortality. The HD transgenic lines exhibited a significantly higher yield than the MD and LD transgenic lines owing to their high CPB resistance. CONCLUSION: Commercially available transgenic potato plants with above 0.1% Cry3A of total soluble protein and NT control refugia could control damage, delay adaptation and halt dispersion eastwards. The two HD transgenic lines developed in this study, PAH1 and PAH2, are ideal for use as cultivars or germplasm to breed new cultivars. Copyright © 2012 Society of Chemical Industry     

Efficacy of chemical plant protection products on potato in Ukraine1

The principal potato pests controlled with plant protection products in Ukraine are Leptinotarsa decemlineata and Phytophthora infestons. Results are presented of trials of insecticides and fungicides against these pests. As a result, Sumi-alfa 5% EC (a.s. esfenvalerate), Karate water-soluble granules (a.s. lambda-cyhalothrin) and Betabytroid 2.5% EC (a.s. beta-cyfluthrin) were recommended for full-scale testing against Colorado beetle in Ukraine. Sul'fokarbation and EKhR 19517 were recommended for full-scale testing against potato blight.     

菌株NDBJJ-BFG的鉴定及其对马铃薯甲虫不同虫态的毒力测定

为筛选出对马铃薯甲虫Leptinotarsa decemlineata(Say)具有防治潜力的菌株,本研究从马铃薯甲虫僵虫虫体上分离菌株NDBJJ-BFG,通过形态学特征和分子生物学方法明确其分类地位,采用喷雾法和覆土法测定了不同浓度下该菌株对马铃薯甲虫室内毒力的致死中浓度LC50及半致死时间LT50。结果表明,经形态特征与r DNA ITS序列分析最终确定菌株NDBJJ-BFG为球孢白僵菌Beauveria bassiana(Bals.)Vuill。该菌株对1、2、3和4龄马铃薯甲虫幼虫的LC50分别为0.91×10~6、1.51×10~6、5.09×10~6和6.84×10~6个/m L;采用喷雾法和覆土法处理蛹的LC50分别为1.43×10~7个/m L和8.15×10~6个/m L,成虫的LC50分别为5.08×10~7个/m L和2.97×10~7个/m L。在孢子悬浮液浓度相同时LT50随虫龄的增大而延长,其中成虫的LT50最长,其次是蛹;在相同龄期下LT50随着孢子悬浮液浓度的增大而缩短。表明菌株NDBJJ-BFG对马铃薯甲虫1龄和2龄幼虫具有高毒力,其在马铃薯甲虫的生物防治中将具有较大的应用潜力。     

印楝素对马铃薯甲虫的控制作用

为探讨植物源杀虫剂印楝素对马铃薯甲虫的防治作用,采用叶碟法测定印楝素乳油对其成虫的拒食作用,并通过药效试验测定印楝素乳油对马铃薯甲虫的控制效果。结果显示,印楝素对马铃薯甲虫成虫有较强的非选择性拒食作用,且拒食率随取食时间的增加而升高。在取食36 h后,浓度为6、7.5、10、15和30 mg/L印楝素的拒食率分别为55.5%、59.1%、53.4%、76.1%和63.3%,拒食率与浓度无显著相关性;但印楝素对马铃薯甲虫的选择性拒食率较低,各浓度处理间均无显著差异,取食36 h时,选择拒食率约20%;在田间10 mg/L印楝素溶液施药1 d和3 d后,虫口减退率分别为26.52%和50.76%,显著高于对照组,施药9 d后,马铃薯甲虫的校正防治效果为48.86%。表明印楝素对马铃薯甲虫有一定的控制效果。     

Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction

BACKGROUND: Pediculosis is the most prevalent parasitic infestation of humans. Resistance to pyrethrin‐ and pyrethroid‐based pediculicides is due to knockdown (kdr)‐type point mutations in the voltage‐sensitive sodium channel α‐subunit gene. Early detection of resistance is crucial for the selection of effective management strategies. RESULTS: Kdr allele frequencies of lice from 14 countries were determined using the serial invasive signal amplification reaction. Lice collected from Uruguay, the United Kingdom and Australia had kdr allele frequencies of 100%, while lice from Ecuador, Papua New Guinea, South Korea and Thailand had kdr allele frequencies of 0%. The remaining seven countries investigated, including seven US populations, two Argentinian populations and populations from Brazil, Denmark, Czech Republic, Egypt and Israel, displayed variable kdr allele frequencies, ranging from 11 to 97%. CONCLUSION: The newly developed and validated SISAR method is suitable for accurate monitoring of kdr allele frequencies in head lice. Proactive management is needed where kdr‐type resistance is not yet saturated. Based on sodium channel insensitivity and its occurrence in louse populations resistant to pyrethrin‐ and pyrethroid‐based pediculicides, the T917I mutation appears to be a key marker for resistance. Results from the Egyptian population, however, indicate that phenotypic resistance of lice with single or double mutations (M815I and/or L920F) should also be determined. Copyright © 2010 Society of Chemical Industry