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ABSTRACT: The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is an invasive wood boring beetle that is decimating North America's ash trees (Fraxinus spp.). To find effective and safe indigenous biocontrol agents to manage EAB, we conducted a survey in 2008-2009 of entomopathogenic fungi (EPF) infecting EAB in five outbreak sites in southwestern Ontario, Canada. A total of 78 Beauveria spp. isolates were retrieved from dead and mycosed EAB cadavers residing in the phloem tissues of dead ash barks, larval frass extracted from feeding galleries under the bark of dead trees. Molecular characterization using sequences of the ITS, 5' end of EF1-α and intergenic Bloc region fragments revealed that Beauveria bassiana and Beauveria pseudobassiana were commonly associated with EAB in the sampled sites. Based on phylogenetic analysis inferred from ITS sequences, 17 of these isolates clustered with B. bassiana, which further grouped into three different sub-clades. However, the combined EF1-α and Bloc sequences detected five genotypes among the three sub-clades. The remaining 61 isolates clustered with B. pseudobassiana, which had identical ITS sequences but were further subdivided into two genotypes by variation in the EF1-α and Bloc regions. Initial virulence screening against EAB adults of 23 isolates representing the different clades yielded 8 that produced more than 90% mortality in a single concentration assay. These isolates differed in virulence based on LC(50) values estimated from multiple concentration bioassay and based on mean survival times at a conidia concentration of 2×10(6) conidia/ml. B. bassiana isolate L49-1AA was significantly more virulent and produced more conidia on EAB cadavers compared to the other indigenous isolates and the commercial strain B. bassiana GHA suggesting that L49-1AA may have potential as a microbiological control agent against EAB.
Journal of Invertebrate Pathology 06/2012; 111(1):41-9. · 2.06 Impact Factor
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ABSTRACT: We provide molecular systematics of a microporidian species, Nosema fumiferanae, one of the most common natural enemies of spruce budworm, Choristoneura fumiferana. The uncharacterized flanking region upstream of the large subunit (LSU) rRNA and the complete rRNA cistron of N. fumiferanae was 4,769 bp long. The organization of the rRNA gene was 5'-LSU rRNA-ITS-SSU rRNA-IGS-5S-3' and corresponded primarily to most insect (i.e. lepidopteran) Nosema species identified and classified to date. Phylogenetic analysis based on the complete rRNA cistron indicated that N. fumiferanae is closely related to Nosema plutellae and is correctly assigned to the "true" Nosema group. Suggestions were provided on a criterion to delineate the "true" Nosema from other microsporidian species.
Journal of Eukaryotic Microbiology 12/2011; 59(1):93-6. · 2.66 Impact Factor
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ABSTRACT: A microsporidium was isolated from the bronze birch borer, Agrilus anxius Gory (Coleoptera: Buprestidae), collected near Sudbury and Sault Ste Marie, Canada. Light and electron microscopic investigations showed that gross pathology and ultrastructure of the investigated Cystosporogenes species was similar to those characterized and described for other Cystosporogenes species. Small subunit rRNA gene sequence data and comparative phylogenetic analysis confirmed that the microsporidian species from A. anxius is most closely related to the genus Cystosporogenes clade of microsporidia. Infection average in the Sudbury and Sault Ste Marie beetle populations was >80% and relatively stable in 2006-2007 but declined in 2008. Field prevalence of the A. anxius isolate, mechanisms that may potentially be involved in its horizontal (autoinfection) and vertical (transovarial) transmission, and disease dynamics are discussed. The congeneric relationship between Agrilus planipennis and A. anxius makes it imperative to study the virulence of this Cystosporogenes species in A. planipennis.
Journal of Invertebrate Pathology 12/2010; 107(1):1-10. · 2.06 Impact Factor
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ABSTRACT: 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 approximately 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.
Journal of Invertebrate Pathology 06/2008; 99(2):166-72. · 2.06 Impact Factor
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ABSTRACT: We examined isolates from 4 commercial bioinsecticides based on different strains of Bacillus thuringiensis subspecies (kurstaki, israelensis, aizawai, and tenebrionis) for the presence of genes encoding proteins with known enterotoxigenicity (nhe, hbl, cytk, ces) and various other putative virulence genes (piplc, sph, bceT, entFM, entS, entT). The piplc and bceT sequences were present in all the isolates; sph was found in aizawai and israelensis; entFM only in israelensis; and entS in kurstaki, israelensis, and tenebrionis. Our results corroborate previous findings that isolates used in commercial products contain all nhe and hbl component genes but not the ces gene. We ascertained that the cytK gene present in the kurstaki-, israelensis-, and aizawai-based products belongs to the cytK-2 type and not the more toxigenic cytK-1 variant originally isolated from enterotoxic Bacillus cereus. We provide the first evidence that hemolytic (hblA) and nonhemolytic (nheA, nheB, nheC) enterotoxin genes are expressed during septicemia in a target insect. This opens the door for their possible participation in pathogenesis in target insects. If enterotoxins do not contribute to bacterial pathogenesis in target insects, their genes could be deleted from commercial production strains to pre-empt perceptions of public health risks.
Canadian Journal of Microbiology 01/2008; 53(12):1283-90. · 1.36 Impact Factor
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ABSTRACT: We have isolated a microsporidium from a laboratory colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Light and electron microscopic investigations showed that gross pathology and ultrastructure of our isolate are similar to those described for Cystosporogenes legeri from the European grape vine moth, Lobesia botrana. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods revealed perfect homology with the C. legeri sequence. The microsporidian was infectious to other Choristoneura species, as well as Malacosoma disstria, Lymantria dispar, and Lambdina fiscellaria. Incubation of infected egg masses at 41 degrees C for 20 min followed by 30 min in 33% formaldehyde did not reduce disease incidence in larval offspring. Exposure of one or two generations to fumagillin at 6000 ppm or higher eliminated infection in adult moths, but also reduced colony fitness. A clean colony was established by conducting individual matings and selecting disease-free offspring.
Journal of Invertebrate Pathology 10/2004; 87(1):16-28. · 2.06 Impact Factor
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ABSTRACT: 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.
Entomologia Experimentalis et Applicata 05/1997; 83(3):253-262. · 1.53 Impact Factor
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ABSTRACT: We modified a neonate droplet-feeding technique to investigate lethal dose requirements for the spruce budworm, Choristoneura fumiferana (Clemens), without the confounding influence of instar-specific feeding rates and feeding behavior. Individual 4th, 5th, and 6th instars consumed known doses of 3 commercial Bacillus thuringiensis variety kurstaki products suspended in a buffer containing sucrose. The droplet volume was adjusted for each instar to ensure complete uptake. Experiments with 32p indicated that 98-99% of the presented volume was taken up during imbibing and that 89-98% of this was actually ingested. The LD50s expressed in international units per larva ranged from 1.6 to 1.8 for 4th, 2.2 to 2.9 for 5th, and 5.1 to 7.5 for 6th instars. The decrease in larval susceptibility in later instars was not caused by an increase in larval weight; on a per unit weight basis, 6th instars were 4.5- fold more susceptible than 4th instars (0.32 versus 1.44 IU/mg fresh weight). The relationships between larval stage and susceptibility was the same for Dipel 48AF, Foray 48B, and Foray 76B. Droplet sizes that are theoretically required to deliver a lethal dose in the form of 1 droplet per spruce needle ranged from 55 to 96 µm for an LD50 and from 129 to 192 µm for an LD95, depending on instar and potency of the product. These calculations suggest that droplets in the size range that is most commonly encountered on coniferous foliage after aerial application contain at best little more than an LD50.
Journal of Economic Entomology 03/1997; 90(2):560-565. · 1.70 Impact Factor
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ABSTRACT: Nosema isolates from five lepidopteran forest defoliators, Nosema fumiferanae from spruce budworm, Choristoneura fumiferana; a Nosema sp. from jack pine budworm, Choristoneura pinus pinus and western spruce budworm, Choristoneura occidentalis (Nosema sp. CPP and Nosema sp. CO, respectively); Nosema thomsoni from large aspen tortrix, Choristoneura conflictana; and Nosema disstriae, from the forest tent caterpillar, Malacosoma disstria were compared based on their small subunit (SSU) ribosomal RNA (rRNA) gene sequences. Four of the species sequenced, N. fumiferanae, Nosema sp. CPP, Nosema sp. CO, and N. disstriae have a high SSU rDNA sequence identity (0.6%-1.5%) and are members of the "true Nosema" clade. They all showed the reverse arrangement of the (large subunit [LSU]-internal transcribed spacer [ITS]-SSU) of the rRNA gene. The fifth species, N. thomsoni has the usual (SSU-ITS-LSU) arrangement and is not a member of this clade showing only an 82% sequence similarity. We speculate, therefore, that a genetic reversal may have occurred in the common ancestor to the "true Nosema" clade. Although, the mechanism for rearrangement of the rRNA gene subunits is not known we provide a possible explanation for the localization. N. fumiferanae, Nosema sp. CPP, and Nosema sp. CO clustered together on the inferred phylogenetic tree. The high sequence similarities, the reverse arrangement in the rRNA gene subunits, and the phylogenetic clustering suggest that these three species are closely related but separate species.
Journal of Eukaryotic Microbiology 55(1):51-8. · 2.66 Impact Factor
