Insecticidal activity of avidin combined with genetically engineered and traditional host plant resistance against Colorado potato beetle (Coleoptera: Chrysomelidae) larvae

Colorado potato beetle, Leptinotarsa decemlineata (Say), is a destructive pest of potato, Solanum tuberosum (L.), in North America. It is renowned for adapting to insecticides. With the arsenal of effective insecticides decreasing, it is important to consider alternative forms of control. Biotin is an essential coenzyme for insect growth and development. Avidin is a protein found in chicken egg that sequesters biotin and has shown insecticidal properties against a range of insect. We assessed the effectiveness of avidin against the Colorado potato beetle neonates in a no-choice detached leaf bioassay at 0, 17, 34, 51, 102, and 204 microg avidin/ml over 12 d. The LC50 was 136 microg avidin/ml (108-188 95% CL). The combined effects of avidin (136 microg avidin/ml) with Bt-Cry3A or leptines were evaluated with neonates and third instars over 12 and 6 d, respectively. Three potato lines were used: susceptible line, a line engineered to express Cry3A from Bacillus thuringiensis, and a line expressing the natural resistance factor leptines. The addition of avidin at the LC50 concentration significantly reduced consumption by neonates, but it did not affect consumption by third instars feeding on the susceptible line and the leptine line. Survival of neonates feeding on the susceptible line with avidin was significantly reduced compared with the susceptible line. Survival of third instars on the Bt-Cry3A with avidin was significantly reduced after 3 d compared with survival on the Bt-Cry3A, suggesting the addition of avidin may increase susceptibility to Bt-Cry3A.     

Within field movement of overwintered Colorado potato beetle: a patch-based approach

Abstract:  After comparing the persistence of four marking techniques, a mark–release–resight study was performed to characterize mid-season movement of the Colorado potato beetle [ Leptinotarsa decemlineata (Say); Col., Chrysomelidae] simultaneously in a fallow and in a wheat field. Isolated patches of potatoes were installed in a random spatial arrangement on both fields similarly. Overwintered beetles were individually marked and released. Beetles showed limited inter-patch movement activity (15.9% of recovery events) with an overall mean daily dispersal of 0.309 m (0.0–7 m). There was a significant difference in the insects' movement distance between the fallow and wheat field but there was no difference between the movement distances of males and females. The distance between the patches varied between 1 and 7.81 m, and inter-patch movement was infrequent (15.9%). Results suggest that surrounding fields by wheat rather than fallow grounds should be studied as a possible strategy to reduce the movement of overwintered beetles between potato fields.     

间作玉米对马铃薯甲虫种群及天敌昆虫的影响

在农田尺度下,如何提升农田生态系统自身的控害能力,探索有利于害虫生态调控的农田种植模式是时下的研究热点之一,而间作作为一种能提高农田生态系统生物多样性以及控害能力的种植模式,已被广泛的应用于农作物生产.为进一步明确马铃薯-玉米间作种植模式对马铃薯甲虫种群动态的影响,在马铃薯甲虫发生期,对马铃薯-玉米2:2间作、马铃薯-玉米4:2间作、马铃薯-玉米-马铃薯2:2:2间作、马铃薯单作4种作物种植模式进行调查.结果表明:在第一代高龄幼虫为害高峰期(6月28日-7月19日)与第一代高龄幼虫为害末期(7月26日-8月16)时,单作田高龄幼虫数量均高于3种间作田,差异不显著(P>0.05).在第一代成虫羽化高峰期(7月12日-8月2日)与第一代成虫羽化末期(8月2日-8月23日)时,单作田成虫数量均高于3种间作田,差异不显著(P>0.05).整个调查期间,就高龄幼虫与成虫而言,3种马铃薯间作田虫量均小于单作田.此外,对3种间作田的天敌昆虫种群调查表明,优势天敌多异瓢虫的平均虫量马铃薯-玉米-马铃薯2:2:2间作田比马铃薯-玉米4:2间作田高,且差异显著(P<0.05).在马铃薯-玉米间作后期,间作田的马铃薯甲虫种群数量低于单作田,通过间作会对迁移能力较强的高龄幼虫与成虫有一定阻隔与调控作用.     

