Entomopathogenic fungus Beauveria bassiana-based bioinsecticide suppresses severity of powdery mildews of vegetables by inducing the plant defense responses.

The entomopathogenic fungus Beauveria bassiana is used commercially as a microbial insecticides against a wide range of agricultural insect pests. Some strains of B. bassiana protect the plants from pathogens, but the underlying mechanisms are largely unknown. Here, we found that prophylactic sprays of commercial bioinsecticide Botanigard on cucumber, tomato, and strawberry plants suppressed the severity of economically damaging powdery mildews. On leaf surfaces, hyphal elongation and spore germination of cucumber powdery mildew, Podosphaera xanthii, were inhibited, but B. bassiana strain GHA, the active ingredient isolated from Botanigard, only inhibited hyphal elongation but had no effect on spore germination of P. xanthii. In addition, strain GHA suppressed powdery mildew symptoms locally, not systemically. Treatment with Botanigard and strain GHA induced a hypersensitive response (HR)-like cell death in epidermal cells of the cucumber leaves in a concentration-dependent manner and inhibited penetration by P. xanthii. Transcriptome analysis and mass spectrometry revealed that GHA induced expression of salicylic acid (SA)-related genes, and treatment with Botanigard and GHA increased the SA level in the cucumber leaves. In NahG-transgenic tomato plants, which do not accumulate SA, the biocontrol effect of tomato powdery mildew by GHA was significantly reduced. These results suggested that B. bassiana GHA induces SA accumulation, leading to the induction of HR-like cell death against powdery mildew and subsequent suppression of fungal penetration. Thus, Botanigard has the potential to control both insect pests and plant diseases.

Antifungal roles of adult-specific cuticular protein genes of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.

Involvement of laccase2 and yellow-e genes in antifungal host defense of the model beetle, Tribolium castaneum.

We previously reported that the moderate knockdown of chitin synthase 1 gene of the model beetle Tribolium castaneum impairs the host defense against entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, which infect host insects via the direct penetration of cuticular integuments (Hayakawa et al., 2017). In this study, we focused on the antifungal roles of laccase2 (Lac2) as well as yellow-e (Y-e) genes, both of which are shown to be important to the establishment of stable cuticular structures in this beetle species. The expression profiles of the two genes somewhat resembled each other, peaking in late prepupae and mid to late pupae, while the transcript levels of Lac2 were higher than Y-e throughout. The knockdown of Lac2 gene at the prepupal and pupal peaks with relatively small amounts of dsRNA resulted in pupae with a lighter color and adults with a lighter color and dimpled/wrinkled elytra, respectively. Meanwhile, similar gene knockdown of Y-e but with 10 times more dsRNA compared to Lac2 resulted in pupae having a normal appearance and adults with a darker color. We conducted fungal infection assays with B. bassiana and M. anisopliae using these knockdown animals. The knockdown of Y-e gene had no or limited effects in both pupae and adults in terms of the antifungal host defense. Similarly, the knockdown of Lac2 gene did not change significantly the defense phenotypes of the resulting pupae. By sharp contrast, the host defense of the adult beetles against the two fungal species was almost totally destroyed by the moderate knockdown of Lac2 gene, suggesting its indispensable role in antifungal host defense presumably through the construction of sound cuticles of the adults. Finally, we investigated the maturation of host defense against fungal infection in the Lac2 knockdown adults and found that while the day 10 adults were still susceptible to M. anisopliae infection with some delay of death in comparison with day 1 adults, they exhibited complete refractoriness to B. bassiana.

Chitin synthase 1 gene is crucial to antifungal host defense of the model beetle, Tribolium castaneum.

The importance of the insect cuticle as a primary protective barrier against entomopathogens has long been noted. In the present study, we addressed this issue by utilizing an experimental infection system composed of the model beetle T. castaneum and two entomopathogenic fungal species, Beauveria bassiana and Metarhizium anisopliae. The pupae were relatively susceptible to these fungi by the natural route of infection, with some refractoriness developed with age, while the adults exhibited much higher refractoriness. Whereas M. anisopliae exhibited seemingly higher infectivity to the pupae compared to B. bassiana when the natural conidium infection was employed, direct inoculation of cultured hyphal body cells into the hemocoel was found highly and equally virulent in the pupae for the both fungal species. These results collectively suggest an important role of the cuticular integument in antifungal host defense, and we subsequently conducted the knockdown of chitin synthase 1 gene (CHS1). We targeted the prepupal and mid-pupal peaks of its expression respectively by using injection of the dsRNA at very low dosages to avoid lethality. The resulting pupae looked normal, but the adults showed a mild phenotype with dimpled/wrinkled elytra. The CHS1 gene knockdown compromised significantly host defense against the fungal infection via the natural route, except the configuration of knockdown pupae and M. anisopliae, suggesting an indispensable role of CHS1.

Prophenoloxidase genes and antimicrobial host defense of the model beetle, Tribolium castaneum.

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum.