Optimizing ethanol-baited traps for monitoring damaging ambrosia beetles (Coleoptera: Curculionidae, Scolytinae) in ornamental nurseries

The exotic ambrosia beetles Xylosandrus crassiusculus (Motschulsky) and Xylosandrus germanus (Blandford) (Coleoptera: Curculionidae: Scolytinae) are serious pests in ornamental tree nurseries. To optimize bottle-traps as a monitoring system for X. crassiusculus and X. germanus in nurseries, we tested whether increasing the rate of commercial ethanol lures improved captures or early detection of these species. Experiments were conducted in Ohio (2008 and 2009) and Virginia (2008), two states that have experienced significant damage from X. crassiusculus, X. germanus, or both. There were four treatments: no-lure (unbaited control), 1-ethanol lure, 2-ethanol lures and 1 + 1-ethanol lures (one lure in the trap and one suspended 0.5 m above the trap). Captures of X. crassiusculus and X. germanus were higher in all ethanol treatments than unbaited controls, and were generally higher in treatments with two lures versus one. There was no difference in beetle captures between the 2-lure and 1 + 1-lure treatments. First detection of X. crassiusculus and X. germanus occurred more consistently in the treatments with two lures than one lure. Xyleborinus saxesenii (Ratzeburg), Anisandrus sayi Hopkins, Hypothenemus dissimilis Zimmermann, and Hypothenemus eruditus Westwood were also more attracted to traps baited with ethanol than unbaited controls. X. saxesenii was captured in higher numbers in the treatments with two lures than one in Virginia but not in Ohio. There was no difference in captures of the other species among ethanol treatments. The current research shows that ethanol release rates influence sensitivity of traps for detecting emergence of overwintered ambrosia beetles.     

Low beta diversity of ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) in lowland rainforests of Papua New Guinea

We assessed the effect of geographical distance on insect species turnover in a situation where other major environmental factors, including host plant species, altitude, and climate, were constant. We sampled ambrosia beetles (Coleoptera, Curculionidae: Scolytinae and Platypodinae) from four tree species: Artocarpus altilis , Ficus nodosa , Leea indica and Nauclea orientalis , at three sites forming a 1000 km transect in lowland rainforests of northern Papua New Guinea. A standardized volume of wood from trunk, branches and twigs was sampled for ambrosia beetles from three individuals of the four tree species at each site. Each tree was killed standing and left exposed to beetle colonization for 20 days prior to sampling. We obtained 12 751 individuals from 84 morphospecies of ambrosia beetles. We surveyed most of the local species richness at each site, predicted by Chao 2 species richness estimates. The similarity of ambrosia beetle communities, estimated by Chao-Sorensen index, was not correlated with their geographical distance. Likelihood analysis and Q-mode analysis using Monte Carlo-generated null distribution of beetles among sites supported the hypothesis that the assemblages of ambrosia beetles at different sites are drawn from the same species pool, regardless of their geographical distance. Tree part (trunk, branch, or twig) was more important predictor of the composition of ambrosia beetle communities than was the host species or geographical location. All three variables, however, explained only a small portion of variability in ambrosia assemblages. The distribution of ambrosia beetles among tree parts, tree species and study sites was mostly random, suggesting limited importance of host specificity or dispersal limitation.     

Pathogens of bark beetles (Curculionidae: Scolytinae) and other beetles in Bulgaria

The presence and diversity of beetle pathogens associated with different tree species in Bulgaria was investigated. In total, 818 specimens belonging to 22 beetle species were examined. Pathogens occurred in 9 host species. The infections were found in the gut (virus, nematodes, protozoans, microsporidia) and haemolymph (nematodes) of the infected insects. The following pathogen species: ItEPV, Beauveria bassiana, B. brongniartii, Isaria farinosa, Gregarina typographi, Gregarina spp., Chytridiopsis typographi, Chytridiopsis sp., and nematodes were revealed. Insects with mycoses were mummified and filled up with fungal structures, formed pseudosclerotium. Most pathogens were established in bark beetles (Scolytinae). For the first time, the fungus B. bassiana was reported in Tomicus piniperda, Orthotomicus longicollis, O. erosus, X. spinole, Taphrorychus villifrons and Phylobius sp. in Bulgaria. We also present the first records of gregarines in O. longicollis, Acanthocinus aedilis, Rhagium inquisitor, Pyrochroa coccinea, and of the microsporidium Chytridiopsis sp. in O. longicollis.     

Evaluation of insecticides for protecting Southwestern ponderosa pines from attack by engraver beetles (Coleoptera: Curculionidae: Scolytinae)

Insecticides that might protect pine trees from attack by engraver beetles (Ips spp.) have not been rigorously tested in the southwestern United States. We conducted two field experiments to evaluate the efficacy of several currently and potentially labeled preventative insecticides for protecting high-value ponderosa pine, Pinus ponderosa Dougl ex. Laws., from attack by engraver beetles. Preventative sprays (0.19% permethrin [Permethrin Plus C]; 0.03, 0.06, and 0.12% bifenthrin [Onyx]; and 1.0 and 2.0% carbaryl [Sevin SL] formulations) and systemic implants (0.875 g per capsule acephate [Acecap] and 0.650 g per capsule dinotefuran) were assessed on bolts (sections of logs) as a surrogate for live trees for a period of 13 mo posttreatment. The pine engraver, Ips pini (Say), was the most common bark beetle found attacking control and treated bolts, but sixspined ips, Ips calligraphus (Germar), and Ips lecontei Swain also were present. After approximately 13 mo posttreatment in one experiment, the spray treatments with 2.0% carbaryl, 0.19% permethrin, and 0.06 or 0.12% bifenthrin prevented Ips attack on the bolts at a protection level of > or = 70%. The acephate and dinotefuran systemic insecticides, and the 0.03% bifenthrin spray, provided inadequate (< or = 36%) protection in this experiment. For the other experiment, sprayed applications of 1.0% carbaryl, 0.19% permethrin, and 0.06% bifenthrin prevented beetle attack at protection levels of > or = 90, > or = 80, and > or = 70%, respectively, when bolts were exposed to Ips beetle attack for approximately 9-15 wk posttreatment. The sprays with 0.19% permethrin and 0.06% bifenthrin also provided > or = 90% protection when bolts were exposed for approximately 15-54 wk posttreatment. We concluded that under the conditions tested, 1.0 and 2.0% carbaryl, 0.19% permethrin, and 0.06 and 0.12% binfenthrin were acceptable preventative treatments for protecting ponderosa pine from successful engraver beetle attack for one entire flight season in the U.S. Southwest.     

Influence of temperature on spring flight initiation for southwestern ponderosa pine bark beetles (Coleoptera: Curculionidae, Scolytinae)

Determination of temperature requirements for many economically important insects is a cornerstone of pest management. For bark beetles (Coleoptera: Curculionidae, Scolytinae), this information can facilitate timing of management strategies. Our goals were to determine temperature predictors for flight initiation of three species of Ips bark beetles, five species of Dendroctonus bark beetles, and two genera of bark beetle predators, Enoclerus spp. (Coleoptera: Cleridae) and Temnochila chlorodia (Mannerheim) (Coleoptera: Ostomidae), in ponderosa pine forests of northcentral Arizona. We quantified beetle flight activity using data loggers and pheromone-baited funnel traps at 18 sites over 4 yr. Ambient air temperature was monitored using temperature data loggers located in close proximity to funnel traps. We analyzed degree-day accumulation and differences between minimum, average, and maximum ambient temperature for the week before and week of first beetle capture to calculate flight temperature thresholds. Degree-day accumulation was not a good predictor for initiation of beetle flight. For all species analyzed other than D. adjunctus Blandford, beetles were captured in traps only when springtime temperatures exceeded 15.0 degrees C. D. adjunctus was collected when maximum temperatures reached only 14.5 degrees C. Once initial flights had begun, beetles were often captured when maximum ambient air temperatures were below initial threshold temperatures. Maximum and average air temperatures were a better predictor for beetle flight initiation than minimum temperature. We establish a temperature range for effective monitoring of bark beetles and their predators, and we discuss the implications of our results under climate change scenarios.     

Lack of genetic differentiation in aggressive and secondary bark beetles (Coleoptera: Curculionidae, Scolytinae) from Arizona

Bark beetles (Coleoptera: Curculionidae, Scolytinae) play an important role as disturbance agents in ponderosa pine (Pinus ponderosa Douglas ex Lawson) forests of Arizona. However, from 2001 to 2003, elevated bark beetle activity caused unprecedented levels of ponderosa pine mortality. A better understanding of the population structure of these species will facilitate analysis of their dispersal patterns and improve management strategies. Here, we use fluorescently labeled amplified fragment length polymorphism (fAFLP) analysis to resolve genetic variation among and within sampling locations in northcentral Arizona of Ips pini (Say), Dendroctonus brevicomis LeConte, and D. frontalis Zimmermann. We generated genetic fingerprints for >500 beetle specimens and analyzed genetic diversity. For all species, gene flow estimates among sampling locations were high, and significant population subdivision was not discernible across a large portion of ponderosa pine forests in Arizona. However, a weak relationship was detected with I. pini population structure and elevation. Because of the lack of genetic differentiation detected throughout the large study area, our findings suggest these insects are capable of long distance dispersal and exhibit a high degree of gene flow across a broad region. We conclude that our results are consistent with strong dispersal patterns and large population sizes of all three species.     

Efficacy of systemic insecticides for protection of loblolly pine against southern pine engraver beetles (Coleoptera: Curculionidae: Scolytinae) and wood borers (Coleoptera: Cerambycidae)

We evaluated the efficacy of the systemic insecticides dinotefuran, emamectin benzoate, fipronil, and imidacloprid for preventing attacks and brood production of southern pine engraver beetles (Coleoptera: Curculionidae: Scolytinae) and wood borers (Coleoptera: Cerambycidae) on standing, stressed trees and bolt sections of loblolly pine, Pinus taeda L., in eastern Texas. Emamectin benzoate significantly reduced the colonization success of engraver beetles and associated wood borers in both stressed trees and pine bolt sections. Fipronil was nearly as effective as emamectin benzoate in reducing insect colonization of bolts 3 and 5 mo after injection but only moderately effective 1 mo after injection. Fipronil also significantly reduced bark beetle-caused mortality of stressed trees. Imidacloprid and dinotefuran were ineffective in preventing bark beetle and wood borer colonization of bolts or standing, stressed trees. The injected formulation of emamectin benzoate was found to cause long vertical lesions in the sapwood-phloem interface at each injection point.     

Additions to the check list of bark beetles (Coleoptera,Curculionidae: Scolytinae) from Leningrad Province,Russia

The field work of the authors during the last 16 years in both the western and the eastern parts of Leningrad Province, Russia, has resulted in the addition of 7 species to the regional list of bark beetles, namely: Hylurgus ligniperda F., Crypturgus subcribrosus Egg., Pityogenes irkutensis Egg., Scolytus mali Bechst., S. scolytus F., S. pygmaeus F., and Xyleborinus attenuatus Blandf. Of these species, P. irkutensis occurs only in the eastern districts of Leningrad Province, C. subcribrosus, over the whole territory, and the remaining species, only in the western districts. We suggest excluding Polygraphus proximus Blandf. from the regional faunal list. As a result, the updated list of the bark beetle species of Leningrad Province comprises 75 taxa. New data on findings of rare species in the region are considered.     

The influence of forest stand and site characteristics on the composition of exotic dominated ambrosia beetle communities (Coleoptera: Curculionidae: Scolytinae)

Economic and biological consequences are associated with exotic ambrosia beetles and their fungal associates. Despite this, knowledge of ambrosia beetles and their ecological interactions remain poorly understood, especially in the oak-hickory forest region. We examined how forest stand and site characteristics influenced ambrosia beetle habitat use as evaluated by species richness and abundance of ambrosia beetles, both the native component and individual exotic species. We documented the species composition of the ambrosia beetle community, flight activity, and habitat use over a 2-yr period by placing flight traps in regenerating clearcuts and older oak-hickory forest stands differing in topographic aspect. The ambrosia beetle community consisted of 20 species with exotic ambrosia beetle species dominating the community. Similar percentages of exotic ambrosia beetles occurred among the four forest habitats despite differences in stand age and aspect. Stand characteristics, such as stand age and forest structure, influenced ambrosia beetle richness and the abundances of a few exotic ambrosia beetle species and the native ambrosia beetle component. Topographic aspect had little influence on ambrosia beetle abundance or species richness. Older forests typically have more host material than younger forests and our results may be related to the amount of dead wood present. Different forms of forest management may not alter the percent contribution of exotic ambrosia beetles to the ambrosia beetle community.     

危害猕猴桃的新害虫——端齿材小蠹

【目的】本文描述了首次在贵州猕猴桃上发生的一种害虫——端齿材小蠹Anisandrus apicalis(Blandford)。【方法】通过对当地修文县谷堡乡平滩村猕猴桃病树上小蠹的观察和形态研究,进行分类和鉴定。【结果】此次发现的端齿材小蠹是贵州省境内第一次报道,也是首次发现在猕猴桃病枝上发生。本文提供了该小蠹的分类和鉴别,附有相关近缘种的形态特征图和检索表,并介绍了其寄主和分布,以及在贵阳修文猕猴桃的危害和发生等情况。【结论】此次发生在猕猴桃上的端齿材小蠹是贵州省境内的首例,本文结果可以为当地植物检疫和猕猴桃种植管理提供参考。虽然此次端齿材小蠹在猕猴桃上发生,但是未发现其会直接为害猕猴桃健康植株,需要在将来进行深入的实验调查。     

Phylogeny of haplo–diploid,fungus‐growing ambrosia beetles (Curculionidae: Scolytinae: Xyleborini) inferred from molecular and morphological data

Cognato, A. I., Hulcr, J., Dole, S. A. & Jordal, B. H. (2010). Phylogeny of haplo‐diploid, fungus‐growing ambrosia beetles (Curculionidae: Scolytinae: Xyleborini) inferred from molecular and morphological data. —Zoologica Scripta, 40, 174–186. The ambrosia beetle tribe Xyleborini currently contains 30 genera and approximately 1200 species which are distributed throughout worldwide forests with most diversity located in the tropics. They also represent the most invasive scolytines in North America. Despite economic concerns and biological curiosity with this group, a comprehensive understanding of generic boundaries and the evolutionary relationship among species is lacking. In this study, we include 155 xyleborine species representing 23 genera in parsimony and Bayesian analyses using 3925 nucleotides from mitochondrial (COI) and nuclear genomes (28S, ArgK, CAD, EF‐1α) and 39 morphological characters. The phylogenies resulting from the parsimony analyses, which treated gap positions either as missing or fifth character states, and the Bayesian analysis were generally similar. Clades with high support or posterior probabilities were found in all trees, while those with low support were not recovered by all analyses. Fourteen of the 23 genera were monophyletic although not all relationships among the genera were resolved. We show monophyly of several species groups associated with particular morphological and biological characters and suggest recognition of these groups as genera. Most interesting was the monophyly of South and Central American species representing several genera. This finding suggests recent and fast radiation of xyleborines in the New World accompanied by morphological and biological diversification.     

Secondary structure alignment and direct optimization of 28S rDNA sequences provide limited phylogenetic resolution in bark and ambrosia beetles (Curculionidae: Scolytinae)

Phylogenetic relationships in Scolytinae were reconstructed from 107 DNA sequences that spanned the D2 and D3 expansion segments, and related core regions of the nuclear large ribosomal subunit (28S). Sequences were analysed by parsimony and Bayesian analyses of aligned sequences aided by a new secondary structure model for the D2–D3 domains. Direct optimization was performed on ambiguous alignment regions in combination with fixed states optimization of unambiguous regions, but performed poorly compared to the Bayesian and parsimony analyses. Generally, the phylogenetic signal mainly resolved relationships within tribes, while deeper divergences were either not resolved or received marginal support. In addition to confirming several previously established clades, we found that Micracini formed the sister group to Cactopinus , a group of mainly cactus feeding scolytine beetles. Furthermore, Ipini was monophyletic with Pseudips and Acanthotomicus subtending to the most basal node of that clade. The monophyly of Corthylini, which consists of the bark and cone feeding Pityophtorina and the ambrosia fungus-feeding Corthylina, was supported in some of the analyses. A close relationship was found between Phloeotribus and the two Phloeosinini genera Chramesus and Pseudochramesus , suggesting an evolutionary trajectory for the origin of a lamellate antennal club in Phloeotribus .     

