isolation-behavioral-identification-pathogenicity-assessment

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

Huzhou University

Lund University

Microbial symbionts are known to assist exotic pests in their colonization of new host plants. However, there has been little evidence linking symbiotic invasion success to mechanisms for mitigation of native plant resistance. The red turpentine beetle (RTB) was introduced with a fungus, Leptographium procerum, to China from the United States and became a destructively invasive symbiotic complex in natural Pinus tabuliformis forests. Here, we report that three Chinese-resident fungi, newly acquired by RTB in China, induce high levels of a phenolic defensive chemical, naringenin, in pines. This invasive beetle-fungus complex is suppressed by elevated levels of naringenin. However, cryptic microbiotas in RTB galleries strongly degrade naringenin, and pinitol, the main soluble carbohydrate of P. tabuliformis, is retained in L. procerum-infected phloem and facilitate naringenin biodegradation by the microbiotas. These results demonstrate that cryptic microbiota mitigates native host plant phenolic resistance to an invasive symbiotic complex, suggesting a putative mechanism for reduced biotic resistance to symbiotic invasion.

Does cryptic microbiota mitigate pine resistance to an invasive beetle-fungus complex? Implications for invasion potential.

Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an important destructive disease of pine forests worldwide. In addition to behaving as a plant-parasitic nematode that feeds on epithelial cells of pines, this pest relies on fungal associates for completing its life cycle inside pine trees. Manipulating microbial symbionts to block pest transmission has exhibited an exciting prospect in recent years; however, transforming the fungal mutualists to toxin delivery agents for suppressing PWN growth has received little attention.

Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene.

There is growing evidence that some devastating biotic invasions are facilitated by microbial symbionts. The red turpentine beetle (RTB), an innocuous secondary insect attacking weakened trees in North America, has formed an invasive complex with the fungus Leptographium procerum in China, and this invasive beetle-fungus symbiotic complex is capable of attacking and killing healthy pines. A previous study demonstrated that three Chinese-resident fungi, newly acquired by RTB in China, induce high levels of a phenolic defensive chemical, naringenin, in pines and this invasive beetle-fungus complex is suppressed by elevated levels of naringenin while the beetle uses its gallery as an external detoxification system in which particular yeast-like fungi and bacterial species biodegrade naringenin. However, the functional roles of key microbial players in the symbiosis, contained within the microbiome of the bark beetle gallery, have not been well elucidated.

Bacterial microbiota protect an invasive bark beetle from a pine defensive compound.

Zika virus (ZIKV; ) is a devastating virus transmitted to humans by the mosquito Aedes aegypti. The interaction of the virus with the mosquito vector is poorly known. The double-stranded RNA (dsRNA)-mediated interruption or activation of immunity-related genes in the Toll, IMD, JAK-STAT, and short interfering RNA (siRNA) pathways did not affect ZIKV infection in A. aegypti. Transcriptome-based analysis indicated that most immunity-related genes were upregulated in response to ZIKV infection, including leucine-rich immune protein (LRIM) genes. Further, there was a significant increment in the ZIKV load in , , and -silenced A. aegypti, suggesting their function in modulating viral infection. Further, gene function enrichment analysis revealed that viral infection increased global ribosomal activity. Silencing of  and , two ribosomal large subunit genes, increased mosquito resistance to ZIKV infection.  fat body culture assay revealed that the expression of  and  was responsive to the Juvenile hormone (JH) signaling pathway. These two genes were transcriptionally regulated by JH and its receptor methoprene-tolerant (Met) complex. Silencing of  also inhibited ZIKV infection in A. aegypti. This suggests that ZIKV enhances ribosomal activity through JH regulation to promote infection in mosquitoes. Together, these data reveal A. aegypti immune responses to ZIKV and suggest a control strategy that reduces ZIKV transmission by modulating host factors.  Most flaviviruses are transmitted between hosts by arthropod vectors such as mosquitoes. Since therapeutics or vaccines are lacking for most mosquito-borne diseases, reducing the mosquito vector competence is an effective way to decrease disease burden. We used high-throughput sequencing technology to study the interaction between mosquito Aedes aegypti and ZIKV. Leucine-rich immune protein (LRIM) genes were involved in the defense in response to viral infection. In addition, RNA interference (RNAi) silencing of  and , two JH-regulated ribosomal large subunit genes, suppressed ZIKV infection in A. aegypti. These results suggest a novel control strategy that could block the transmission of ZIKV.

Juvenile Hormone-Sensitive Ribosomal Activity Enhances Viral Replication in Aedes aegypti.

The red turpentine beetle (RTB) is one of the most destructive invasive pests in China and solely consumes pine phloem containing high amounts of d-pinitol. Previous studies reported that d-pinitol exhibits deterrent effects on insects. However, it remains unknown how insects overcome d-pinitol during their host plant adaptation. We found that d-pinitol had an antagonistic effect on RTB, which mainly relied on gallery microbes to degrade d-pinitol to enhance host adaptation with mutualistic  and two symbiotic bacteria,  and , responsible for this degradation. Genomic, transcriptomic, and functional investigations revealed that all three microbes can metabolize d-pinitol via different branches of the inositol pathway. Our results collectively highlight the contributions of symbiotic microbes in RTB's adaptation to living on pine, thereby facilitating outbreaks of RTB in China. These findings further enrich our knowledge of symbiotic invasions and contribute to the further understanding of plant-insect interactions.

Symbiotic microbes aid host adaptation by metabolizing a deterrent host pine carbohydrate d-pinitol in a beetle-fungus invasive complex.

Entomopathogen-based biocontrol is crucial for blocking the transmission of vector-borne diseases; however, few cross-latitudinal investigations of entomopathogens have been reported for vectors transmitting woody plant diseases in forest ecosystems. The pine sawyer beetle  is an important wood borer and a major vector transmitting pine wilt disease, facilitating invasion of the pinewood nematode  (PWN) in China. Due to the limited geographical breadth of sampling regions, species diversity of fungal associates (especially entomopathogenic fungi) on .  adults and their potential ecological functions have been markedly underestimated. In this study, through traditional fungal isolation with morphological and molecular identification, 640 fungal strains (affiliated with 15 genera and 39 species) were isolated from 81 beetle cadavers covered by mycelia or those symptomatically alive across five regional populations of this pest in southern China. Multivariate analyses revealed significant differences in the fungal community composition among geographical populations of . , presenting regionalized characteristics, whereas no significant differences were found in fungal composition between beetle genders or among body positions. Four region-representative fungi, namely,  (Zhejiang),  (Sichuan),  (Fujian), and .  (Guangxi), as well as the three fungal species , , and , showed significantly stronger entomopathogenic activities than other fungi. Additionally, insect-parasitic entomopathogenic fungi (. , . , . , and . ) exhibited less to no obvious phytopathogenic activities on the host pine , whereas . , , and certain species of  spp.-isolated from .  body surfaces-exhibited remarkably higher phytopathogenicity. Our results provide a broader view of the entomopathogenic fungal community on the vector beetle . , some of which are reported for the first time on  spp. in China. Moreover, this beetle might be more highly-risk in pine forests than previously considered, as a potential multi-pathogen vector of both PWN and phytopathogenic fungi.

Discovery of entomopathogenic fungi across geographical regions in southern China on pine sawyer beetle  and implication for multi-pathogen vectoring potential of this beetle.

Chihang Cheng

Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province

Huzhou University

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer