Serovar Aizawai Research Articles

Selectivity of Bacillus thuringiensis (Bacillales: Bacillaceae) to the polyphagous predator Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae)

AbstractBioinsecticides based on Bacillus thuringiensis (Bt) are widely used to control agricultural pests. However, there are concerns about their impact on non‐target organisms, with for example, the green lacewing Ceraeochrysa claveri, a polyphagous predator with important value in the biological control of insect pests in agroecosystems. This study aimed to analyse the effects of Agree® bioinsecticide (containing Bt serovar aizawai GC‐91) on the post‐embryonic development of C. claveri. For this, neonate larvae of C. claveri were fed with Diatraea saccharalis eggs contaminated with different concentrations of Agree® (0.62, 1.25, 2.50 and 5.00 g L−1) until their pupation. Samples of the midgut of larvae were collected for histological and ultrastructural analysis. The larval and pupal inviability data were collected at the end of the respective developmental stages. After the emergence of adult insects, were considered inviable those with morphological alterations. To evaluate the reproduction, adult insect couples were formed, and the oviposition was collected. No significant differences in the viability of eggs, larvae, pupae and adults of C. claveri contaminated with Agree® were reported. Midgut analyses did not reveal cellular damage in the epithelium, indicating a possible lack of insecticidal proteins receptors. The oviposition and their viability were similar between treated and control groups. Our results demonstrated that Agree® bioinsecticide did not affect the post‐embryonic development of C. claveri. This illustrates that the combined use of Bt and C. claveri for the biological control of arthropod pests can be a sustainable strategy for integrated pest management.

Encapsulation of Bacillus thuringiensis in an inverse Pickering emulsion for pest control applications

Here, we present an inverse Pickering emulsion-based formulation for Bacillus thuringiensis serovar aizawai (BtA) encapsulations utilized towards pest control applications. The emulsification was carried out by high shear homogenization process via ULTRA-TURRAX®. The water-in-mineral oil emulsions were stabilized by commercial hydrophobic silica. Different silica contents and water/oil ratios were studied. Stable emulsions were obtained at 2 and 3 wt% silica at 30% and 20% water volumes, respectively. The structure of the Pickering emulsions were characterized by laser scanning confocal microscopy and cryogenic scanning electron microscopy. The BtA cells, spores and crystals were encapsulated in the water droplets of the inverse Pickering emulsions. An emulsion composed of 3 wt% silica and 30% water was found to be the most suitable for encapsulation. The pest control efficiency of the encapsulated BtA against Spodoptera littoralis first instar larvae was tested. The studied BtA/emulsion system exhibited a mortality rate of 92%. However, the non-formulated BtA has shown 71% mortality, and the emulsion alone resulted in only 9% mortality. These findings confirm that an emulsion with encapsulated BtA can function as an efficient formulation for biopesticides.

Influence of tannic acid on the insecticidal activity of a Bacillus thuringiensis serovar aizawai formulation against Spodoptera litura fabricius (Lepidoptera: Noctuidae)

We previously reported that tannic acid reduces the insecticidal activity of Bacillus thuringiensis serovar aizawai (Bta) against Spodoptera litura in a concentration-dependent manner. In this study, we further evaluated the biochemical mechanisms involved in the interactions between tannic acid and Bta. Application of tannic acid to the Bta insecticidal crystal protein (ICP) activated with trypsinization under alkaline conditions led to the formation of aggregates. Furthermore, the binding between the activated ICP and S. litura midgut brush border membrane vesicles (BBMVs) was evaluated in the presence of tannic acid. The binding of the activated ICPs to BBMVs decreased in a dose-dependent manner after treatment with tannic acid. As polyethylene glycol (PEG) forms strong bonds with tannic acid, its effect on the insecticidal activity of the Bta formulation was evaluated in S. litura third instar larvae. Although tannic acid reduced the insecticidal activity of Bta, the application of a Bta and PEG mixture improved the insecticidal activity, even in the presence of tannic acid. Tannic acid could also inhibit the growth of Bta in artificial media. Taken together, these results suggest that tannic acid inhibits the binding of ICP to BBMVs and the growth of Bta, which reduces its insecticidal activity.

