Esteya vermicola is a nematophagous fungus with strong parasitic ability against the pinewood nematode (Bursaphelenchus xylophilus) and shows great potential for the biological control of pine wilt disease. However, this fungus is highly sensitive to environmental stress factors and often exhibits early necrosis when cultured on conventional nutrient-rich media, limiting its large-scale application. In this study, we optimized the long-term cultivation and conidiation conditions of E. vermicola CBS115803 by supplementing minimal medium (MM) with amino acids, and evaluated its stress tolerance and infectivity against the pinewood nematode. Among 20 tested amino acids, histidine significantly increased total conidia production, while arginine, glutamine, and proline markedly promoted the formation of lunate conidia. The combination of arginine, histidine, glutamine, and proline (AHGP) produced the highest overall conidia yield and lunate conidia proportion. The MM + AHGP medium maintained long-term colony viability, whereas colonies on PDA and CM media showed obvious degeneration. This formulation also improved mycelial growth, total conidiation, and the proportion of lunate conidia. Moreover, conidia produced on MM + AHGP exhibited the highest germination rates and infectivity under various stress conditions, including cold, heat, oxidative, osmotic, and UV stresses. Conidia germination was significantly enhanced following treatment at 0 °C, suggesting that low temperatures may activate dormancy-breaking pathways. This amino acid-optimized medium offers an effective technical foundation for stable large-scale production and storage of E. vermicola conidia, providing a new avenue for the biocontrol of pine wilt disease.

Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD), poses a severe global threat to coniferous forests. Esteya vermicola, an endoparasitic nematophagous fungus, exhibits promising biocontrol potential against this pinewood nematode. The vesicular transport system, evolutionarily conserved in eukaryotes, is essential for fungal pathogenicity. Based on our genome sequence of E. vermicola CBS115803, we identified EvSec22, a gene encoding a SNARE protein implicated in vesicular transport process. This study investigates the role of EvSec22 in E. vermicola during nematode infection, utilizing our optimized gene knockout methodology. Infection assays revealed that EvSec22 deletion significantly impaired the pathogenicity of E. vermicola against B. xylophilus. Phenotypic analyses revealed that the ΔEvSec22 mutant exhibited suppressed hyphal growth, reduced conidiation, and abnormal septal spacing. Furthermore, the mutant showed significantly diminished tolerance to osmotic stress (sorbitol) and oxidative stress (hydrogen peroxide). Overall, the EvSec22 gene is associated with the virulence of E. vermicola CBS115803 against B. xylophilus, and its deletion also affects the normal growth of E. vermicola and its tolerance to abiotic stress. This study providing new insights into SNARE protein functions in fungal biocontrol agents.

Pine wilt disease caused by pine wood nematodes (PWN) have been reported to give severe damage in many countries worldwide. Many measures of PWN management have been implemented, including using the nematophagous fungus Esteya vermicola as a biological control agent. However, understanding on the interaction between this fungus and pine trees is still limited. This study aimed to evaluate the effects of resinous compounds on spore germination and mycelial growth of E. vermicola in vitro. Research results showed that resinous compounds from pine trees significantly affect spore germination and mycelial growth of E. vermicola. In which, (+)α-pinene, (-)α-pinene, and (-)limonene completely inhibited spore germination and mycelial growth, with 100% of spore germination inhibition rate (SGIR) and mycelial growth inhibition rate (MGIR). The mixture of 14 resinous compounds also indicated potent inhibition of spore germination and mycelial growth of E. vermicola, with 100% and 91.8% SGIR and MGIR, respectively. These results suggest that trunk injection of spore suspension of E. vermicola to pine tree could be unsuccessful and ineffective for the control of pine wilt disease.

Esteya vermicola Fxy121 strain, isolated from live pine trees, exhibited high adhesion and infection activity against Bursaphelenchus xylophilus. In this study, we studied the biological characteristics of Esteya vermicola Fxy121, and screened species-specific primers used for its colonization detection on pine trees. The liquid fermentation formula with higher blastospore production was obtained by adjusting the ratio of carbon and nitrogen and adding mineral salts, blastospore concentration was 5.5 × 108 spores mL-1 cultured for 6 days using new formula. The germination of blastospores was not affected when suspension was placed at 4°C-30°C for 1-8 h. The number of lunate spores increased first and then decreased with the increase of initial blastospore concentration of E. vermicola Fxy121, medium concentration of blastospores suspension showed higher infectivity. The evF/evR primer pair successfully amplified the except fragment size of 522 bp from isolates of E. vermicola and from pine needles sprayed with blastospores suspension of E. vermicola. This study suggests that blastospores concentration of E. vermicola Fxy121 significantly affect the production of lunate spores and infectivity against B. xylophilus. The species-specific primers allowed fast and reliable detection of E. vermicola directly from tissues of pine trees sprayed and injected with spore suspension.

Pine wilt disease (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is one of the most serious threats to pine forests worldwide. The fungus Esteya vermicola, with its lunate conidia capable of parasitizing the PWN, has shown promise as an efficient biological control agent against PWD. Solid-state fermentation (SSF) is preferred for large-scale applications in the field, as it facilitates microbial agent transport and ensures a long shelf life. However, research on enhancing the yield of lunate conidia from E. vermicola through SSF is limited. In this study, we initially achieved a yield of 3.04 × 108 conidia/g using a basic SSF medium composed of wheat bran, corn flour, and soybean flour. To improve this yield, we employed an orthogonal experimental design (OED) to identify the optimal medium composition, which required a wheat bran-to-corn flour-to soybean flour ratio of 7:2:1 (w/w/w), a substrate-to-water ratio of 1:0.7 (w/v), and the addition of 1.33% (w/w) glucose, 1.33% (w/w) yeast extract fermentation, and 1.33% (w/w) MgSO4. Using the response surface methodology (RSM), we calculated the optimal fermentation conditions, which were 24.9 °C, 78.0% relative humidity (RH), an inoculation volume of 16.3% (v/w), and a fermentation time of 7.1 days. Under these conditions, the yield of lunate conidia reached a maximum of 16.58 × 108 conidia/g, a 4.45-fold increase after optimization. This study improved the yield of E. vermicola lunate conidia and provides insights for developing biopesticides based on this strain.

Unraveling the interactions of Esteya vermicola, pinewood nematode, and pine hosts: Insights into population dynamics and molecular responses

The pinewood nematode (Bursaphelenchus xylophilus) causes severe damage to pine trees. The nematophagous fungus, Esteya vermicola, exhibits considerable promise in the biological control of Bursaphelenchus xylophilus due to its infectivity. Notably, the lunate conidia produced by E. vermicola can infect Bursaphelenchus xylophilus. In the study, we aim to investigate the genes involved in the formation of the lunate conidia of E. vermicola CBS115803. Esteya vermicola CBS115803 yielded 95% lunate conidia on the complete medium (CM) and 86% bacilloid conidia on the minimal medium (MM). Transcriptomic analysis of conidia from both media revealed a significant enrichment of differentially expressed genes in the pathway related to 'cellular amino acid biosynthesis and metabolism'. Functional assessment showed that the knockout of two arginine biosynthesis genes (EV232 and EV289) resulted in defects in conidia germination, mycelial growth, lunate conidia formation, and virulence of E. vermicola CBS115803 in Bursaphelenchus xylophilus. Remarkably, the addition of arginine to the MM improved mycelial growth, conidiation and lunate conidia formation in the mutants and notably increased conidia yield and the lunate conidia ratio in the wild-type E. vermicola CBS115803. This investigation confirms the essential role of two arginine biosynthesis genes in lunate conidia formation in E. vermicola CBS115803. The findings also suggest that the supplementation of arginine to the culture medium can enhance the lunate conidia yield. These insights contribute significantly to the application of E. vermicola CBS115803 in managing Bursaphelenchus xylophilus infections. © 2023 Society of Chemical Industry.

Pine wilt disease, which is caused by the nematode Bursaphelenchus xylophilus, is one of the most destructive forest diseases worldwide. Esteya vermicola, a nematophagous fungus, has emerged as a promising biological control agent. However, the limited availability of gene function analysis techniques hinders further genetic modification of this fungus. In this study, we employed a combination of enzymes (driselase, snailase, and cellulase) to enzymatically degrade the cell wall of the fungus, resulting in a high yield of protoplasts. Furthermore, by utilizing 0.6M sucrose as an osmotic pressure stabilizer, we achieved a significant protoplast regeneration rate of approximately 31%. Subsequently, we employed the polyethylene glycol-mediated protoplast transformation method to successfully establish a genetic transformation technique for E. vermicola CBS115803. Additionally, through our investigation, we identified the Olic promoter from Aspergillus nidulans, which effectively enhanced the expression of the DsRed gene encoding a red fluorescent protein in E. vermicola CBS115803. Moreover, we successfully implemented a split-marker strategy to delete the EvIPMD gene in E. vermicola CBS115803. In summary, our findings present valuable experimental methodologies for gene function analysis in E. vermicola CBS115803.

Abstract Pine wilt disease is a devastating forest disease worldwide caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus). Esteya vermicola is widely recognized as a promising bio‐control agent effective against PWN. During a survey of associated microorganisms of Pinus yunnanensis in Southwest China, a novel isolate of E. vermicola was obtained. This isolate produced two types of conidia. One type of conidia was hyaline, unicellular, bacilloid, and non‐adhesive. The second type of conidia is solitary, hyaline, unicellular asymmetric elliptic or lunate, concave inward, ending slightly apiculate, adhesive on the concave surface, and contained an ovoid endospore‐like structure. Only the lunate conidia showed infection activity against PWN. This novel isolate produced a high proportion of infective lunate conidia and exhibited high adhesion and infection activity against PWN by adhering to the nematode within 48 h and killing 74.5% of the tested PWN individuals within 4 days. Given that the isolate was obtained from P. yunnanensis, it may be well adapted to the endogenous environment of pine trees. The isolate shows potential as a bio‐control agent against a plant‐parasitic nematode in the field.

Esteya vermicola is the first recorded endoparasitic nematophagous fungus with high infectivity capacity, attacking the pinewood nematode Bursaphelenchus xylophilus which causes pine wilt disease. Endosymbionts are found in the cytoplasm of E. vermicola from various geographical areas. We sequenced the genome of endobacteria residing in E. vermicola to discover possible biological functions of these widespread endobacteria. Multilocus phylogenetic analyses showed that the endobacteria form a previously unidentified lineage sister to Phyllobacterium myrsinacearum species. The number of genes in the endobacterium was 4542, with 87.8% of the proteins having a known function. It contained a high proportion of repetitive sequences, as well as more Acyl-CoA synthetase genes and genes encoding the electron transport chain, compared with compared with plant-associated P. zundukense Tri 48 and P. myrsinacearum DSM 5893. Thus, this symbiotic bacterium is likely to be more efficient in regulating gene expression and energy release. Furthermore, the endobacteria in nematophagous fungi Esteya vermicola contained multiple nematicidal subtilase/subtilisin encoding genes, so it is likely that endobacteria cooperate with the host to kill nematodes.

Abstract Esteya vermicola is a potential biological agent of the pinewood nematode (PWN), Bursaphelenchus xylophilus, due to its high infectivity. However, knowledge of E. vermicola colonization in the host pine tree is less known. To reveal the distribution pattern of E. vermicola inside the pine tree and the interactions between fungus and PWN, biocontrol tests were conducted on 10 years old Pinus densiflora. A green fluorescence protein (GFP)‐tagged E. vermicola was used to observe the fungal hyphae. Real‐time TaqMan PCR quantification was applied to quantify the fungal hyphae in host tree. The results suggest that inoculation with the fungus significantly improved the survival rate of tested trees. Besides, the number of PWN extracted from fungal inoculated tested trees significantly decreased. Moreover, the fungal hyphae and lunate conidia grown in tested trees were observed with the GFP‐tagged E. vermicola. The real‐time quantification of E. vermicola illustrated the differential distribution of E. vermicola in wilted, wilting and healthy pine trees. Additionally, the promotion of fungal hyphal dispersion by B. xylophilus was found in the pine trees artificially infected with PWN. This study reveals the distribution of E. vermicola and PWN during biocontrol of pine wilt disease and provides direct evidence of the effectiveness of this fungus to control PWN. The migration of PWN infected by E. vermicola was supposed to improve the fungal extension in host pine tree.

Esteya vermicola is an endoparasitic fungus producing lunate conidia, which kill pine wood nematode (PWN), and PWN could cause pine wilt disease (PWD). The aims of this study were to increase production and confirm the resistance (temperature and UV irradiation) of lunate conidia, and further determine the effective concentrations of conidia infecting PWN. In this study, rice was used as a carrier to absorb conidial suspension to propagate conidia. The optimal conditions for lunate conidia production were 25°C temperature, 9days of culture time, 2:1 rice/distilled water ratio and 10% inoculum size. The germination rate of E. vermicola cultured on potato dextrose agar was influenced by UV irradiation, similar to growth on rice. Esteya vermicola cultured on rice under heat stress might be more suitable for application in the field. The concentration (1×108 conidia per ml) to kill PWN had the highest infectivity among the four conidia concentrations tested after 3days of inoculation. This study showed a rice substrate-supported high-quality conidia production and the optimal infectivity concentration of E. vermicola. These results provide the necessary process of an economical and efficient biological control strategy against PWD.

As the causal agent of pine wilt disease, Bursaphelenchus xylophilus, is a serious pathogen of forest pine trees. Esteya vermicola is a nematophagous fungus of B. xylophilus and exhibits great potential as a biological control agent. However, the in vivo infection mechanism of E. vermicola on B. xylophilus is unclear. Experiments were conducted to study the colonization of host plant and infection of B. xylophilus by E. vermicola inside pine tree xylem. A green fluorescent protein (GFP)-tagged E. vermicola transformant was constructed as a biomarker to study the in vivo colonization and infection of B. xylophilus in pine trees. The in vitro infection of B. xylophilus by E. vermicola was observed through GFP expression. The bacilloid conidia produced by trophic hyphae in the body of the nematode are described. Additionally, the monitoring of in vivo colonization by GFP-tagged E. vermicola showed the germination and hyphal extension of this fungus after inoculation. Moreover, B. xylophilus infected by this biocontrol agent were extracted from healthy seedlings and observed in the xylem of trees that were wilting due to pine wilt disease. Evidence of fungal colonization and infection of B. xylophilus by E. vermicola is provided to improve our understanding of the in vivo infection mechanisms used by this nematophagous fungus against B. xylophilus. The infection of B. xylophilus by E. vermicola was inferred to begin with the implantation of propagules, and this inference will require future investigation. The colonization of Esteya vermicola in host pine tree xylem and the in vivo infection of pinewood nematode by E. vermicola were investigated using the green fluorescence protein transformant. © 2020 Society of Chemical Industry.

Esteya vermicola has been used as an effective biocontrol agent for the management of the pinewood nematode, Bursaphelenchus xylophilus. Tools for monitoring the colonization and parasitism patterns of E. vermicola are required for the development of highly effective biocontrol strategies. Because the TaqMan PCR technique is effective for quantification of species in environmental samples, a real-time PCR-based methodology was developed for absolute quantification of E. vermicola via internal standard addition and extrapolation of DNA quantity to hyphal length. Primers and a probe for the 28S ribosomal RNA gene of E. vermicola were designed, and nested TaqMan real-time PCR-based quantification was performed. In addition, internal standard-based yield measurement was correlated to the absolute quantity of target genomic DNA. Moreover, an extrapolation curve obtained by optical microscopy and image analysis of the mycelia was constructed for the measurement of fungal hyphal length. The absolute quantification method developed in the present study provides a sensitive and accurate technique to quantify fungal density in either wood or other substrate samples and can be used as an effective tool for future studies of biocontrol agents.

Esteya vermicola is an endophytic fungus of pine wood nematode(s) (PWN) Bursaphelenchus xylophilus and a promising biocontrol agent. However, until now, only the lunate spores of E. vermicola have been demonstrated to be highly effective against pine wilt disease. In the present study, blastospores of E. vermicola were successfully used as a biocontrol agent through injection into large pine trees in Aewol-eup, Jeju, South Korea. When pine trees were injected with a 6–8 mL blastospore suspension of E. vermicola (108 mL−1) 1 month before inoculating them with PWN, 73.77% of the trees remained alive after one year. When injected with blastospores of E. vermicola 2 and 4 weeks after PWN infection, 40% of the trees were saved. As a control, the pine trees inoculated with PWN only all died. Moreover, a hypothesis of the mechanism of biocontrol within the pine tree was proposed based on the population density of PWN over time along with the presence of E. vermicola in the pine tree. The hypothesis involved a cycle that included the growth of E. vermicola without PWN infection, attraction and adhesion to PWN, movement and death of infected PWN, production of a second generation of lunate spores, and attraction of other migratory PWN.

