Abstract
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.
Keywords: Biological control, nematophagous fungus, pine wilt disease, resinous compounds
1. Introduction
Symptoms of pine wilt disease were first discovered in the early twentieth century in several areas on Kyushu Island, Japan. Initially, it was believed that insects were responsible for causing the symptoms. However, in 1968, it was confirmed that the cause of pine wilt was a nematode belonging to the genus Bursaphelenchus [1,2]. Further research revealed that the spread of this disease was facilitated by an insect vector, namely the Monochamus beetle [3,4]. After that point, the disease cycle of pine wilt was determined [5]. In Korea, pine wilt disease was first reported in Busan in 1988, and this disease caused severe damage to pine plantations [6]. Several subsequent studies have been conducted on the biology, distribution, virulence, incidence, and severity of Bursaphelenchus nematodes [7].
Esteya vermicola had been identified as a nematophagous fungus in 1999 and this fungus might be a pathogen for Aphelenchida nematodes than Tylenchida nematodes [8,9]. The potential of E. vermicola as a biocontrol agent against the pine wood nematode has been demonstrated [10]. E. vermicola may emit volatile organic compounds (VOCs) similar to those found in host pine trees, which can attract the pine wood nematode [11]. However, the growth and sporulation of E. vermicola can be influenced by various biotic or abiotic factors [10]. Hence, a comprehensive understanding on biological properties of E. vermicola is necessary.
It is known that various resinous compounds from pine trees have already been reported [12–15] and generally have inhibitory effects on the mycelial growth of most fungi [16,17]. Therefore, the interaction between the nematophagous fungus and the host pine tree might affect the parasitic ability of E. vermicola for the pine wood nematode within the tree. In this study, we investigated the effects of 14 resinous compounds on spore germination and mycelial growth of a nematophagous fungus, E. vermicola.
2. Materials and methods
2.1. Preparation of resinous compounds
Detailed information on 14 resinous compounds used in the experiment are provided in Table 1. These compounds were solved in the acetone and used in different experiments.
Table 1.
List of resinous compounds from pine trees used in the experiment.
| No. | Compounds | Purity | Supplier | CAS No. |
|---|---|---|---|---|
| 1 | Pinene, (1 R)-(+)-α-C10H16 | >95 | TCI | 7785-70-8 |
| 2 | Pinene, (1S)-(-)-α- | 99 | Sigma-Aldrich | 7785-26-4 |
| 3 | Camphene, DL-(contains ca. 20% Tricyclene) | >78 | TCI | 79-92-5 |
| 4 | Pinene, (-)-β- | >94 | TCI | 127-91-3 |
| 5 | Myrcene (stabilized with BHT) | >70 | TCI | 123-35-3 |
| 6 | Phellandrene, α- | >65 | TCI | 99-83-2 |
| 7 | Carene, 3- | 95 | Sigma-Aldrich | 13466-78-9 |
| 8 | Terpinene, α- | 85 | Fluka | 99-86-5 |
| 9 | Limonene, (-)- | >95 | TCI | 5989 |
| 10 | Limonene, (R)-(+)- | 97 | Sigma-Aldrich | 5989-27-5 |
| 11 | Terpinolene | >85 | TCI | 586-62-9 |
| 12 | Bornyl acetate (contains ca. 20% isobornyl acetate) | >70 | TCI | 76-49-3 |
| 13 | Caryophyllene, β- | >90 | TCI | 87-44-5 |
| 14 | Humulene, α- | >96 | Sigma-Aldrich | 6753-98-6 |
2.2. Isolation of Esteya vermicola
Pure culture of E. vermicola was obtained from G810® (DaedukBio, Korea), which is a commercial product of spore suspension of E. vermicola. To obtain a single spore isolate, 0.1 mL of suspension was spread on Potato dextrose agar (PDA), and a plug containing growing hyphae from a single spore was transferred on sterile culture medium. The pure culture isolate was confirmed as E. vermicola by phylogenetic analyses based on LSU gene sequence (Figure 1).
Figure 1.
Phylogenetic tree based on neighbor-joining analysis a data set of LSU gene sequence for Esteya vermicola and its close relates. Numbers above or below the nodes represent bootstrap support (1,000 replicates). The isolates obtained in this study are shown in bold.
2.3. Effects of resinous compounds on spore germination of Esteya vermicola
Antifungal activity of each compound against E. vermicola was tested separately. Resinous compounds were added to the artificial culture media of E. vermicola. After solving 100 µL of each resinous compound completely by adding 0.5 mL of acetone as the solvent, 0.1 mL of the solution was added on the surface of the solidified culture media (PDA), and spread uniformly with the sterilized glass rod. After drying the added resinous compounds, the spore suspension of E. vermicola was inoculated on culture media and kept in the dark at 25 °C for five days. Spore germination was measured based on the number of germinated spores and compared with the untreated control. Spore germination inhibition rate (SGIR) was calculated according to the following formula.
where C is the number of germinated spores of E. vermicola in the untreated control plate and T is the number of germinated spores of E. vermicola in the treated plate.
2.4. Effects of resinous compounds on mycelial growth of Esteya vermicola
For investigating the effects of resinous compounds on mycelial growth of E. vermicola, agar disks containing mycelium were taken from agar plate of E. vermicola by using cork borer (5 mm diameter), placed on the center of culture media supplemented with each resinous compound, kept in the dark at 25 °C for 10 days. Mycelial growth was measured in each treatment, and compared with the untreated control. Mycelial growth inhibition rate (MGIR) was calculated using the formula mentioned above, where C is the mycelial growth of E. vermicola in the control plate and T is the mycelial growth of E. vermicola in the treated plate.
2.5. Effects of the mixture of resinous compounds on spore germination and mycelial growth of Esteya vermicola
Afterward, the mixtures of all resinous compounds were used to evaluate the inhibitory ability against the nematophagous fungus. Based on the relative composition ratios [17], all resinous compounds used in this experiment were mixed together by the estimated ratios (Table 2). Major components of resinous substance in the needles of Pinus densiflora and P. thunbergii were α-pinene, β-pinene, (-)limonene, and bornyl acetate, which showed composition ratios as 25.26%, 5.67%, 12.89%, and 5.78%, respectively. Other compounds, β-caryophyllene, myrcene, terpinolene, camphene, and α-humulene also showed 10.7%, 5.14%, 4.3%, 3.7%, and 1.79%, respectively. Among 14 compounds used in the previous experiment, 11 compounds comprised upto 75.23% of total composition in the resinous substance (Table 2). Effects of the mixture on spore germination and mycelial growth of E. vermicola were evaluated by following the methods conducted in the previous experiment.
Table 2.
Composition ratios of the resinous compounds in the mixture of resinous substance.
| No. | Resin compound | Composition ratio | Estimated ratio | Volume (mL) |
|---|---|---|---|---|
| 1 | (+)α-Pinene | 25.26 | 2.5 | 0.25 |
| 2 | (-)α-Pinene | 2.5 | 0.25 | |
| 3 | Camphene | 3.7 | 1 | 0.1 |
| 4 | β-Pinene | 5.67 | 1 | 0.1 |
| 5 | Myrcene | 5.14 | 1 | 0.1 |
| 6 | α-Phellandrene | 0 | 0 | 0 |
| 7 | 3-Carene | 0 | 0 | 0 |
| 8 | α-Terpinene | 0 | 0 | 0 |
| 9 | (-)Limonene | 12.89 | 1.25 | 0.125 |
| 10 | (+)Limonene | 1.25 | 0.125 | |
| 11 | Terpinolene | 4.3 | 1 | 0.1 |
| 12 | Bornyl acetate | 5.78 | 1 | 0.1 |
| 13 | β-Caryophyllene | 10.7 | 2 | 0.2 |
| 14 | α-Humulene | 1.79 | 0.4 | 0.04 |
2.6. Statistics analysis
The following statistical analysis was conducted to determine the significant difference in inhibition rates against E. vermicola among compounds: one-way ANOVA followed by Tukey’s HSD test with a 5% probability level. All the graphs and statistical analyses were performed in IBM SPSS Statistics version 27 for Windows.
