The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana, are key environmental pathogens of insects that have been exploited for biological control of insect pests. Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, mediating responses to the environment. Bbslt2, encoding for an Slt2 family MAPK, was isolated and characterized from B. bassiana. Gene disruption of Bbslt2 affected growth, caused a significant reduction in conidial production and viability, and increased sensitivity to Congo Red and fungal cell wall degrading enzymes. ΔBbslt2 mutants were altered in cell wall structure and composition, which included temperature dependent chitin accumulation, reductions in conidial and hyphal hydrophobicity, and alterations in cell surface carbohydrate epitopes. The ΔBbslt2 strain also showed hypersensitivity to heat shock and altered trehalose accumulation, which could only be partially attributed to changes in the expression of trehalase (ntl1). Insect bioassays revealed decreased virulence in the ΔBbslt2 strain using both topical and intrahemoceol injection assays. These results indicate that Bbslt2 plays an important role in conidiation, viability, cell wall integrity and virulence in B. bassiana. Our findings are discussed within the context of the two previous MAP kinases characterized from B. bassiana.     

The Hog1-like MAPK Mpk3 collaborates with Hog1 in response to heat shock and functions in sustaining the biological control potential of a fungal insect pathogen

The mitogen-activated protein kinase (MAPK) Mpk3/MpkC resembles the MAPK Hog1 but does not necessarily function in some filamentous fungi. Here, we compared functions of Mpk3 and Hog1 in Beauveria bassiana, a filamentous fungal insect pathogen, by multi-phenotypic analyses of their single/double deletion mutants. Growth defects of Δmpk3 were moderate on all 14 minimal media with different carbon or nitrogen sources and less severe than those of Δhog1 on most media tested. The double deletion mutant suffered significantly more severe growth defects than those observed in Δmpk3 and Δhog1, suggesting overlapping and collaborative roles of Mpk3 and Hog1 in uptake of six carbon and four nitrogen sources during normal growth. Despite little impact on conidiation capacity, mpk3 deletion slowed down conidial germination as much as hog1 or double deletion. Conidial resistance to UV-B irradiation decreased less in Δmpk3 than in Δhog1 or in the double mutant. The fungal virulence was similarly attenuated for all deletion mutants against Galleria mellonella larvae through normal cuticle infection. Intriguingly, the Δmpk3 mutant displayed null response to high osmolarity and fludioxonil fungicide, to which both Δhog1 and double mutants were hypersensitive and highly resistant, respectively, but it was more sensitive to a 3-h heat shock at 40 °C than Δhog1 during normal incubation. Western blot hybridization demonstrated that Mpk3 could collaborate with Hog1 in response to heat shock rather than to the chemical stresses. Altogether, Mpk3 collaborates with Hog1 only in response to heat shock and functions in sustaining the pest control potential of B. bassiana.     

Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidylinositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and virulence

Beauveria bassiana is a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes β-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putative B. bassiana GPI-anchored β-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized. B. bassiana targeted gene knockouts lacking Bbgas1 were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including β-1,3-glucans, in the ΔBbgas1 strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1 conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1 conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1 knockout showed reduced virulence in Galleria mellonella insect bioassays in both topical and intrahemocoel-injection assays. B. bassiana ΔBbgas1 strains complemented with the complete Bbgas1 open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. The Bbgas1 gene did not complement the phenotype of a Saccharomyces cerevisiae β-1,3-glucanosyltransferase Δgas1 mutant, indicating that this family of enzymes likely possess discrete cellular functions.     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

