Development of a Loop-Mediated Isothermal Amplification Assay as an Early-Warning Tool for Detecting Emerald Ash Borer (Coleoptera: Buprestidae) Incursions.

The emerald ash borer (EAB), Agrilus planipennis (Fairmaire), is the most destructive invasive insect species of ash (Fraxinus spp.) in North America. An accurate method for early detection of this noxious insect pest is indispensable to providing adequate warning of A. planipennis infestation. A loop-mediated isothermal amplification (LAMP) assay (EAB-LAMP) was developed based on mitochondrial cytochrome c oxidase subunit I (COI) gene. The EAB-LAMP required only 30 min at 65°C to amplify A. planipennis DNA from specimens collected from geographically distinct locations. There was no cross-reactivity with other Agrilus and insect species. The developed EAB-LAMP differentially detected traces of A. planipennis genome (COI) within frass from various Fraxinus species. EAB-LAMP was also able to distinguish among A. planipennis DNA and other Agrilus species and nontarget insect species in trap captures. By detecting A. planipennis DNA in two additional trap captures (in situ), the EAB-LAMP was more sensitive and reliable than visual inspection. We tested the quantitative nature of the assay by evaluating pooled trap samples and demonstrated that the EAB-LAMP was capable of functioning optimally using a pool size of at least five individual trap samples. This potentially circumvents the need to perform large-scale individual analysis for processing trap samples. Considering its performance, specificity, sensitivity, and repeatability, the developed EAB-LAMP could be a valuable tool to support strategy and operation of large-scale surveillance for A. planipennis and could profitably be used in routine monitoring programs for effective management of A. planipennis.

Characterization and virulence of Beauveria spp. recovered from emerald ash borer in southwestern Ontario, Canada.

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.

Complete rRNA sequence, arrangement of tandem repeated units and phylogeny of Nosema fumiferanae from spruce budworm, Choristoneura fumiferana (Clemens).

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.

Morphology, molecular characteristics and prevalence of a Cystosporogenes species (Microsporidia) isolated from Agrilus anxius (Coleoptera: Buprestidae).

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.

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 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.

Molecular data and phylogeny of Nosema infecting lepidopteran forest defoliators in the genera Choristoneura and Malacosoma.

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.

Detection of Bacillus cereus virulence factors in commercial products of Bacillus thuringiensis and expression of diarrheal enterotoxins in a target insect.

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.

Occurrence of Cystosporogenes sp. (Protozoa, Microsporidia) in a multi-species insect production facility and its elimination from a colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae).

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.