Sublethal effects of Bacillus thuringiensis on the spruce budworm, Choristoneura fumiferana

Exposing larvae of the spruce budworm, Choristoneura fumiferana (Clemens), to sublethal ( 50% lethal dose) levels of Bacillus thuringiensis subsp. kurstaki at various stages of their development significantly increased development time to the pupal stage and reduced pupal size and number of eggs laid per female, but did not affect the proportion of embryonated eggs. The changes in larval development time, pupal weight and fecundity depended on the larval stage that was treated. Exposure of fourth instars delayed larval development and reduced only male pupal weights with no effects on fecundity. Exposure of sixth instars delayed larval development to a lesser extent than exposure of fourth instars but had a pronounced effect on weight of both male and female pupae. The effect on pupal weight was sex dependent, as males tended to be more affected than females. The reduction in male pupal weight did not appear to influence fecundity, because the effect of exposure was explained by the change in female pupal weight. Effects on larval growth and pupal weight were proportional to the dose that was ingested during exposure, and were observed at doses as low as one-tenth of the LD₅₀. Ingestion of an LD₅₀ caused a 29 or 45% delay in development of, respectively, female or male larvae when exposed as fourth instars and a 30% reduction in female pupal weight when larvae were exposed as sixth instars.     

Effects of bud phenology and foliage chemistry of balsam fir and white spruce trees on the efficacy of Bacillus thuringiensis against the spruce budworm, Choristoneura fumiferana

Abstract 1 Efficacy of commercial formulations of Bacillus thuringiensis ssp. kurstaki (Btk) against spruce budworm Choristoneura fumiferana was investigated in mixed balsam fir‐white spruce stands. Btk treatments were scheduled to coincide with early flaring of balsam fir shoots, and later with flaring of white spruce shoots. Btk efficacy on the two host trees was compared and examined according to the foliar content of nutrients and allelochemicals and the insect developmental stage at the time of spray. 2 Larvae fed white spruce foliage were less vulnerable to Btk ingestion than larvae fed balsam fir foliage. Higher larval survival on white spruce, observed 10 days after spray, was related to higher foliage content in tannins and a lower N/tannins ratio, which might have induced inactivation of Btk toxins. 3 Larval mortality due to Btk did not depend on spruce budworm larval age. 4 Foliage protection of both host trees was similar in plots treated with Btk: larval mortality due to Btk treatment reduced insect grazing pressure on balsam fir trees; meanwhile, suitability of white spruce foliage seemed to decrease very rapidly, which induced high larval mortality among spruce budworm fed on white spruce trees. Nevertheless, following Btk sprays, 50% more foliage remained on white spruce than on balsam fir trees, because of the higher white spruce foliage production. 5 Both spray timings achieved similar protection of white spruce trees, but Btk treatments had to be applied as early as possible (i.e. during the flaring of balsam fir shoots to optimally protect balsam fir trees in mixed balsam fir‐white spruce stands).     

High tolerance against Chrysomela tremulae of transgenic poplar plants expressing a synthetic cry3Aa gene from Bacillus thuringiensis ssp tenebrionis

Hybrid poplars (Populus tremula ×Populus tremuloides) have been genetically engineered viaAgrobacterium tumefaciens, to express a syntheticcry3Aa gene derived from the native Bacillusthuringiensis subsp. tenebrionis cry3Aa gene.The presence and the expression of the transgene have been verified in fourtransgenic poplar lines, using Southern, northern and western analyses. Thetransgenic poplar's toxicity towards the phytophagous beetleChrysomela tremulae (Coleoptera, Chrysomelidae) has beenassessed on six month-old greenhouse-grown selected plants in laboratoryconditions. Laboratory experiments consisted of feeding tests of fresh detachedleaves on C. tremulae at all developmental stages. Ourresults indicate that the transgenic poplar leaves, expressing a Cry3Aa proteinamount in a range of 0.05–0.0025% of total soluble protein, weredefinitely deleterious for C. tremulae, regardless of thedevelopmental stage.     

Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter

We produced 49 broccoli plants (Brassica oleracea L. ssp. italica) containing a Bacillus thuringiensis cry1Ab gene under control of the chemically inducible PR-1a promoter from tobacco. Most of them showed substantial or complete control of neonate diamondback moth larvae, regardless of whether the transgene was induced or not. Ten plants were selected for detailed study via northern and western analysis and insect bioassays. They expressed the cry1Ab gene and gave complete insect control when treated with the chemical inducers INA (2,6-dichloroiso-nicotinic acid) or BTH (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester); however, leaves treated with water alone were also partially or completely protected from insect damage. Transgenic progeny plants showed greater inducibility than primary transformants at the molecular level. Two progeny lines produced cry1Ab mRNA and Cry1Ab protein and gave insect control only after induction, both when detached leaves and intact plants were tested. The relevance of these results to resistance management strategies is discussed.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Expression of a cry2Aa gene in an acrystalliferous Bacillus thuringiensis strain and toxicity of Cry2Aa against Helicoverpa armigera

In the recent past research has been mainly focused on the expression of cry1 genes of Bacillus thuringiensis (Bt) to engineer lepidopteran insect resistance in plants. Search for structurally different toxins is necessary for the management of resistance development in insects. The intact cry2Aa operon (3.95 kb) of a new isolate of Bt, 47-8, was subcloned into a Bt shuttle vector, pHT3101 (6.7 kb). Recombinant pHT3101 containing the cry2Aa operon of Bt strain 47-8 was named as pTN2Aa and used to transform acrystalliferous Bt strain 4Q7 by electroporation. Phase contrast microscopic observation revealed the presence of crystalline inclusions in the transformants of Bt strain 4Q7 harbouring pTN2Aa. SDS–PAGE of a spore–crystal mixture prepared from transformants of acrystalliferous Bt strain 4Q7 harbouring pTN2Aa showed a single band of about 65 kDa alone confirming the expression of the cloned cry2Aa. Bioassay with Helicoverpa armigera showed 71.4% mortality caused by the proteins encoded by the newly cloned cry2Aa gene (at the concentration of 2.3 g/l) on the seventh day and all the survivors that escaped from Cry2Aa toxicity showed severe (81–99%) inhibition in larval growth.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.     

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

Bacillus thuringiensis Cry1Ba endotoxin, which was shown to exhibit a tenfold lower lethal concentration 50 (LC50) than Cry1Ac in a Striped Stem Borer (SSB) diet incorporation assay. The 1.950-bp synthetic cry1B gene, possessing an overall GC content of 58 %, was cloned under the control of the maize ubiquitin promoter first intron and first exon regions. The resulting vector, designated as pUbi-cry1B, was transferred to two commercial Mediterranean cultivars of rice, Ariete and Senia, using microprojectile acceleration-mediated transformation. Thirty-two and 47 T0 events were generated in cvs. Ariete and Senia, respectively. Southern blot and immunoblot analyses allowed the identification of 7 Senia and 1 Ariete events harbouring both an intact gene cassette and expressing Cry1B at a level ranging from 0.01% to 0.4% of the total soluble proteins. Three Senia and 1 Ariete events were found to be protected against second instar SSB larvae in whole plant feeding assays, exhibiting 90–100% mortality 7 days after infestation. Spatial and temporal variation in transgene expression was further examined in resistant event 64 of cv. Ariete. Stable accumulation of Cry1B, representing 0.4% of the total soluble proteins, was observed over the T2 to T4 generations in leaf tissue 20, 40, 70 and 90 days after germination in both young and old leaves and in internodes. Ariete event 64 was found to be fully protected from attacks of third and fourth instar SSB larvae over subsequent generations. Received: 7 February 2000 / Revision received: 18 May 2000 / Accepted: 29 May 2000     

Cloning, characterization and expression of a new cry1Ab gene from Bacillus thuringiensisWB9

A new cry 1Ab-type gene, cry 1Ab17, was cloned from Bacillus thuringiensis WB9 by PCR. Nucleotide sequence indicated that the open reading frames (ORFs) consists of 3471 bases and encodes a protein of 1156 residues with a calculated molecular weight of 130.5 kDa and an pI value of 5.04. Homology comparison revealed that the deduced amino acid sequence of Cry1Ab17 had 95.4% to 99.7% identity with those of the known Cry1Ab proteins. The Cry1Ab17 was one residue longer than the known Cry1Ab (except for Cry1Ab2). Domain I (Tyr(33) to Arg(253)), II (Arg(265) to Phe(462)), III (Asn(464) to Thr(610)) of the Cry1Ab17 were 96.8%, 68.2% and 100% identical to the corresponding domains of Cry1Aa. Additionally, the cry 1Ab17 gene was expressed in Escherichia coli BL21 under the control of T7 promoter and the Cry1Ab17 isolated from the culture medium was toxic to 3rd instar Plutella xylostella larvae.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.     

Inheritance and expression of the cry1Ab gene in Bt ( Bacillus thuringiensis) transgenic rice

The inheritance and expression patterns of the cry1Ab gene were studied in the progenies derived from different Bt (Bacillus thuringiensis) transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F₂ populations. Crosses between japonica intra-subspecies had no significant effect on the segregation ratios of the cry1Ab gene, but crossing between japonica and indica inter-subspecies led to distorted segregation of the cry1Ab gene in the F₂ population. Field-release experiments indicated that the cry1Ab gene was stably transmitted in an intact manner via successive sexual generations, and the concentration of the Cry1Ab protein was kept quantitatively stable up to the R₆ generation. The cry1Ab gene, driven by the maize ubiquitin promoter, displayed certain kinds of spatial and temporal expression patterns under field conditions. The content of the Cry1Ab protein varied in different tissues of the main stems, the primary tillers and the secondary tillers. Higher levels of the Cry1Ab protein were found in the stems, leaves and leaf sheaths than in the roots, while the lowest level was detected in grains at the maturation stage. The content of the Cry1Ab protein in the leaves peaked at the booting stage and was lowest at the heading stage. Furthermore, the Cry1Ab content of cry1Ab expression in different tissues of transgenic rice varied individually with temperature. Received: 17 April 2001 / Accepted: 7 May 2001     

Nutritional physiology of the eastern spruce budworm,Choristoneura fumiferana,infected with Nosema fumiferanae,and interactions with dietary nitrogen

Summary Female eastern spruce budworm larvae, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae), inoculated with a medium lethal spore dosage of the microsporidium Nosema fumiferanae (Thomson) exhibited significant reductions in consumptive index (CI), nitrogen consumptive index (NCI), relative growth rate (RGR), and gross (ECI) and net (ECD) production effeciencies when compared to microsporidian-free larvae. Diseased larvae also exhibited significant increases in approximate digestibility (AD), N utilization efficiency (NUE), and larval moisture content. Both healthy and diseased insects were reared on 2.5% N and 4.5% N diets. Those on the 2.5% N diet showed significant increases in CI, although NCI was still lower than NCI measured for larvae reared on 4.5% N. NUE was also higher on the 2.5% N diet. Diseased cohorts reared on 2.5% N diet had significantly greater mortality than those reared on 4.5% N diet. Pupal weight and development time of infected individuals did not respond to dietary N concentration. However, healthy insects achieved greater pupal weights in a shorter time on the 4.5% N diet than those on the 2.5% N diet. Mortality of healthy insects was unaffected by dietary N.     

Taste receptor activity and feeding behaviour reveal mechanisms of white spruce natural resistance to Eastern spruce budworm Choristoneura fumiferana

The pattern of feeding of Eastern spruce budworm Choristoneura fumiferana (Clem.) (Lepidoptera, Tortricidae) is compared on foliage from white spruce Picea glauca (Moench) Voss. (Pinaceae) trees previously determined to be susceptible and resistant to defoliation by budworm. No differences are observed in electrophysiological responses from taste sensilla to aqueous extracts of the two foliage types, nor is there a preference for either extract type in a choice test. Acetone extracts from the two foliage types are both preferred to a control sucrose solution, although neither elicits a preference relative to the other. These results suggest that there is no difference in phagostimulatory power of internal leaf contents of the two foliage types. Longer‐term observation of feeding behaviour in a no‐choice situation shows no difference in meal duration, confirming the lack of difference in phagostimulatory power. However, on average, intermeal intervals are twice as long on the resistant foliage, leading to an overall lower food consumption during the assay. This result suggests an anti‐digestive or toxic effect of the resistant foliage that slows behaviour and limits food intake. Previous research has shown that waxes of the resistant foliage deter initiation of feeding by the spruce budworm and that this foliage contains higher levels of tannins and monoterpenes. The data suggest that the resistant foliage contains a post‐ingestive second line of defence against the spruce budworm.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2–3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.     

On the Adaptive Nature of the Dissociation Process in Bacillus thuringiensis

The process of dissociation into variants that differ in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40°C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.