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.
Keywords: Esteya vermicola, pinewood nematode, long-term growth, conidiation, amino acids, stress tolerance
1. Introduction
Pine wilt disease (PWD), caused by the pinewood nematode [Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle], presents a significant threat to pine forests globally. The nematode is primarily transmitted by insect vectors, such as the pine sawyer beetle (Monochamus spp.) (Coleoptera: Cerambycidae) [1]. When these beetles feed or oviposit on pine trees, the nematodes emerge from the insect body and invade the host through wounds created by the insect [2]. Once inside the tree, the nematodes proliferate and migrate mainly through resin canals [3]. Their feeding activity and mechanical damage to plant tissues trigger host defense responses, leading to abnormal resin secretion, vessel blockage, and ultimately disruption of water and nutrient transport [3]. As a result, affected trees wither rapidly and die within a short period, causing extensive mortality in pine stands. Due to its fast spread and high lethality, PWD has become one of the most challenging forest diseases worldwide and the PWN is listed as a quarantine pest in many countries [4,5].
Physical control has long been a traditional method for limiting the spread of PWD. Measures such as felling and destroying diseased trees and using traps to reduce vector populations can lower pathogen and insect densities and slow disease progression [6]. However, these methods are costly, labor-intensive, and logistically challenging in large or topographically complex forest areas. Hence, physical control alone is insufficient for sustainable management [7]. Chemical control, including spraying nematicides and injecting systemic insecticides into tree trunks, is currently the most widely used strategy [6]. Although these methods can quickly reduce nematode and vector populations, their long-term and large-scale use has caused serious problems, including harm to non-target organisms and the development of resistance in nematodes and beetles [6,7].
Given these drawbacks, biological control has gained increasing attention as an environmentally friendly and sustainable alternative. Esteya vermicola, an endoparasitic fungus specifically targeting B. xylophilus, is considered a promising biocontrol agent due to its strong infection capability [8]. At least eight strains have been identified, among which CBS115803 is the most extensively studied [8,9]. A key morphological characteristic of E. vermicola is the production of two distinct types of conidia: lunate and bacilloid. Only lunate conidia are infective to the pinewood nematode [10]. Therefore, both the yield and viability of lunate conidia are critical indicators in evaluating its biocontrol potential [11].
Previous studies have shown that E. vermicola achieves high infection rates, causing rapid nematode mortality. For example, pretreatment with the fungus prior to inoculation with the pine wood nematode increased the survival rate of pine seedlings to 90.0% [12]. Moreover, E. vermicola can adhere to beetle cuticles, providing sustained control effects [13]. In addition, E. vermicola releases volatile organic compounds such as α-pinene, β-pinene, and camphor, which mimic the odors emitted by host pines and effectively attract nematodes toward fungal hyphae or conidia, thereby increasing infection opportunities [14]. The surface of lunate conidia is coated with adhesive substances that strongly adhere to the nematode cuticle [15]. Upon adhesion, lunate conidia germinate and form infection pegs that penetrate the cuticle through mechanical pressure and enzymatic degradation, typically within 18–24 h. Once inside the nematode body cavity, fungal hyphae rapidly grow and consume host nutrients, eventually killing the nematode. Following host death, E. vermicola hyphae emerge from the cadaver, form new conidiophores, and produce both lunate and bacilloid conidia, initiating a new infection cycle [9].
The nutritional composition of the culture medium strongly affects E. vermicola growth and conidiation. On nutrient-poor media such as minimal medium (MM: 7.4 mM KH2PO4, 2 mM MgSO4·7H2O, 6.7 mM KCl, 23.5 mM NaNO3, 58.4 mM sucrose) and water agar (15 g/L agar), hyphal growth and conidiation are limited, with strain CBS115803 producing mainly bacilloid conidia (86%). In contrast, nutrient-rich media like potato dextrose agar (PDA: potato 200 g, glucose 20 g, agar 15 g/L) and complete medium (CM: 18.8 mM yeast extract, 29.2 mM sucrose, 11.1 mM casein hydrolysate) promote vigorous growth and up to 95% lunate conidia [10]. Supplementing PDA with glycine, L-leucine, or ammonium nitrate further enhances growth, adhesion, and nematode mortality [16]. Arginine supplementation also stimulates lunate conidia formation via its metabolic pathway [10].
Studies have shown that culture conditions and formulation processes significantly affect the conidia yield and biocontrol efficacy of E. vermicola. Factors such as carbon-to-nitrogen ratio, pH, temperature, and water activity determine mycelial growth and conidiation, while appropriate conditions enhance conidia viability and infectivity against B. xylophilus [17]. Solid-state fermentation using rice or substrates composed of wheat bran, corn flour, and soybean flour can produce highly active conidia with strong resistance to heat and ultraviolet radiation [11]. Moreover, alginate-clay encapsulation combined with stabilizing additives (e.g., glycerol, yeast extract) effectively prolongs shelf life while maintaining viability [18].