Effect of temperature and poultry litter in Beauveria bassiana (bals.) Vuill. and Metarhizium anisopliae (Metsch) virulence against the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate the influence of temperature and poultry litter on germination vegetative growth virulence and conidial production of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates on larvae and adults of the lesser mealworm (Alphitobius diaperinus) (Panzer). The vegetative growth and conidial production were evaluated on culture media. Virulence was studied submerging larvae and adults in a conidial suspension (1 x 10(8) conidia/ml). All the experiments were carried out in growth chamber (26 degrees C and 32 degrees C and 14h photophase). Fungus-killed insects were daily collected and used for microscopic conidial counts. The poultry litter effect was evaluated by submerging the insects in a fungal suspension (10(8) conidia/ml) and then transferring them to cups containing poultry litter (new and used). B. bassiana isolates were more sensitive than M. anisopliae to high temperature because conidia viability, vegetative growth and virulence were negatively affected (P < 0.05). The conidial production was higher to B. bassiana in 26 degrees C (7 to 11 x 10(8) conidia/larval cadaver and 8 x 10(8) conidia/adult cadaver) (P < 0,05). Larval stage was about 10 times more sensitive to M. anisopliae at 26 degrees C than adults stage. Regarding B. bassiana, differences on sensitivity between larval stages and adults were not observed at this temperature. However, at 32 degrees C, larval stage was more sensitive for CB116 and UEL50 isolates. Mortality was higher when larvae and adults (15.7 and 66.7% respectively) were treated by B. bassiana and maintained on new poultry litter at 26 degrees C) (P < 0.05).     

球孢白僵菌羧基转运蛋白基因RNA干扰的沉默效应

羧基转运蛋白基因是近年来分离出的新基因,在虫生真菌中可能与穿透昆虫体壁时的能量代谢有关。构建了针对该基因的双链RNA干扰载体,采用芽生孢子转化法将载体质粒转入球孢白僵菌,并通过RT-PCR检测转化前后BbJEN1的表达情况。RT-PCR检测结果显示,球孢白僵菌转化子BbJEN1基因的表达水平显著下降,表明干扰载体具有明显的沉默效应。在两种培养基上,转化子与原始菌株的生长速度和产孢量差异均不显著,而分生孢子萌发则显著滞后于原始菌株。使用马尾松毛虫幼虫对转化子和原始菌株进行生物测定,原始菌株的半致死浓度、半致死剂量和半致死时间分别为2.79×106个孢子/mL、84.12个孢子/mm2和6.49d,而转化子的半致死浓度、半致死剂量和半致死时间则分别增加到1.27×107个孢子/mL、382.92个孢子/mm2和8.09d,毒力显著下降。由此表明BbJEN1基因与球孢白僵菌分生孢子发芽以及毒力均有关。     

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.     

生防真菌分生孢子抗氧化能力与多糖含量的关系

作为重要的丝孢类昆虫病原真菌,球孢白僵菌和玫烟色棒束孢因其易于生产和环境友好等优点而在害虫生防防治中受到广泛青睐。为初步探求孢子耐氧化力及其与孢子多糖含量的关系,球孢白僵菌和玫烟色棒束孢11株菌经胁迫后的残存指数随氧化剂H2O2浓度增加而减小。所有菌株的残存指数均能良好地与Logistic方程拟合,并计算出各菌株在氧化胁迫条件下的半致死浓度。结果显示玫烟色棒束孢孢子的耐氧化力强于球孢白僵菌。两种真菌的分生孢子耐氧力与各自多糖含量呈现良好线性正相关。培养基碳源成分和浓度变化可影响球孢白僵菌孢子耐氧化力,但耐氧化力与多糖含量依旧呈现线性正相关。由此可见,生防真菌分生孢子的耐氧化力的确与多糖积累有关,并在一定程度上受培养条件的调节。研究结果有望为提高生防真菌孢子环境稳定性提供新的策略。     

Community composition, host range and genetic structure of the fungal entomopathogen Beauveria in adjoining agricultural and seminatural habitats

