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

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 Monochamus alternatus is an important wood borer and a major vector transmitting pine wilt disease, facilitating invasion of the pinewood nematode Bursaphelenchus xylophilus (PWN) in China. Due to the limited geographical breadth of sampling regions, species diversity of fungal associates (especially entomopathogenic fungi) on M. alternatus 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 M. alternatus, presenting regionalized characteristics, whereas no significant differences were found in fungal composition between beetle genders or among body positions. Four region-representative fungi, namely, Lecanicillium attenuatum (Zhejiang), Aspergillus austwickii (Sichuan), Scopulariopsis alboflavescens (Fujian), and A. ruber (Guangxi), as well as the three fungal species Beauveria bassiana, Penicillium citrinum, and Trichoderma dorotheae, showed significantly stronger entomopathogenic activities than other fungi. Additionally, insect-parasitic entomopathogenic fungi (A. austwickii, B. bassiana, L. attenuatum, and S. alboflavescens) exhibited less to no obvious phytopathogenic activities on the host pine Pinus massoniana, whereas P. citrinum, Purpureocillium lilacinum, and certain species of Fusarium spp.—isolated from M. alternatus body surfaces—exhibited remarkably higher phytopathogenicity. Our results provide a broader view of the entomopathogenic fungal community on the vector beetle M. alternatus, some of which are reported for the first time on Monochamus 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

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

Abstract Background 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. Results In the present study, a nematicidal gene cry5Ba3, originally from a soil Bacillus thuringiensis (Bt) strain, was codon-preferred as cry5Ba3Φ and integrated into the genome of a fungus eaten by PWN, Botrytis cinerea, using Agrobacterium tumefaciens-mediated transformation. Supplementing wild-type B. cinerea extract with that from the cry5Ba3Φ transformant significantly suppressed PWN growth; moreover, the nematodes lost fitness significantly when feeding on the mycelia of the cry5Ba3Φ transformant. N-terminal deletion of Cry5Ba3Φ protein weakened the nematicidal activity more dramatically than did the C-terminal deletion, indicating that domain I (endotoxin-N) plays a more important role in its nematicidal function than domain III (endotoxin-C), which is similar to certain insecticidal Cry proteins. Conclusions Transformation of Bt nematicidal cry genes in fungi can alter the fungivorous performance of B. xylophilus and reduce nematode fitness. This finding provides a new prospect of developing strategies for breaking the life cycle of this pest in pines and controlling pine wilt disease

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 period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits. Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs

Symbiotic microbes aid host adaptation by metabolizing a deterrent host pine carbohydrate <scp>d</scp> -pinitol in a beetle-fungus invasive complex

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 Leptographium procerum and two symbiotic bacteria, Erwinia and Serratia , 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. </jats:p

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

Abstract Background 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. Results In this report, the symbiotic naringenin-degrading microbiota were found to increase RTB survivorship in the presence of induced host defenses, and potential genes associated with degradation pathways were discovered. While fungi in the gallery microbiota had little involvement in naringenin degradation, bacterial community structure within the beetle gallery was highly correlated to naringenin degrading activity. Phylotypes of the Gram-negative bacterial genus Novosphingobium, which possessed genes involved in degradation pathways, were highly correlated to naringenin degradation activities and RTB associated with an isolated species of this genus acquired protection against naringenin and gained fitness. Conclusions Our results demonstrated that symbiotic bacterial community of RTB galleries enhances the survivorship and overall fitness of invasive beetles by degrading the host phenolic naringenin, ultimately overcoming the tree defenses and facilitating the success of the invasive beetle-fungi complex. This dynamic interplay between the invasive insect pest and multipartite microbes suggests a putative mechanism in invasion ecology for mitigating biotic resistance to symbiotic invasion

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

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 Monochamus alternatus is an important wood borer and a major vector transmitting pine wilt disease, facilitating invasion of the pinewood nematode Bursaphelenchus xylophilus (PWN) in China. Due to the limited geographical breadth of sampling regions, species diversity of fungal associates (especially entomopathogenic fungi) on M. alternatus 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 M. alternatus, presenting regionalized characteristics, whereas no significant differences were found in fungal composition between beetle genders or among body positions. Four region-representative fungi, namely, Lecanicillium attenuatum (Zhejiang), Aspergillus austwickii (Sichuan), Scopulariopsis alboflavescens (Fujian), and A. ruber (Guangxi), as well as the three fungal species Beauveria bassiana, Penicillium citrinum, and Trichoderma dorotheae, showed significantly stronger entomopathogenic activities than other fungi. Additionally, insect-parasitic entomopathogenic fungi (A. austwickii, B. bassiana, L. attenuatum, and S. alboflavescens) exhibited less to no obvious phytopathogenic activities on the host pine Pinus massoniana, whereas P. citrinum, Purpureocillium lilacinum, and certain species of Fusarium spp.—isolated from M. alternatus body surfaces—exhibited remarkably higher phytopathogenicity. Our results provide a broader view of the entomopathogenic fungal community on the vector beetle M. alternatus, some of which are reported for the first time on Monochamus 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.</jats:p

MOESM1 of Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene

Additional file 1. This file includes: Methods. Figure S1. Phylogenetic analysis of Cry5Ba3 with other homologous cry5 subfamily proteins. Figure S2. Codon modification of cry5Ba3 to cry5Ba3ÎŚ. Figure S3. Fungal colony morphologies. Figure S4. Fungal colony morphologies of Botrytis cinerea transformants with different lengths of cry5Ba3ÎŚ