Amino acids, as nitrogen sources and regulatory signaling molecules, play a significant role in the growth, development, and metabolism of fungi. Studies have demonstrated that basic amino acids such as arginine and asparagine can promote biomass accumulation in Rhizomucor pusillus AUMC 11616.A and Mucor circinelloides AUMC 6696.A, while cysteine may exhibit inhibitory effects [19]. Supplementing PDA with glycine, L-leucine, or ammonium nitrate further enhances fungal growth, adhesion, and nematode mortality [16]. Compared to single nitrogen sources, the synergistic use of amino acid combinations optimizes fungal cultivation. For instance, a combination of lysine, β-alanine, arginine, and glutamic acid can restore the vitality of degenerated strains and increase spore production by up to five times [20]. Our research also reveals that arginine plays a crucial role in the growth and virulence of the parasitic fungus E. vermicola, significantly promoting mycelial growth, conidia formation, and enhancing the infectivity of lunate conidia, particularly by increasing the proportion of lunate conidia [10]. Therefore, optimizing culture media is essential for the efficient production and application of E. vermicola. The aim of this study was to screen and optimize amino acid combinations for the long-term cultivation and conidium production of E. vermicola CBS115803, providing a foundation for its practical application.
2. Materials and Methods
2.1. Screening of Culture Media
Amino acid screening: Twenty amino acids were individually added to 20 groups of MM solid medium at a final concentration of 100 μg/mL and autoclaved. Each medium (10 mL) was poured into sterile Petri dishes with a diameter of 60 mm. MM without amino acid addition was used as the control, resulting in a total of 21 types of medium. Sterilized toothpicks were used to inoculate E. vermicola mycelial blocks at the center of each plate. The plates were sealed, inverted, and incubated at 25 °C for 14 days. Colonies were then rinsed with sterile water to obtain conidia suspensions corresponding to each of the 21 media. Conidia counts were performed using a hemocytometer under a microscope, recording both the total number of conidia and the number of lunate conidia, and calculating the percentage of lunate conidia. Each experiment was repeated five times.
Screening of optimal amino acid concentrations: Based on the initial screening results, four amino acids (arginine, histidine, glutamine, and proline) were further tested at three concentration gradients (100 μg/mL, 200 μg/mL, and 400 μg/mL). After autoclaving, MM media containing these concentrations were poured into plates. Sterilized toothpicks were used to inoculate E. vermicola mycelial blocks at the center. Plates were sealed, inverted, and incubated at 25 °C for 14 days. Colonies were rinsed with sterile water to obtain conidia suspensions. Conidia counts were performed microscopically using a hemocytometer, and both growth performance and conidia proportions were analyzed.
2.2. Combination Screening of Amino Acids
Arginine, histidine, glutamine, and proline were individually added to MM at the optimal concentration (400 μg/mL) determined in the previous step. In addition, considering the distinct advantages of arginine and histidine in promoting lunate conidia and total conidia, respectively, the arginine-histidine combination (AH) was prepared, along with the combination of the four most advantageous amino acids, arginine-histidine-glutamine-proline (AHGP). MM without amino acids served as the control. After autoclaving, 5 mL of medium was poured into Petri dishes. Mycelial plugs of E. vermicola CBS115803 were inoculated at the center of each plate using sterile toothpicks. Plates were sealed with parafilm, inverted, and incubated at 25 °C for 14 days. Colonies were rinsed with sterile water to prepare conidia suspensions, which were observed under a microscope to count total conidia and lunate conidia. Each experiment was repeated five times and each repetition included three Petri dishes.
2.3. Analysis of Conidia Germination Under Stress Conditions
To further investigate conidia germination under stress conditions, we tested low temperature (0 °C), high temperature (45 °C), osmotic stress (1 M sorbitol), oxidative stress (0.025% H2O2), and UV irradiation. Wild-type E. vermicola CBS115803 colonies were cultured on PDA medium for 8 days and then rinsed with sterile MM liquid medium, MM supplemented with 1 M sorbitol, or MM supplemented with 0.025% H2O2. The resulting suspensions were filtered and adjusted to a final concentration of 5 × 106 conidia/mL. To evaluate germination under stress conditions, six treatments were applied. Untreated conidia suspension served as the control and was inoculated into MM liquid medium containing different amino acid conditions, including MM alone, MM supplemented with arginine (Arg), histidine (His), glutamine (Gln), proline (Pro), an arginine-histidine combination (AH), and an arginine-histidine-glutamine-proline combination (AHGP), each at a final amino acid concentration of 400 μg/mL. For cold stress, conidia suspensions were incubated at 0 °C for 1 h before inoculation into the same seven media. For heat stress, suspensions were incubated at 45 °C for 1 h, mixed, and divided into seven aliquots prior to inoculation. Ultraviolet stress was simulated by exposing conidia suspensions to UV irradiation for 12 min under a sterile workbench, after which they were inoculated into the same set of media. In addition, suspensions obtained by rinsing colonies with 1 M sorbitol MM or 0.025% H2O2 MM were each divided into seven aliquots and inoculated into the amino acid-containing MM media described above. All cultures were incubated at 25 °C, and conidia germination rates were determined microscopically using a hemocytometer on days 1, 2, 3, and 4.