3. Results
3.1. Effects of resinous compounds on spore germination of Esteya vermicola
All of the 14 resinous compounds tested showed inhibitory effects on the spore germination of E. vermicola. Among them, (+)α-pinene, (-)α-pinene, β-pinene, and (-)limonene inhibited spore germination completely, and thus SGIR of them were 100%, followed by bornyl acetate (97.5%), α-phellandrene (60.4%), α-terpinene (57.2%), terpinolene (54.2%), β-caryophyllene (52.9%), 3-carene (47.5%), camphene (47.4%), myrcene (37.9%), (+)limonene (37.4%), and α-humulene (36.9%), while the solvent, acetone, showed no inhibition for spore germination. These results indicated that the resinous compounds tested showed strong inhibitory effects on the spore germination in the range from 36.9% to 100%, and averaged 66.4% (Figures 2 and 3).
Figure 2.
Spore germination inhibition rate (SGIR) of resin compounds against Esteya vermicola on PDA containing 0.1 mL of resin compound after incubating in the dark at 25 °C for 5 days. Different letters indicate a significant difference (p < 0.05) among treatments by Tukey’s HSD test.
Figure 3.
Spore germination inhibition (SGI) of resin compounds against Esteya vermicola on PDA containing 0.1 m of resin compounds after incubating in the dark at 25 °C for 5 days.
3.2. Effects of resinous compounds on mycelial growth of Esteya vermicola
All of the fourteen resinous compounds showed inhibitory effects on the mycelial growth of E. vermicola. (+)α-pinene, (-)α-pinene, and (-)limonene inhibited mycelial growth completely, and thus MGIR of them were 100%. MGIRs were followed by β-pinene (97.2%), myrcene (45.8%), α-phellandrene (44.0%), bornyl acetate (36.6%), 3-carene (34.6%), (+)limonene (23.5%), α-humulene (23.5%), α-terpinene (10.3%), camphene (9.4%), and terpinolene (5.8%), while, β-caryophyllene and the solvent, acetone, did not showed no inhibition for mycelial growth. These results indicated that the resinous compounds tested showed comparative inhibition on the mycelial growth in the range from 0% to 100%, and averaged 45.1% (Figures 4 and 5).
Figure 4.
Mycelial growth inhibition rate (MGIR) of resin compounds against Esteya vermicola on WA containing 0.1 mL of resin compound after incubating in the dark at 25 °C for 10 days. Different letters indicate a significant difference (p < 0.05) among treatments by Tukey’s HSD test.
Figure 5.
Mycelial growth of Esteya vermicola on WA containing 0.1 mL of resinous compounds after incubating in the dark at 25 °C for 10 days.
3.3. Effects of mixture of resinous compounds on spore germination and mycelial growth of Esteya vermicola
The mixture of resinous compounds showed very strong inhibition on spore germination and mycelial growth of E. vermicola with 100% and 91.8% SGIR and MGIR, respectively (Figures 6 and 7). These results indicated that resinous compounds from pine trees strongly inhibit spore germination and mycelial growth of E. vermicola. Based on the results by in vitro assay, it could be inferred that spore germination and mycelial growth of trunk-injected spore suspension of E. vermicola into pine trees for the management of pine wilt disease will be greatly inhibited by widely distributed resinous compounds and exuded when the injection holes were made by drilling within pine trees, and thus the expected control efficacy for preventing and curing of pine wood nematode could not be obtained.
Figure 6.
