Evaluating Beauveria bassiana Strains for Insect Pest Control and Endophytic Colonization in Wheat

Hong S., Gao H., Chen H., Wang C.

Similar to the physiological importance of gut microbiomes, recent works have shown that insect ectomicrobiotas can mediate defensive colonization resistance against fungal parasites that infect via cuticle penetration. Here we show that engineering the entomopathogenic fungus Metarhizium robertsii with a potent antibacterial moricin gene from silkworms substantially enhances the ability of the fungus to kill mosquitos, locusts, and two Drosophila species. Further use of Drosophila melanogaster as an infection model, quantitative microbiome analysis reveals that engineered strains designed to suppress insect cuticular bacteria additionally disrupt gut microbiomes. An overgrowth of harmful bacteria such as the opportunistic pathogens of Providencia species is detected that can accelerate insect death. In support, quantitative analysis of antimicrobial genes in fly fat bodies and guts indicates that topical fungal infections result in the compromise of intestinal immune responses. In addition to providing an innovative strategy for improving the potency of mycoinsecticides, our data solidify the importance of both the ecto- and endo-microbiomes in maintaining insect wellbeing. Genetic engineering of Metarhizium robertsii with a silkworm antibacterial gene substantially boosts fungal potency against different insects. Both the cuticle and gut microbiomes can be disrupted to accelerate insect death after fungal infection.

Dai J., Tang X., Wu C., Liu S., Mi W., Fang W.

Plant-derived sugars and lipids are key nutritional sources for plant associated fungi. However, the relationship between utilization of host-derived sugars and lipids during development of the symbiotic association remains unknown. Here we show that the fungus Metarhizium robertsii also needs plant-derived lipids to develop symbiotic relationship with plants. The fatty acid binding proteins FABP1 and FABP2 are important for utilization of plant-derived lipids as the deletion of Fabp1 and Fabp2 significantly reduced the ability of M. robertsii to colonize rhizoplane and rhizosphere of maize and Arabidopsis thaliana. Deleting Fabp1 and Fabp2 increased sugar utilization by upregulating six sugar transporters, and this explains why deleting the monosaccharide transporter gene Mst1, which plays an important role in utilization of plant-derived sugars, had no impact on the ability of the double-gene deletion mutant ΔFabp1::ΔFabp2 to colonize plant roots. FABP1 and FABP2 were also found in other plant-associated Metarhizium species, and they were highly expressed in the medium using the tomato root exudate as the sole carbon and nitrogen source, suggesting that they could be also important for these species to develop symbiotic relationship with plants. In conclusion, we discovered that utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by M. robertsii.

Sui L., Lu Y., Zhou L., Li N., Li Q., Zhang Z.

IntroductionEntomopathogenic fungi (EPF) can colonize and establish symbiotic relationships with plants as endophytes. Recently, EPF have been reported to suppress plant pathogens and induce plant resistance to diseases. However, the potential mechanisms via which EPF as endophytes control major plant diseases in situ remain largely unknown.MethodsPot and field experiments were conducted to investigate the mechanisms via which an EPF, Beauveria bassiana, colonizes tomato, under Botrytis cinerea infection stress. B. bassiana blastospores were inoculated into tomato plants by root irrigation. Tomato resistance to tomato gray mold caused by B. cinerea was evaluated by artificial inoculation, and B. bassiana colonization in plants and rhizosphere soil under B. cinerea infection stress was evaluated by colony counting and quantitative PCR. Furthermore, the expression levels of three disease resistance-related genes (OXO, CHI, and atpA) in tomato leaves were determined to explore the effect of B. bassiana colonization on plant disease resistance performance in pot experiments.ResultsB. bassiana colonization could improve resistance of tomato plants to gray mold caused by B. cinerea. The incidence rate, lesion diameter, and disease index of gray mold decreased in both the pot and field experiments following B. bassiana colonization. B. bassiana was more likely to accumulate in the pathogen infected leaves, while decreasing in the rhizosphere soil, and induced the expression of plant resistance genes, which were up-regulated in leaves.DiscussionThe results indicated that plants could “recruit” B. bassiana from rhizosphere soil to diseased plants as directional effects, which then enhanced plant growth and resistance against pathogens, consequently inhibiting pathogen infection and multiplication in plants. Our findings provide novel insights that enhance our understanding of the roles of EPF during pathogen challenge.