2.4. Nematode Infection Activity Assay
A suspension of B. xylophilus was prepared by mixing nematodes with streptomycin and penicillin in an 8:1:1 ratio. The treated nematodes were then inoculated onto Botrytis cinerea T7 mycelium-rich medium. Sterile water (100 μL) was sprayed onto the Petri dish lids, and after the nematodes had consumed the fungal mycelia, they accumulated in the water droplets on the lids. The plates were sealed, inverted, and incubated in the dark at 25 °C for 5 days. Nematodes were carefully collected from the droplets using pipette tips with trimmed ends to obtain a nematode suspension for subsequent assays.
For infection assays, colonies of wild-type E. vermicola CBS115803 were cultured on PDA medium for 8 days, rinsed with sterile water, and the resulting conidia suspension was filtered and adjusted to a final concentration of 5 × 106 conidia/mL. Aliquots (20 μL) of the conidia suspension were evenly spread with a sterile spreader onto seven types of solid MM media (MM, MM + Arg, MM + His, MM + Gln, MM + Pro, MM + AH, and MM + AHGP), serving as the experimental group. For the control group, 20 μL of sterile water was spread onto the same seven media without conidia suspension. All plates were sealed and incubated at 25 °C for 5 days. Subsequently, 20 μL of nematode suspension (approximately 300 nematodes) was evenly applied onto each plate, with five biological replicates per treatment. Nematode mortality was then recorded daily from day 1 to day 5 under an inverted microscope.
2.5. Long-Term Culture on Solid Media
Five types of solid media (PDA, CM, MM, MM + AH, and MM + AHGP) were prepared, and approximately 5 mL of each medium was poured into 90 mm Petri dishes. After cooling, blocks of E. vermicola CBS115803 were inoculated onto the center of each plate using sterilized toothpicks. The plates were then incubated for two months under standard culture conditions. Mycelial growth was periodically examined using a Nikon stereomicroscope (Nikon Corporation, Tokyo, Japan).
2.6. FDA/PI Staining for Hyphal Viability Assessment
The viability of E. vermicola hyphae was evaluated using fluorescein diacetate (FDA) and propidium iodide (PI) double staining [21]. Fresh hyphae were harvested and washed twice with sterile phosphate-buffered saline (PBS). The samples were then incubated with FDA (final concentration of 10 μg/mL) and PI (final concentration of 5 μg/mL) in the dark at room temperature for 15 min. After staining, the hyphae were observed under a fluorescence microscope. Viable hyphae exhibiting esterase activity emitted green fluorescence due to FDA hydrolysis, whereas non-viable hyphae with compromised cell membranes were stained red by PI. The proportion of live and dead hyphae was determined based on fluorescence signals. The hyphal was observed under a fluorescence microscope (DM3000, Leica, Wetzlar, Germany).
2.7. RNA-Sequencing and Transcriptome Analysis
The E. vermicola CBS115803 strain was maintained on complete medium, both supplemented with 1.5% agar, and incubated for eight days. Following cultivation, colonies were gently rinsed with sterile ddH2O, and both lunate and bacilloid conidia were collected. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA yield and integrity were examined with a NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA).
Poly(A)+ mRNA was isolated from the total RNA using oligo(dT)-conjugated magnetic beads and fragmented in Illumina (San Diego, CA, USA) fragmentation buffer under elevated temperature. First-strand cDNA synthesis was performed with random hexamer primers and SuperScript II, followed by second-strand synthesis using DNA polymerase I in the presence of RNase H. DNA ends were polished to generate blunt termini, then adenylated at the 3′ ends prior to adaptor ligation. Size selection (400–500 bp) was carried out with the AMPure XP system (Beckman Coulter, Brea, CA, USA), and the adaptor-ligated products were amplified in 15 PCR cycles with Illumina primers. After purification, library concentration and insert size distribution were assessed with an Agilent Bioanalyzer 2100 (Santa Clara, CA, USA). Sequencing was performed on the NovaSeq 6000 platform (Illumina) by Shanghai Personalbio Technology Co., Ltd. (Shanghai, China). Clean reads were filtered from raw data and aligned to the E. vermicola reference genome.
Gene expression levels were normalized and quantified with DESeq2. Differentially expressed genes (DEGs) were identified under the criteria |log2 fold change| ≥ 1 and p < 0.05. GO functional categorization and KEGG pathway enrichment were then conducted to interpret the biological roles of the DEGs.
2.8. Statistical Analysis
All experimental data are presented as mean ± standard deviation (SD). Significant differences were determined by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test for multiple comparisons using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA).
3. Result
3.1. Rich Nutrient Media Induce Early Necrosis in E. vermicola
Commonly used media for fungal culture include PDA, CM, and MM. However, we found that E. vermicola CBS115803 exhibited necrosis when cultured on nutrient-rich PDA and CM media. On CM medium, colony necrosis began at the center after approximately 7 days of incubation, and by 18 days the colonies were almost completely necrotic. Similarly, colonies grown on PDA also showed central necrosis by this stage. In contrast, necrosis was less pronounced on the nutrient-poor MM. However, hyphal growth was sparse and insufficient to support high mycelial biomass or conidia production (Figure 1A,B). These findings showed that conventional media were insufficient for maintaining abundant and long-term growth of E. vermicola CBS115803, limiting its potential as a biocontrol agent.
