Diet Incorporation Assay Research Articles

Characterization of Resistance to the Mexican Rice Borer (Lepidoptera: Crambidae) among Sugarcane Cultivars.

Simple SummaryThe Mexican rice borer, Eoreuma loftini, is one of the most important insect pests of sugarcane in the US. Management strategies that are environmentally friendly and economically profitable are needed to mitigate the pest’s impact. Cultivar resistance can become a key management strategy for Mexican rice borer control in sugarcane because it is inexpensive and easy for sugarcane growers to implement. Resistance is compatible with almost all other management strategies and minimizes further input costs incurred by the grower. However, the mechanisms of resistance are not completely understood. Elucidating which mechanisms and traits confer resistance can help sugarcane breeding programs increase the resistance level of future cultivar releases. We found that the currently grown commercial cultivars L 01-299 and HoCP 85-845 had the least amount of borer-related injury, while L 12-201 and HoCP 00-950 had the greatest level of injury. These differences appear to be related to the ability of recently hatched larvae to establish feeding rather than female preference to lay eggs on specific cultivars. Further work is needed to determine the influence of environmental factors on resistance expression and identify plant characteristics that are responsible for Mexican rice borer resistance.Cultivar resistance is an essential management strategy for the Mexican rice borer, Eoreuma loftini (Dyar), in sugarcane in the USA, but resistance mechanisms are poorly understood. Resistance was evaluated among Louisiana’s (USA) commercial sugarcane cultivars and experimental clones through field screenings, greenhouse trials, and a diet incorporation assay. Cultivars L 01-299 and HoCP 85-845 had the lowest borer injury levels, while HoCP 00-950 and L 12-201 were among the most heavily injured in field and greenhouse trials. The variability of results between the two field trials suggests that a genotype × environment interaction might affect the expression of resistance. Oviposition did not differ among evaluated cultivars in the greenhouse choice study. Results from the no-choice experiment showed that neonatal establishment differed among cultivars by up to 3-fold. In a diet incorporation assay, all cultivars reduced larval weight up to 86.5% and increased days to pupation by 1.8-fold relative to the diet-only control. Collectively, these results suggest that Louisiana’s sugarcane breeding germplasm contains various resistance levels to E. loftini, emphasizing the importance of screening cultivars before they are released to growers. Future studies should try to determine the influence of environmental factors on resistance expression.

Management of Stemborers (Lepidoptera: Crambidae) Using Foliar-Applied Chlorantraniliprole in Rice

Rice is one of the most important crops in Louisiana and Texas, USA. It is subjected to various insect pests throughout the growing season. Lepidopteran stem-boring pests including the Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae), are increasing in economic importance as insect pests in rice production, particularly in southwest Louisiana, USA. Field trials were conducted in Louisiana and Texas to examine the efficacy of foliar-applied insecticides (chlorantraniliprole and the pyrethroids ζ-cypermethrin and λ-cyhalothrin) and chlorantraniliprole seed treatment for control of rice stemborers during 2018 and 2019. Furthermore, a diet incorporation assay assessed the toxicity of chlorantraniliprole to laboratory-reared E. loftini. Results of field experiments in Louisiana showed foliar applications of chlorantraniliprole, at 22 to 35 d after permanent flood was established, reduced stemborer injury to rice by 57.9 to 96.5% in 2018 and 73.9 to 87.5% in 2019 compared to nontreated or pyrethroid-treated field plots. However, rice yields in 2019 did not vary among insecticide-treated and nontreated plots. In the Texas field experiments, chlorantraniliprole applied as seed treatment reduced stemborer injury, but was not effective in reducing injury when sprayed directly to soil after planting or when applied at permanent flood. The LC50 and LC90 (lethal concentration that produced 50% and 90% mortality, respectively) of chlorantraniliprole on third-instar E. loftini were 0.09 ± 0.03 and 0.53 ± 0.17 µg a.i. mL-1, respectively, at 6 d after exposure. At 10 d after exposure, LC50 and LC90 were 0.04 ± 0.02 and 0.16 ± 0.04 µg a.i. mL-1, respectively. In addition to chlorantraniliprole seed treatment, foliar application of chlorantraniliprole in rice could provide a new tool for management of damaging stemborer infestations.