Bioactivity of Bacillus thuringiensis (Bacillales: Bacillaceae) on Diatraea saccharalis (Lepidoptera: Crambidae) eggs.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that synthesizes specific protein toxins, which can be exploited for control of various insect pests, including Diatraea saccharalis, a lepidopteran that severely damages sugarcane crops. Although studies have described the effects of Bt in the larval phases of D. saccharalis, few have examined its effect on insect eggs. Herein, we studied the entomopathogenic potential of Bacillus thuringiensis serovar Aizawai GC-91 (Bta) during D. saccharalis embryo development with the aim of understanding the entomopathogenic mechanism and developing new biological control techniques for target insects. Bta concentrations of 5, 10 and 20 g L-1 demonstrated the strongest bioactivity, reducing D. saccharalis egg viability by 28.69%, 33.91% and 34.98%, respectively. The lethal concentrations (LCs) were estimated as: LC50 = 28.07 g L-1 (CI 95% = 1.89-2.38) and LC90 = 65.36 g L-1 (CI 95% = 4.19-5.26). Alterations in egg coloration, melanization and granule accumulation were observed at 24 h, persisting until 144 h. The embryo digestive systems were severely damaged, including narrowing of the intestinal lumen, vesiculations and degenerated cells, causing embryonic death. The toxicity caused by Bta in D. saccharalis embryos demonstrated its potential as a biological control agent and as a sustainable alternative for integrated management of D. saccharalis infestation. © 2020 Society of Chemical Industry.

Potential of bioinsecticidal Bacillus thuringiensis inoculum to suppress gray mold in tomato based on induced systemic resistance

AbstractThe potential of the active ingredient of a commercial bioinsecticide, XenTari® (Bacillus thuringiensis [BT] serovar aizawai strain ABTS‐1857), to suppress gray mold in tomato plants was elucidated. First, a suspension of the active ingredient of XenTari® and a liquid culture of the bacterial strain as BT inocula were sprayed onto detached leaves or drenched into pots of tomato seedlings, and then, propagules of the gray mold fungus, Botrytis cinerea, were inoculated onto the leaves. The gray mold disease was significantly suppressed when rhizospheres were drenched with either inoculum, but not when inocula were sprayed onto detached leaves of seedlings. Both BT inocula were verified not to directly inhibit the mycelial growth of B. cinerea based on in vitro culture plate assays. Additionally, real‐time RT‐PCR analysis verified that the active ingredient increased the expression levels of defence‐related genes, such as PR‐1(P6) and P4, in the leaves of tomato seedlings. These results suggest that the active ingredient has the potential to suppress gray mold disease in tomato, not through direct antagonistic interactions with B. cinerea, but rather through systemic activation of the plant defence system by increased expression of several defence‐related genes.

Action of Bacillus thuringiensis (Bacillales: Bacillaceae) in the midgut of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae)

The relevance of Bacillus thuringiensis in pest control strategies have been increasing in recent decades. This entomopathogenic microorganism is considered safe, and its action is highly selective to target organisms. Its toxic effect is attributed to the toxins synthesized during sporulation. The nature of the produced toxins depends on the bacterial variety. Some varieties of B. thuringiensis can synthesize from one to eight distinct toxins, which have specific effects on different orders of susceptible insects. The sugarcane borer, Diatraea saccharalis, is a holometabolous lepidopteran that causes severe damage to sugar cane cultures during its larval phase. Therefore, to evaluate B. thuringiensis serovar Aizawai GC-91 efficiency and its effects on the midgut of first instar D. saccharalis larvae, we reared the insects with contaminated artificial diet for 144 h. Larvae mortality during this period revealed that 5, 10, and 20 g/L concentrations were the most efficient, resulting in 100% mortality of treated larvae, with LC50 and LC90 estimated at 0.307 and 1.330 g/L, respectively. Furthermore, histological and ultrastructural analyses revealed damage to midgut cells. Within the first 24 h of treatment, the midgut of infected insects presented peritrophic membrane degeneration, cytoplasmic vacuolization, and cellular hypertrophy; spherites and calcium granules were also observed. At 48 h of treatment, the digestive system had collapsed, with microvilli degeneration, basement membrane and muscular fiber bundles disruption, and cellular lysis. These results confirm the toxicity of the B. thuringiensis serovar Aizawai GC-91 on D. saccharalis larvae and its potential use as a biocontroller against this pest species.