ABSTRACTThe present study evaluated the protective effects of the nematophagous fungus Esteya vermicola on the large pine trees of Mt. Wora, Jinju, South Korea for six years. When pine trees were treated with E. vermicola 110 days before artificial normal pinewood nematode (PWN) infection, 30–50% of the trees survived for six years. When pine trees were treated with E. vermicola one week after artificial normal PWN infection, 40% of the trees were saved. In contrast, all of the control trees were killed by pine wilt disease in the first year. Although it has been more than six years since the beginning of this experiment, the existence of E. vermicola inside the treated pine trees was successfully detected using a PCR method with two pairs of specific primers for E. vermicola. These results suggest that E. vermicola possesses great potential as a biocontrol agent to combat the disastrous pine wilt disease. This is the first report of using nematophagous fungi to control pine wilt disease in the field for a duration of over five years.

Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly understood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fungal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphological features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed similarity in large number of transporters and secondary metabolite coding genes. Noteworthy, expanded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theoretical foundation for further research and application.

Symbioses have played pivotal roles in biological, ecological, and evolutionary diversification. Symbiotic bacteria affect the biology of hosts in a number of ways. Esteya vermicola, an endoparasitic nematophagous fungus, has high infectivity in the pine wood nematode (PWN), which causes devastating ecological damage and economic losses in Asia and Europe. An integration of molecular, phylogenetic, and morphological analyses revealed that surface-sterilized E. vermicola with septate hyphae from different geographic locations harbor bacterial endosymbionts. 16S rRNA gene sequences from four fungal strains all clustered in a well-supported monophyletic clade that was the most closely related to Pseudomonas stutzeri and affiliated with Gammaproteobacteria. The existence and intracellular location of endobacteria was revealed by fluorescent in situ hybridization (FISH). Our results showed that endobacteria were coccoid, vertically inherited, as yet uncultured, and essential symbionts. Ultrastructural observations indicated that young and old endobacteria differed in cell size, cell wall thickness, and the degree of reproduction. The results of the present study provide a fundamental understanding of the endobacteria inside E. vermicola and raise questions regarding the impact of endobacteria on the biology, ecology, and evolution of their fungal host.

The complete mitochondrial genome of the Nematophagous fungus Esteya vermicola CBS 115803 was determined using the PacBio RS II sequencing technology. The circular molecule is 47,282bp in length with a GC content of 24.85%. Annotated genes including 14 conserved protein-coding genes, the large and the small rRNA subunit (rnl and rns) and 27 tRNAs. The phylogenetic analysis showed that E. vermicola had close genetic relationship with the genus Sporothrix.

Esteya vermicola (Ophiostomataceae) is an endoparasitic fungus that has great potential as a biological control agent against the pinewood nematode Bursaphelenchus xylophilus which causes pine wilt disease. We tested E. vermicola for control of pine wilt disease by spraying E. vermicola conidia on artificial wounds on pine seedlings, and the optimum E. vermicola treatment density and application time were also investigated in the greenhouse. The wounds were similar to those made by sawyer beetles (Coleoptera: Cerambycidae). Esteya vermicola treatments significantly increased the survival rate of pine seedlings that were infected by pinewood nematodes. Wounded plants sprayed with 107 CFU/ml E. vermicola had a 73.0% greater survival rate than nonwounded pine seedlings treated similarly. The treatment of pine seedlings with 107 CFU/ml E. vermicola 14 d before nematode infection increased their survival rate by 90.0%. The number of pinewood nematodes isolated from dead pine seedlings sprayed with E. vermicola was 76% less than the number of pinewood nematodes in the controls. Moreover, infected nematodes and the hyphae of E. vermicola were detected in the dead or wilting pine seedlings. Therefore, spraying E. vermicola on the wounds of pine seedlings made by sawyer beetles provides good control of the pine wilt disease that is caused by pinewood nematodes.