Bursaphelenchus xylophilus is killed by homologues of 2-(1-undecyloxy)-1-ethanol

2-(1-Undecyloxy)-1-ethanol, monochamol, is a male-produced aggregation pheromone of the Monochamus species, which are efficient vectors of the pine wood nematode (PWN), Bursaphelenchus xylophilus, which cause devastating damage to pines worldwide. The nematicidal activity of synthetic monochamol and its homologues (ROEtOH: R = C7-C13) were investigated to find potential alternatives to the currently used PWN control agents abamectin and emamectin. Compounds with C7-C13 chain length alkyl groups exhibited 100% nematicidal activity at a concentration of 1000 mg/L. At a concentration of 100 mg/L, 2-(1-nonyloxy)-1-ethanol (C9OEtOH), 2-(1-decyloxy)-1-ethanol (C10OEtOH), 2-(1-undecyloxy)-1-ethanol (C11OEtOH), and 2-(1-dodecyloxy)-1-ethanol (C12OEtOH) showed 100% nematicidal activity, but the others showed weaker activities. C11OEtOH showed similar nematicidal activity to abamectin in terms of LD90 values, which were 13.30 and 12.53 mg/L, respectively. However, C9OEtOH, C10OEtOH, and C12OEtOH (LC90 values: 53.63, 38.18, and 46.68 mg/L, respectively) were less effective than C11OEtOH and abamectin. These results indicate that monochamol could be an effective alternative agent against PWN. The relationship of insecticidal and nematicidal activity to different carbon chain lengths in compounds is discussed.

the monochamol and its homologues to find potential alternatives to currently used PWN control agents. This case is the first known instance of B. xylophilus being killed by homologues of 2-(1-undecyloxy)-1-ethanol.

Results and Discussion
The nematicidal activities of 2-(1-alkyloxy)-1-ethanol homologues, undecanol, and abamectin are shown in Table 1. There was a significant difference in nematicidal activities according to chain length. All 2-(1-alkyloxy)-1-ethanol homologues and undecanol showed 100% mortality at a concentration of 1000 mg/L. 2-(1-Alkyloxy)-1-ethanol homologues with C 9 -C 12 chain length in the alkyl group and undecanol exhibited 100% mortality even at a concentration of 100 mg/L. However, other homologues with shorter or longer chain lengths, such as C 7 OEtOH, C 8 OEtOH, and C 13 OEtOH, showed less than 40% of mortality at the same concentration. At a concentration of 10 mg/L, only C 11 OEtOH and undecanol showed more than 80% nematicidal activity. Undecanol has been reported to show nematicidal activity against PWN 23 . Our synthetic monochamol also contained 1% undecanol. The mortality of PWN at 100 ppm of C 11 OEtOH which was containing 1 ppm undecanol was higher than that at 1 ppm of undecanol (40%) ( Table 1). Therefore, the mortality of C 11 OEtOH was mainly due to C 11 OEtOH itself.
Based on the mortality data (Table 1), LC 50 and LC 90 values were calculated to compare the toxicity of 2-(1-alkyloxy)-1-ethanol homologues and undecanol in relation to the number of carbon atoms, along with abamectin, which is in practical use in trunk injection [9][10][11] . The C 11 OEtOH and undecanol showed significantly higher nematicidal activity (13.30 and 15.78 mg/L, respectively) than the rest in terms of LC 90 values, with no significant difference from abamectin (12.53 mg/L) ( Table 2), followed by C 10 EtOH, C 12 OEtOH and C 9 OEtOH. The homologues C 13 OEtOH, C 8 OEtOH, and C 7 OEtOH showed weaker nematicidal activities than the rest.
The nematicidal activity of the test compounds varied with carbon chain length. Alkanols with C 8 -C 11 carbon chain length and alkylamine with C 16 showed significantly higher nematicidal activity against PWN than compounds with shorter or longer carbon chain lengths 23,24 . In this study, too, C 11 OEtOH showed higher nematicidal activity than other 1-(2-alkyloxy)-2-ethanol homologues with shorter or longer carbon chains.
The relationship between the structure of aliphatic carboxylic acids and their toxicity to nematodes has been sparsely reported. Among linear carboxylic acids with C 4 -C 10 carbon atom chains, octanoic acid (C 8 chain length) was the most toxic to two Drosophila species 25 . The authors suggested that its higher toxicity might be linked to its structural characteristics, allowing an easy transfer of the compounds through the insect cuticle. Li et al. suggested that the steric hindrance of longer-chain analogs affects the toxicity of aliphatic isothiocyanates 26 1 Confident limit. 2 Degree of freedom. 3 The same letters in a column are not significantly different.
toxicity of compounds increased when the electron population or electron accessibility (richness) increased 27 . In our study, C 11 OEtOH exerted the most effective nematicidal activity, even though its total length is equivalent to tetradecanol (C 14 OH) which showed weak nematicidal activity in a previous study 23 . The oxy group (-O-) in the structure of C 11 OEtOH possibly contributed to the nematicidal activity. The addition of an oxy group to the structure would increase toxicity by increasing the compound's transferability through insect cuticles. However, the mechanism of nematicidal activity of some homologues of 2-(1-alkyloxy)-1-ethanol remains unknown. The different susceptibilities of invertebrates and vertebrates to these compounds provide an acceptable therapeutic ratio for pesticide use. In order to overcome resistance development of a pest against pesticides, the activities of the compounds must have different modes of action 28 . The mode of action of avermectin is related to the GABA-gated chloride channel 29 . Kang et al. 30 estimated the inhibitory activity of C 11 OH against acetylcholinesterase (AChE) and glutathione S-transferase (GST) of PWN to elucidate the mode of action 30 . Although C 11 OH showed relatively high nematicidal activity, it showed no or little inhibition activity against AChE and GST of PWN 30 . This result suggests that AChE and GST may not be the targets of C 11 OH. For the safe practical use of C11OEtOH as a nematicide, the mode of action of C 11 OEtOH should be investigated.
It is unclear why the pheromone of the Monochamus vector species has strong nematicidal activity against PWN, a parasite of Pinus spp. The reasons for PWN to spread through the Monochamus vector species that produce an aggregation pheromone that can kill them are worthy of future study.
For a trunk injection agent to be effective against PWN, it is necessary for the injected agent to be translocated throughout the tree at an effective concentration. For this purpose, the agent should have adequate water solubility and diffuse to every part of the tree. C 11 OEtOH showed as effective a nematicidal activity as abamectin, which is in practical use as a trunk injection agent. Although C 11 OEtOH has the advantage of being easily synthesized, it shows very low water solubility. Therefore, to develop C 11 OEtOH as a commercial trunk injection against PWN, a water-soluble preparation of C 11 OEtOH should be formulated.
In conclusion, 2-(1-undecyloxy)-1-ethanol (monochamol) can be used as an effective nematicide against pine wood nematode, and water-soluble monochamol formulations are required along with the identification of an effective concentration (dose) for the control of pine wilt disease. Studying the mechanisms of the nematicidal activity of monochamol is also valuable.

