Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Influence of pod maturity and level of domestication on biochemical components in wild and cultivated pigeonpea (Cajanus cajan)

Variations in the trypsin inhibitors and lectin content in the developing pods of thirty accessions of Cajanus scarabaeoides , a wild relative of pigeonpea, from wide geographical locations and six cultivated genotypes were estimated at juvenile, immature and mature stages of pod development. Genotypes differed at all three stages for these two biochemical components. Total protein and trypsin inhibitor contents were higher in the wild accessions than in the cultivated genotypes. Although lectin content in the juvenile stage of pod development in the wild accessions ICPW 138 and ICPW 98 was highest, it was absent in the mature pods in both the cultivated and the wild genotypes. Very high broad-sense heritability estimates indicated the possibility of involvement of few genes in the inheritance of these biochemical components, which could be easily introgressed into the elite agronomic background.     

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

Brazilian strains of Bacillus thuringiensis, namely S701, S764 and S1265 were analysed regarding their cry gene and protein contents, crystal type, and activity against larvae of the lepidopteran fall armyworm (Spodoptera frugiperda Smith), the velvet caterpillar (Anticarsia gemmatalis), the dipterans (Culex quinquefasciatus and Aedes aegypti) and the coleopteran (Tenebrio molitor). The LC₅₀ of the strains against second instar larvae of S. frugiperda or A. gemmatalis revealed a high potency against those insect species. The spore–crystal mixtures of the isolates were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and showed similar protein pattern as the B. thuringiensis subsp. kurstaki strain HD‐1 (proteins approximately 130 and 65 kDa) for isolates S701 and S764, respectively, and only one major protein of approximately 130 kDa for isolate S1265. The polymerase chain reaction (PCR) using total DNA of the isolates and general and specific primers showed the presence of cry1Aa, cry1Ac, cry1Ia and cry2Ab genes in the two isolates serotyped as B. thuringiensis kurstaki (S701 and S764) and the presence of cry1D and cry2Ad in B. thuringiensis morrisoni S1265 strain. Scanning electron microscopy of strains S701 and S764, showed the presence of bipyramidal, cuboidal and round crystals, like in strain HD‐1 and bipyramidal and round crystals like in strain S1265.     

Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests

The insecticidal activities and specificities of the Vip3Aa proteins derived from different Bt strains are very different, although the similarities between these proteins are higher than 95%. In this study, we hypothesised that the differences in Vip3Aa11 and Vip3Aa39 C-terminal amino acids determine their differences in insecticidal activity against three Lepidoptera insects. To find the amino acid residues associated with insecticidal activity, nine different amino acid residues of Vip3Aa11 were substituted with the corresponding amino acid residues from Vip3Aa39 by site-directed mutagenesis. The toxicity of each protein was estimated by bioassays, and the results demonstrated that the mutant Y784N lost its insecticidal activity against three insects (Agrotis ipsilon, Helicoverpa armigera, and Spodoptera exigua). The insecticidal activity of S543N, I544L, and S686R against S. exigua increased 5-fold, 2.65-fold, and 8.98-fold, while the toxicity to H. armigera and A. ipsilon slightly decreased compared with that of the Vip3Aa11 protein. These findings indicate that the amino acid residues Ser⁵⁴³, Ile⁵⁴⁴, Thr⁶⁸⁵, Ser⁶⁸⁶, Arg⁷⁰⁴, Ile⁷⁸⁰, and Tyr⁷⁸⁴ may be insecticidal activity-related residues. Additionally, the trypsin activation of the four mutants indicated that all proteins can form a 62-kDa core fragment, except Y784N. A possible association between the insecticidal activity and trypsin sensitivity of Vip3A proteins is suggested.     

Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae)

The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDS–PAGE analysis, cry gene contents, H-serotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDS–PAGE in Tp6 and Tp14 isolates, respectively. According to H-serotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests.     

Use of two formulations and two application techniques to deliver Bacillus thuringiensis var. israelensis for the control of Musca domestica (Diptera: Muscidae) larvae and adults in poultry houses

A field study was carried out for 6 wks to assess, from both an efficiency and economic perspective, the effect of individual and integrated success of feeding and topical applications of two formulations of Bacillus thuringiensis var. israelensis (Bti) in controlling house fly (Musca domestica L.) larvae and adults in poultry houses. There was no significant difference between the 1 g and 2 g L^?1 spray applications of Bti. In the absence of spray applications, no significant differences in larval mortalities were observed between the 250 mg and 500 mg kg^?1 feed applications. The percentage mortality of larvae accomplished as a result of using a combination of 250 mg kg^?1Bti feed and 2 g L^?1 spray applications was equivalent to that obtained as a result of combining 500 mg kg^?1Bti and 1g L^?1 spray applications. Treatment with Bti caused significant reductions in the emergence (up to 74%) of house fly adults compared to the control. The fact that the emergence of adult house flies was affected by Bti treatments implies that Bti has sublethal effects on house fly larvae. The cost–benefit analysis (expressed in terms of mortality of larvae growing) indicated that the most effective combination for house fly larvae and adult house fly emergence control was the 500 mg kg^?1 of feed and 2 g L^?1 spray application combination that resulted in 67% larval mortality and a 74% decrease in adult house fly emergence. This study presents commercial users with various alternatives for possible combinations of the two Bti formulations.     

Novel strategies for the biocontrol of noctuid pests (Lepidoptera) based on improving ascovirus infectivity using Bacillus thuringiensis

Identifying novel biocontrol agents and developing new strategies are urgent goals in insect pest biocontrol. Ascoviruses are potential competent insect viruses that may be developed into bioinsecticides, but this aim is impeded by their poor oral infectivity. To improve the per os infectivity of ascovirus, Bacillus thuringiensis kurstaki (Btk) was employed as a helper to damage the midgut of lepidopteran larvae (Helicoverpa armigera, Mythimna separata, Spodoptera frugiperda, and S. litura) in formulations with Heliothis virescens ascovirus isolates (HvAV-3h and HvAV-3j). Btk and ascovirus mixtures (Btk/HvAV-3h and Btk/HvAV-3j) were fed to insect larvae (3rd instar). With the exception of S. frugiperda larvae, which exhibited low mortality after ingesting Btk, the larvae of the other tested species showed three types of response to feeding on the formulas: type I, the tested larvae (H. armigera) were killed by Btk infection so quickly that insufficient time and resources remained for ascoviral invasion; type II, both Btk and the ascovirus were depleted by their competition, such that neither was successfully released or colonized the tissue; type III, Btk was eliminated by the ascovirus, and the ascovirus achieved systemic infection in the tested larvae. The feeding of Btk/ascovirus formulas led to a great reduction in larval diet consumption and resulted in a significant decrease in the emergence rate of H. armigera, M. separata, and S. litura larvae, which suggested that the formulas exerted marked oral control effects on both the contemporary individuals and the next generation of these tested pest species.     

The effect of oxygen tension on the production of Bacillus thuringiensis subsp. israelensis toxin active against Aedes aegypti larvae

An optimized batch production of Bacillus thuringiensis subsp. israelensis was made in a stirred Bioflo III reactor using a previously selected medium, and operating conditions in the range of 100–500 rev/min stirrer speeds and 0.2–1 air flow/culture medium volume/minute (v/v/m) aeration rates, including five combinations; at the end of fermentation, dry powders were recovered and evaluated against Aedes aegypti larvae at 0.05 mg/l. Later, the lethal concentration inducing 50% mortality (LC₅₀) was determined for the two most toxic powders. The bioinsecticide yields varied from 9.1 to 15.7 g/l and the total fermentation times ranged between 18 and 30.3 h. The toxicity varied for two powders much more than for the others. These were for combinations with 300 rev/min:1 v/v/m and 500 rev/min:0.6 v/v/m, giving mortality percentages of 47.2 and 59.7, with LC₅₀ values of 0.2675 and 0.0685 mg/l, respectively. A t test showed no significant difference. However, the larvicidal powder produced with 300 rev/min:1 v/v/m gave more variable toxicity than the powder obtained with 500 rev/min:0.6 v/v/m.     

Comparative bioassay of different isolates of Bacillus thuringiensis subsp. kurstaki on the third larval instars of diamondback moth,Plutella xylostella (L.) (Lep.: Plutellidae)

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most important pests of cruciferous plants throughout the world. In recent years, this insect has been a serious pest for cabbage fields in Tehran province. Resistance of P. xylostella to all main groups of insecticides has been recorded and it is ranked in the 20 most resistant pest species reported up to now. According to many researchers, to eliminate the problem of pest resistance to chemical pesticides, an integrated pest management programme should be used. In line with this, the uses of microbial control agents (MCAs) are discussed. The bacterium, Bacillus thuringiensis (Bt) is one of microbial control agents of pests. It is characterised by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the pathogenicity of some Bt isolates, including Dipel, 20, 29, 79 and 87, was tested against P. xylostella and the lethal concentrations (LC₅₀) of their crystal proteins to P. xylostella third larval instar was determined. The experiment was designed in factorial in randomised complete design with 5 treatments (different concentrations including 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ CFU/ml and 5 replications and with 10 third larval instars. Spore–crystal complex was applied to the surface of natural diets (cabbage leaves) and the mortality of P. xylostella larvae was assessed 120?h after exposure of Bt toxin in each treatment. Results showed that percentage of survival was significantly higher for control treatment. Results also showed that after 5?days, LC₅₀ for isolates of Dipel, 20, 29, 79 and 87 were equal to 1?×?10⁶, 1?×?10⁵, 5?×?10⁵, 4?×?10⁵ and 1?×?10⁴ CFU/ml, respectively. LT₅₀ were equal to 93.71, 48.04, 71, 40.49 and 75.28?h. Of and most the percentage larval mortality relate to attendance 87 and also at least percentage mortality is related to the groom Dipel.     

Laboratory evaluation of entomopathogenic fungi against the white grubs,Holotrichia oblita and Anomala corpulenta (Coleoptera: Scarabaeidae) from the field of peanut,Arachis hypogaea

We evaluated the pathogenicity of nine isolates of entomopathogenic fungi, including six isolates of Metarhizium anisopliae, one of Beauveria bassiana, and two of Beauveria brongniartii, against eggs and various larval instars of two scarab-beetle species, Holotrichia oblita and Anomala corpulenta, under laboratory conditions. The fungal isolates differed in pathogenicity. Generally, the isolates were more pathogenic to A. corpulenta than to H. oblita. Some of the isolates prevented egg hatching and caused larval death. Isolates M2-2 and Br5-8 caused 39 and 27% egg mortality, respectively, and produced 23 and 24% viewable fungal-infection rates in H. oblita. Three isolates had no significant effect on egg hatchability. Three isolates (Br5-8, Br232818, and M200614) caused about 40% mortality in H. oblita first instars. In A. corpulenta, all isolates except M200614 caused more than 60% egg mortality, and M2-2, Br232818 and Br5-8 caused egg-infection rates greater than 50%. M2-2 caused 47% infection and 100% mortality in first-instar larvae of A. corpulenta, while Br5-8 and Br232818 yielded over 80% mortality of the larvae. The three most virulent isolates, M2-2, Br232818 and Br5-8, were selected for further bioassays against second- and third-instar larvae. In addition, seven graduated concentrations of a Br5-8 conidial suspension were assayed against H. oblita second instars. Larval mortality was positively correlated with fungal dosage, and the LC₅₀ was 4.49×10⁶ conidia/mL. These three virulent isolates may be used to prevent H. oblita and A. corpulenta larval infestations in field crops.     

Field and laboratory studies on water conditions affecting the potency of VectoBac (Bacillus thuringiensis serotype H-14) against larvae of the blackfly, Simulium damnosum

River water conditions that might influence the efficacy of VectoBac, a formulation of the microbial insecticide Bacillus thuringiensis H-14 Berliner against Simulium damnosum sensu lato Theobald (Diptera: Simuliidae) larvae were investigated. A standard formulation was assayed 130 times over 15 months using a mini-gutter system at a field station beside the River Pra in Ghana. The lethal concentration (LC) values, river temperature, conductivity, turbidity and pH were analysed using univariate and multivariate statistics to identify which of these parameters influenced its performance. River temperature, conductivity and turbidity (in that order) were identified as having direct effects on the potency of VectoBac. Water temperature and conductivity were negatively correlated, whereas turbidity and pH were positively correlated with LC values. Analyses of river water samples revealed that despite observed differences in total solids, sodium and potassium cations and chloride concentrations, all the parameters measured did not differ significantly between wet and dry seasons. A simple method for rearing S. damnosum s.l. in the laboratory was then adopted to study the effect of conductivity on potency of VectoBac under controlled conditions. Increasing the conductivity of the water medium up to 3,000 microS enhanced potency by about 42%, whereas increasing that of the insecticide alone raised it by 37%. The results obtained suggest that for effective use of VectoBac for blackfly control in West Africa, river temperature, conductivity and turbidity should be taken into consideration, perhaps by only selecting rivers with optimal conditions for treatment. The laboratory-based system developed for assaying the product overcomes the vagaries associated with field conditions and also the demand for huge logistic requirements of the mini-gutter system, which has to be sited near rivers.