Zeng S., Lin Z., Yu X., Zhang J., Zou Z.

Mycoinsecticides are essential for the development of integrated pest management as substitutes to chemical insecticides, but their usage is limited by their inferior virulence. Thus, genetically engineered bioinsecticides, including recombinant entomopathogenic fungi, have been regarded as a breakthrough to rapidly control pests.

Sani I., Jamian S., Saad N., Abdullah S., Mohd Hata E., Jalinas J., Ismail S.I.

Entomopathogenic fungi (EPF) are natural enemies which affect insect population and have long been recognized as biological control agents against many insect pests. Some isolates have also been established as endophytes, benefiting their host plants without causing any symptoms or negative effects. Here we demonstrated two entomopathogenic fungal species, Isariajavanica (Frieder. & Bally) Samson & Hywel-jone 2005 and Purpureocillium lilacinum (Thom) Luangsa-ard, Hou-braken, Hywel-Jones & Samson (2011) as endophytes in tomato plants by using the seed inoculation method and examined their effect on plant growth, B. tabaci mortality, and adult emergence. Our study indicated that tomato seeds treated with a fungal suspension of I. javanica and P. lilacinum enabled their recovery from plant tissues (root, stem and leaf) up to 60 days after inoculation (DAI). Both endophytic isolates also caused significant mortality of adult B. tabaci on seedlings inoculated with, I. javanica (51.92±4.78%), and P. lilacinum (45.32±0.20%) compared to the control treatment (19.29±2.35). Adult emergence rates were significantly high in the control treatments (57.50±2.66%) compared to I. javanica (15.00±1.47%) and P. lilacinum (28.75±4.78%) treatments. This study provides evidence that endophytic isolates of I. javanica and P. lilacinum have a biocontrol potentials for used against whiteflies and could also explored as plant growth promoters.

Wang L., Lai Y., Chen J., Cao X., Zheng W., Dong L., Zheng Y., Li F., Wei G., Wang S.

Entomopathogenic fungi infect insects by penetrating through the cuticle into the host body. To breach the host cuticle, some fungal pathogens produce specialized infection cells called appressoria, which develop enormous turgor pressure to allow cuticle penetration. However, regulatory mechanisms underlying appressorium turgor generation are poorly understood. Here, we show that the histone lysine methyltransferase ASH1 in the insecticidal fungus Metarhizium robertsii , which is strongly induced during infection of the mosquito cuticle, regulates appressorium turgor generation and cuticle penetration by activating the peroxin gene Mrpex16 via H3K36 dimethylation. MrPEX16 is required for the biogenesis of peroxisomes that participate in lipid catabolism and further promotes the hydrolysis of triacylglycerols stored in lipid droplets to produce glycerol for turgor generation, facilitating appressorium-mediated insect infection. Together, the ASH1–PEX16 pathway plays a pivotal role in regulating peroxisome biogenesis to promote lipolysis for appressorium turgor generation, providing insights into the molecular mechanisms underlying fungal pathogenesis.

Cafarchia C., Pellegrino R., Romano V., Friuli M., Demitri C., Pombi M., Benelli G., Otranto D.

• Mosquitoes and ticks are a threat for animal health, acting as vectors of pathogens. • Their management mainly rely to the employ of synthetic insecticides and acaricides. • We reviewed the use of entomopathogenic fungi (EPF) for mosquito and tick control. • Strengths and weaknesses of EPF formulations and delivery methods are discussed. • Solutions to improve the EPF delivery and effectiveness in the field are proposed. Insects, ticks, and mites represent a threat to animal health globally, mainly due to their role as vectors of pathogens. Among the most important diseases, those transmitted by mosquitoes (e.g., malaria and arboviral infections) and ticks (e.g., Lyme borreliosis, babesiosis, and viral haemorrhagic fever) have a huge impact on human health. The principal methods available for reducing the public health burden of most vector-borne diseases are vector-based intervention relying to insecticides and acaricides. However, the use of these products is challenged by the introduction of invasive species, the quick development of physiological insecticide and acaricide resistance, and their non-target effects on human health and environment. In this scenario, insecticide/acaricide-free control approaches based on the employment of entomopathogenic fungi (EPFs) are currently considered a promising tool in Integrated Pest/Vector Management, even if their large-scale use is still limited. In this article, we provide an overview on current knowledge about the role of EPFs for mosquito and tick management to assess solutions improving the delivery and efficacy of EPFs in the field. Laboratory research provided solid evidence that EPFs represent a next-generation control tool to manage mosquito and tick populations. However, the viability, infectivity, and persistence of fungal spores under field conditions are still inadequate. Herein we also discuss the development and optimization of EPF-based lure and kill approaches through biopolymers to improve cost-competitive, safety and eco-friendly pest and vector control tools.

González-Guzmán A., Rey M., Froussart E., Quesada-Moraga E.

Microbial control of insect and mite pests is a key tool to develop integrated pest management (IPM) and sustainable agriculture. Entomopathogenic fungi (EF) may have associations with the plants, playing additional ecological roles in the rhizosphere, in the phylloplane, and as plant endophytes. Beauveria bassiana 04/01TIP and Metarhizium brunneum 01/58Su are two strains that showed very good results either in pest control or plant growth promotion and would be good candidates to develop mycoinsecticides as an alternative to pesticides.

Hong S., Sun Y., Chen H., Wang C.

Insects can assemble defensive microbiomes on their body surfaces to defend against fungal parasitic infections. The strategies employed by fungal pathogens to combat host cuticular microbiotas remains unclear. Here, we report the identification and functional characterization of the defensin-like antimicrobial gene BbAMP1 encoded by the entomopathogenic fungus Beauveria bassiana. The mature peptide of BbAMP1 can coat fungal spores and can be secreted by the fungus to target and damage Gram-positive bacterial cells. Significant differences in insect survival were observed between the wild-type and BbAMP1 mutant strains during topical infection but not during injection assays that bypassed insect cuticles. Thus, BbAMP1 deletion considerably reduced fungal virulence while gene overexpression accelerated the fungal colonization of insects compared with the wild-type strain in natural infections. Topical infection of axenic Drosophila adults evidenced no difference in fly survivals between strains. However, the gnotobiotic infections with the addition of Gram-positive but not Gram-negative bacterial cells in fungal spore suspensions substantially increased the survival of the flies treated with ∆BbAMP1 compared to those infected by the wild-type and gene-overexpression strains. Bacterial colony counts and microbiome analysis confirmed that BbAMP1 could assist the fungus to manipulate insect surface bacterial loads. This study reveals that fungal defensin can suppress the host surface defensive microbiomes, which underscores the importance to extend the research scope of fungus-host interactions.

Yu L., Xu S., Tong S., Ying S., Feng M.

Solar ultraviolet (UV) irradiation is harmful to formulated conidia as active ingredients of fungal pesticides and hence restrains their field application in sunny days of summer, a season requiring frequent pest controls. This conflict makes it necessary to explore optimal strategies for the application of fungal pesticides to suppress pest populations but avoid solar UV damage during summer.The conidia of Beauveria bassiana, a wide-spectrum fungal pesticide, were tolerable to UVB (major solar UV wavelengths) damage of ≤0.5 J cm-2 . The damage of this upper limit caused a loss of conidial viability and infectivity if not photoreactivated by light exposure after irradiation. Intriguingly, the light exposure resulted in a high photoreactivation rate of UVB-inactivated conidia and an insignificant or marginal difference in insecticidal activity between normal conidia and those photoreactivated. Modeling analysis of solar UVB intensity recorded hourly over the daylight of five sunny summer days from 5:00 am to 7:00 pm at 30° 17'57'' N and 120°5'7'' E revealed a variation of daily accumulated UVB dose from 2.07 to 2.78 J cm-2 , which was far beyond the upper limit. A more tolerable dose of ~0.2 J cm-2 appeared between 3:00 pm and 5:00 pm, and no harmful dose accumulated between 5:00 pm and 7:00 pm.Fungal UVB tolerance, fungal photoreactivation capability and the daily accumulation pattern of solar UV irradiation are based to propose an optional strategy for low-risk or non-risk application of fungal pesticides after 3:00 or 5:00 pm during summer. © 2022 Society of Chemical Industry.

Camara I., Yin Y., Cao K., Sangbaramou R., Um-e-Hani, Cao C., Tan S., Shi W.