Spore germination inhibition rate (SGIR) and mycelial growth inhibition rate (MGIR) of mixture against Esteya vermicola on WA containing 0.1 mL mixture of resinous compounds after incubating in the dark at 25 °C for 10 days, respectively. *indicates a significant difference (p < 0.05) by two-sample t-test.
Figure 7.
Spore germination of Esteya vermicola on PDA containing 0.1 mL mixture of resinous compounds after incubating in the dark at 25 °C for 5 days.
4. Discussion
Nematicidal mechanism of a nematophagous fungus, E. vermicola, is known for direct contact of germinated hyphae onto the nematode surface, penetration into the body, infection, and killing of the nematode [8,10]. Thus, in order to show nematicidal activity of E. vermicola, the spores in the injected spore suspension should germinate, grow and survive successfully within pine trees. However, plenty of various resinous compounds exist in pine woods and needles, and are exuded when drill holes are made to inject spore suspension into pine trees. Moreover, exudate resinous compounds in pine trees usually inhibit fungal growth by antifungal activity as well as injection of chemicals by physical plugging of entry points [18]. Therefore, trunk injection of pesticides or nutrients into coniferous trees are conducted during winter season, when resin exudation is almost stopped [19]. According to the obtained results, 14 resinous compounds inhibited spore germination of E. vermicola in the range of 36.9% to 100%. Among tested compounds, (+)α-pinene, (-)α-pinene, β-pinene, and (-)limonene showed 100%, bornyl acetate 97.5%, α-phellandrene 60.4%, α-terpinene 57.2%, terpinolene 54.2%, β-caryophyllene 52.9%, 3-carene 47.5%, camphene 47.4%, myrcene 37.9%, (+)limonene 37.4%, and α-humulene 36.9% in SGIR. Resinous compounds also inhibited mycelial growths of E. vermicola in the range of 0% to 100%. (+)α-pinene, (-)α-pinene, and (-)limonene 100%, β-pinene 97.2%, myrcene 45.8%, α-phellandrene 44.0%, bornyl acetate 36.6%, 3-carene 34.6%, (+)limonene 23.5%, α-humulene 23.5%, α-terpinene 57.2%, camphene 47.4%, terpinolene 5.8%. β-caryophyllene and acetone 0% in MGIR.
Since these resinous compounds normally exist as the mixed state in pine wood, spore germination and mycelial growth of E. vermicola were evaluated after making a mixture of resinous compounds according to the composition ratio in pine wood [17]. The results indicated that SGIR and MGIR were 100% and 91.8%, respectively. The mixture of 14 resinous compounds used in this experiment comprised 75.23% of the total resinous compounds in pine wood. It is considered that these high inhibition rates in both spore germination and mycelial growth of E. vermicola are caused by the inhibiting effect of resinous compound, which exist in the mixture at high concentrations. Thus, we can infer that the spores in trunk-injected spore suspension of E. vermicola cannot easily germinate and grow successfully within pine trees, in spite of high spore germination rate and active mycelial growth are prerequisite for the effective control of pine wood nematode in the treated trees. In conclusion, the use of E. vermicola, a biocontrol agent, by trunk injection for preventing or curing pine wilt disease will not be promising in pine trees.
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- 1.Kiyohara T, Tokushige Y.. Inoculation experiments of a nematode, Bursaphelenchus sp., onto pine trees. J Jpn For Soc. 1971;53:210–218. [Google Scholar]
- 2.Tokushige Y, Kiyohara T.. Bursaphelenchus sp. in the wood of dead pine trees. J Jpn For Soc. 1969;51:193–195. [Google Scholar]
- 3.Mamiya Y, Enda N.. Transmission of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae). Nematol. 1972;18(2):159–162. doi: 10.1163/187529272X00395. [DOI] [Google Scholar]
- 4.Morimoto K, Iwasaki A.. Role of Monochamus alternatus (Coleoptera: Cerambycidae) as a vector of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae). J Jpn For Soc. 1972;54:177–183. [Google Scholar]
- 5.Mamiya Y. Pine wilting disease caused by the pine wood nematode, Bursaphelenchus lignicolus, in Japan. Jpn Agric Res Q. 1976;10:206–211. [Google Scholar]
- 6.Shin S-C. Pine wilt disease in Korea. In: Pine wilt disease. Tokyo: Springer; 2008. p. 26–32. [Google Scholar]
- 7.Lee J-P, Sekhon SS, Kim JH, et al. The pine wood nematode Bursaphelenchus xylophilus and molecular diagnostic methods. Mol Cell Toxicol. 2021;17:1–13. [Google Scholar]
- 8.Liou JY, Shih JY, Tzean SS.. Esteya, a new nematophagous genus from Taiwan, attacking the pinewood nematode (Bursaphelenchus xylophilus). Mycol Res. 1999;103(2):242–248. doi: 10.1017/S0953756298006984. [DOI] [Google Scholar]
- 9.Wang X, Wang T, Wang J, et al. Morphological, molecular and biological characterization of Esteya vermicola, a nematophagous fungus isolated from intercepted wood packing materials exported from Brazil. Mycoscience. 2014;55(5):367–377. doi: 10.1016/j.myc.2014.01.002. [DOI] [Google Scholar]
- 10.Chu WH, Dou Q, Chu HL, et al. Research advance on Esteya vermicola, a high potential biocontrol agent of pine wilt disease. Mycol Progress. 2015;14(12):115. doi: 10.1007/s11557-015-1137-9. [DOI] [Google Scholar]
- 11.Lin F, Ye J, Wang H, et al. Host deception: predaceous fungus, Esteya vermicola, entices pine wood nematode by mimicking the scent of pine tree for nutrient. PLoS One. 2013;8(8):e71676. doi: 10.1371/journal.pone.0071676. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kim Y-B, Kim S-M, Kang M-K, et al. Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-d-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes. Tree Physiol. 2009;29(5):737–749. doi: 10.1093/treephys/tpp002. [DOI] [PubMed] [Google Scholar]
- 13.Lee J, Cho KS, Jeon Y, et al. Characteristics and distribution of terpenes in South Korean forests. J Ecol Environ. 2017;41(1):19. doi: 10.1186/s41610-017-0038-z. [DOI] [Google Scholar]
- 14.Moon B-H, Yoo K-S.. Characterization of volatile essential oil from needle leaves by gas chromatography-mass spectrometry. J Korean Soc Environ Anal. 2005;8:181–185. [Google Scholar]
- 15.Yatagai M, Sato T.. Terpenes of leaf oils from conifers. Biochem Syst Ecol. 1986;14(5):469–478. doi: 10.1016/0305-1978(86)90004-9. [DOI] [Google Scholar]
- 16.Kim Y-S, Shin D-H.. Volatile components and antibacterial effects of pine needle (Pinus densiflora S. and Z.) extracts. Food Microbiol. 2005;22(1):37–45. doi: 10.1016/j.fm.2004.05.002. [DOI] [Google Scholar]
- 17.Park J, Lee G.. Volatile compounds and antimicrobial and antioxidant activities of the essential oils of the needles of Pinus densiflora and Pinus thunbergii. J Sci Food Agric. 2011;91(4):703–709. doi: 10.1002/jsfa.4239. [DOI] [PubMed] [Google Scholar]
- 18.Perveen K, Bokhari NA, Iram S, et al. Antifungal activity of essential oil of Commiphora molmol oleo gum resin. J Essent Oil Bear Plants. 2018;21(3):667–673. doi: 10.1080/0972060X.2018.1492975. [DOI] [Google Scholar]
- 19.Berger C, Laurent F.. Trunk injection of plant protection products to protect trees from pests and diseases. Crop Prot. 2019;124:104831. doi: 10.1016/j.cropro.2019.05.025. [DOI] [Google Scholar]