Resistance to the Sugarcane Borer (Lepidoptera: Crambidae) in Louisiana Sugarcane Cultivars.

Cultivar resistance is a key management strategy for the sugarcane borer, Diatraea saccharalis (F.), the primary pest in Louisiana sugarcane, but mechanisms of resistance are not well understood. This research evaluated the potential mechanisms of cultivar resistance to D. saccharalis among commercially produced sugarcane cultivars and experimental lines through three field screenings, two greenhouse experiments, and one diet incorporation assay. The resistant standards HoCP 85-845, HoCP 04-838, and L 01-299 were among the cultivars with the lowest D. saccharalis injury levels in both field and greenhouse trials. Cultivars HoCP 00-950 and L 12-201 were among the most heavily injured in both trials. Differences in oviposition among cultivars in the greenhouse choice study were not detected, suggesting adult preference is not a key factor in resistance. This was also supported by the no-choice greenhouse experiment in which up to 9-fold differences in neonate establishment among cultivars were detected. Larval injury among cultivars in greenhouse experiments was consistent with field studies suggesting traits that affect neonate establishment (e.g., rind hardness) help to confer resistance in the field. In the diet incorporation assay, lower larval weights and longer time to pupation were observed on resistant cultivar Ho 08-9003, but no differences were found among current commercial cultivars. Continuous evaluation of cultivar resistance to D. saccharalis is important in developing effective integrated pest management strategies for this pest. More research into plant characteristics (e.g., leaf sheath tightness and pubescence) associated with resistance is needed.

Insecticidal activity of marigold Tagetes patula plants and foliar extracts against the hemipteran pests, Lygus hesperus and Bemisia tabaci.

The western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae) and the whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) are key hemipteran pests of numerous crop plants throughout the western United States and Mexico. Management in the U.S. currently relies on only a few insecticides and is threatened by the evolution of resistance. New chemistries or alternative management strategies are needed to reduce selection pressure on current insecticides and enhance control. Here, we investigated the bio-insecticidal toxicity of the French marigold, Tagetes patula Linnaeus (Asterales: Asteraceae), against both L. hesperus and B. tabaci. Assays indicated significantly reduced survival of both pest species on T. patula plants, and in diet incorporation assays containing aqueous and methanolic marigold foliar extracts. Mortality was concentration-dependent, indicating the presence of one or more extractable toxicants. These data suggest that T. patula plants have insecticidal constituents that might be identified and developed as novel alternatives to conventional chemical treatments.

Biopesticide synergy when combining plant flavonoids and entomopathogenic baculovirus

Four crop plants known to be hosts for the lepidopteran Trichoplusia ni (soybean, green bean, cotton, and cabbage) were treated with the biopesticide AfMNPV baculovirus in a dosage response assay. Treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Leaf trichomes on soybeans were not found to be responsible for the observed increase of insecticidal activity. Three flavonoid compounds (daidzein, genistein, and kaempferol) were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The individual flavonoid compounds did not cause T ni. mortality in no-virus assays when incorporated into artificial insect diet. The combination of the three flavonoid compounds at leaf level concentrations significantly increased baculovirus activity in diet incorporation assays. When the daidzein, genistein, and kaempferol were added to artificial diet, at 3.5–6.5 × leaf level concentrations, virus activity increased 1.5, 2.3, and 4.2-fold for each respective flavonoid. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against T. ni.

Insecticide resistance and enhanced cytochrome P450 monooxygenase activity in field populations of Spodoptera litura from Sichuan, China

Spodoptera litura (Fabricius) is a widely distributed worldwide insect pest, particularly of vegetable crops. To determine the sensitivity of S. litura to insecticides, the toxicities of beta-cypermethrin, chlorpyrifos, emamectin benzoate, methoxyfenozide, and indoxacarb were tested from 2014 to 2016 using a diet incorporation assay on populations of S. litura collected from different districts in Sichuan Province and a susceptible strain (Lab-HN). Compared to the Lab-HN strain, field populations showed high levels of resistance to beta-cypermethrin (267.3- to 1789.2-fold). Field populations exhibited low or moderate resistance to chlorpyrifos in 2014 (8.9- to 24.4-fold) and high levels in 2016 (440.5- to 950.5-fold). All populations collected in 2014 and 2016 were susceptible or had a low or moderate level of resistance to indoxacarb (2.2- to 31.0-fold). No field populations exhibited resistance to emamectin benzoate or methoxyfenozide (0.7- to 1.5-fold and 0.9- to 1.4- fold) in 2014, whereas the resistance in 2016 increased to 29.8- to 55.2-fold and 38.1- to 59.0- fold, respectively. A synergist experiment indicated that the Lab-HN strain and field-collected population from Shuangliu in 2016 (SL16 population) to beta-cypermethrin may be related to cytochrome P450 monooxygenases (P450s), with the highest synergism ratios (SR) of piperonyl butoxide (PBO) of 34.4- and 73.3-fold, but not to carboxylesterase (CarE) or glutathione-S- transferase (GST). We detected the activities of detoxification enzymes and found that the ethoxycoumarin O-deethylase activities were also the strongest in the Lab-HN strain and the SL16 population with the highest SR values of 2.6- and 4.1-fold, respectively. Non-denaturing polyacrylamide gel electrophoresis (native PAGE) indicated that the inhibition of in vivo P450 activity caused by PBO was due to the binding of the synergist to the E5 or E6 isozyme, which were absent in the Lab-HN strain.

Effect of different sowing dates on expression of resistance to Helicoverpa armigera in different genotypes of chickpea across four plantings using diet incorporation assay under laboratory conditions

Effect of different sowing dates on expression of resistance to Helicoverpa armigera in different genotypes of chickpea across four plantings using diet incorporation assay under laboratory conditions

Effect of plant secondary metabolites on common cutworm, Spodoptera litura (Lepidoptera: Noctuidae)

AbstractThe effect of various flavonoids, lectins and phenyl β‐D‐glucoside on larval survival, weights and the activities of digestive (total serine protease and trypsin) and detoxifying (esterase and glutathione‐S‐transferase) enzymes of Spodoptera litura larvae at 7 days after treatment was studied through diet incorporation assay. Flavonoids (rutin, chlorogenic acid, quinic acid, caffeic acid, naringenin, quercitin, kaempferol, myricetin, catechin, and ferulic acid) were incorporated in artificial diet at 100, 500 and 1000 ppm, lectins: groundnut leaf lectin (GLL), concavalin A (ConA) and phenyl β‐D‐glucoside at 1, 2 and 5 μg/mL. Flavonoids such as rutin, quercitin and kaempferol at 1000 ppm were more toxic to S. litura larvae than quinic acid, caffeic acid, naringenin, myricetin, catechin, and ferulic acid. Larval growth and development were significantly reduced in S. litura larvae fed on a diet with GLL and ConA at 5 μg/mL compared to the larvae fed at 2 and 1 μg/mL concentrations. The larvae fed on flavonoid‐treated diets showed significant reduction in serine protease, trypsin and esterase activities. The flavonoids such as rutin, chlorogenic acid, quinic acid, naringenin, quercitin, kaempferol and myricetin, and lectins, GLL and ConA can be utilized in insect control programs.

Herbivore-responsive cotton phenolics and their impact on insect performance and biochemistry

Insect damage to plants can have several impacts on plant biochemical composition and such changes may influence the insect performances. Here, we examined herbivore-responsive alterations in the phenolic profile of cotton plant using three cotton genotypes, Bc-68-2, Gcot-16 and Coker-312, belonging to G. hirsutum sp. during their infestation with two biting and chewing insects viz., Helicoverpa armigera and Spodoptera litura, separately for 96h. Both insects had similar effects on phenolic induction in cotton plants (Gossypium sp.) at 96h post infestation (hpi) however, the phenolic accumulation was genotype specific. We measured 13 phenolic compounds, out of which four phenolic acids viz., gallic acid (GA), cinnamic acid (CA), p-coumaric acid (PA) and salicylic acid (SA) displayed higher accumulation in response to insect herbivory at 96hpi. The subsequent impact of four herbivore-responsive phenolics on insect performance was evaluated through diet incorporation assay. Insect larvae displayed significant reduction in larval weights and substantial mortality on 10th day in case of CA and PA incorporated diets at higher concentrations. Physiological examination of treated larvae on 5th and 10th day post feeding at CA and PA containing diets revealed oxidative stress, reduced lactate dehydrogenase enzyme activity, altered glucose and protein level and enhanced glutathione-S-transferase enzyme activity in larvae feeding on treatment diets as compared to controls. Our results suggest that the defensive chemistry of phenolics has important effect on the insect fitness and physiology and the flavonoids such as CA and PA can be utilized in pest control programmes.

Evaluation of no-choice cage, detached leaf and diet incorporation assays to screen chickpeas for resistance to the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae)

It i.s difficult to compare genotypic resistance to insects across seasons and locations because of the variation in the onset and severity of insect infestation. Therefore, in this study, we used the no-choice cage technique and detached leaf and artificial diet incorporation assays for evaluating chickpea genotypes for resistance to the beet armyworm Spodopteraexigua (Hubner). The results indicated that the no-choice cage technique was not useful for evaluating chickpea genotypes for resistance to S. exigua. In the detached leaf assay, leaf feeding by S. exigua larvae was significantly lower in ICC 12 475 and RIL 20 genotypes at the vegetative stage and in ICC 10 393, ICC 12 475, KAK 2, RIL 20 and RIL 25 genotypes at the flowering stage, while larval weight gain was lower in insects reared on EC 583264, ICC 10 393, ICC 12 475 and RIL 20 genotypes at the vegetative stage; and in those reared on ICC 10 393, ICC 12 475, EC 583264, ICCL 86 111, KAK 2, RIL 20 and RIL 25 genotypes at the flowering stage in plants raised under greenhouse conditions. In plants raised under field conditions, the EC 583260, ICC 12 475, ICCL 86 111, ICCV 10, KAK 2, RIL 20 and RIL 25 genotypes in the November sowing and the KAK 2, ICC 3137, ICCL 86 111 and RIL 25 genotypes in the December sowing suffered low leaf damage at the vegetative stage; and EC 58 320, EC 583264, ICC 12 745 and RIL 25 genotypes in the November sowing and the EC 583264, ICC 3137, ICC 12 475, 1CCL 86 111, KAK 2, RIL 20 and RIL 25 genotypes in the December sowing suffered low leaf damage at the flowering stage, while low larval weights were recorded in insects reared on the ICC 12 475, EC 583264, ICCL 86 111 and RIL 25 genotypes at the flowering stage. In the diet incorporation assay, the survival of S. exigua larvae reared on diets with leaf powder of the ICC 12 475, ICC 10 393 and RIL 25 genotypes was significantly lower, while a significant reduction in larval weights was recorded in those reared on diets with leaf powder of the ICC 10 393, ICC 12 475, ICCL 86 111, KAK 2, RIL 25 and ICC 3137 genotypes. The fecundity of insects was also reduced in insects reared on diets with leaf powder of the RIL 25, RIL 20, ICCV 10, ICCL 86 111, ICC 12 475, ICC 3137, KAK 2 and ICC 10 393 genotypes. The results suggest that detached leaf assay could be used for large-scale screening of chickpea genotypes for resistance to S. exigua, while the diet incorporation assay could be used to gain additional information on the antibiosis mechanism of resistance to this insect.

Effect of plant secondary metabolites on legume pod borer, Helicoverpa armigera

The effect of various flavonoids, lectins and phenyl β-d-glucoside on larval survival, weights and the activities of digestive (total serine protease and trypsin) and detoxifying [esterase and glutathione-S-transferase] enzymes of Helicoverpa armigera larvae at 5 and 10 days after treatment (DAT) was studied through diet incorporation assay. Flavonoids (quercetin, cinnamic acid, caffeic acid, chlorogenic acid, catechin, trihydroxyflavone, gentisic acid, ferulic acid, protocatechuic acid and umbelliferone) were incorporated in artificial diet at 100, 500 and 1000 ppm, lectins: groundnut leaf lectin (GLL), concavalin (ConA) and phenyl β-d-glucoside at 2.5 and 5 μg mL−1. Flavonoids such as chlorogenic acid, caffeic acid and protocatechuic acid at 1,000 ppm were more toxic to H. armigera larvae at 10 DAT than quercetin, catechin, cinnamic acid, trihydroxyflavone, gentisic acid, ferulic acid and umbelliferone. Larval growth and development were significantly reduced in H. armigera larvae fed on a diet with GLL and ConA at 5 μg mL−1 compared to the larvae fed at 2.5 and 1.25 μg mL−1 concentrations. The enzyme activities of the larvae were significantly reduced in flavonoid-treated diets. The flavonoids such as chlorogenic acid, caffeic acid, gentisic acid, trihydroxyflavone, catechin and protocatechuic acid, and lectins, GLL and ConA can be utilized in insect control programs.

Susceptibility of field populations of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) in China to chlorantraniliprole and the activities of detoxification enzymes

The cutworm Spodoptera litura is a major insect pest of vegetables and cotton in China, and has been reported to develop resistance to various classes of insecticides. Chlorantraniliprole, which has been registered recently in China, provides a novel option for control of this pest. The susceptibilities of S. litura collected from Southeast China to chlorantraniliprole were determined by diet incorporation assay with neonates. The susceptibility variation among 12 field populations was low (<4-fold), with median lethal concentration (LC50) values varying from 28.4 to 102.5 μg/l. However, all the 12 field populations were less susceptible to chlorantraniliprole than a laboratory susceptible population. The most tolerant populations were sampled from Guangdong and Anhui Provinces where S. litura had been frequently challenged by insecticides. However, no correlation was found between LC50 values and the number of applications of this chemical. Biochemical assays were performed to determine the potential mechanisms involved in the tolerance observed in field populations. Most field populations showed significantly enhanced activities of mixed function oxidase enzymes compared with the susceptible strain. Only a few populations had higher activities of esterase and glutathione-S-transferase than the susceptible strain. No correlations were observed between activities of metabolic enzymes and chlorantraniliprole toxicity, suggesting that these detoxification enzymes were not the main cause of the field tolerance observed in this study, and there might be other mechanisms conferring the tolerance variation to chlorantraniliprole in S. litura.

Monitoring of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) resistance to chlorantraniliprole in China

Beet armyworm, Spodoptera exigua is a major insect pest of vegetables in China, and has been reported to develop resistance to many broad-spectrum insecticides. Recently registered chlorantraniliprole provides a novel option for control of this pest resistant to other conventional insecticides. The susceptibilities of field collected populations were measured by diet incorporation assay with neonate, obvious variation of susceptibility was observed among the 18 field populations with LC 50 values varying from 0.039 to 0.240 mg/liter. Moderate resistant level was discovered in 8 of 18 field populations, other 8 populations had become low level tolerance to chlorantraniliprole, and only one population in all the field colonies remained susceptible. Biochemical assays were performed to determine the potential mechanisms involved in tolerance variation. Field populations displayed varied detoxification enzyme activities, but the regression analysis between chlorantraniliprole toxicities and enzyme activities demonstrated each field population might have specific biochemical mechanisms for tolerance. Artificial selection in laboratory with chlorantraniliprole was carried out, 23 generations of continuous selections resulted in 11.8-fold increase in resistance to chlorantraniliprole, and 3.0-fold and 3.7-fold increases in mixed function oxidase and esterase, respectively. Compared with the susceptible strain kept in laboratory the selection strain had developed 128.6-fold resistance to this insecticide. Synergism assays showed the detoxification enzymes might not involved in the resistance observed in field collected populations and the selected strain.

Susceptibility of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) to Vip3A insecticidal protein expressed in VipCot™ cotton

Susceptibility of laboratory and field colonies of Helicoverpa zea (Boddie) and Heliothis virescens F. to Vip3A insecticidal protein was studied in diet incorporation and diet overlay assays from 2004 to 2008. Responses of field populations were compared to paired responses of University of Arkansas laboratory susceptible H. zea (LabZA) and H. virescens (LabVR) colonies. After 7 d of exposure, observations were made on number of dead larvae ( M) and the number of larvae alive but remaining as first instars (L1). Regression estimates using M (LC 50) and M plus L1 (MIC 50) data were developed for laboratory and field populations. Susceptibility of laboratory and field populations exposed to Vip3A varied among different batches of protein used over the study period. Within the same batch of Vip3A protein, susceptibilities of laboratory colonies of both species (LabZA and LabVR) were similar. Field colonies were significantly more susceptible to Vip3A than the respective reference colonies of both species. Within field populations, susceptibility to Vip3A varied up to 75-fold in H. zea and 132-fold in H. virescens in LC 50 estimates. Variabilities in MIC 50s were up to 59- and 11-fold for H. zea and H. virescens, respectively.

Activity of spinosad on stored‐tobacco insects and persistence on cured tobacco strips

Every year raw tobacco and manufactured tobacco products are lost to two major storage pests, the cigarette beetle, Lasioderma serricorne (F) and the tobacco moth, Ephestia elutella (Hiibner). Post-harvest management of both insects is achieved through sanitation, insect monitoring and fumigation with phosphine. However, insect resistance to phosphine and control failures have been reported, and fumigants are under constant regulatory pressure. Here we report the evaluation of spinosad, a bioinsecticide derived from the fermentation of the soil micro-organism Saccharopolyspora spinosa Mertz & Yao. Spinosad was first registered in 1997 and is now widely used as a field pest control agent on many crops, including tobacco. The insecticidal activity of the fermentation product (technical spinosad, TS) was measured by diet incorporation assays against L serricorne and E elutella larvae. Mortality levels were determined on newly hatched larvae and over the whole insect life cycle. For both species, no emergence of adult insects was observed in cured tobacco sprayed with 50mg TS kg(-1) and inoculated with eggs or newly hatched larvae. These results indicated that spinosad has potential for the control of both species in stored tobacco, since 100% control of both pests could be achieved at 50 mg TS kg(-1), and with almost full control (90-95%) at 10 mg kg(-1). We also monitored the stability of the product on cured tobacco. The original concentration of the main active component of TS, spinosyn A, did not change significantly over 18 months, indicating no loss of spinosad during a typical leaf storage period of time. Bioassays against larvae confirmed that the bioinsecticidal activity of spinosad was retained.

Toxicity and Repellency of Tephrosia candida to Larval and Adult Diaprepes Root Weevil (Coleoptera: Curculionidae)

Leaves of the tropical legume Tephrosia candida DC deterred feeding by adults of the Diaprepes root weevil, Diaprepes abbreviatus (L.), compared with leaves of Citrus macrophylla Wester, a common citrus rootstock, or T. vogelii Hook. f. When larvae were placed in pots containing plants of the three species for 28 d in a growth chamber, larval survival and weight gain were significantly reduced in pots containing plants of T. candida compared with larvae in pots with C. macrophylla or T. vogelii. Diet incorporation of lyophilized roots of T. candida into an artificial diet increasingly inhibited the growth of larvae and increased larval mortality with increased concentration of roots, whereas roots from C. macrophylla or T. vogelii had no effect compared with the diet-only control. T. candida, but not T. vogelii, contains at least one constituent that acts as an antifeedant toward adult D. abbreviatus and as a toxicant toward larvae. No antifeedant effect of roots of T. candida toward larvae was observed in no-choice pot tests or in a diet incorporation bioassay. In pots, larval feeding damage to roots of T. candida was evident. In the diet incorporation assay, 97% of larvae survived 29 d on a diet of cellulose powder (a nutritionally inert filler) despite losing weight. We conclude that decreased survival and weight gain of larvae-fed fresh or lyophilized roots of T. candida were the result of ingestion of a toxicant and not deterrence from feeding.

Toxicity and repellency of Tephrosia candida to larval and adult Diaprepes root weevil (Coleoptera: Curculionidae).

Leaves of the tropical legume Tephrosia candida DC deterred feeding by adults of the Diaprepes root weevil, Diaprepes abbreviatus (L.), compared with leaves of Citrus macrophylla Wester, a common citrus rootstock, or T. vogelii Hook. f. When larvae were placed in pots containing plants of the three species for 28 d in a growth chamber, larval survival and weight gain were significantly reduced in pots containing plants of T. candida compared with larvae in pots with C. macrophylla or T. vogelii. Diet incorporation of lyophilized roots of T. candida into an artificial diet increasingly inhibited the growth of larvae and increased larval mortality with increased concentration of roots, whereas roots from C. macrophylla or T. vogelii had no effect compared with the diet-only control. T. candida, but not T. vogelii, contains at least one constituent that acts as an antifeedant toward adult D. abbreviatus and as a toxicant toward larvae. No antifeedant effect of roots of T. candida toward larvae was observed in no-choice pot tests or in a diet incorporation bioassay. In pots, larval feeding damage to roots of T. candida was evident. In the diet incorporation assay, 97% of larvae survived 29 d on a diet of cellulose powder (a nutritionally inert filler) despite losing weight. We conclude that decreased survival and weight gain of larvae-fed fresh or lyophilized roots of T. candida were the result of ingestion of a toxicant and not deterrence from feeding.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Bacillus thuringiensis (Bt) for the Control of Insect Pests in Stored Tobacco: A Review

Abstract Among the insect species causing infestations and serious damages to stored commodities, the cigarette beetle, Lasiodermaserricorne (F.) and the tobacco moth, Ephestiaelutella (Hübner) are the major pests of both raw and manufactured tobacco. Post-harvest tobacco control is achieved through sanitation, insect monitoring, and fumigation with phosphine. However, insect resistance to phosphine and control failures have been reported, and increasing regulatory pressure is being exerted on fumigants. Biological control agents such as Bacillus thuringiensis (Bt) appear to be environmentally sound and potentially viable alternatives to chemical control. Bt is a bacterium that produces insecticidal crystal proteins during the sporulation phase and has been, for more than 40 years, the microorganism of choice for the biocontrol of phytophagous insect pests. It produces insecticidal crystal proteins that display specific activity against certain orders of insects and become active upon ingestion by the insect. Our laboratory has conducted extensive research and worldwide surveys to evaluate the presence of Bt in stored tobacco and has confirmed previous findings indicating that Bt may be considered part of the naturally occurring phylloplanemicroflora. Several Bt strains were isolated from tobacco and characterized by DNA and protein profiling. The insecticidal activity of selected strains and of two commercial products against the larvae of L. serricorne was determined by diet incorporation assays. Moreover, the stability of Bt spores and crystal proteins on cured tobacco leaves was assessed over a storage period of time of 30 months. Cigarette prototypes were made with Bt-treated tobacco. Standard cigarette and smoke evaluations did not show any significant difference between the test and control cigarettes. Although the tested Bt strains and products did not yield satisfactory levels of mortality at the required times and doses, the experimental results summarized in the present review indicate that B. thuringiensishas potential for the control of the cigarette beetle. The integration of conventional control approaches with novel systems based on biological agents with different modes of action should offer new avenues for the effective management of stored tobacco pests in line with integrated pest management (IPM) concepts.

Expression of a Bacillus thuringiensis cry1B synthetic gene protects Mediterranean rice against the striped stem borer.

Cry1Ba endotoxin, which was shown to exhibit a tenfold lower lethal concentration 50 (LC50) than Cry1Ac in a Striped Stem Borer (SSB) diet incorporation assay. The 1.950-bp synthetic cry1B gene, possessing an overall GC content of 58 %, was cloned under the control of the maize ubiquitin promoter first intron and first exon regions. The resulting vector, designated as pUbi-cry1B, was transferred to two commercial Mediterranean cultivars of rice, Ariete and Senia, using microprojectile acceleration-mediated transformation. Thirty-two and 47 T0 events were generated in cvs. Ariete and Senia, respectively. Southern blot and immunoblot analyses allowed the identification of 7 Senia and 1 Ariete events harbouring both an intact gene cassette and expressing Cry1B at a level ranging from 0.01% to 0.4% of the total soluble proteins. Three Senia and 1 Ariete events were found to be protected against second instar SSB larvae in whole plant feeding assays, exhibiting 90-100% mortality 7 days after infestation. Spatial and temporal variation in transgene expression was further examined in resistant event 64 of cv. Ariete. Stable accumulation of Cry1B, representing 0.4% of the total soluble proteins, was observed over the T2 to T4 generations in leaf tissue 20, 40, 70 and 90 days after germination in both young and old leaves and in internodes. Ariete event 64 was found to be fully protected from attacks of third and fourth instar SSB larvae over subsequent generations.