Effects of Entomopathogenic fungi and Bacillus thuringiensis‐based biopesticides on Spoladea recurvalis (Lepidoptera: Crambidae)

Spoladea recurvalis (Fabricius) is one of the most devastating pests of amaranths causing severe yield losses of 60%–100% to the crop. Unfortunately use of chemical pesticides is the most common control strategy that vegetable farmers rely on to control the pest. However, it is not effective and harmful to environmental and human health. Aiming to provide more environmentally friendly alternatives, this study evaluated the effects of various entomopathogenic fungal isolates and commercial based Bacillus thuringiensis Subsp. kurstaki product Halt^®, on the pest. Twenty‐four entomopathogenic fungal (EPF) isolates from three genera (14 Metarhizium anisopliae, 9 Beauveria bassiana and 1 lsaria fumosorosea) were screened in the laboratory to assess their pathogenicity against second instar larvae of S. recurvalis. Only M. anisopliae ICIPE 30 reached a moderate threshold, causing 58.3% larval mortality. All the 11 isolates (8 M. anisopliae, 2 B. bassiana and 1 l. fumosorosea) tested against adult S. recurvalis were pathogenic, with M. anisopliae ICIPE 30 and B. bassiana ICIPE 725 causing the highest mortality of 92% and 83%, respectively. Metarhizium anisopliae ICIPE 30 had the shortest LT₅₀ value of 4.8 days. Bacillus thuringiensis Subsp. kurstaki product Halt^® caused <50% mortality on S. recurvalis larvae. A consecutive application of M. anisopliae ICIPE 30 and Bt did not cause a significant increase in larval mortality compared to separate applications of both products. Results of this study suggest that M. anisopliae ICIPE 30 was the most potent candidate and could be used in an autodissemination approach for management of adult S. recurvalis.     

Interaction of acid exudates in chickpea with biological activity of Bacillus thuringiensis towards Helicoverpa armigera

The gram pod borer, Helicoverpa armigera, is one of the most important constraints to chickpea production. High acidity of chickpea exudates is associated with resistance to pod borer, H. armigera; however, acidic exudates in chickpea might influence the biological activity of the bacterium, Bacillus thuringiensis (Bt), applied as a foliar spray or deployed in transgenic plants for controlling H. armigera. Therefore, studies were undertaken to evaluate the biological activity of Bt towards H. armigera on chickpea genotypes with different amounts of organic acids. Significantly lower leaf feeding, larval survival and larval weights were observed on ICC 506EB, followed by C 235, and ICCV 10 across Bt concentrations. Leaf feeding by the larvae and larval survival and weights decreased with an increase in Bt concentration. However, rate of decrease in leaf feeding and larval survival and weights with an increase in Bt concentration was greater on L 550 and ICCV 10 than on the resistant check, ICC 506EB, suggesting that factors in the resistant genotypes, particularly the acid exudates, resulted in lower levels of biological activity of Bt possibly because of antifeedant effects of the acid exudates. Antifeedant effects of acid exudates reduced food consumption and hence might reduce the efficacy of Bt sprays on insect‐resistant chickpea genotypes or Bt‐transgenic chickpeas, although the combined effect of plant resistance based on organic acids, and Bt had a greater effect on survival and development of H. armigera than Bt alone.     

Effect of different additives on the persistence and insecticidal activity of native strains of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The persistence and insecticidal activity of native strains of Bacillus thuringiensis was evaluated in formulations containing different additives such as arrow-root powder, carboxy methyl cellulose (CMC), gum acacia, non-food grade (NFG) starch, and soluble starch. Persistence of B. thuringiensis varied with different additives used in the formulations. Among the different additives used, NFG starch provided maximum protection to B. thuringiensis and native strain 42 showed maximum persistence (83%) which was higher than that obtained in commercial formulation. In commercial formulation, the persistence of B. thuringiensis was 47% only after 3 d of spray. The feeding trials conducted on second instar larvae of H. armigera using leaves sprayed with NFG starch formulation revealed 70% larval mortality while commercial formulation showed 50% mortality during the same period.     