• Nodulation response in locusts decreased following injection with the antipyretic drug. • Phenidone increased mortality rate in infected locusts. • Phenidone reduced the therapeutic effect of thermoregulation on the survival of infected locusts. The use of eicosanoids biosynthesis inhibitors (EBIs) as immune disruptors in host-pathogen interaction is a promising tool in insect pest control. In this study, co-applying the dual cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor phenidone (104 µg/larva) with the entomopathogen Beauveria bassiana (10 8 spores/mL) on Locusta migratoria manilensis , sharply declined the nodulation response. The reduction of nodulation response by phenidone proved that the dual COX/LOX is involved in nodules formation in L. migratoria manilensis . We investigated the effect of the antipyretic drug on the fungus virulence under different thermal conditions (thermoregulation and non-thermoregulation). Interestingly, a significant increase in the mortality rate of fungus-phenidone treated locusts was recorded. The drug suppressed the therapeutic effect of thermoregulation in infected locusts combined with phenidone tailoring a mortality rate of 70% by 14 days post-infection. Whereas 40% of the mortality rate was observed in those infected locusts under the same thermal condition but not treated with phenidone. These findings indicated that phenidone can cripple the immune defense of L. migratoria manilensis and additionally increase the virulence of B. bassiana under certain ecological conditions.

Tang Y., Zhang Y., Zhang Q., Chen R., Gong L., Wei X., Yang J., Wu K., Huang W., Li S., Toufeeq S., Liu Q., Ling E.

Prophenoloxidase (PPO), an important immunity protein in insects, is mainly produced by hemocytes and released into the hemolymph upon cell lysis. In addition, PPO can also be produced by epidermal cells in the foregut to detoxify the toxic plant secondary metabolites and in the hindgut to kill pathogens through PPO-induced melanization. Previously, we noticed a pair of tubes extended from the larval hindgut became melanized upon staining in dopamine dissolved in 30% ethanol. However, the structure and function of these tubes are largely unknown. In this study, we performed staining of the tubes and the neighboring Malpighian tubule for further confirmation. Eventually, we detected PPO inside epidermal cells of the tubes, and called them as PPO-positive tubes. We observed that the PPO-positive tubes are physically derived from the hindgut but strongly adhere to the Malpighian tubule. Inside the PPO-positive tubes, there is an acellular peritrophic membrane to protect the epidermal cells. Furthermore, the PPO-positive tubes act like a doorkeeper to firstly detoxify the metabolite wastes collected by the Malpighian tubule from the hemolymph.

Wang X., Yang X., Zhou F., Tian Z.Q., Cheng J., Michaud J.P., Liu X.

• Cuticular bacterial diversity of Grapholita molesta was investigated by 16S rDNA sequencing. • The dominant bacteria on the cuticle of Grapholita molesta were cultured and identified. • Cuticular bacterium Pantoea sp. exhibited high antifungal activity against Beauveria bassiana. The oriental fruit moth, Grapholita molesta (Lepidoptera: Tortricidae), is a destructive orchard pest native to the Asian subcontinent that has spread throughout many fruit-growing regions of the world. The entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) is widely employed for control of insect pests, including G. molesta . Various studies have shown that microorganisms present on the insect cuticle can inhibit fungal spore germination and growth, thus inhibiting their pathogenicity and biological control efficacy. We conducted bioassays on larvae pre-treated with antibiotics to test whether cuticular microbiota could reduce the pathogenicity of B. bassiana to G. molesta . Control larvae (untreated with antibiotics) had higher survival than antibiotic-treated larvae following inoculation with B. bassiana , indicating that cuticular microbiota inhibited infection of G. molesta by B. bassiana . The bacterial diversity present on the cuticle of G. molesta larvae was characterized by 16S rDNA amplicon sequencing. The dominant bacterium isolated was Pantoea sp. and its antagonism toward B. bassiana was assayed in vitro using the inhibition zone method. Inhibitory effects on the pathogenicity of Pantoea sp. against B. bassiana were demonstrated by reintroducing the bacterium to G. molesta larvae that had been pre-treated with antibiotic, which increased their survival in comparison to antibiotic-treated larvae following inoculation with B. bassiana . This study provides potentially valuable insights for maximizing the efficacy of B. bassiana applications for biological control of G. molesta .

Ye X., Xiong S., Teng Z., Yang Y., Wang J., Yu K., Wu H., Mei Y., Xue C., Yan Z., Yin C., Wang F., Yao H., Fang Q., Song Q., et. al.

A fundamental feature of parasitism is the nutritional exploitation of host organisms by their parasites. Parasitoid wasps lay eggs on arthropod hosts, exploiting them for nutrition to support larval development by using diverse effectors aimed at regulating host metabolism. However, the genetic components and molecular mechanisms at the basis of such exploitation, especially the utilization of host amino acid resources, remain largely unknown. To address this question, here, we present a chromosome-level genome assembly of the parasitoid wasp Cotesia chilonis and reconstruct its amino acid biosynthetic pathway. Analyses of the amino acid synthetic pathway indicate that C. chilonis lost the ability to synthesize ten amino acids, which was confirmed by feeding experiments with amino acid-depleted media. Of the ten pathways, nine are known to have been lost in the common ancestor of animals. We find that the ability to synthesize arginine was also lost in C. chilonis because of the absence of two key genes in the arginine synthesis pathway. Further analyses of the genomes of 72 arthropods species show that the loss of arginine synthesis is common in arthropods. Metabolomic analyses by UPLC-MS/MS reveal that the temporal concentrations of arginine, serine, tyrosine, and alanine are significantly higher in host (Chilo suppressalis) hemolymph at 3 days after parasitism, whereas the temporal levels of 5-hydroxylysine, glutamic acid, methionine, and lysine are significantly lower. We sequence the transcriptomes of a parasitized host and non-parasitized control. Differential gene expression analyses using these transcriptomes indicate that parasitoid wasps inhibit amino acid utilization and activate protein degradation in the host, likely resulting in the increase of amino acid content in host hemolymph. We sequenced the genome of a parasitoid wasp, C. chilonis, and revealed the features of trait loss in amino acid biosynthesis. Our work provides new insights into amino acid exploitation by parasitoid wasps, and this knowledge can specifically be used to design parasitoid artificial diets that potentially benefit mass rearing of parasitoids for pest control.

Vázquez-Benito J.A., Santillán-Galicia M.T., Guzmán-Franco A.W., Hernández-Domínguez C., Romero-Rosales F., Ortiz-Osuna Á., Rodríguez-Maciel J.C.

• Brevipalpus yothersi is the vector of the Citrus leprosis virus. • B. yothersi was susceptible to infection by fungal pathogens. • Eggs were the preferred prey of the predatory mites Neoseiulus californicus and Amblyseius swirskii , followed by nymphs and then larvae . • Predatory mites contaminated with fungal conidia achieved greater B. yothersi mortality than predatory mites alone. • Combined use of predatory mites and fungal conidia could represent a more efficient biological control strategy for B. yothersi . The mite Brevipalpus yothersi (Baker) (Acari: Tenuipalpidae), vector of Citrus leprosis virus, is typically controlled using synthetic pesticides. However, alternative control strategies are required to reduce pesticide use for more sustainable citrus production. The mortality of B. yothersi following combined application of fungal pathogens and predatory mites was evaluated. First, we quantified susceptibility of B. yothersi adults to infection when treated with conidial suspensions (1 × 10 8 conidia mL −1 ) of three fungal isolates, Bb88 ( Beauveria bassiana ), Ma129 ( Metarhizium anisopliae ) and Pfr4 ( Cordyceps fumosorosea ). Then, we evaluated the feeding preference (choice and no choice) of the predatory mites Neoseiulus californicus and Amblyseius swirskii for eggs, larvae, nymphs and adults of B. yothersi . In addition, we evaluated the searching capacity of both predators when foraging for eggs of B. yothersi . Finally, the mortality of B. yothersi adults exposed to predatory mites contaminated with conidia of two fungal isolates was determinated. Isolates Bb88 and Ma129 caused the highest mortality in B. yothersi . Both predatory mites preferred to consume eggs, followed by larvae and then nymphs, but not adults of B. yothersi, in both experiments (choice and no choice). The predatory mite A. swirskii had a greater search capacity than N. californicus . The greatest mortality of B. yothersi adults was achieved when predatory mites contaminated with conidia were used (especially isolate Ma129 [approx. 80%]), compared with treatments of predatory mites alone (approx. 35%). The predatory mite A. swirskii and the fungus M. anisopliae (Ma129) was the most promising combination for further evaluation for biological control of B. yothersi .