Persistence and inheritance of costs of resistance to imidacloprid in Colorado potato beetle

Reduced fitness among resistant versus susceptible individuals slows resistance evolution and makes it easier to manage. A loss of resistance costs could indicate novel adaptations or mutations contributing to resistance. We measured costs of resistance to imidacloprid in a Massachusetts resistant population compared with a Massachusetts susceptible population in 1999 in terms of fecundity, hatching success, egg development time, and sprint speed. Resistance was additive and seemed to be polygenic with high heritability. The fecundity cost appeared overdominant in 1999, and the hatch rate cost was partly recessive in 1999, but neither was significantly different from dominant or recessive. In 2004, we repeated our measures of resistance costs in Massachusetts in terms of fecundity and hatching success, and we added a new resistant population from Maine. In 2005, we compared development time of Maine resistant and the laboratory susceptible colony eggs. Significant fecundity costs of resistance were found in both population in both 1999 and 2004, and significant egg developmental time costs were found in 1999 and 2005. However, the hatching success costs of resistance were significant in 1999 and not apparent in 2004, suggesting some modification or replacement of the resistance genes in the intervening time.     

Glandular trichomes of Solanum berthaultii and resistance to the Colorado potato beetle

The defensive mechanisms of the wild potato, solanum berthaultii Hawkes, to larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say), were studied by selective removal of glandular trichomes and trichome exudates from leaflets, and by comparing performance on S. berthaultii and on the cultivated potato, S. tuberosum L., which lacks defensively active type A and B glandular trichomes. Removal of type A trichomes increased the proportion of larvae that fed on S. berthaultii. Removal of the exudate from type B trichomes increased the proportion of larvae that fed and led to a decrease in mortality. The predominant active compounds in type B exudate, i.e. fatty acid esters of sucrose, were only effective in the presence of type A trichomes. Sucrose esters did not affect larval feeding on S. tuberosum leaflets or on S. berthaultii leaf discs from which the type A trichomes had been removed. Growth of surviving larvae was not significantly affected by removing type A trichomes or type B exudate. Growth of larvae was significantly increased when S. berthaultii leaflets were presented in artificial diet which eliminated the physical barrier of the type B stalks. Growth was no different on artificial diet containing either S. berthaultii or S. tuberosum leaf material (fresh or lyophilized powder) but was poorer on these diets than on S. tuberosum leaflets. The presence of type A trichomes is a fundamental requirement for expression of S. berthaultii resistance to L1 L. decemlineata. Type B droplets containing sucrose esters increase the expression of resistance in the presence of defensively-active type A trichomes.

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Résumé Les mécanismes de défense de la pomme de terre sauvage, S. berthaultii Hawkes, aux larves de Leptinotarsa decemlineata Say, ont été étudiés par ablation sélective des trichomes glandulaires et par l'élimination de leur exsudat des folioles, et par comparaison avec S. tuberosum L. qui a perdu les trichomes glandulaires défensifs A et B. L'ablation des trichomes A a augmenté la proportion de larves ayant consommé S. berthaultii. L'élimination de l'exsudat des trichomes B a augmenté la proportion de consommatrices et réduit la mortalité. Les principaux composés actifs de l'exsudat B, c'est-à-dire des esters d'acides gras de sucrose, n'étaient actifs qu'en présence de trichomes A. Les esters de sucrose n'ont pas modifié la consommation larvaire sur folioles de S. tuberosum, ou sur disques de feuilles de S. berthaultii dont les trichomes A avaient été enlevés. La croissance des larves survivantes n'a pas été modifiée significativement par l'ablation des trichomes A ou l'élimination de l'exsudat de B. La croissance des larves a été significativement augmentée quand les folioles de S. berthaultii ont été incorporés dans l'aliment artificiel après élimination de la barrière physique due aux pédoncules B. La croissance a été de même importance sur aliments artificiels contenant des feuilles (fraiches ou en poudre lyophylisée) de S. berthaultii ou de S. tuberosum, mais plus faible que sur folioles de S. tuberosum. La présence de trichomes A est indispensable à la résistance de S. berthaultii aux L, de L. decemlineata. Les gouttelettes de type B contenant des esters de sucrose augmentent l'expression de la résistance en présence d'une défense active par trichomes A.

     

Orientation of Colorado potato beetle to natural and synthetic blends of volatiles emitted by potato plants

1 Behavioural responses of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), to volatiles emitted from solanaceous host plants (potato and tomato), a non‐host legume (soybean), and 13 synthetic blends or three individual chemicals emitted by potato plants were investigated in laboratory bioassays. 2 Both male and female CPB were attracted to volatiles emitted by mechanically damaged potato foliage, but not to mechanically damaged tomato foliage; CPB offered a choice between the two damaged solanaceous plants did not show a preference. 3 Among 16 odourous blends or individual chemical components of potato plant emissions tested, six blends were attractive, two were repellent, and eight elicited no preference in laboratory bioassays. Volatile blends containing relatively high amounts of the green leaf volatiles (E)‐2‐hexen‐1‐ol and (Z)‐3‐hexen‐1‐ol, or the sesquiterpene β‐ caryophyllene, were unattractive or repellent. Minimal blends attractive to CPB were comprised of (Z)‐3‐hexenyl acetate (±)‐linalool and methyl salicylate: the combination of all three chemicals elicited sexually dimorphic attraction of males; two component blends comprised of (Z)‐3‐hexenyl acetate and either (±)‐linalool and methyl salicylate attracted both sexes. Individual compounds were inactive. No significant difference was noted between two attractive blends, or an attractive synthetic blend vs. mechanically damaged potato foliage. 4 These results show that CPB are attracted to blends of specific chemicals emitted by their host plants and provide a basis for the use of plant attractants as a component of integrated management of pestiferous populations.     

非寄主植物间套作对马铃薯甲虫种群及其天敌的影响

农田作物布局作为害虫生态调控的重要内容,一直是保护性生物防治的研究热点。为进一步明确马铃薯田块作物间套作种植模式对马铃薯甲虫种群动态的影响,探索马铃薯甲虫可持续防控的新思路与新方法,本研究在马铃薯甲虫发生期对马铃薯-玉米、马铃薯-向日葵、马铃薯单作3种作物间套作模式进行田间种群数量调查,分析比较不同种植模式下的马铃薯甲虫种群动态差异。结果表明：在马铃薯甲虫发生期,马铃薯单作第二代幼虫为害高峰期出现晚于两种间套作模式,第一代成虫为害高峰期早于两种间套作模式。第二代幼虫为害低峰期（8月26日-9月7日）时,马铃薯-玉米间套作幼虫量显著低于马铃薯单作（P<0.05）,马铃薯-玉米间套作幼虫量显著低于马铃薯-向日葵（P<0.05）,整个调查期间,马铃薯单作虫量要大于两种间套作。此外,天敌昆虫群落调查表明：间套作玉米异色瓢虫量显著高于马铃薯单作（P<0.05）,间套作向日葵的草蛉、食蚜蝇虫量高于马铃薯单作。间套作向日葵或玉米对越冬代马铃薯甲虫的扩散有影响,马铃薯播种初期间套作向日葵或玉米能在一定程度上阻隔马铃薯甲虫的定殖扩散。     

Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae

Commercial biopesticides based on the fungal pathogen Beauveria bassiana strain GHA and the bacterial pathogen Bacillus thuringiensis tenebrionis were applied alone and in combination (tank mixed) against larval populations of the Colorado potato beetle, Leptinotarsa decemlineata, in small plots of potatoes over three field seasons. Interactions between the two products were evaluated in terms of pest-control efficacy. B. bassiana (formulated as Mycotrol) was applied at low and medium label rates of 1.25 and 2.5 x 10(13) conidia/ha, and B. thuringienis (formulated as Novodor) was applied at low and high label rates of 40.3 and 120.8 x 10(6) Leptinotarsa units/ha. Two weekly applications of the bacterial pesticide alone provided 50-85% control of beetle larvae within 14 days after the initial application, while applications of the mycopesticide alone produced no greater than 25% control. Maximum control, in nearly all tests, was produced by the combination of the two products. The combined treatments produced a statistically significant 6-35% greater reduction in larval populations than would have been predicted had the two biopesticides acted independently. This low-level synergistic interaction was observed during all field seasons and resulted from combinations at all rates, including, in one of two tests, the low rates of each product. These results indicate that B. thuringiensis and B. bassiana have strong potential for integrated biologically based management of Colorado potato beetle.     

Colorado potato beetle larvae on potato plants expressing a locust proteinase inhibitor

The natural defence system of plants often involves inhibitors of digestive enzymes of their pests. Modem and environmental-friendly methods try to increase this plant resistance by expressing heterologous protease inhibitors in crops. Here we report the effects of expressing a gene from desert locust (Schistocerca gregaria) encoding two serine protease inhibitors in potato on Colorado potato beetle (Leptinotarsa decemlineata) larvae. The gene encoding both peptides on a single chain was used for Agrobacterium-mediated transformation of potato plants. The presence of the active inhibitor protein in the leaves was verified. The feeding bioassays in the laboratory showed that despite the low level of the peptide in leaves, CPB larvae on transgenic plants have grown slightly but significantly more slowly than those on control potato plants. The results support the notion that expression of multifunctional proteinase inhibitors of insect origin in plants might be a good strategy to improve insect resistance.     

The genetic base and phenotypic manifestations of Colorado potato beetle resistance to organophosphorus insecticides

A high level of resistance to organophosphorus insecticides was recorded in a local population of the Colorado potato beetle (Ufa district, Bashkortostan). Toxicological analysis demonstrated that the fraction of susceptible individuals did not exceed 0.025. DNA analysis of the polymorphism of acetylcholinesterase gene (AChE) according to the presence of mutation 980A>G in the sample of Colorado potato beetle imagoes demonstrated the absence of wild-type (sensitive, SS) homozygotes and prevalence (0.79) of the homozygotes with mutant resistant type (RR). The phenetic analysis of the variation of the integument pattern phenes demonstrated statistically significant differences between the imago phenotypes in the ratio of SS and RR genotypes.     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.     