Comparison Study Of Toxicity <i>Bacillus thuringensis</i> from Soil Isolate and Mulberry Leaves in Indonesia

Background and Objective: Most strains of B. thuringiensis produce delta-endotoxin crystals toxic to lepidopteran insects such moth. But some strains of B. thuringiensis produce delta-endotoxin crystals toxic to dipteran insects such as mosquitoes and blackflies. To compare B. thuringiensis isolate from soil and mulberry leaves. Material and Methods:One gram of soil samples was suspended in 9 ml of sterile distilled water and shaken for 5 min. the upper layer of the soil suspension was transferred to a test tube and heated at 80°C for 5 min in water bath to kill non-spore-forming organism and vegetative cells to prepare the sporulated culture, bacteria were grown on nutrient agar pH 7.2, at 30°C for 4 days. The isolates were obtained from mulberry leaves collected in West Java, Indonesia, using the leaf-lift technique. Leaves were trimmed to fit inside a 100 mm petri dish. Abaxial leaf surfaces were placed in contact with nutrient agar, and a sterile, per forated stainless steel disk was placed on the leaf sections to ensure maximum contact with the agar. After the sample was coated with carbon and gold, it was observed and photographed with (SEM). However they had differences form crystal protein, but they had unique as insecticidal to control the same orders (mosquitocidal). Results:The study found that B. thuringiensis serovar entomocidus INA288 produced large cuboidal-form crystals and B. thuringiensis serovar aizawai BUN 1-14 a little homology with serovar entomocidus INA288 but, B. thuringiensis israelensis ONR60A has irregular shaped crystal protein. Toxicity results that serovar entomocidus INA288, serovar aizawai BUN 1-14 and serovar israelensis ONR60A showed toxic to mosquito insects. which was composed of major protein of 130 kDa peptides. B. thuringiensis serovar entomocidus INA288 has 70 kDa and aizawai BUN 1-14 had 69 kDa. Conclusion: B. thuringiensis strain had different crystal protein form but had same toxicity.

Characterization of a novel mosquitocidal strain of Bacillus thuringiensis serovar aizawai which harbors a rolling-circle replication plasmid, pBt1–3

Bacillus thuringiensis 1–3, isolated from a Korean soil sample, was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test, and produced bipyramidal-shaped crystal proteins with a molecular weight of 130kDa. PCR analysis with specific cry gene primers showed that B. thuringiensis 1–3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A genes, differing from that of serovar of aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D genes. In contrast to the reference strain, B. thuringiensis aizawai showed insecticidal activity against Plutella xylostella larvae, the B. thuringiensis 1–3 showed insecticidal activity against not only P. xylostella, but also Aedes aegypti, owing to its Cry2A crystal protein. In this study, we modified the plasmid capture system (PCS) through in vitro transposition to clone small cryptic plasmids from B. thuringiensis 1–3. Fifty-three clones were acquired, and their sizes were approximately 10kb. Based on the sequence analysis, they were classified into four groups, showing similarities with four known B. thuringiensis plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of the pGI3-like clones, pBt1–3, was fully sequenced, and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1–3 showed high similarity with pGI3, which is of the rolling-circle replication (RCR) group VI family.

Toxicity of Bacillus thuringiensis d-exotoxins and b-endotoxins to Drosophila melanogaster, Ephestia kuhniella and human erythrocytes

A total of 73 Bacillus thuringiensis (Bt) strains were screened for the presence of non-hemolytic insecticidal β -exotoxin-free δ -endotoxins. Out of them, 45 Bt strains produced δ -endotoxins with specific insecticidal activity against Drosophila melanogaster and/or Ephestia kuhniella larvae. The thermostable β -exotoxin was observed only in 15 Bt strains and appeared to exhibit dual non-specific insecticidal activity against both D. melanogaster and E. kuhniella larvae and showed in vitro hemolysis for human erythrocytes. It was found that β -exotoxin was produced by Bt strains belonging to five serovars ( israelensis , kenyae , kurstaki , pakistani , and tohokuensis ) and two non-serotypable strains. This result suggests that β-exotoxin production is a strain-specific property rather than a serovar-specific property. To our knowledge, this is the first study that demonstrates β -exotoxins production association with Bt strains belonging to serovars israelensis, pakistani, and tohokuensis. The plasmid DNA profiles of some β -exotoxin producing Bt strains shared large plasmid patterns which may have the common β -exotoxin regulatory gene(s). It was found that 16 local Bt strains, 15 of which belonged to five serovars ( aizawai , israelensis , kurstaki , morrisoni , and pakistani ) and one was autoagglutinated strain, produced non-hemolytic insecticidal β -exotoxin-free δ -endotoxins. Based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), the genotypic relatedness among these 16 Bt strains was investigated. The strains were grouped into two clusters. Bt strains within serovars israelensis were grouped in two subclusters, Bt strains within serovars aizawai were genomically homogeneous and clustered together, while the other serovars were grouped together in one subcluster. The autoagglutinated strain was clustered within serovar israelensis . Thus, these δ -endotoxins can be developed for the use in Bt-based insecticidal preparations. Keywords: Endotoxin, exotoxin, hemolytic, serovar, thuringiensis