Although intensively investigated for biological control of insect pests, little is known about the ecology of the fungal entomopathogenic genus Beauveria in natural or agricultural habitats. In this study, we used molecular phylogenetic and genotypic information to infer species diversity, reproductive potential and genetic structure of Beauveria occurring within a single arable field and bordering hedgerow in Denmark. Isolates were sampled from cultivated field and hedgerow soils, from insects harbouring latent fungal infections, and from the phylloplanes of three plant species common in the hedgerow flora. A nuclear phylogeny of this local Beauveria assemblage resolved seven phylogenetic species, including (i) five phylogenetic species within Beauveria bassiana sensu stricto ; (ii) Clade C, a taxonomically uncharacterized species that is morphologically indistinguishable but phylogenetically distant from B. bassiana s.s. ; and (iii) Beauveria brongniartii. All seven species were present throughout the hedgerow habitat, including as infections in insects. Significantly, only B . bassiana s.s. phylogenetic species Eu_1 was isolated from tilled soils. Mating type polymerase chain reaction assays demonstrated that all five B. bassiana s.s. phylogenetic species possess bipolar outcrossing mating systems. Of these, only the Eu_1 population contained two mating types; however, a 31:2 skew in MAT1:MAT2 mating types suggests a low frequency of sexual reproduction in this population. The four remaining B. bassiana s.s. phylogenetic species were fixed for single mating types and these populations are evidently clonal. Multilocus microsatellite genotyping revealed polymorphism in all five phylogenetic species of B. bassiana s.s. ; however, all show evidence of clonal genetic structure.     

Antagonism between Beauveria bassiana and imidacloprid when combined for Bemisia argentifolii (Homoptera: Aleyrodidae) control

Imidacloprid and the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin are both used to control the whitefly Bemisia argentifolii Bellows & Perring. We tested whether the two control strategies acted additively, synergistically, or antagonistically when combined for whitefly control. We found antagonism in that B. bassiana inhibited the effectiveness of imidacloprid. When B. bassiana was combined with imidacloprid, insect response was either less than or similar to (depending on B. bassiana rates) that when imidacloprid was used alone. Adding imidacloprid to B. bassiana treatments always increased mortality, but the increase was less than additive. Beauveria bassiana spore germination and colony formation were not inhibited by imidacloprid in vitro, and B. bassiana did not adsorb or degrade imidacloprid in a tank mix. We hypothesize that B. bassiana caused a behavioral response that reduced insect feeding and uptake of imidacloprid.     

A Proposed Role for the Cuticular Fatty Amides of Liposcelis bostrychophila (Psocoptera: Liposcelidae) in Preventing Adhesion of Entomopathogenic Fungi with Dry-conidia

Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We investigated several of L. bostrychophila's cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophila's tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.     

Molecular investigation on strain genetic relatedness and population structure of Beauveria bassiana

Triplicate molecular methods, i.e. polymerase chain reaction-restriction fragment length polymorphism of the pr1 gene, microsatellite markers and 28S rDNA haplotyping by detecting the presence or absence of group I introns, were used for population study of the entomopathogenic fungus, Beauveria bassiana. The findings showed that the average genetic diversity index of geographical populations was significantly smaller than that of populations derived from insect host orders, indicating that the genetic relatedness of B. bassiana strains was highly associated with geographical locality rather than insect host species. The reproductive style of all the B. bassiana populations was found to be non-clonal. Population structure analysis revealed that the average divergent coefficient among populations of B. bassiana was far below 1 (0.1112), which indicated that there was no significant genetic differentiation between populations, and that the overall genetic diversity mainly resulted from the genetic variations within geographical populations. Statistically, genetic distances between populations were positively correlated with geographical distances, suggesting that geographical separation poses an obstacle to the possibility and frequency of genetic exchanges between populations. On the other hand, gene flow was indirectly established to occur between B. bassiana populations.     

Diatomaceous earth increases the efficacy of Beauveria bassiana against Tribolium castaneum larvae and increases conidia attachment

This research tested the suppressive ability of Beauveria bassiana (Balsamo) Vuillemin alone and in combination with diatomaceous earth against the red flour beetle, Tribolium castaneum (Herbst). Adults did not show a dose response to B. bassiana, and the addition of diatomaceous earth (DE) did not result in a significant increase in mortality. Against larvae, however, DE at 190 mg/kg grain enhanced the efficacy of B. bassiana at all concentrations ranging from 33 to 2,700 mg of conidia per kilogram of grain. The presence of DE resulted in 17- and 16-fold decreases in the median lethal concentration of B. bassiana at 56 and 75% RH, respectively. No significant differences in larval mortality in response to B. bassiana and diatomaceous earth alone or in combination were found between 56 and 75% RH. Conidial attachment to larvae was significantly greater with 190 mg/kg DE than without it. The partial analysis of lipids taken up by DE from the larvae revealed the removal of phospholipids and long-chain fatty acids. These results support the hypothesis that diatomaceous earth enhances the efficacy of B. bassiana against larval T. castaneum, at least in part by damaging the insect cuticle, thus increasing conidial attachment and making nutrients more available to conidia for their germination.     

球孢白僵菌种群在松林中的寄主转移及遗传多样性对松毛虫持续控制的影响

通过林间接种式放菌及其后一周年的野外调查,从林间采集到30种寄主昆虫及从土壤、落叶和气流中分离到119株球孢白僵菌．酯酶同工酶分析表明,它们属于32个不同酯酶型,呈现出丰富的遗传多样性．释放菌株所属的酯酶型02包括从11种昆虫上分离出的18个菌株,表明林间释放的菌株已成功地在不同寄主昆虫种群中宿存下来,并以常发的地方病状态存在于松毛虫及松灰象甲等12种昆虫种群之中;当林间目标寄主缺乏时,其它寄主可将食物链维系下去．其它酯酶型分别包括1～23个菌株．一周年内的寄主转移动态结果表明,球孢白僵菌在松林生态系统中不同寄主间可转移寄生．每个酯酶型中的菌株对松毛虫的毒力相差很大,表明球孢白僵菌在松林中的延续和扩散流行不是1条路线,每个酯酶型至少代表食物网上的1条支链．有些环节的寄主连接了不同的酯酶型,使松林中食物网变得十分复杂,另外。从土壤、枯枝落叶层、林冠层和空气中分离到的球孢白僵菌分属于不同的酯酶型,表明松林中还存在着复杂的腐生食物链,有利于松毛虫及其它害虫的持续控制。     

CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in entomopathogenic fungus Beauveria bassiana

Infection of insects by the entomopathogenic fungus Beauveria bassiana proceeds via attachment and penetration of the host cuticle. The outermost epicuticular layer or waxy layer of the insect represents a structure rich in lipids including abundant amounts of hydrocarbons and fatty acids. A member of a novel cytochrome P450 subfamily, CYP52X1, implicated in fatty acid assimilation by B. bassiana was characterized. B. bassiana targeted gene knockouts lacking Bbcyp52x1 displayed reduced virulence when topically applied to Galleria mellonella, but no reduction in virulence was noted when the insect cuticle was bypassed using an intrahemoceol injection assay. No significant growth defects were noted in the mutant as compared with the wild-type parent on any lipids substrates tested including alkanes and fatty acids. Insect epicuticle germination assays, however, showed reduced germination of ΔBbcyp52x1 conidia on grasshopper wings as compared with the wild-type parent. Complementation of the gene-knock with the full-length gene restored virulence and insect epicuticle germination to wild-type levels. Heterologous expression of CYP52X1 in yeast was used to characterize the substrate specificity of the enzyme. CYP52X1 displayed the highest activity against midrange fatty acids (C12:0 and C14:0) and epoxy stearic acid, 4-8-fold lower activity against C16:0, C18:1, and C18:2, and little to no activity against C9:0 and C18:0. Analyses of the products of the C12:0 and C18:1 reactions confirmed NADPH-dependent regioselective addition of a terminal hydroxyl to the substrates (ω-hydroxylase). These data implicate CYP52X1 as contributing to the penetration of the host cuticle via facilitating the assimilation of insect epicuticle lipids.     

Mortality of Colorado potato beetle (Leptinotarsa decemlineata) after sublethal stress with the CryIIIA delta-endotoxin of Bacillus thuringiensis and subsequent exposure to Beauveria bassiana

Acute or chronic sublethal exposure of Colorado potato beetle larvae to the CryIIIA delta-endotoxin of Bacillus thuringiensis Berliner did not significantly (P > 0.05) alter their subsequent susceptibility to Beauveria bassiana (Balsamo) Vuillemin. During the period of exposure to B. bassiana there was continued mortality from previous exposure to delta-endotoxin, and B. bassiana also caused significant mortality. Acute and chronic exposure to delta-endotoxin significantly prolonged larval development. The weights of prepupae and adults were significantly reduced by exposure to delta-endotoxin, with the greatest effect being from chronic exposure. Separation of the manifestations of stress in time (feeding vs soil stages) and space (toxin damage to the insect gut vs fungal penetration of the cuticle and activity in the hemocoel) may have precluded alteration of insect susceptibility to infection by B. bassiana. Endemic populations of B. bassiana are not expected to influence the development of resistance in the Colorado potato beetle to the delta-endotoxin of B. thuringiensis.     

Entomopathogenic Fungi Associated with Natural Populations of the Moroccan Locust Dociostaurus maroccanus (Thunberg) (Orthoptera: Gomphocerinae) and Other Acridoidea in Spain

The entomopathogenic fungi Beauveria bassiana Vuill . and Metarhizium anisopliae (Metschnikoff) have been found in natural populations of the Moroccan locust Dociostaurus maroccanus (Thunberg) and other species of acridoids that cohabit the same locust breeding areas in southern Spain . Infection levels of B. bassiana on insects collected in the field and maintained under laboratory conditions were relatively high (1 . 6 - 20 . 5%) . The prevalence of the disease extended from spring to summer in the three consecutive years monitored . A local isolate of this fungus demonstrated high virulence (LD = 440 conidia / insect) against the 50 locust D. maroccanus in the laboratory bioassay . The relatively wide host range of B. bassiana and its adaptation to the dry and hot conditions dominating the locust breeding area suggest that this isolate could be considered in the development of a biological control programme against D. maroccanus.     

A phosphoketolase Mpk1 of bacterial origin is adaptively required for full virulence in the insect-pathogenic fungus Metarhizium anisopliae

Pentose metabolism through the phosphoketolase pathway has been well characterized in bacteria. In this paper, we report the identification of a phosphoketolase homologue Mpk1 in the insect-pathogenic fungus Metarhizium anisopliae . Phylogenetic analysis showed that fungal phosphoketolases are of bacterial origin and diverged into two superfamilies. Frequent gene loss or lack of acquisition is evident in specific fungal lineages or species. The mpk1 gene is highly expressed when grown in trehalose-rich insect haemolymph but poorly induced by insect cuticle or carbohydrate-rich plant root exudate. In addition, mpk1 gene expression and enzyme activity could be upregulated by different sugars including xylose, trehalose, glucose or sucrose. mpk1 null mutants generated by homologous recombination grew similar to the wild type of M. anisopliae on medium amended with xylose as a sole carbon source. However, insect (tobacco hornworm, Manduca sexta ) bioassays showed significantly reduced virulence in Δ mpk1 . The results of this study suggest that the horizontally transferred Mpk1 in M. anisopliae plays an important niche adaptation role for fungal propagation in insect haemocoel. Following the carbohydrate flux from plants to plant-feeding insects and insect pathogenic fungi, a tritrophic relationship is discussed in association with the requirement of fungal phosphoketolase pathway.     

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.