Phylogeography of spruce beetles (Dendroctonus rufipennis Kirby) (Curculionidae: Scolytinae) in North America

Tree-feeding insects that are widespread in north temperate regions are excellent models for studying how past glaciations have impacted differentiation and speciation. We used mitochondrial DNA (mtDNA) sequences and allele frequencies at nine microsatellite loci to examine genetic population structure across the current range of the spruce beetle (Dendroctonus rufipennis), an economically important insect in North America. Two major haplotype groups occur across northern North America, from Newfoundland to Alaska, on white spruce (Picea glauca), and a third distinctive haplotype group occurs throughout the Rocky Mountains on Engelmann spruce (Picea engelmannii). The two mtDNA lineages found in northern populations are 3-4% divergent from each other and from the lineages found in the Rocky Mountains. Analyses of microsatellite data also suggest the existence of major population groupings associated with different geographical regions. In the Pacific Northwest, concordant contact zones for genetically distinct populations of spruce beetles and their principal hosts appear to reflect recent secondary contact. Although we could detect no evidence of historical mtDNA gene flow between allopatric population groups, patterns of variation in the Pacific Northwest suggest recent hybridization and introgression. Together with the pollen record for spruce, they also suggest that beetles have spread from at least three glacial refugia. A minimum estimate of divergence time between the Rocky Mountain and northern populations was 1.7 Myr (million years), presumably reflecting the combined effects of isolation during multiple glacial cycles.     

DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用

[目的]DNA条形码技术已成为生物分类鉴定的有力工具.DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性.我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类.[方法]针对大小蠹昆虫设计引物以提高PCR扩增效率.运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定.[结果]使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段.将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种.通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种(核苷酸序列一致性为100％),与形态鉴定结果一致.[结论]结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类.该检测技术可以应用于其他经济重要性有害生物的检测鉴定.     

External microstructure of the ambrosia beetle Platypus koryoensis (Coleoptera: Curculionidae: Platypodinae)

Platypus koryoensis is a minute ambrosia beetle found in forests. It can cause significant economic damage to oak trees. Recently in Korea, it has been reported as a major pest of oak trees, because it causes sooty mold of oak by introducing the pathogenic fungus Raffaelea sp. In this paper, we demonstrate the fine structural aspects of the external body of the ambrosia beetle using field emission scanning electron microscopy, as a part of basic research into this pest so that strategies for its control might be developed. This beetle has a sensory system well developed to respond to both visual and chemical stimuli. Both sexes have a pair of faceted compound eyes and a pair of knobbed antennae, but simple eyes are absent. The mouthparts on its distinct snouts are effective devices for penetration and for boring holes. The mouthparts consist of the labrum, a pair of mandibles, a pair of maxillae and the labium. Both the maxillary and the labial palpi have the function of directing the food to the mouth and holding it while the mandibles chew the food. The distal ends of these palpi are flattened and have shovel‐like setae. The thorax has a particularly hard exoskeleton and hard elytra, including powerful muscles that operate both the wings and the legs. The legs are multi‐segmented and have a strong femur and tibia, including one pair of claws on the end of each tarsal segment. Characteristically, both male and female beetles have mycangial cavities for storing spores and other microorganisms, but only females have three pairs of large depressions on their dorsal thorax.     