Seasonal occurrence of amaranth Lepidopteran defoliators and effect of attractants and amaranth lines in their management

Lepidopteran defoliators are the most important pests of cultivated amaranths causing severe losses in cultivated fields worldwide. Leaf‐webbers, whose larvae fold, web or glue amaranth leaves using their silken webs as they feed and leaf‐worms which cause windowing but do not glue or fold leaves are mainly reported. Sustainable management strategies for these pests are still lacking given the adverse effects of synthetic pesticides. Field experiments were conducted during two seasons at two different sites in Central Kenya, to assess amaranth lepidopteran pests and their natural enemies’ population dynamics, evaluate the efficacy of phenylacetaldehyde (PAA) floral lure as attractant and the effects of three amaranth lines (Abuk1, Abuk2 and Abuk8) on the pests’ abundance and damage. Abundance of leaf‐webbers (p = .537), leaf‐worms (p = 1) and their associated parasitoids (p = .083) did not differ between the dry and wet seasons. The parasitoids Atropha tricolor and Apanteles sp. caused parasitism of 6.2% and 33.3% on Spoladea recurvalis and Choristoneura sp., respectively. PAA incorporated traps attracted moths that were largely unrelated to the damaging larvae observed on the crops with only 0.5% of total trap catches being S. recurvalis. Sub‐sites in which PAA were incorporated had significantly higher number of leaf‐webber larvae on the crops compared to control sub‐sites (p = .014). Amaranth lines studied had significant (p = .007) effect on lepidopteran defoliators’ abundance and damage, with fewer leaf‐webbers and lower severity of damage recorded on Abuk2 compared to Abuk8. The implication of these findings for the control of lepidopteran defoliators in East Africa is discussed.     

Performance of the newly identified endoparasitoid Cotesia icipe Fernandez‐Triana & Fiaboe on Spodoptera littoralis (Boisduval)

Cotesia icipe Fernandez‐Triana & Fiaboe is a solitary koinobiont larval endoparasitoid, recently discovered in Kenya and new to science, that parasitizes select lepidopteran herbivores of amaranth. We investigated its host range on five commonly encountered amaranth lepidopteran defoliators. Cotesia icipe accepted, successfully and aggressively parasitized the amaranth noctuid defoliators Spodoptera littoralis (Boisduval) and Spodoptera exigua (Hübner), but failed to parasitize Herpetogramma bipunctalis (F.), Spoladea recurvalis (F.) and Udea ferrugalis (Hübner) all in Crambidae family. On S. littoralis, C. icipe was highly efficient, with 95% of females successfully ovipositing during 2 hr of exposure. Parasitism rate and larval and pupal non‐reproductive mortalities were significantly higher at higher parasitoid density. A single female of C. icipe parasitized 42.99 ± 2.66% of the 50 exposed larvae for oviposition in 24 hr, whereas a cohort of five females of C. icipe conferred 85.59 ± 1.46% parasitism rate. The efficiency ratio per female was much higher in single releases than in cohort releases while a balanced sex ratio was obtained in F1 offspring regardless of the density of female released. The potential use of C. icipe for conservation and augmentative biological control of S. littoralis in amaranth as well as its potential use against other Noctuid moths and in other commodities is discussed.     

Insecticidal potential of Bacillus thuringiensis for the biological control of neotropical brown stink bug

Euschistus heros (Hemiptera: Pentatomidae) is considered a major insect pest in soybean production in Brazil. The indiscriminate application of pesticides to fields leads to reduction in biodiversity, selection of resistant populations, emergence of new pest outbreaks, and damage to non‐target organisms. The objective of this research was to evaluate the effects of exposure to Cry proteins present in new isolates of Bacillus thuringiensis Berliner, commercial formulations, and Bt soybean on E. heros development. The feeding preference of E. heros for Bt‐ vs. non‐Bt soybean was also evaluated. All treatments caused significant mortality to E. heros except the Bt soybean. Tests with combinations of isolated Bt‐toxins indicated that combinations had greater efficacy than other treatments (>98% mortality). The results demonstrate that E. heros is susceptible to B. thuringiensis toxins, which may contribute to the management of this insect in soybean agro‐ecosystems.     

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.     

Suspension concentrate formulation of Bacillus thuringiensis var. kurstaki for effective management of Helicoverpa armigera on sunflower (Helianthus annuus)

A local strain DOR Bt-1 belonging to Bacillus thuringiensis var. kurstaki (Bt.k) was multiplied through solid state fermentation and the resulting technical powder was milled and sieved to obtain particles of various sizes. Efficacy of Bt.k. against larvae of Helicoverpa armigera was found to increase with the decrease in particle size. Boric acid was found to be synergistic to DOR Bt-1 technical powder. LC₅₀ of the Bt and boric acid mixture (75:25) was lower at 89.63 mg/100 mL in comparison to Bt alone at 116.75 mg/100 mL. A suspension concentrate formulation with DOR Bt-1 technical was developed using boric acid as an adjuvant. The formulation was found to be highly effective against H. armigera in laboratory bioassays with an LC₅₀ of 185 µL (containing 53.36 mg Bt). The formulation gave effective control of H. armigera on sunflower within 3 days after spray even at the lowest dose of 1.0 mL/L under field conditions.     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains and selection of an isolate active against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> and <Emphasis Type="Italic">Peridroma saucia</Emphasis>

Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate—Bt RT—produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity.     

