# Amino Acid-Optimized Medium Improves Long-Term Growth, Conidiation, Stress Tolerance, and Biocontrol Performance of Esteya vermicola Against Pinewood Nematode

## TL;DR

A new amino acid-based medium improves the growth and stress tolerance of a fungus used to control a harmful nematode in pine trees.

## Contribution

An amino acid-optimized medium enhances long-term viability and biocontrol performance of Esteya vermicola.

## Key findings

- Histidine, arginine, glutamine, and proline together maximize conidia yield and lunate conidia proportion.
- The MM + AHGP medium maintains long-term colony viability and improves stress tolerance of conidia.
- Low temperatures enhance conidia germination, suggesting activation of dormancy-breaking pathways.

## Abstract

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.