Association analysis between serotype, cry gene content, and toxicity to Helicoverpa armigera larvae among Bacillus thuringiensis isolates native to Spain

Serotyping, cry gene content, and toxicity to Helicoverpa armigera were determined for 178 isolates of Bacillus thuringiensis native to Spain. A total of 13 different cry1 and cry2 genes were detected when isolates were screened by PCR analysis. Results showed that cry2 and cry1Ia were the most frequent cry genes in the collection (74 and 57%, respectively); whereas cry1D, cry1Aa, cry1Ab, and cry1C were only moderately abundant (49, 48, 47, and 36%, respectively). The most uncommon cry genes were cry1Ac, cry1E, cry1B, cry1Ib, cry1Ad, cry1F, and cry1G, with frequencies of 24, 14, 13, 8, 5, 5, and 1%, respectively. The distribution of some cry genes was somewhat associated with particular serovars. For example, genes cry1C and cry1D were especially frequent in the serovar aizawai, while cry1B was very frequent in the serovar thuringiensis. Bioassays against H. armigera larvae showed a wide variation in the insecticidal potency, even among strains sharing the same set of cry genes and within the same serotype.

Population structure and evolution of the Bacillus cereus group.

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.

Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content

Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA. There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains. Serovars canadensis and entomocidus were heterogeneous. Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement. For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi. These data suggest a clonal population structure to B. thuringiensis with some consistency of Cry-plasmid composition within PFGE types.

Characterization of flagellar antigens and insecticidal activities of Bacillus thuringiensis populations in animal feces.

In total, 287 Bacillus thuringiensis isolates, recovered from feces of 28 zoo-maintained animal species, were examined for flagellar (H) antigenicity and insecticidal activity. Serologically, 209 isolates (72.8%) were allocated to the 8 H serogroups, 4 were untypable, and 74 were untestable. Among the 8 H serotypes detected, H3abc (serovar kurstaki) predominated at a high frequency of 88.0%, followed by H6 (serovar entomocidus) with a frequency of 7.7%. Insecticidal activity was associated with 67.2% of the fecal populations: 188 isolates were toxic to both Bombyx mori (Lepidoptera: Bombycidae) and Aedes aegypti (Diptera: Culicidae), 2 isolates were specific for B. mori, and 3 isolates were toxic to A. aegypti only. Of the isolates with dual toxicity, 97.9% belonged to the serovar kurstaki, producing bipyramidal parasporal inclusions. All of the H7 (serovar aizawai) isolates were toxic to both insects.

Molecular Typing of Bacillus thuringiensis Serovars by RAPD-PCR

One hundred and twenty-six strains of Bacillus thuringiensis representing 57 serovars were allocated to 58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns. Serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, morrisoni, pakistani, sotto, thuringiensis and tolworthi each encompassed identical or closely related strains. Despite this genomic homogeneity, most of these serovars also included at least one variant strain. Serovars aizawai, canadensis, entomocidus and sotto biotype dendrolimus, on the other hand, were genomically heterogeneous. Of the 57 serovars examined, 31 contained at least one strain with a closely related or identical RAPD pattern to a strain from a different serovar. We conclude that while the species is genomically diverse, the homogeneous serovars represent clonal lineages of successful insect pathogens.

Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production.

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.

Characterization of two Bacillus thuringiensis isolates from South Brazil and their toxicity against Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Abstract The entomopathogenic bacterium Bacillus thuringiensis (Bt) is the most widely used biopesticide. The specific toxic activity to insects and other organisms is related to the presence of crystals that have different morphologies, sizes, numbers and compositions according to the Bt strain. The crystals contain different proteins, which are encoded by distinct cry (crystal) genes. In this work, we have characterized two novel Bt strains isolated from the soils of south Brazil (named UNI498 and UNI872) and determined the lethal concentrations (LC50) of their crystal proteins to Anticarsia gemmatalis larvae. Serotyping of the new isolates was performed and the results indicated that UNI872 is a Bt serovar kurstaki and UNI498 is a Bt serovar aizawai. By transmission electron microscopy, we observed that UNI498 has crystals with cubic morphology whereas UNI872 has crystals with a bipyramidal shape. Polymerase chain reactions (PCRs) revealed the presence of cry1-like sequences in both isolates and a cry2-like gene in UNI 872. The cry1 fragments were sequenced and showed identities of 96 and 97% to known cry1A genes (UNI872 and UNI498, respectively). The presence of Cry1 proteins in both isolates was confirmed by SDS–PAGE. Bioassays to determine the LC50 were performed with A. gemmatalis third instar larvae using purified proteins. Results showed LC50s of 1.69 ng / cm 2 for UNI498 and 0.49 ng / cm 2 for UNI872.