Figure 1.
Growth of Esteya vermicola CBS115803 on PDA (potato dextrose agar), CM (complete medium), and MM (minimal medium) media after 18 days of incubation (A). Mycelia were stained with FDA/PI to assess cell viability (B). Bright-field images and the corresponding fluorescence images of FDA (viable cells, green) and PI (non-viable cells, red) were acquired. Green arrows indicate viable mycelial segments on PDA medium, whereas red arrows indicate dead mycelial segments.
3.2. Effects of Amino Acid Supplementation on Mycelial Growth and Conidia Production
To improve growth, we supplemented MM with 20 kinds of L-type amino acids, including glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. In all cases, E. vermicola exhibited enhanced mycelial growth and increased hyphal density compared to unsupplemented MM (Figure 2A). Total conidia production was also significantly elevated, with histidine supplementation yielding the highest conidia count, up to 2.1 × 107 CFU/mL (Figure 2B).
Figure 2.
Effects of amino acid supplementation on E. vermicola CBS115803 mycelial growth and conidiation. (A) Colony morphology on MM (minimal medium) with different amino acids after 7 days of incubation. (B) Total conidia count after 14 days. (C) Percentage of lunate conidia in MM with amino acid supplementation. Statistical significance was evaluated by comparing the amino acid–supplemented media with MM. Data are presented as mean ± SD (Standard Deviation) (n = 6). Error bars indicate standard deviation. ns, not significant (p > 0.05); ** and **** indicate statistical significance at p < 0.01 and p < 0.0001, respectively.
The proportion of lunate conidia varied depending on the amino acid added. Among non-polar amino acids, only proline markedly increased the lunate conidia percentage (22.4%) compared with the control (9.8%), whereas other non-polar amino acids (Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tryptophan, Methionine) resulted in a decrease. Among polar uncharged amino acids, only glutamine supplementation led to a higher lunate conidia percentage than the control; supplementation with serine or asparagine showed no significant differences, while glycine, threonine, cysteine, and tyrosine led to reductions. For polar charged amino acids, responses varied: arginine significantly increased the lunate conidia percentage to 51.4%, lysine and aspartic acid had no significant effects, whereas histidine and glutamic acid reduced the proportion of lunate conidia (Figure 2C).
3.3. Effects of Amino Acid Combinations on Mycelial Growth and Conidiation of E. vermicola
Among the two types of conidia produced by E. vermicola, only lunate conidia are capable of infecting pinewood nematodes, making the proportion of lunate conidia a critical indicator for evaluating culture media. Considering both mycelial growth and conidiation, four amino acids (arginine, histidine, glutamine, and proline) were selected as candidates for subsequent combination assays. At a concentration of 400 μg/mL in MM, all four amino acids significantly increased the proportion of lunate conidia. However, for arginine, histidine, and proline, higher concentrations were associated with reduced total conidia numbers. Based on the combined evaluation of mycelial growth, total conidia count, and lunate conidia percentages, 400 μg/mL was chosen as the working concentration for further experiments (Figure S1).
To balance total conidia production and lunate conidia proportion, arginine and histidine were tested in combination (AH), along with a four-amino-acid combination of arginine, histidine, glutamine, and proline (AHGP). Seven media were evaluated: MM, MM + Arg, MM + His, MM + Gln, MM + Pro, MM + AH, and MM + AHGP. All amino acid-supplemented media supported better mycelial growth than MM alone (Figure 3A). In terms of conidiation, MM + His yielded the highest total conidia count but with a relatively low lunate conidia percentage. In contrast, both MM + Arg and MM + AHGP showed the highest lunate conidia proportions, while MM + AHGP produced nearly twice as many total conidia as MM + Arg. Taken together, these results suggest that MM + AHGP is the most effective formulation for enhancing conidiation and lunate conidia production (Figure 3B).
Figure 3.
Effects of amino acid combinations on E. vermicola CBS115803 growth and conidiation. (A) Colony morphology on MM (minimal medium), MM + Arg, MM + His, MM + Gln, MM + Pro, MM + AH, and MM + AHGP after 14 days of incubation. (B) Total conidia count and lunate conidia percentages. (C) Growth of E. vermicola after 2 months of incubation on PDA, CM, MM, MM + AH, and MM + AHGP. Data are presented as mean ± SD (n = 6). Statistical significance was evaluated by comparing the amino acid–supplemented media with MM. Error bars indicate standard deviation. *** and **** indicate statistical significance at p < 0.001 and p < 0.0001, respectively. AHGP represents the four amino acids: Arg, His, Gln, and Pro, respectively.
To further assess whether amino acid combinations promote long-term culture, E. vermicola was grown on PDA, CM, MM, MM + AH, and MM + AHGP for two months. Colonies on PDA and CM underwent complete necrosis, confirming that these nutrient-rich media are unsuitable for long-term maintenance. Colonies grown on MM showed sparse hyphae and partial darkening, indicating limited preservation capacity. By contrast, colonies on MM + AH and MM + AHGP remained viable, with MM + AHGP showing more abundant mycelial growth and significantly larger colony diameters than MM + AH (Figure 3C). FDA/PI staining further confirmed the viability status of the hyphae (Figure S2). These results demonstrate that MM + AHGP medium provides superior conditions for both short-term conidiation and long-term preservation of E. vermicola CBS115803.
3.4. Amino Acid Supplementation Enhances Conidia Germination of E. vermicola
To evaluate germination capacity, conidia of E. vermicola CBS115803 were prepared from seven media: MM, MM + Arg, MM + His, MM + Gln, MM + Pro, MM + AH, and MM + AHGP. Under standard conditions (25 °C), conidia from MM (control) showed a significantly lower germination rate (5.7%) compared with all amino acid-supplemented treatments. In particular, conidia from MM + Arg, MM + His, MM + AH, and MM + AHGP showed significantly high on rates (p < 0.05), with MM + AHGP consistently exhibiting the highest performance (Table 1).
Table 1.
The Effect of Different Treatments and Culture Media on Conida Germination of E. vermicola CBS115803.
| Treatment Methods | MM | MM + Arg | MM + His | MM + Gln | MM + Pro | MM + AH | MM + AHGP |
|---|---|---|---|---|---|---|---|
| 25 °C | 5.70 ± 0.14 e | 8.50 ± 0.41 c | 9.94 ± 0.44 b | 7.74 ± 0.42 d | 7.84 ± 0.16 cd | 10.12 ± 0.34 b | 12.51 ± 0.74 a |
| 0 °C Treatment | 16.42 ± 0.39 e | 17.98 ± 0.33 d | 22.21 ± 0.61 c | 18.37 ± 0.49 b | 18.53 ± 0.50 b | 24.04 ± 0.20 c | 25.31 ± 0.27 a |
| 45 °C Treatment | 4.44 ± 0.41 e | 7.19 ± 0.22 bd | 7.60 ± 0.15 b | 7.03 ± 0.28 d | 7.61 ± 0.08 b | 8.60 ± 0.20 b | 10.11 ± 0.41 a |
| Sorbitol Treatment | 4.61 ± 0.54 e | 7.01 ± 0.05 c | 6.47 ± 0.45 cd | 6.41 ± 0.34 d | 6.44 ± 0.28 cd | 7.64 ± 0.36 b | 8.59 ± 0.36 a |
| UV Treatment | 4.44 ± 0.30 e | 7.75 ± 0.57 c | 6.53 ± 0.12 d | 6.72 ± 0.37 d | 6.35 ± 0.10 d | 8.80 ± 0.32 b | 10.60 ± 0.32 a |
| H2O2 Treatment | 4.73 ± 0.42 f | 6.45 ± 0.25 e | 7.71 ± 0.45 c | 7.77 ± 0.17 c | 7.04 ± 0.20 d | 9.22 ± 0.34 b | 10.24 ± 0.21 a |
Data in the table represent the percentage of conidia germination (%) and are presented as mean ± SD (n = 6). Statistical significance was set at p < 0.05, and different lowercase letters (a–f) denote significant differences at this level (one-way ANOVA followed by Duncan’s multiple range test). AHGP represents the four amino acids: Arg, His, Gln, and Pro, respectively.
Across all stress treatments, the germination pattern was similar to that observed at 25 °C: amino acid supplementation enhanced germination rates compared with MM alone, with MM + AHGP yielding the highest values under each condition. Unexpectedly, conidia pretreated at 0 °C exhibited markedly higher germination rates than under any other condition, with a 2- to 4-fold increase across all media types (Table 1).
Transcriptome analysis of conidia treated at 0 °C and 25 °C showed that a total of 1220 differentially expressed genes (DEGs) were identified, including 497 upregulated and 723 downregulated genes. KEGG pathway analysis revealed that upregulated genes were enriched in protein synthesis, signal transduction, transcription factor activity, and lipid metabolism pathways, whereas downregulated genes were primarily involved in amino acid metabolism, carbohydrate metabolism, and vitamin metabolism. The downregulated genes were primarily involved in routine metabolic functions, whereas the upregulated genes included regulators of signaling and transcription (Figure S3).
3.5. Effects of Amino Acid Combinations on the Nematode Infection Ability of E. vermicola
On MM, pinewood nematode mortality reached 84.67% by day 4 post-infection with E. vermicola. Previous studies have indicated that the infection efficiency of E. vermicola varies depending on the culture medium; therefore, we compared the effects of single amino acids (Arg, His, Gln, Pro) and amino acid combinations on nematode infection capacity. By day 4 post-infection, nematode mortality rates for E. vermicola cultured on MM supplemented with arginine, histidine, glutamine, and proline were 90.67%, 87.67%, 84.67%, and 80.67%, respectively. Compared with the MM control, arginine and histidine significantly enhanced infection capacity, whereas proline reduced it. The AH (arginine and histidine) combination resulted in a mortality rate of 91.00%, slightly higher than either amino acid alone. Strikingly, when arginine, histidine, glutamine, and proline were combined (AHGP), the nematode mortality reached 98.33% (Figure 4). These findings demonstrate that supplementation with all four amino acids synergistically enhances the infection capacity of E. vermicola CBS115803, yielding superior results compared with single or dual amino acid treatments.
Figure 4.
Amino acid supplementation significantly enhances the infection capacity of E. vermicola against pinewood nematodes. Statistical significance was evaluated by comparing the amino acid–supplemented media with MM (minimal medium) medium. Data are presented as mean ± SD (n = 6). Error bars indicate standard deviation. ns, not significant (p > 0.05); *, **, ***, and **** indicate significance levels at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Results were recorded at 4 days post-inoculation. AHGP represents the four amino acids: Arg, His, Gln, and Pro, respectively.
4. Discussion
PDA and CM are nutrient-rich media commonly used in fungal research and have also been frequently applied for the cultivation of E. vermicola CBS115803 under laboratory conditions. However, we found that such highly nutritious media (e.g., PDA or protein- and nitrogen-rich combinations) often led to colony degeneration or even autolysis of E. vermicola over prolonged culture periods. This phenomenon is consistent with previous observations in certain filamentous fungi, where initially vigorous growth under high amino acid or protein concentrations is followed by hyphal aggregation and death at later stages [22].
Our previous work has demonstrated that amino acids play a crucial role in regulating hyphal growth and conidiation in E. vermicola. Based on this, we supplemented a MM with various amino acids to identify conditions that could sustain long-term solid culture of E. vermicola while maintaining high conidial yield and quality, including a high proportion of lunate conidia and improved pathogenicity. The results showed that all tested amino acids promoted, to some extent, mycelial growth and total conidia production. However, with respect to lunate conidial formation, only arginine, glutamine, and proline significantly enhanced the proportion of lunate conidia, while other amino acids even caused a reduction. Similar patterns have been reported in Alternaria species, where aspartate and glutamate stimulated spore germination, whereas cysteine, tryptophan, and phenylalanine inhibited it [23]. These findings suggest that amino acids function not only as nitrogen or carbon sources but also as signaling molecules that influence fungal differentiation and germination pathways.
Further investigation revealed that supplementation with a mixture of four amino acids AHGP (arginine, histidine, glutamine, and proline) resulted in better hyphal growth than any single amino acid treatment. After two months of cultivation, colonies grown on MM + AHGP remained active and vigorous, whereas those on PDA and CM were completely degenerated. This indicates that MM + AHGP is an optimal solid medium for E. vermicola, suitable for sustained growth and large-scale production of viable conidia for biological control of the pinewood nematode.
For field application in pinewood nematode control, E. vermicola CBS115803 must be prepared as conidia suspensions and injected directly into tree trunks, or supplied continuously via infusion bags for large trees. Thus, the germination capacity and stress tolerance of conidia in suspension are critical determinants of their biocontrol efficiency. Interestingly, under stress conditions (heat, cold, osmotic stress, and oxidative stress), the germination rate of conidia from the MM + AHGP group was significantly higher than that of the MM control, the single amino acid-supplemented media, and even MM + AH. Similarly, other studies have shown that E. vermicola exhibits enhanced stress resistance, including UV and drought tolerance, when cultured with glycine, L-leucine, or ammonium nitrate [16]. Unexpectedly, across all culture conditions, conidia germination after 0 °C treatment was higher than at 25 °C. Other studies have similarly shown that low temperatures can significantly prolong E. vermicola conidia shelf life while maintaining biocontrol efficacy against B. xylophilus during long-term storage [18]. From a practical standpoint, this finding is beneficial for production and storage of conidia suspension and it can be temporarily stored in refrigeration before use without losing viability. Similar effects have been reported in arbuscular mycorrhizal fungi (AMF), where cold storage at 4 °C improves the germination and colonization of AMF Rhizophagus irregularis DAOM197198 propagules by breaking dormancy and activating enzymes, particularly enhancing hyphal germination and long-type germ tubes [24]. For Claviceps purpurea (Fr.) Tul., cold preconditioning markedly enhanced sclerotial germination, with optimal responses depending on temperature and exposure duration [25]. The downregulated genes were primarily involved in routine metabolic functions, while the upregulated genes were associated with protein synthesis, signal transduction, transcription factor activity, and lipid metabolism pathways.
Moreover, conidia obtained from MM + AHGP medium exhibited the highest infection and mortality rates against the pinewood nematode. This may result from their enhanced viability and germination capacity, leading to more effective penetration and colonization. A comparable trend has been reported in Fusarium oxysporum, where 18 amino acids were systematically tested for their effects on colony growth, conidiation, and secondary metabolism. Acidic amino acids (Asp, Glu) and sulfur-containing amino acids (Cys, Met) inhibited growth and toxin synthesis, whereas histidine, isoleucine, and tyrosine activated fungal growth and metabolism through the TORC1–Tap42 pathway [26]. Our transcriptomic analysis results indicate that the downregulated genes were primarily associated with routine metabolic functions, which may suggest a reduction in the metabolic activity of E. vermicola under low temperature conditions. In contrast, the upregulated genes included regulators of signaling and transcription, and their upregulation may be related to the enhanced conidia germination ability. However, further functional analysis of these genes is required to confirm this association.
