Control efficacy of Bacillus thuringiensis and a new granulovirus isolate against Cydia pomonella in orchards

Abstract

Codling moth, Cydia pomonella, is one of the most serious pests of apple and pear worldwide. This study evaluates the efficacies of a granulovirus, a Bacillus thuringiensis (Bt) strain and their combination in the control of C. pomonella in China. A Cydia pomonella granulovirus (CpGV) was isolated from C. pomonella cadavers in an orchard in Gansu, China. Droplet-feeding bioassays showed the median lethal concentration (LC₅₀) of this CpGV isolate (CpGV-C1) against the third instar C. pomonella larvae was 770 OBs µl^?1. The LC₅₀ values of Bt C-33 and kurstaki HD-1 against the third instar larvae were 26.3 µg ml^?1 and 15.7 µg ml^?1, respectively. Field tests indicated the control efficacies of CpGV-C1 and the combination of CpGV-C1 and Bt against C. pomonella larvae in apple orchards were similar to that of beta-cypermethrin. Our data demonstrated that the combination of CpGV and Bt might effectively protect apple fruits from the damage of C. pomonella larvae and had the potential to be developed as a low-cost, highly effective insecticide.     

苹果蠹蛾颗粒体病毒CpGV-CJ01的分离和鉴定

苹果蠹蛾颗粒体病毒(Cydia pomonella granulovirus,CpGV)是控制苹果蠹蛾Cydia pomonella(L.)种群的重要生防因子。商业化产品病毒株单一,抗性的快速产生会降低CpGV的防效,高毒力或与之具有遗传差异的新病毒株可用于延缓病毒抗性的产生或发展,对苹果蠹蛾的控制策略具有重要作用。本研究从中国酒泉地区苹果园中的苹果蠹蛾野生种群及其实验室种群发病虫体中分离获得一株致病病毒,通过幼虫发病特征、病毒包涵体的形态学及分子生物学方法共同证实该病毒株为CpGV,且与CpGV-M相似,将其命名为CpGV-CJ01。其幼虫的发病体征具有CpGV感染的典型症状,包涵体的电镜形态学观察结果符合CpGV结构特征,利用分子生物学方法克隆得到Granulin基因和凋亡抑制蛋白基因(inhibitor of apoptosis protein,iap)均与CpGV-M具有99%以上的相似性,但是对于毒力的准确定量需要做进一步研究。我国新病毒株的发现为我国开展苹果蠹蛾颗粒体病毒的生物学防治创造了先决条件,并可作为延缓世界范围内CpGV抗性的候选菌株。     

Field resistance of codling moth against Cydia pomonella granulovirus (CpGV) is autosomal and incompletely dominant inherited

The current appearance of local codling moth populations with resistance to Cydia pomonella granulovirus (CpGV) is an impediment to continuous CpGV application. Therefore, crossing experiments have been performed in order to gain information about the inheritance of resistance. Evidence is presented that the observed field resistance is stably inherited even under non-selective conditions in the laboratory. Offspring of reciprocal F(1) crosses between a susceptible ('S') and a resistant ('R') strain and backcrosses between F(1) and S were bioassayed at different CpGV concentrations. The resistant strain showed 100 times lower susceptibility in 7-day bioassays. The responses of the reciprocal crosses (male S x female R and female S x male R) did not differ significantly, indicating that resistance is autosomally inherited. The median lethal concentration for the F(1) progeny was intermediate between those of its parental strains. Mortality data obtained from the backcrosses suggested that inheritance of resistance is due to a non-additive, polygenic trait.     

Overcoming the resistance of codling moth against conventional Cydia pomonella granulovirus (CpGV-M) by a new isolate CpGV-I12

Recently, codling moth (CM, Cydia pomonella L.) populations with a significantly reduced susceptibility to C. pomonella granulovirus (CpGV) products have been observed in Germany. A novel CpGV isolate, designated CpGV-I12, is able to overcome the CpGV resistance. CpGV-I12 originated from Iran and showed superior efficacy in laboratory bioassays against a resistant CM strain (CpR), which has a 100-fold reduced susceptibility to commercially used isolate CpGV-M. Determination of the median lethal concentration (LC₅₀) indicated that CpGV-I12 is nearly as efficient in resistant CpR as CpGV-M in a susceptible CM strain (CpS). Beyond, CpGV-I12 caused superior mortality in CpS. Infection experiments showed that the resistance breaking effect can be observed in all instars of CpR. CpGV-I12 is a promising alternative control agent of CM in orchards where conventional CpGV products fail. In addition, we demonstrate in bioassays with recombinant expressed Cry1Ab that cross-resistance to CpGV and Bacillus thuringiensis products is not likely.     

Characterisation of Cydia pomonella granulovirus from codling moths in a laboratory colony and in orchards of British Columbia*

Sequence analyses indicate that a granulovirus isolated from a laboratory colony of codling moth in British Columbia, Canada is identical or very similar to the Mexican isolate of Cydia pomonella granulovirus (CpGV-M). In addition to codling moth, the vims was found to cause mortality of secondary leafroller pests of North American orchards, including the fruit tree leafroller and the obliquebanded leafroller. A survey in apple growing regions of British Columbia indicates that 23% of the wild population of codling moth larvae carry CpGV.     

一种增强苹果蠹蛾颗粒体病毒田间防效方法的建立

【背景】苹果蠹蛾是危害苹果和梨等蔷薇科果实的世界性重要蛀果害虫之一,目前已侵入我国新疆、甘肃、黑龙江、宁夏、内蒙古、吉林、辽宁等7省市,正在向苹果主产区扩散,严重威胁我国苹果产业的发展。欧美国家的田间应用证明,Cypo GV是防治苹果蠹蛾效果最佳的生防制剂。研究开发本土Cypo GV商业化制剂,对保证我国苹果业健康发展有重大意义。【方法】用室内和田间生测方法,在半人工饲料中添加被紫外线处理过的氧化铁和Cypo GV-ZY混合悬液,其中氧化铁的添加浓度分别为3、5、7、10、12 mg·m L~(-1),并完成其对苹果蠹蛾初孵幼虫的生物测定。【结果】在病毒悬液中添加7 mg·m L~(-1)的氧化铁能显著提高Cypo GV在田间的活性和持久性,与未添加氧化铁处理相比较,初孵幼虫死亡率分别提高了53.88%和96.64%。【结论与意义】添加低剂量氧化铁,显著增强了Cypo GV对田间强日光、强紫外线的适应性,提高了该病毒在田间的活性和持久性,说明氧化铁可作为商业化Cypo GV制剂的助剂,提高田间Cypo GV对苹果蠹蛾防治效果。本研究为我国Cypo GV制剂生产过程中添加助剂提供了依据。     

苹果蠹蛾老熟幼虫诱杀技术

本文对简易杀虫诱集带诱杀苹果蠹蛾Cydia pomonella(L.)老熟幼虫的效果进行了研究。发现在编织袋,旧衣物,瓦楞纸3种材料中,旧衣物对苹果蠹蛾的老熟幼虫捕获效果最好,其诱捕量占总诱捕量的68.42%;在诱集带中添加的农药中(包括乐斯本、敌敌畏、白僵菌、灭幼脲3号、高效氯氰菊酯等),乐斯本的杀虫效果最好,幼虫死亡率达到98.20%;在不同浓度的乐斯本溶液中,500倍乐斯本杀虫效果最好,幼虫死亡率达到91.61%,且对幼虫没有驱避作用;500倍乐斯本+50倍白僵菌药杀虫诱集带有效期长达40多天,37d后对幼虫的杀伤效果依然达到96%以上,作为老熟幼虫的防治措施十分理想。     

New method for testing solar sensitivity of commercial formulations of the granulovirus of codling moth (Cydia pomonella, Tortricidae: Lepidoptera)

A method for screening codling moth granulovirus (CpGV) formulation sensitivity to sunlight using specially prepared half apples and a solar simulator is described. The half apple preparation allows an even coverage of virus over the surface of the fruit that would not be possible using whole apples. Leaves and artificial medium were not usable for extended periods of exposure in the solar simulator due to excess drying. Fruit was sprayed with 10⁻³ and 10⁻⁵ dilutions of three commercial formulations of CpGV (Carpovirusine, Cyd-X, and Virosoft) and infested with codling moth neonates. Half of the sprayed fruit was exposed to 650 W/m² for 4 h in an Atlas Suntest CPS solar simulator resulting in an accumulated radiant energy of 9.36 × 10⁶ J/m² before they were infested with neonate codling moth larvae. Spraying non-irradiated fruit with the 10⁻³ dilution of Cyd-X and Virosoft resulted in nearly 100% mortality of neonate larvae. Irradiation reduced viral activity by 71-98% at the 10⁻³ dilution and by up to 32% at the 10⁻⁵ dilution relative to non-irradiated fruit. The procedures utilized enabled good preservation of the fruit throughout the incubation period and minimized invasion of the fruit by plant pathogens and saprophytic organisms. This laboratory method for screening candidate formulations and potential UV protectants could conserve time and resources by eliminating adjuvants with less potential in laboratory tests and field testing only the most promising candidates. It also enables year-round testing.     

Horizontal Escape of the Novel Tc1-Like Lepidopteran Transposon TCp3.2 into Cydia pomonella Granulovirus

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons. Received: 27 February 1997 / Accepted: 16 May 1997     

不同剂型迷向剂处理对苹果蠹蛾控制效果比较

【背景】苹果蠹蛾是我国重要的检疫性害虫,目前从新疆迅速向东部扩散。苹果蠹蛾性信息素胶条迷向剂对防控苹果蠹蛾高效、安全,但成本较高。【方法】2013年在宁夏中卫市常乐镇马路滩村果园使用3种不同剂型的迷向剂对苹果蠹蛾进行了防治试验,每个处理果园面积3.3 hm~2,分别使用性诱剂处理后,用诱捕器监测处理后的诱蛾量,在生长期调查果实为害率,比较防治效果。【结果】通过整个生长季诱捕器监测,悬挂点以900个·hm~(-2)双层处理,胶条迷向剂在开花前处理1次,整个生长季处理区中心部位没有监测到苹果蠹蛾成虫;透明塑管迷向剂分别在开花前和6月底处理2次,整个生长季中心部位平均每个诱捕器诱到3.3头雄蛾;膏体迷向剂分别在开花前和6月底处理2次,整个生长季中心部位平均每个诱捕器诱到12头雄蛾。在6月下旬和果实采收前进行果实为害率调查,胶条迷向剂区平均蛀果率分别为0和0.56%,透明塑管迷向剂区蛀果率分别为0.89%和1.38%,膏体迷向剂处理区蛀果率分别为0.79%和1.38%。常规农药防治区2个时期蛀果率分别为1.44%和3.13%。【结论与意义】综合考虑防治效果和使用成本,在虫口密度高、效益高的果园可以使用胶条迷向剂,膏体迷向剂和透明塑管迷向剂只有在虫口密度较低的情况下使用。     

Isolation of microsatellite loci in the codling moth,Cydia pomonella (Lepidoptera: Tortricidae)

Cydia pomonella L. is an important insect pest of pome fruits worldwide. We have isolated and characterized 17 microsatellite loci from the enriched genomic libraries constructed using a biotin/streptavidin capture protocol. Among these loci, 11 were scored polymorphic when 50 individuals from a laboratory population were screened. Their alleles numbered two to four, with the observed heterozygosity (H_O) ranging from 0.114 to 0.404. Successful amplification was obtained for all these loci when the designed primers were tested, showing the promise of use in genetic mapping and population studies.     

苹果蠹蛾体内wolbachia wsp基因克隆与序列分析

【目的】对苹果蠹蛾Cydia pomonella L.体内共生菌Wolbachia进行分子生物学鉴定,确定该虫体内Wolbachia的进化位置,为进一步探讨Wolbachia对其生殖作用的调控机制提供理论依据。【方法】应用Wolbachia的wsp基因特异引物,通过PCR扩增法检测了苹果蠹蛾10个地理种群(新疆伊犁、吐鲁番、和田、石河子、奎屯、哈密、库尔勒、阿拉尔、喀什、和甘肃张掖)感染Wolbachia的状况,并对阿拉尔种群体内的Wolbachia的wsp基因进行测序和序列分析。【结果】苹果蠹蛾10个地理种群全部感染了tWolbachia,利用wsp基因的特异性引物从阿拉尔种群体内扩增出了617 bp的Wolbachia的wsp基因片段(GenBank登录号为KC832324),系统发育分析结果表明,苹果蠹蛾体内感染的Wolbachia属于A群Dor亚群,与锤角细蜂亲缘关系较近。【结论】苹果蠹蛾体内普遍感染了Wolbachia,属于A群Dor亚群。     

Spatial discrimination of pheromones and behavioural antagonists by the tortricid moths Cydia pomonella and Adoxophyes orana

Male moths responding to their species-specific sex pheromone, may cease their upwind flight when pheromone components of sympatric species are added to the mixture. The interspecific interaction between the pheromone response of the tortricid moths Cydia pomonella and Adoxophyes orana was investigated in field-trapping and wind-tunnel studies. Addition of the A. orana pheromone [(Z9)-tetradecenylacetate and (Z11)-tetradecenylacetate] to a source containing the C. pomonella pheromone [(E8, E10)-dodecadienol] resulted in a significant inhibition of attraction by male C. pomonella. It is demonstrated that this behavioural antagonist for C. pomonella must be emitted from the same point source to induce this inhibitory effect. A spatial separation of the two interspecific pheromones (at 14 cm, 5 cm and 0.5 cm crosswind) restored the attraction of the conspecific pheromone for male C. pomonella. In contrast to C. pomonella, male A. orana were not inhibited by point sources releasing both the C. pomonella and A. orana pheromone. We suggest that the discrepancy in the interspecific pheromone interaction between these two tortricids can be explained if we consider the evolutionary ecology of interspecific pheromone communication in C. pomonella and A. orana. Accepted: 18 July 1999     