Host range and gene contents of Bacillus thuringiensis strains toxic towards Spodoptera exigua

AbstractThirty‐five strains of the entomopathogenic bacterium Bacillus thuringiensis active on Spodoptera exigua, were characterized by means of serological identification and determination of cry gene contents by PCR. The insecticidal activity of these 35 strains was further confirmed against S. exigua and tested against two other species of the same genus: S. littoralis and S. frugiperda. The results indicate that serovars aizawai, thuringiensis, and kurstaki were the most frequent within S. exigua ‐active strains and that serovar aizawai had the highest number of strains exhibiting toxicity against the three species bioassayed. The presence in cry genes as determined by PCR suggests a non random distribution of some cry genes among serovars. Genes cry1C, cry1D, and cry1E, which are known to code for proteins toxic against Spodoptera species, were very common within S. exigua‐active strains, specially in those belonging to serovar aizawai. However, some strains harbouring one or more of these genes were not toxic to S. littoralis or S. frugiperda; and some strains lacking all of the Spodoptera‐active genes were found to be toxic to all three species. This suggests differences in the expression levels among strains bearing toxic genes and the involvement of other genes toxic to Spodoptera species. Since strains sharing the same cry genes exhibited different host ranges, the results indicate the need to perform toxicity bioassays in addition to other tests (serological identification and PCR) in order to determine the insecticidal activity of B. thuringiensis strains.

Molecular and insecticidal characterization of a Bacillus thuringiensis strain isolated during a natural epizootic.

A new Bacillus thuringiensis strain belonging to the serovar aizawai was isolated from a dead larva of the lepidopteran Mythimna loreyi collected in a corn crop in Spain during a natural epizootic. This strain, which was named Leapi01, was compared with the kurstaki and aizawai strains isolated from Dipel(R) and Xentari(R), by electron microscopy, SDS-PAGE, plasmid pattern, PCR and insecticidal activity. This strain showed similar morphological and biochemical characteristics to the standard strains. The content in cry genes of Leapi01 was analysed with a set of general and specific primers recognizing most of the cry genes reported to date. DNA amplification was obtained with primers corresponding to six genes and, to clearly determine the identity of the genes, the amplified fragments were sequenced and corresponded to cry1Aa, cry1Ab, cry1Ca, cry1Da, cry2Ab and cry1Ia. However, the proteins encoded by two of these genes, Cry2 and Cry1I, were not detected in the SDS-PAGE of the purified parasporal bodies. The insecticidal activity of Leapi01 was determined by bioassays against two Lepidoptera species, Helicoverpa armigera and Spodoptera littoralis, that were found to be very susceptible to Leapi01 purified crystals. Since two of the cry genes identified in Leapi01 appear to be silent, other factors may be involved in the toxicity of the strain. As a result of this study, the potential of Leapi01 as biological control agent is discussed, with special emphasis on the high toxicity and relatively broad spectrum activity compared with two B. thuringiensis strains that are the active ingredients of commercial preparations commonly used as bioinsecticides.

Natural Coprevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis in the Host, Delia radicum

Adult cabbage root flies (Delia radicum) from three Danish localities were diagnosed microscopically for the natural prevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis. C. deliaradicae was significantly coprevalent with S. castrans. B. thuringiensis sporangia were diagnosed in the hemolymph in two D. radicum which were also infected with S. castrans and proved to belong to serovar aizawai and serovar balearica. The biological characterization of S. castrans proved that at 17.5°C flies developed an abdominal hole 7.9 days (mean) after infection and that 5.7 days (mean) passed from the emergence of the hole to the death of the infected host. No mortality effect among D. radicum subjected to B. thuringiensis serovar aizawai, balearica, and kurstaki isolates was detected. RAPD with DNA proved that six B. thuringiensis serovar balearica isolates (all from the same fly) were indistinguisable. This indicates that proliferation of B. thuringiensis in the abdomen of an S. castrans-infected D. radicum may be due to just one genotype. The profiles of one isolated aizawai strain did not correspond to the profiles of other serovar aizawai strains used for comparison. The biological significance of the interaction between the involved pathogens is discussed.

Novel Bacillus thuringiensis serovar aizawai strains isolated from mulberry leaves in Indonesia

Novel Bacillus thuringiensis serovar aizawai strains isolated from mulberry leaves in Indonesia