In summary, our study identifies MM + AHGP as an optimized solid medium suitable for the long-term cultivation and preservation of E. vermicola CBS115803. This formulation supports sustained mycelial activity, high conidial productivity, enhanced conidia quality, and improved stress tolerance, providing a practical foundation for large-scale production of E. vermicola conidia in pinewood nematode biocontrol applications.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof12020107/s1, Figure S1. Effects of four amino acids (arginine, histidine, glutamine, and proline) on the total conidia count and the proportion of lunate conidia of E. vermicola. Figure S2. Growth of E. vermicola after 2 months of incubation on PDA, CM, MM, MM + AH, and MM + AHGP media. Figure S3. KEGG enrichment results of the transcriptomic analysis of E. vermicola conidia germination under 0 °C/25 °C treatment conditions.
Author Contributions
Conceptualization, X.P. and C.X.; methodology, X.P. and Y.W.; software, X.P. and L.H.; investigation, X.P., Z.C. and T.Y.; resources, P.L. and C.X.; writing—original draft preparation, X.P. and C.X.; visualization, X.P. and J.L.; supervision and funding acquisition, C.X. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was funded by the National Natural Science Foundation of China [32271898].
Footnotes
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References
- 1.Togashi K., Akbulut S., Matsunaga K., Sugimoto H., Yanagisawa K. Transmission of Bursaphelenchus xylophilus between Monochamus alternatus and Monochamus saltuarius through Interspecific Mating Behaviour. J. Appl. Entomol. 2019;143:483–486. doi: 10.1111/jen.12604. [DOI] [Google Scholar]
- 2.Dropkin V.H., Linit M. Pine Wilt-A Disease You Should Know. Arboric. Urban For. 1982;8:1–6. doi: 10.48044/jauf.1982.001. [DOI] [Google Scholar]
- 3.Cardoso J.M.S., Manadas B., Abrantes I., Robertson L., Arcos S.C., Troya M.T., Navas A., Fonseca L. Pine Wilt Disease: What Do We Know from Proteomics? BMC Plant Biol. 2024;24:98. doi: 10.1186/s12870-024-04771-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Li M., Li H., Ding X., Wang L., Wang X., Chen F. The Detection of Pine Wilt Disease: A Literature Review. Int. J. Mol. Sci. 2022;23:10797. doi: 10.3390/ijms231810797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Back M.A., Bonifácio L., Inácio M.L., Mota M., Boa E. Pine Wilt Disease: A Global Threat to Forestry. Plant Pathol. 2024;73:1026–1041. doi: 10.1111/ppa.13875. [DOI] [Google Scholar]
- 6.Faria J.M.S., Barbosa P., Vieira P., Vicente C.S.L., Figueiredo A.C., Mota M. Phytochemicals as Biopesticides Against the Pinewood Nematode Bursaphelenchus Xylophilus: A Review on Essential Oils and Their Volatiles. Plants. 2021;10:2614. doi: 10.3390/plants10122614. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Fan Y., Liu L., Wu C., Yu G., Wang Z., Fan J., Tu C. The Effect of Regulating Soil pH on the Control of Pine Wilt Disease in a Black Pine Forest. Forests. 2023;14:1583. doi: 10.3390/f14081583. [DOI] [Google Scholar]
- 8.Wang C.Y., Fang Z.M., Sun B.S., Gu L.J., Zhang K.Q., Sung C.K. High Infectivity of an Endoparasitic Fungus Strain, Esteya Vermicola, Against Nematodes. J. Microbiol. 2008;46:380–389. doi: 10.1007/s12275-007-0122-7. [DOI] [PubMed] [Google Scholar]
- 9.Pires D., Vicente C.S.L., Inacio M.L., Mota M. The Potential of Esteya spp. for the Biocontrol of the Pinewood Nematode, Bursaphelenchus xylophilus. Microorganisms. 2022;10:168. doi: 10.3390/microorganisms10010168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Chen C., Hu Z., Zheng X., Yuan J., Zou R., Wang Y., Peng X., Xie C. The Essential Role of Arginine Biosynthetic Genes in Lunate Conidia Formation, Conidiation, Mycelial Growth, and Virulence of Nematophagous Fungus, Esteya vermicola CBS115803. Pest Manag. Sci. 2024;80:786–796. doi: 10.1002/ps.7809. [DOI] [PubMed] [Google Scholar]
- 11.Zhu Y., Mao Y., Ma T., Wen X. Effect of Culture Conditions on Conidia Production and Enhancement of Environmental Stress Resistance of Esteya Vermicola in Solid-State Fermentation. J. Appl. Microbiol. 2021;131:404–412. doi: 10.1111/jam.14964. [DOI] [PubMed] [Google Scholar]