Efficacy of <Emphasis Type="Italic">Steinernema feltiae</Emphasis> against the leek moth <Emphasis Type="Italic">Acrolepiopsis assectella</Emphasis> in laboratory and field conditions

The susceptibility of larvae of the leek moth, Acrolepiopsis assectella Zeller (Lepidoptera: Acrolepiidae) to different concentrations of an autochthonous strain of Steinernema feltiae (Rhabditida: Steinernematidae) was examined in laboratory experiments using Petri dishes. The efficacy of this strain in pots and field experiments was also evaluated. High mortality (80%–100%) of leek moth larvae was observed when these larvae were exposed to low concentrations (3 × 10³ to 1 × 10⁴ IJs/m²) of S. feltiae under laboratory conditions. Foliar application of 30,000 IJs/leek in pot experiments caused a 98% reduction in leek moth larvae. Field experiments showed a 87.7% reduction of leek moth larvae with the nematode treatment, significantly higher than the 22% reduction with the Bacillus thuringiensis treatment. The efficacy of the treatments with S. feltiae in relation to the microhabitat of the leek moth larvae between the interfolded leaves of the leek is discussed.     

Comparing the predatory performance of green lacewing on cotton bollworm on conventional and Bt cotton

We compared the survival of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) eggs and larvae on Bt and conventional cotton, in the presence or absence of the generalist predator, green lacewing larvae, Mallada signatus, (Schneider) (Neuroptera: Chrysopidae). In small arenas, green lacewings consumed a similar number of H. armigera eggs (ave. 15.8 ± 1.3 on conventional, 12.6 ± 1.4 on Bt cotton per predator over 24 h) and larvae (ave. 6.8 ± 0.7 conventional, 6.5 ± 0.8 Bt per predator over 24 h) whether on Bt or conventional cotton leaves. Likewise, similar numbers of eggs were consumed by each lacewing larva searching whole plants of either Bt (ave. 15.5 ± 0.6 of 49 over 24 h) or conventional (ave. 13.6 ± 1.1 of 49 over 24 h). On conventional plants over 72 h, survival of H. armigera larvae was 72.8% and decreased to 37.7% when lacewings were present, giving a net consumption rate of 35.1% (8.6 prey per predator over 72 h). On Bt cotton plants, 13.6% of the H. armigera larvae survived after 72 h and this decreased to 1.7% when lacewings were present. This combination of mortality factors operated synergistically. Helicoverpa armigera larvae moved to fruiting structures on conventional or Bt cotton but failed to survive in the squares (young flower buds) when the impacts of Bt and lacewings were combined. The removal of first to second instar H. armigera larvae from squares of Bt cotton by predators has the potential to reduce immediate pest damage and, perhaps more importantly, remove potentially Bt‐resistant genotypes.     

Ovicidal effects of entomopathogenic fungal isolates on the invasive Fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)

Maize is a major staple food for over 300 million people in sub‐Saharan Africa. Sustainable productivity of this primary crop has been recently threatened by Fall armyworm (FAW), Spodoptera frugiperda invasion. Due to lack of environmentally safe management strategies, immediate responses by growers and governments to tackle FAW are based on rampant use of pesticides. Looking for efficient biopesticides, twenty entomopathogenic fungal isolates (14 Metarhizium anisopliae and 6 Beauveria bassiana) were screened for their efficacy against eggs and second instar larvae of FAW. A single discriminating concentration of 1 × 10⁸ conidia ml^?1 and four replicates per treatment were used in all experiments. Isolates were assessed for their ability to cause mortality of FAW second instar larvae, eggs and the neonate larvae that emerged from treated eggs. Among the isolates tested, only B. bassiana ICIPE 676 caused moderate mortality of 30% to second instar larvae. Metarhizium anisopliae ICIPE 78, ICIPE 40 and ICIPE 20 caused egg mortalities of 87.0%, 83.0% and 79.5%, respectively, and M. anisopliae ICIPE 41 and ICIPE 7 outperformed all the others by causing 96.5% and 93.7% mortality to the neonate larvae, respectively. The cumulated mortality of eggs and neonates was highest with M. anisopliae ICIPE 41 (97.5%), followed by M. anisopliae ICIPE 7, 655, 40, 20 and 78 with total mortality of 96.0%, 95.0%, 93.5%, 93.0% and 92.0%, respectively. These isolates with high cumulated mortality (≥92%), especially ICIPE 78 and 7, which are already commercialized for spider mites and ticks control respectively, would be good candidates for development as biopesticides for management of FAW in Africa if further evidence of their efficacy is obtained in the field.     

Development of a model for prediction of target insect mortality following field application of a Bacillus thuringiensis formulation

A model for predicting mortality of Schizura concinna larvae on Cercis occidentalis trees following application of Bacillus thuringiensis formulations is based on an estimation of the effective dose. This is calculated from the initial application level and field persistence characteristics of the pathogen and from the feeding rate of the target insects. Using the estimated effective dose and the time the pathogen was on the host plant, the probability of larval mortality is calculated by means of a multiple logistic dose-response model. Predicted mortalities for B. thuringiensis applications at concentrations 0.00025, 0.0025, 0.025, and 0.25%, $\text{w}\text{v}$, were 7.7, 23.3, 46.2, and 87.1%, respectively. Corresponding, observed mortalities adjusted for larval loss due to forces unrelated to the B. thuringiensis application were 6, 54, 66, and 100%.     

Resistant cabbage cultivars change the susceptibility of Plutella xylostella to Bacillus thuringiensis

1 Laboratory studies demonstrated that the susceptibility of larvae of the lepidopteran crucifer pest Plutella xylostella to the insect pathogen Bacillus thuringiensis (Bt) was influenced by the host plant. 2 Larvae reared on the resistant cabbage cultivars Minicole F₁ and Red Drumhead were significantly more susceptible to Bt (the LC₅₀ fell to one half) than larvae fed leaves of susceptible cultivars. 3 However, a third resistant cultivar, Aquarius F₁, had no synergistic effect on Bt‐related mortality. 4 Actual uptake of Bt was monitored in the bioassays, as a preliminary experiment showed that the plant resistance reduced consumption of Bt‐treated leaf discs. However, differences in feeding rate did not explain the observed differences in mortality.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (<Emphasis Type="Italic">Apis mellifera L.</Emphasis>) larvae

Morphological alterations, by means of histological and ultrastructural analysis, have been used to determine the effects of boric acid and fipronil on midgut tissues of honeybee worker, Apis mellifera L. larvae. In order to observe possible morphological alterations in the midgut, two groups of bioassays were performed. In the first one, the larvae were chronically treated with different concentrations of boric acid added to the food (1.0, 2.5 and 7.5 mg/g). In the second group, the larvae were fed with diets containing different concentrations of fipronil (0.1 and 1 μg/g) and compared with control groups without these chemical compounds. In the first bioassay, the larvae were collected on day 3 and in the second bioassay on day 4, when the mortality rate obtained in the toxicological bioassay was not very high. The larval midguts were removed and processed for morphological analyses using a light and transmission electron microscopy. We observed cytoplasmic vacuolizations, with the absence of autophagic vacuoles, and chromatinic compacting in most of the cells in the groups treated with pesticides. The morphological alterations were far greater in the larvae treated with boric acid than in the larvae treated with fipronil. Our data suggest that the midgut cell death observed was in response to boric acid and fipronil action. This study significantly improves the understanding of the toxicological effect of these insecticides from the ecotoxicological perspective.     

<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">tenebrionis</Emphasis> control of synanthropic mites (Acari: Acaridida) under laboratory conditions

Bacillus thuringiensis (Bt) toxins present a potential for control of pest mites. Information concerning the effect of Bt and its possible application to the biocontrol of synathropic mites is rare. The toxic effect of Bacillus thuringiensis var. tenebrionis producing Cry3A toxin was tested on the mites Acarus siro L., Tyrophagus putrescentiae (Schrank), Dermatophagoides farinae Hughes, and Lepidoglyphus destructor (Schrank) via feeding tests. Fifty mites were reared on Bt additive diets in concentrations that ranged from 0 to 100 mg g⁻¹ under optimal conditions for their development. After 21 days, the mites were counted and the final populations were analyzed using a polynomial regression model. The Bt diet suppressed population growth of the four mite species. The fitted doses of Bt for 50% suppression of population growth were diets ranging from 25 to 38 mg g⁻¹. There were no remarkable differences among species. Possible applications of Bt for the control of synanthropic mites are discussed.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.