Mycangia of the ambrosia beetle, Austroplatypus incompertus (Schedl) (Coleoptera: Curculionidae: Platypodinae)

Abstract  Ambrosia beetles have an obligate relationship with the ambrosia fungi that they feed on. This requires that the beetles have means to transport those fungi when they colonise new hosts. Some ambrosia beetles have special structures called mycangia to transport fungi in. This paper describes the mycangia of the ambrosia beetle Austroplatypus incompertus and illustrates how the mycangical hairs are probably used by the beetle to acquire fungal spores for transport. The mycangia and probable method of fungal acquisition of this species are compared with those of other ambrosia beetles.     

Phylogeography of the bark beetle Dendroctonus mexicanus Hopkins (Coleoptera: Curculionidae: Scolytinae)

Dendroctonus mexicanus is polyphagous within the Pinus genus and has a wide geographical distribution in Mexico and Guatemala. We examined the pattern of genetic variation across the range of this species to explore its demographic history and its phylogeographic pattern. Analysis of the mtDNA sequences of 173 individuals from 25 Mexican populations allowed to us identify 53 geographically structured haplotypes. High haplotype and low nucleotide diversities and Tajima’s D indicate that D. mexicanus experienced rapid population expansion during its dispersal across mountain systems within its current range. The nested clade phylogeographic analysis indicates that the phylogeographic pattern of D. mexicanus is explained by continuous dispersion among lineages from the Sierra Madre Occidental, the Sierra Madre Oriental and the Trans-Mexican Volcanic Belt. However, we also observed isolation events among haplotypes from the Cofre de Perote/Trans-Mexican Volcanic Belt/Sierra Madre Oriental and the Trans-Mexican Volcanic Belt/Sierra Madre del Sur, which is consistent with the present conformation of mountain systems in Mexico and the emergence of geographical barriers during the Pleistocene.     

Alimentary canal and reproductive tract of Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae, Scolytinae)

The alimentary canal and the reproductive tract of males and females of Hypothenemus hampei Ferrari are described. The alimentary canal of H. hampei showed the crop with several spine-like structures and the midgut with few gastric caeca. We evidenced for the first time that adult females need to feed on coffee in order to produce viable eggs before and during oviposition period. The presence of air bubbles inside the anterior midgut may be due to starvation rather than the response of the environment during flying as previously reported. Two ovaries and the same structures and arrangements common to individuals of the Curculionidae beetles composed the female reproductive system. The male reproductive tract showed a significant difference with respect to other Curculionidae and even other Scolytinae species as it showed a sclerotized aedeagus with several pore-like structures in the terminal portion where sperm is released during mating.     

Isolation,morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae)

The coffee berry borer Hypothenemus hampei Ferrari, 1876 (Coleoptera: Curculionidae: Scolytinae) is considered the most serious pest of the coffee crop and is controlled primarily with the use of chemical insecticides. An alternative to this control method is the use of the entomopathogenic bacterium, Bacillus thuringiensis Berliner, 1911. Therefore, the objective of this work was to select strains of B. thuringiensis virulent against H. hampei and characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains of B. thuringiensis underwent a selective bioassay to evaluate their toxicity to H. hampei first-instar larvae. Among the strains tested, 11 and the standard B. thuringiensis subspecies israelensis (IPS-82) caused mortality above 90%. Then, the median lethal concentration (LC₅₀) was estimated for these strains followed by characterization using morphological, biochemical and molecular methods. The lowest LC₅₀ was obtained for strain BR58, although this concentration did not differ significantly from that of the standard strain IPS-82 or from that of strains BR137, BR80 and BR67. The molecular characterization detected cry4A, cry4B, cry10, cry11 and cyt1 genes in 10 of the most virulent strains (BR58, BR137, BR80, BR81, BR147, BR135, BR146, BR138, BR139, BR140). Strain BR67 differed completely from the others and amplified only the cry3 gene. This strain was more virulent than BR135, BR146, BR138, BR139 and BR140, but it did not differ from BR58, BR137, BR80, BR81 and BR147. The protein profile revealed proteins of 28, 65, 70 and 130 kDa, and the morphological analysis identified spherical crystalline inclusions in all strains. The results showed that the 11 strains studied have potential for use as a gene source for insertion into coffee plants for the control H. hampei, especially the cry3, cry4A, cry4B, cry10, cry11 and cyt1 genes, that were repeated in the most virulent isolates.