- 12.Wang Z., Zhang Y., Wang C., Wang Y., Sung C. Esteya Vermicola Controls the Pinewood Nematode, Bursaphelenchus Xylophilus, in Pine Seedlings. J. Nematol. 2017;49:86–91. doi: 10.21307/jofnem-2017-048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Wang H.H., Wang Y.B., Yin C., Gao J., Tao R., Sun Y.L., Wang C.Y., Wang Z., Li Y.X., Sung C.K. In Vivo Infection of Bursaphelenchus Xylophilus by the Fungus Esteya vermicola. Pest Manag. Sci. 2020;76:2854–2864. doi: 10.1002/ps.5839. [DOI] [PubMed] [Google Scholar]
- 14.Lin F., Ye J., Wang H., Zhang A., Zhao B. Host Deception: Predaceous Fungus, Esteya vermicola, Entices Pine Wood Nematode by Mimicking the Scent of Pine Tree for Nutrient. PLoS ONE. 2013;8:e71676. doi: 10.1371/journal.pone.0071676. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Wang R., Dong L., Chen Y., Qu L., Wang Q., Zhang Y. Esteya Vermicola, a Nematophagous Fungus Attacking the Pine Wood Nematode, Harbors a Bacterial Endosymbiont Affiliated with Gammaproteobacteria. Microbes Environ. 2017;32:201–209. doi: 10.1264/jsme2.ME16167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Wang Z., Wang C.Y., Gu L.J., Wang Y.B., Zhang Y.A., Sung C.K. Growth of Esteya Vermicola in Media Amended with Nitrogen Sources Yields Conidia with Increased Predacity and Resistance to Environmental Stress. Can. J. Microbiol. 2011;57:838–843. doi: 10.1139/w11-068. [DOI] [PubMed] [Google Scholar]
- 17.Xue J.J., Zhang Y.G., Wang C.Y., Wang Y.Z., Hou J.G., Wang Z., Wang Y.B., Gu L.J., Sung C.K. Effect of Nutrition and Environmental Factors on the Endoparasitic Fungus Esteya vermicola, a Biocontrol Agent Against Pine Wilt Disease. Curr. Microbiol. 2013;67:306–312. doi: 10.1007/s00284-013-0365-y. [DOI] [PubMed] [Google Scholar]
- 18.Xue J.J., Hou J.G., Zhang Y.A., Wang C.Y., Wang Z., Yu J.J., Wang Y.B., Wang Y.Z., Wang Q.H., Sung C.K. Optimization of storage condition for maintaining long-term viability of nematophagous fungus Esteya vermicola as biocontrol agent against pinewood nematode. World J. Microbiol. Biotechnol. 2014;30:2805–2810. doi: 10.1007/s11274-014-1704-2. [DOI] [PubMed] [Google Scholar]
- 19.Mohamed H., Awad M.F., Shah A.M., Nazir Y., Naz T., Hassane A., Nosheen S., Song Y. Evaluation of Different Standard Amino Acids to Enhance the Biomass, Lipid, Fatty Acid, and γ-Linolenic Acid Production in Rhizomucor pusillus and Mucor circinelloides. Front. Nutr. 2022;9:876817. doi: 10.3389/fnut.2022.876817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Yang H., Qiu H.-L., Tian L.-Y., Xiao L.-N., Ling S.-Q., Qin C.-S., Xu J.-Z. Amino acids promote the rejuvenation of degenerated Metarhizium anisopliae. Biol. Control. 2024;198:105639. doi: 10.1016/j.biocontrol.2024.105639. [DOI] [Google Scholar]
- 21.Arévalo-Jaimes B.V., Torrents E. Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from Candida parapsilosis. J. Fungi. 2024;10:209. doi: 10.3390/jof10030209. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.White S., McIntyre M., Berry D.R., McNeil B. The Autolysis of Industrial Filamentous Fungi. Crit. Rev. Biotechnol. 2002;22:1–14. doi: 10.1080/07388550290789432. [DOI] [PubMed] [Google Scholar]
- 23.Daigle D.J., Cotty P.J. The Influence of Cysteine, Cysteine Analogs, and Other Amino Acids on Spore Germination of Alternaria Species. Can. J. Bot. 1991;69:2353–2356. doi: 10.1139/b91-296. [DOI] [Google Scholar]
- 24.Liu X., Ye G., Feng Z., Zhou Y., Qin Y., Yao Q., Zhu H. Cold Storage Promotes Germination and Colonization of Arbuscular Mycorrhizal Fungal Hyphae as Propagules. Front. Plant Sci. 2024;15:1450829. doi: 10.3389/fpls.2024.1450829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Uppala S.S., Wu B.M., Alderman S.C. Effects of Temperature and Duration of Preconditioning Cold Treatment on Sclerotial Germination of Claviceps purpurea. Plant Dis. 2016;100:2080–2086. doi: 10.1094/pdis-02-16-0215-re. [DOI] [PubMed] [Google Scholar]
- 26.Deng Y., Wang R., Zhang Y., Li J., Gooneratne R. Effect of Amino Acids on Fusarium oxysporum Growth and Pathogenicity Regulated by TORC1-Tap42 Gene and Related Interaction Protein Analysis. Foods. 2023;12:1829. doi: 10.3390/foods12091829. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.