Materials and Methods
Collection of pine wood nematodes. PWN

Synthesis of the monochamol and its homologues. 2-(1-Undecyloxy)-1-ethanol (C 11 OEtOH) and
its homologues were synthesized following the method by Loffredo et al. (Fig. 1) 33 . A general procedure followed was; to a 3-necked round-bottom flask, provided with dropping funnel, a reflux condenser, and inlet for nitrogen was added 80 mmole of ethylene glycol (Daejung, Hwasung, Korea). Sodium (23 mmole, Alfa Aesar) was added in small portion to the ethylene glycol with care under vigorous magnetic stirring and the mixture was heated to 60 °C until added sodium dissolved completely. The appropriate 1-bromoalkane (0.20 mmole, Alfa Aesar/ Sigma-Aldrich) was added and the solution was then kept heating at 60 °C for 4-6 h. After cooling and adding water, the solution was extracted with diethyl ether three times. The organic layer was washed with 2N HCl and brine, and dried over MgSO 4 . After the solvent was removed, the residue was subjected to silica gel column chromatography to obtain desired compounds (35% diethyl ether in hexane fraction). The NMR data of synthesized compounds (data not shown) were in good agreement with literature values 19,33 . The MS data are listed below. NMR data are available in Supplementary Information.  were prepared by serial dilution with distilled water containing BFC30 (200 mg/L). The stock solutions were of 1000 ppm, which were then, diluted by 1/10 times in sequence in each step, if a concentration found somewhat effective to the PWN. Undecanol, which was reported to exhibit nematicidal activity 23 was selected as one of the positive controls because of its 1.0% share in the synthetic monochamol. The stock solution of abamectin (1.8%) was 100 ppm (active ingredient: 18 mg/L) which was also serially diluted. Test solutions (1 mL) were introduced into wells of 24-well plates (Falcon, USA). Each well was then inoculated with about 100 mixed stages (male: female: juvenile = 1:1.2:9.5) of PWN in 10 μ L of distilled water. Controls were treated only with BFC30 solutions. The well plates were held under the same conditions as used for nematode colony maintenance. Mortality of nematodes was recorded after 24 h under a stereoscopic microscope. Nematodes were considered as dead if their bodies were straight and when they did not move, even after transferring to clean water. Two to four trials with three to four replicates were performed on different days. Data were pooled for analysis. Statistical analyses. Nematode mortality was corrected using Abbott's formula 34 and corrected mortality was transformed to arcsine square root values for analysis of variance (ANOVA). Treatment means were compared and separated by Tukey-Kramer HSD test. The LC 50 value was estimated by probit analysis with dose-response data. Differences in LD 50 and LD 90 values between treatments were considered significant if the 95% confidence intervals did not overlap. Statistical analyses were performed using JMP ver. 9.0.2 (SAS Institute Inc., Cary, NC, USA). Mean (± SEM) values of untransformed data are reported.