Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus

J assim , H.K., F oster , H.A. & F airhurst , C.P. 1990. Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus. Journal of Applied Bacteriology 69 , 563–568.
The effects of exposing fifth instar larvae of Scolytus scolytus and S. multistriatus to spore suspensions of Bacillus spp. were investigated. Bacillus thuringiensis ser 3a, 3b increased the mortality of larvae cultured on an artificial medium from approximately 20% in control cultures to over 80% in cultures exposed to the bacteria. The mortality was dose-dependent for S. multistriatus and the approximate LC₅₀ value was 2.2 times 10³ spores/ml. Different serotypes of B. thuringiensis caused different levels of mortality: H6 produced the highest mortality and H1 the lowest. Bacillus alvei and B. cereus were also pathogenic but B. megaterium was not. The results are discussed in relation to the mechanism of pathogenicity and the potential for the use of B. thuringiensis for the control of the vectors of Dutch elm disease.     

Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets

Bacillus thuringiensis screening programs based on the official potency bioassay using third-instar larvae and on a neonate bioassay were developed for Heliothis armigera, Earias insulana, and Spondoptera littoralis. In these bioassays, the diets were standardized to be suitable, with minor modifications, for feeding of the three lepidopterans. The bioassay protocol was based on determination of the LC50 of the microbial standard HD-1-S-80 in the insects susceptible to B. thuringiensis var. kurstaki strains. This was followed by preliminary screening of B. thuringiensis strains at the LC50 of the B. thuringiensis standard. The B. thuringiensis strains causing 100% mortality at this LC50 in the larvae were selected for potency determinations. The neonate bioassay was suitable for accurate determinations of potencies also in S. littoralis--a representative of insects weakly susceptible to the HD-1 standard. The role of the official and the neonate bioassays in developing microbial control programs is discussed.     

三种杀虫剂亚致死剂量对草地贪夜蛾细胞色素P450基因表达的影响

【目的】为明确杀虫剂亚致死剂量对草地贪夜蛾Spodoptera frugiperda细胞色素P450基因表达的影响。【方法】本研究采用叶片浸渍法测定了3种杀虫剂[氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和苏云金杆菌Bacillus thuringiensis(Bt)]对草地贪夜蛾2龄幼虫的毒力,以及通过实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)技术测定了这3种杀虫剂亚致死剂量(LC₁₀)处理后48 h时草地贪夜蛾2龄幼虫16个P450基因的表达量。【结果】氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和Bt对草地贪夜蛾2龄幼虫的LC₁₀值分别为0.931, 0.283和1 089.688 mg/L。2龄幼虫受LC₁₀氯虫苯甲酰胺胁迫后,13个P450基因(CYP4G75,CYP6AB12,CYP6B50,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP9A59,CYP9A58,CYP6AE44及CYP6AE43)表达上调...     

Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors (Aedes aegypti and Culex pipiens)

Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland) were tested against larvae of Aedes aegypti L. and Culex pipiens L. (Culicidae, Diptera). The crystals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B. thuringiensis subsp. japonensis, yoso, jinghongiensis) killed from 68 +/- 7% to 84 +/- 7% of the fourth instar larvae of A. aegypti. The crystals of two other strains (KpF3 and KpC1) of this group caused mortality between 3 +/- 2% and 70 +/- 7%. The LC50 ranged from 3.2 +/- 0.4 to 34.1 +/- 4.8 microg/ml. The effect of B. thulringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to 17 +/- 3%. No significant (0%-37 +/- 6%) effect of B. thuringiensis crystals on the larvae of C. pipiens was observed. Our results show that the delta-endotoxins of B. thuringiensis act very specifically.     

Effects of temperature on mortality of larval stable fly (Diptera: Muscidae) caused by five isolates of Bacillus thuringiensis

We examined the effects of temperature on mortality of larval stable fly [Stomoxys calcitrans (L.)] caused by Bacillus thuringiensis tolworthi 4L3, B. t. darmastedensis 4M1, B. t. thompsoni 401, B. t. thuringiensis HD2, and B. t. kurstaki HD945. At moderate doses, mortality caused by all isolates ranged from 87 to 99% at 15 degrees C and declined to 29-63% as temperature increased to 30 degrees C. A similar pattern was seen when a higher dose was used, except that the reduction in mortality at warmer temperatures was not as great as was seen with the moderate doses. Insecticidal activity of each isolate against first-instar larvae was reduced by only 5-15% after 5 d in the medium. Mortality of second- and third-instar larvae ranged from 2 to 21%, suggesting the isolates were less effective against larger larvae.     

