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BACKGROUND 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. RESULTS: 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. CONCLUSION: 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 [ABSTRACT FROM AUTHOR]

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively. [ABSTRACT FROM AUTHOR]

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