Mapping the disjunct distribution of introduced codling moth Cydia pomonella in China

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分子模拟技术及其在苹果蠹蛾代谢杀虫剂分子机制研究中的应用进展

苹果蠹蛾是世界性重要的果树害虫,也是我国一类进境检疫性有害生物,严重威胁着我国黄土高原和环渤海湾优势苹果产区的果业生产。化学防治是当前苹果蠹蛾防治最经济有效的措施,然而频繁使用化学杀虫剂引发的抗药性问题也在世界范围内相继报道。代谢抗性是苹果蠹蛾抗性中重要的机制,苹果蠹蛾解毒酶代谢杀虫剂的分子机制是当前代谢抗性研究的重点方向。基于分子模拟的方法在相关研究中扮演着越来越重要的角色,本文综述了分子模拟涉及的主要方法及其在苹果蠹蛾代谢杀虫剂分子机制研究中的应用,并展望未来可能的研究方向。     

Prospection and molecular analysis of CpGV isolates infecting Cydia pomonella at different geographical locations in Argentina

The occurrence and genetic diversity of Cydia pomonella granulovirus infecting C. pomonella across Argentina were investigated. Larvae were collected from fruit orchards in regions where CpGV‐based biopesticides have been extensively used and then discontinued (Catamarca) or are still being used (Alto Valle). The survey was also extended to regions where few occasional (i.e. Mendoza) or no virus treatments (various locations) have been made. PCR analysis showed an unexpectedly high proportion of infected larvae (31.3–50%) at most sampling sites in Catamarca. Phylogenetic analysis of these samples, based on gran and lef‐8 genes partial sequences, revealed a previously unreported CpGV variant which could not be assigned to any of the known CpGV genome types and formed a well‐defined new cluster. Instead, samples from Alto Valle were genetically similar to isolate CpGV‐M (the active ingredient in commercial formulations). There, the percentage of infected individuals was considerably lower (1.8–21.9%). The virus was not detected in Mendoza or the other sampling regions. Implications of these results on the epidemiology and current classification of CpGV isolates are discussed.     

Evaluation of spray-dried lignin-based formulations and adjuvants as solar protectants for the granulovirus of the codling moth, Cydia pomonella (L)

Commercial formulations of the codling moth, Cydia pomonella L., granulovirus (CpGV) are limited by their short residual activity under orchard conditions in the Pacific Northwest. We evaluated spray-dried lignin-encapsulated formulations of CpGV for improved solar stability based on laboratory bioassays with a solar simulator and in field tests in an infested apple orchard. In laboratory tests, aqueous lignin formulations containing a high dosage of 3 x 10(10) occlusion bodies (OB)/L, with and without the additives titanium dioxide (TiO(2)) and sugar, provided significant solar protection of virus, i.e., mortality of codling moth exposed to lignin formulations that had been irradiated with 9.36 x 10(6) joules/m(2) was 92-94%, compared with 66-67% from a glycerin-stabilized product (Cyd-X) or suspension of pure unformulated virus at the same rates. By comparison, a lower dosage of the lignin formulation (3 x 10(8)OB/L) did not provide significant solar protection. Equivalent dosage-dependent patterns in solar protection were observed in further tests with the lignin formulation, when an intermediate (3 x 10(9)OB/L) as well as the low dosage provided no solar protection. Equivalent rates of a blank lignin formulation (containing no virus) did not affect larval mortality, suggesting a protective effect of the lignin on the virus at the high rate. The use of several spray adjuvants, 'NuFilm-17' and 'Organic Biolink' (sticker-spreaders at 0.06% v/v), 'Raynox' (sunburn protectant at 5% v/v), and 'Trilogy'(neem oil at 1% v/v) did not provide solar protection of a commercial CpGV preparation in laboratory tests. In season long orchard tests (Golden Delicious), the lignin formulation of CpGV applied at 6.57 x 10(12)OB/ha did not significantly improve control of codling moth or protection of fruit compared with Cyd-X at equivalent rates. Our studies show that lignin-based CpGV formulations provided solar protection at relatively high virus dosages. The testing of lignin formulations containing reduced virus concentrations may allow virus solar protection to be achieved at more economical rates.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.