Effects of sublethal concentrations of Bacillus thuringiensis on larval development of Sesamia nonagrioides

In the Mediterranean Basin, developed diapausing larvae of the borer Sesamia nonagrioides Lefèbvre are found consistently during autumn in transgenic corn, Zea mays L., expressing Bacillus thuringiensis Berliner (Bt) toxins derived from event 176. These larvae can feed on sublethal concentrations of Bt toxins because the expression of this corn toxin declines after flowering. To evaluate the possible consequences of this phenomenon, the effect of sublethal concentrations of the natural toxin producer, B. thuringiensis variety kurstaki (Dipel DF) on the performance of S. nonagrioides in the laboratory was examined. Larvae treated with Dipel DF showed higher mortality, longer developmental time, extra molts, and higher sensitivity to critical daylength for diapause induction than the untreated larvae. Accordingly, diapausing larvae collected in October and February in a Bt cornfield exhibited a higher number of supernumerary molts before pupating and longer diapause development than larvae collected in a non-Bt field at the same time. These results demonstrate that deployment of Bt corn in the Mediterranean may affect both the borer performance and the development of Bt resistance in addition to direct mortality caused by the Bt toxin.     

甜菜夜蛾肽聚糖识别蛋白基因SePGRP-SA的克隆及功能鉴定

【目的】本研究旨在阐明甜菜夜蛾Spodoptera exigua幼虫体内肽聚糖识别蛋白(peptidoglycan recognition protein, PGRP)在响应苏云金芽胞杆菌Bacillus thuringiensis(Bt)感染过程中的功能。【方法】利用PCR方法扩增甜菜夜蛾幼虫肽聚糖识别蛋白基因SePGRP-SA全长cDNA;采用qRT-PCR分析SePGRP-SA在甜菜夜蛾不同发育阶段(卵、1-5龄幼虫、预蛹和蛹)及4龄幼虫不同组织(中肠、马氏管、围食膜、脂肪体、血淋巴和表皮)中的表达。通过RNAi技术沉默SePGRP-SA基因72 h后,qRT-PCR检测SePGRP-SA沉默效率及甜菜夜蛾4龄幼虫中肠抗菌肽相关基因(Ceropin, Attacin和Defensin)和细菌载量的变化。RNAi沉默SePGRP-SA 24 h后,以苏云金芽胞杆菌菌株Bt-GS57饲喂甜菜夜蛾4龄幼虫0, 24, 48, 72, 96和120 h,计算幼虫校正死亡率;饲喂甜菜夜蛾4龄幼虫Bt-GS57后0, 24, 48和72 h,利用qRT-PCR检测中肠SePGRP-SA, Ceropin, Attacin和Defensin的相对表达量。【结果】克隆获得甜菜夜蛾SePGRP-SA全长DNA(GenBank登录号：MW265930),开放阅读框长576 bp,编码191个氨基酸,其编码蛋白的预测分子量为21.59 kD。序列分析结果表明,SePGRP-SA具有典型的PGRP和Ami2保守结构域,信号肽为19个氨基酸,为分泌型蛋白;系统进化分析发现,SePGRP-SA与斜纹夜蛾Spodoptera litura的SlPGRP亲缘关系最近,氨基酸序列一致性达91.1%。发育表达谱结果表明SePGRP-SA在甜菜夜蛾4和5龄幼虫、预蛹和蛹中高表达;组织表达谱结果表明,SePGRP-SA在4龄幼虫各组织中均表达,其中以血淋巴中表达量最高。与注射dsEGFP(对照)相比,注射dsSePGRP-SA的甜菜夜蛾4龄幼虫在72 h时中肠SePGRP-SA基因表达量下调了95.26%,Cecropin, Attacin和Defensin表达量显著下调,中肠细菌载量显著升高。注射dsEGFP和dsSePGRP-SA的甜菜夜蛾4龄幼虫饲喂Bt-GS57,72 h时幼虫校正死亡率分别为50.00%和73.33%,表明幼虫对Bt-GS57的敏感性明显增加。甜菜夜蛾4龄幼虫取食Bt-GS57后,中肠SePGRP-SA, Cecropin, Attacin和Defensin表达量在48 h均显著增加,72 h时降低。【结论】Bt侵染能够引起甜菜夜蛾SePGRP SA基因激活抗菌肽相关基因Cecropin, Attacin和Defensin的表达。     

The effect of seston on mortality of Simulium vittatum (Diptera: Simuliidae) from insecticidal proteins produced by Bacillus thuringiensis subsp. israelensis

Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65-52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality.     

Testing of selected insecticides to assess the viability of the integrated pest management of the Eucalyptus snout-beetle Gonipterus scutellatus in north-west Spain

Abstract:  We investigated, by means of topical, residual and field assays, the toxicity of four insecticides ( Bacillus thuringiensis , azadirachtin, flufenoxuron and ethofenprox) to adult, larvae and eggs of the Eucalyptus snout-beetle Gonipterus scutellatus and their side-effects on the adults of the solitary egg parasitoid Anaphes nitens , its natural enemy. In the topical toxicity experiment, both flufenoxuron and ethofenprox severely affected G. scutellatus , causing 100% adult mortality after 7 days, and 75 and 100% larval mortality, respectively. Ethofenprox also caused 93% larval pre-eclosion mortality and 100% parasitoid mortality. In the residual experiment, azadirachtin acted as an effective larvicide when ingested (73% mortality after 7 days). Ethofenprox showed the highest toxicity, with 100% mortality of adults and larvae of G. scutellatus after 24 h. On the day 7 of observation, flufenoxuron resulted in 93% adult mortality and 100% larval mortality. After contact with residuals, only ethofenprox and flufenoxuron had adversely affected A. nitens (98% mortality after 48 h). Bacillus thuringiensis exhibited only a slight activity against the pest or the parasitoid. The field experiment was carried out on three localities, with a preliminary sampling, and three samplings after the applications of the products, on days 7, 14 and 30. We evaluated the parasitism rate, parasitoid sex ratio, parasitoid pre-eclosion mortality, the abundance of G. scutellatus adults, larvae and egg capsules, the pre-eclosion mortality rate of weevil eggs and larvae and the foliar damage. Only fluenoxuron and ethofenprox reduced the pest population and the foliar damages, but ethofenprox also significantly decreased the rate of parasitism (17%) after 7 days. Given our experimental results, we suggest that flufenoxuron and azadirachtin could be used in association with the natural enemy.     

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria

Mortality in adult tsetse, Glossina morsitans morsitans, caused by Pseudomonas aeruginosa, Serratia marcescens, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis H-14, B. thuringiensis 1, B. thuringiensis 5, B. thuringiensis var. insraelensis, and Providentia rettgeri was determined. When bacteria were smeared on rabbit skin and tsetse allowed to feed only once on the contaminated area, mortality 8 days postingestion was significantly higher (P less than 0.01) in tsetse fed on P. aeruginosa, S. marcescens, B. thuringiensis 1, and P. rettgeri and increased when tsetse were allowed to feed for the second time on the contaminated skin. With this smear technique, however, mortalities were generally not remarkable. In artificial membrane feeding experiments using low concentrations of bacteria (-10(6)/ml of blood), the B. thuringiensis strains caused low mortality, except B. thuringiensis H-14, which caused 59% mortality. However, at this concentration, P. aeruginosa, S. marcescens, B. cereus, and P. rettgeri caused highly significant (P less than 0.01) mortality (64-96%). When higher concentrations of bacteria (10(7)/ml) were used, all the bacteria tested, except B. sphaericus, caused high mortality ranging from 70 to 98%. Thus, mortality depended on the species of bacteria, the dose ingested, and time postingestion.     

Efficacy and persistence of Bacillus sphaericus, Bacillus thuringiensis var. israelensis, and methoprene against Culiseta incidens (Diptera: Culicidae) in tires

The efficacy and persistence of Bacillus sphaericus (2362) was compared at three dosage rates in tires that continually contained cadavers of Culiseta incidens (Thomson) versus tires with all dead larvae removed. At treatment rates of 3.75 and 7.5 ppm, the continual presence of cadavers in the tire water resulted in higher mortality rates. At the 15 ppm treatment rate, mortality rates were similar in tires with or without cadavers. Mortality rates increased in all tires 4-6 wk after treatment, suggesting an amplification of the pathogen. The mortality rate did not exceed 90% for greater than 2 wk in any of the tires. At a second site, the efficacy of B. sphaericus, Bacillus thuringiensis var. israelensis, and methoprene was evaluated against C. incidens in tires exposed to full sunlight versus shaded tires. In shaded tires inoculated with B. sphaericus (15 ppm) and B. thuringiensis var. israelensis (15 ppm), mortality exceeded 90% for 5 and 2 wk, and 50% for 10 and 4 wk, for the two bacteria, respectively. Larvae were adequately controlled (greater than 75% mortality) in the sunny tires for approximately 1 wk. The insect growth regulator, methoprene (applied at 2.5 ppm), inhibited the emergence of approximately 90% of the larvae present at the time of treatment, but not of larvae subsequently introduced into either the sunny or shaded tires.     

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinating B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.     

Isolation and characterization of a novel Bacillus strain from coffee phyllosphere showing antifungal activity

AIMS: The isolation and characterization of a novel coffee-associated Bacillus mojavensis strain, designated as strain AB1, and its survival on the coffee phyllosphere. METHODS AND RESULTS: A pair of 16S rDNA primers was designed to amplify a highly variable region within the 16S rDNA gene of Bacillus spp., with the purpose of identifying the AB1 isolate through PCR and sequence analysis. By this method, AB1 was identified as a strain of B. mojavensis. Bioassays were carried out to characterize the broad spectrum antifungal activity of AB1. Plant colonization studies revealed that AB1 could colonize the coffee phyllosphere better than Bacillus thuringiensis. CONCLUSIONS: These studies suggest that AB1 could be a new strain of B. mojavensis. AB1 is also shown to have antifungal activity against a wide spectrum of pathogenic fungi. The antifungal metabolite of AB1 has been partially characterized as a thermostable, protease- and alkali-resistant substance that is secreted into the surrounding medium. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as is known, this is the first strain of B. mojavensis which has been identified as inhabiting the coffee phyllosphere. The study highlights the potential use of AB1 as an antifungal agent in the coffee crop and as a delivery agent of the insecticidal toxin of B. thuringiensis to the coffee phyllosphere. The 16S rRNA identification strategy discussed could also be used in the identification of other new Bacillus strains.     

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.     

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

Analysis of mosquito larvicidal potential exhibited by vegetative cells of Bacillus thuringiensis subsp. israelensis

Vegetative Bacillus thuringiensis subsp. israelensis cells (6 X 10(5)/ml) achieved 100% mortality of Aedes aegypti larvae within 24 h. This larvicidal potential was localized within the cells; the cell-free supernatants did not kill mosquito larvae. However, they did contain a heat-labile hemolysin which was immunologically distinct from the general cytolytic (hemolytic) factor released during solubilization of B. thuringiensis subsp. israelensis crystals. The larvicidal potential of the vegetative cells was not due to poly-beta-hydroxybutyrate. Instead, it correlated with the ability of vegetative cells to sporulate during the bioassays. No toxicity was observed when bioassays were conducted in the presence of chloramphenicol or streptomycin. It is unlikely that the vegetative cells sporulate in the alkaline (pH 9.5 to 10.5) larval guts after ingestion. B. thuringiensis subsp. israelensis is not an alkalophile; we have been unable to grow it in culture at pH values of greater than or equal to 9.5. Moreover, we have been unable to demonstrate formation of a protective capsule. However, bacteria may replicate in the gut fluids of dead or dying mosquito larvae because their alkaline gut pH values drop markedly after exposure to the B. thuringiensis subsp. israelensis crystal toxins.     

Analysis of mosquito larvicidal potential exhibited by vegetative cells of Bacillus thuringiensis subsp. israelensis.

Vegetative Bacillus thuringiensis subsp. israelensis cells (6 X 10(5)/ml) achieved 100% mortality of Aedes aegypti larvae within 24 h. This larvicidal potential was localized within the cells; the cell-free supernatants did not kill mosquito larvae. However, they did contain a heat-labile hemolysin which was immunologically distinct from the general cytolytic (hemolytic) factor released during solubilization of B. thuringiensis subsp. israelensis crystals. The larvicidal potential of the vegetative cells was not due to poly-beta-hydroxybutyrate. Instead, it correlated with the ability of vegetative cells to sporulate during the bioassays. No toxicity was observed when bioassays were conducted in the presence of chloramphenicol or streptomycin. It is unlikely that the vegetative cells sporulate in the alkaline (pH 9.5 to 10.5) larval guts after ingestion. B. thuringiensis subsp. israelensis is not an alkalophile; we have been unable to grow it in culture at pH values of greater than or equal to 9.5. Moreover, we have been unable to demonstrate formation of a protective capsule. However, bacteria may replicate in the gut fluids of dead or dying mosquito larvae because their alkaline gut pH values drop markedly after exposure to the B. thuringiensis subsp. israelensis crystal toxins.