Colonization of potato fields in eastern North Carolina by Colorado potato beetle

Rotated and non-rotated commercial potato fields were sampled intensively to follow Colorado potato beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), colonization and subsequent oviposition patterns in the spring of 1990 and 1991. Maximum densities of colonizing adults ranged from 0 to 14,891/ha and maximum egg mass densities ranged from 0 to 48,451/ha. Crop rotation generally resulted in lower potato beetle populations. Regardless of crop rotation management practices, colonization of fields planted in potatoes began at field edges and progressed inward in all fields for both years. Management of potato beetles is discussed in light of these findings.     

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K_i values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.     

Disrupting spring colonization of Colorado potato beetle to nonrotated potato fields

Overwintering Colorado potato beetles (Leptinotarsa decemlineata (Say)) were concentrated primarily within woody borders, and mortality was lower in borders than in potato fields. After overwintering, only 15–44% of live beetles were in the potato fields. In experiments with small plots, colonization of fields from woody borders was reduced ∼60% by a trap crop, either treated with adulticide or with beetles collected daily. Such trap crops, or simply pitfall traps to prevent colonization from woody borders, could significantly reduce early-season adult numbers and subsequent larval populations. However, success is dependent on the local densities of overwintered beetles, and the prevalence of arrestment behavior in the case of trap crops.     

Quiescence in the Colorado potato beetle, Leptinotarsa decemlineata

Quiescence (death feigning) was induced in the Colorado potato beetle, Leptinotarsa decemlineata by mechanical disturbance of live and artificial plants on which beetles were walking, feeding or grooming. The response consisted of a sudden cessation of activity lasting 12 to 500 s. Recovery from this immobile state was usually preceded by either antennal or leg movement. The time taken for beetles to come out of the immobile state (i.e. recovery time) was not dependent on age or sex of beetles or on stimulus duration, but was dependent on degree of displacement and on satiation level. Repeated stimulations led to longer recovery times and to a smaller proportion of responding beetles. Stimulation within the immobile state also prolonged recovery time. The most effective frequency of mechanical disturbance for startling beetles was 30 Hz. This response is presumed to be part of the extensive defense repertoire of this beetle, but we have so far been unable to discover a natural enemy that will elicit it.     

Combining genetic engineering and traditional breeding to provide elevated resistance in potatoes to Colorado potato beetle

The sustainable deployment of resistant crop varieties is a critical issue for the implementation of biotechnology in crop pest management. Feeding, biomass accumulation, and mortality were evaluated for susceptible, insecticide‐resistant, and Bacillus thuringiensis (Bt) Cry 3A‐selected Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera, Chrysomelidae) larvae fed on: cultivated potato, a Solanum chacoense line expressing leptine glycoalkaloids, a transformed line expressing Bt toxin, or the leptine line transformed to express Bt toxin. Larvae selected for resistance to Bt‐Cry3A performed better on Bt foliage, but not as well on the leptine foliage, compared to susceptible or insecticide‐resistant larvae. Neither leptine nor Bt toxin completely inhibited the feeding and growth of 3rd and 4th instars of all three strains of Colorado potato beetle. However, for all three strains of Colorado potato beetle on leptine + Bt foliage, feeding was almost zero, growth was zero or negative, and mortality was near 100%.     

Potato field colonization by low-density populations of Colorado potato beetle as a function of crop rotation distance

Monitoring of 10 and 12 commercial potato, Solanum tuberosum L., fields in 2004 and 2005, respectively, confirmed for a low-density population of Colorado potato beetle, Leptinotarsa decemlineata (Say), that potato fields nearest to the previous year's potato fields are significantly more colonized by this beetle than more distant fields. This pattern is partially explained by the presence of a reservoir of colonizers estimated at 35% of the season-long colonizing population in 2004 and 2005. These beetles, which emerged before potato plants broke the ground, were ready to establish themselves on nearby potato plants. The colonizing Colorado potato beetles dispersed within the maximum range of 1.5 km over a season, and the colonization risk for the new crop decreased with distance from the previous year's crop. There was no evidence that rotation distance delayed colonization. In terms of pest management, although the findings confirm that only long 1.5-km rotations can prevent Colorado potato beetle colonization, they also demonstrate that short rotations of 100 m or more can make substantial contributions to pest management programs for low-density beetle populations.     

Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata,Say)

Transgenic Research - Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost...