JP7076199B2 - How to detect odorous substances - Google Patents

Description

The present invention relates to a method for detecting an odorant.

Pine wilt disease (PWD) is a serious disease (infectious disease) caused in East Asian and European countries, especially Pinus species (pine) in Japan, Portugal, China and South Korea (Pine). Jianghua, 2005 (Non-Patent Document 1); Kiyohara and Tokushige, 1971 (Non-Patent Document 2); Manuel et al., 1999 (Non-Patent Document 3); Moon, et al., 2013 (Non-Patent Document 4)), It is known to be caused by the pine wood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle.
The pine wood nematode is native to North American countries (Dwinell and Nickle, 1989 (Non-Patent Document 5); Dwinell, 1993 (Non-Patent Document 6)), but does no harm in the country of origin (Linit, 1988 (Non-Patent Document 6)). Document 7)). However, Pinus massoniana is pathogenic to Asian or European pine such as Japanese red pine, Japanese black pine, Ryukyu pine, Taiwan red pine, Korean pine (Kwon et al., 2006 (Non-Patent Document 8); Mamiya, 1988). (Non-Patent Document 9); Niu et al., 2012 (Non-Patent Document 10); Yun et al., 2012 (Non-Patent Document 11)).
Pine nematode disease causes fatal harm in pine forests, but there is no effective tool to combat this disease.

Taxis plays an important role for the pine wood nematode to search for a host (Aikawa 2008 (Non-Patent Document 12)). The pine wood nematode needs to move from a moribund or dead tree to an undamaged pine tree, which requires the pine woodworm (genus Monochamus). Dispersed third-stage larvae of the pine wood nematode develop into the fourth-stage larvae, gather around the pupae of the longhorn beetle, and enter the adult trachea through air holes (Aikawa, 2008 (Non-Patent Document 12). )). Previous studies have shown that LIV formation in the pine wood nematode is induced by fatty acid ethyl ester and that its structure is present in the epidermis of the longhorn beetle (Zhao et al., 2013 (Non-Patent Document 13)). This suggests that the pine wood nematode finds the pine wood nematode and uses chemotaxis to escape from dying or dead trees. In addition, it has been reported that pine wood nematodes are strongly attracted to the pine compounds α-pinene, β-pinene, and longifolene (Yun et al., 2012 (Non-Patent Document 11)).

Jianghua, S. (2005) Forest invasive species: country report -people ’s republic of China. In the unwelcome guets, Proceeding of the Asia, 80-86. Kiyohara, T. and Tokushige, Y. (1971) Inoculation experiments of a nematode, Bursaphelenchus Sp., onto pine trees. JOURNAL OF THE JAPANESE FORESTRY SOCIETY, 53 Manuel, M. M. Helen, B. Maria, A. B. Ana, C. P. Wolfgang, B. Kai, M. and Edmundo, S. (1999) First report of Bursaphelenchus Xylophilus in Portugal and in Europe. Nematology 1, 727-34. Moon, Y. S. Joung, A. S. and Chan, S. J. (2013) Morphometric variation in pine wood nematodes, Bursaphelenchus Xylophilus and B. Mucronatus, isolated from multiple locations in South Korea. The Plant Pathology Journal 29 (3), 344-49. Dwinell, L.D. and Nickle W.R. (1989) An overview of the pine wood nematode ban in North America. United States Department of Agriculture (USDA): Washington, D.C., 1989; USDA Forest Service General Technical Report SE-55 Dwinell, L. D. (1993) First report of pinewood nematode (Bursaphelenchus Xylophilus) in Mexico. Plant Disease, 846. Linit, M. J. (1988) Nematode-vector relationships in the pine wilt disease system. Journal of nematology 20 (2), 227-35. Kwon, T. S. Lim, J. H. Sim, S. J. Kwon, Y. D. Son, S. K. Lee, K. Y. Kim, Y. T. Park, J. W. Shin, C. H. Ryu, S. B. Lee, C. K. Shin, S. C. Chung, Y. J. and Park, Y. S. (2006) Distribution patterns of Mono alternatus and M. Saltuarius (Coleoptera: Cerambycidae) in Korea. Journal of Korean Forest Society 95 (5), 543-50. Mamiya, Y. (1988) History of Pine Wilt Disease in Japan. Journal of nematology 20 (2), 219-26. Niu, H. Zhao, L. Lu, M. Zhang, S. and Sun, J (2012) The ratio and concentration of two monoterpenes mediate fecundity of the pinewood nematode and growth of its associated Fungi. PLoS ONE 7 (2), e31716 Yun, J. E. Kim, J. and Park, C. G. (2012) Rapid diagnosis of the infection of pine tree with pine wood nematode (Bursaphelenchus Xylophilus) by use of host-tree volatiles. Journal of Agricultural and Food Chemistry 60 (30), 7392 -97. Aikawa, T. (2008) Pine wilt disease transmission biology of Bursaphelenchus Xylophilus in relation to its insect vector, 123-124. Zhao, L. Zhang, S. Wei, W. Hao, H. Zhang, B. Butcher, R. A. and Sun, J. (2013) Chemical signals synchronize the life cycles of a plant-parasitic nematode and its vector beetle. Current Biology 23, 2038-2043.

However, the olfactory response of pine wood nematodes to non-pine synthetics has not been tested.
Therefore, an object of the present invention is to provide a method for detecting an odorous substance.

As a result of diligent studies to solve the above problems, the present inventor has succeeded in detecting an odorant having an incentive or repellent action on nematodes by analyzing the olfactory response of nematodes. , The present invention has been completed.

That is, the present invention is as follows.
(1) A method for detecting an odorant, which comprises assaying the tactics of a nematode using a test substance and a nematode and analyzing the olfactory response of the nematode.
(2) The method according to (1), wherein the nematode is a pine wood nematode.
(3) The method according to (1), which measures the taxis index.
(4) The method according to (3), wherein when the taxis index is a positive value, it indicates an attraction to an odorant.
(5) The method according to (3), wherein when the taxis index is a negative value, it indicates repellent to odorous substances.
(6) A substance of the pine wood nematode, which is detected by the method according to any one of (1) to (4) and exhibits an attracting behavior to an odorous substance, is applied to a pine tree. Trap method.
(7) A method for exterminating pine wood nematodes, which comprises treating the pine wood nematode trapped by the method according to (6).
(8) A substance that has shown a repellent behavior against an odorous substance detected by the method according to any one of (1) to (3) and (5) is applied to a pine tree. How to prevent infection of pine wood nematodes.
(9) A substance that has shown a repellent behavior against odorous substances detected by the method according to any one of (1) to (3) and (5) is applied to a pine tree, and the pine wood nematode is applied to the pine tree. A method for exterminating pine wood nematodes, which comprises repelling the pine wood nematode and treating the repelled pine wood nematode.
(10) A trapping agent for pine wood nematodes, which comprises a substance that has shown an attracting behavior to an odorous substance, which is detected by the method according to any one of (1) to (4).
(11) A repellent for pine wood nematodes, which comprises a substance that has shown a repellent behavior against odorous substances, which is detected by the method according to any one of (1) to (3) and (5).

INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to detect an odorant having an incentive or repellent action on a nematode. Substances that have shown repellent behavior to pine wood nematodes can be used as pine defenders, i.e., repellents to pine wood nematodes, while substances that have shown attracting behavior to odorous substances are for trapping pine wood nematodes. Can be used as a trapping agent

It is a figure of the agar plate used for the behavior analysis of nematodes. An odorant was placed on one end of a 9 cm assay plate and a washed nematode was placed near the center of the plate. Sodium azide (NaN3) was used to paralyze nematodes. After 1 hour, the number of nematodes in regions A, B or C was counted and the taxis index was calculated by the following formula. (A-B) / (A + B + C) Taxis index is between +1.0 and -1.0. Positive values indicate attraction to odorants and negative values indicate repellent. It is a figure which shows the behavioral response of Shimabara to an attractive odor. The assay was performed as shown in Figure 1 with 1 μl of undiluted attractant (geraniol) or diluted attractant (other) present. As a control, the ethanol used as the solvent was spotted on one end of the plate. All odorants showed a taxitactic index significantly different from the control (P <0.05, Dunnett's test, N = 3-4). Error bars indicate S.E.M. It is a figure which shows the behavioral response of a nematode to an unpleasant odor. Assays were performed using diluted odorants ( ^10-2 ) as shown in Figure 1. As a control, the ethanol used as the solvent was spotted on one end of the plate. All odorants show a taxitactic index significantly different from the control (P <0.05, Dunnett's test, N = 3-4). Error bars indicate SEM. It is a figure showing different behavioral responses to odorants in Shimabara and S10. White bars indicate S10 and black bars indicate Shimabara. Two types of pine wood nematodes show different behavioral responses to undiluted 3-methyl-1-butanol and four other diluted odorants (10-2) (P <0.05, Mann-Whitney U test). , N = 3-4). Error bars indicate S.E.M.

Hereinafter, the present invention will be described in detail. The following embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to this embodiment. The present invention can be implemented in various forms as long as it does not deviate from the gist thereof.

The present inventor analyzed the olfactory response to 53 kinds of odorants in the isolated pine wood nematode strains Shimabara and S10. As a result, the inventor found that Shimabara was attracted to 14 kinds of odorants. Of these 14 odorants, 1-octanol strongly attracted Shimabara. On the other hand, S10 did not show an attractive behavior to 53 kinds of odorants, but showed a strong repellent behavior to isoamyl isovalerate. Guaiacol and 2-nonanone also made S10 uncomfortable.
These results show the difference in odor selectivity depending on the isolated strain of pine wood nematode.

As mentioned above, pine wilt disease causes serious damage to pine trees in East Asia and European countries. Pine nematode disease is caused by pine wood nematodes, but there are no effective tools to prevent the disease. Past studies have shown that taxis is important for the pine wood nematode to find a host. Here, the two pine wood nematodes strains (Shimabara and S10) isolated by the present inventor were used to test the olfactory response to 53 odorants.
The results showed that Shimabara was attracted to 14 odorants, of which 1-octanol was the strongest. On the other hand, S10 showed an attractive behavior to 53 kinds of odorants, but S10 showed strong repellent to isoamyl isovalerate, guaiacol, and 2-nonanone.
These results suggest that there are genetic variations in odorant preference among the isolated pine wood nematodes.
However, the nematodes applicable to the present invention are not limited to the above two species of pine wood nematode strains (Shimabara and S10), and can also be applied to other nematodes. Other nematodes include agricultural pests such as soybean cyst nematodes, negsare nematodes, and catworm nematodes.

In the present invention, a substance that exhibits an attractive behavior toward an odorous substance can be used as a trap for exterminating pine wood nematodes. For example, a substance that has an incentive behavior toward an odorous substance is applied to a pine trunk or buried in soil or the like to attract pine wood nematodes to the substance. Kill the pine wood nematodes by exterminating them where they have gathered. This can prevent the infection of pine wood nematodes with pine trees. The substance exhibiting incentive behavior is preferably 1-octanol. In addition, examples of the extermination treatment include treatment with an insecticide and incineration.

On the other hand, substances that show repellent behavior against odorous substances can be used as an anti-infective agent for pine wood nematodes. For example, a substance that has shown a repellent behavior against odorous substances may be applied to the trunk of a pine tree or buried in the soil to prevent the pine wood nematode from infecting the pine tree. Alternatively, a substance that has shown a repellent behavior against odorous substances is injected into the trunk of the pine tree. By capturing or exterminating the pine wood nematodes that existed inside the trunk of the pine tree when they escaped from the trunk, it is possible to prevent the pine wood nematode from infecting the pine tree and to eliminate the pine wood nematode. be able to. Examples of substances that show repellent behavior against odorous substances include isoamyl isovalerate, guaiacol, and 2-nonanone.
The usage, dose, etc. when applying a substance that has an incentive behavior to an odorous substance or a substance that has a repellent behavior to an odorous substance to a pine can be appropriately set, and is not particularly limited. ..

Examples Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Culture The isolated pine wood nematode was cultured and maintained on a potato dextrose agar (PDA) at 25 ° C. Botrytis cinerea was used as food. Shimabara and S10 used in the present invention were obtained from the National Research and Development Agency Forest Research Institute (Tsukuba City, Japan).

Behavior Assay Nematodes were placed on 6 cm PDA plates with Botrytis cinerea and cultured for 7 days. The nematodes on the plate cover were collected with distilled water and 70-100 nematodes were spotted in the center of the 9 cm 2% INA agar assay plate. Immediately prior to the assay, 1 μl of odorant and 0.5 μl of 1 M sodium azide were spotted in two places on one side. On the other side, only sodium azide was spotted. The odorant was obtained from T. Hasegawa Co., Ltd.

Smell substance to be tested
Solutions of 3-methyl-1-butanol, diacetyl, acetylacetone, 2-nonanone, pyrazine, geraniol, citronellol and β-pyrazine were used undiluted and other odorants were diluted 1/100.

Room temperature was maintained at 25 ± 1 ° C. The format of the assay plate is shown in Figure 1. The number of nematodes in regions A, B, and C was visually counted 1 hour after the start of the test, and the taxis index was calculated by the following formula.
Taxis index = (AB) / (A + B + C)
A, B and C each represent the number of nematodes in the corresponding region.

Results and Discussion The inventor tested the behavioral response to 53 odorants, including alcohol, phenol, and ketone, using two isolated strains of pine wood nematode. Shimabara was attracted to 14 odorants, including alcohols, esters and aldehydes (Figure 2, Table 1). Shimabara was most strongly attracted to 1-octanol of the 14 odorants. S10 showed no clear attraction to 53 odorants, showed repellent to guaniacol, 2-nonanone, and isoamyl isovalerate (Fig. 3), and was the strongest repellent to isoamyl isovalerate of the three. There were significant differences in the sensory response between Shimabara and S10 to octanol, 3-methyl-1-butanol, octanal, benzaldehyde, and gamma-nononalactone (Figure 4), with 53 common repellents or attractants tested. It was not present in the substance.
These results indicate that the two isolated strains have different preferences for odorants.

The S10 strain is as highly toxic as the Shimabara strain (Akiba et al., 2012). There are two pathogenic groups of nematodes in North America, and females of these pathogenic types have different tail end shapes. One is the R-type with a circular end and the other is the microprotruding M-type (Akiba et al., 2012). Both S10 and Shimabara are R-shaped (Akiba, 2006). The S10 strain was established from nematodes extracted from dead red pine in Shimane Prefecture, Japan, and Shimabara was collected in Nagasaki Prefecture, Japan. Shimane Prefecture is located in Honshu, Japan, while Nagasaki Prefecture is located in Kyushu. Therefore, it is considered that the difference in the response of S10 and Shimabara to the odorant was caused by the geographical isolation. In Japan, the internal transcribed spacer sequences of pine wood nematode ribosomal DNA differ between toxic and non-toxic (Akiba, 2006; Iwahori et al., 1998). Therefore, the olfactory response to odorants in the non-toxic isolated form requires further testing.

In the present invention, the inventor tested odor selectivity at only two levels of concentration (100-fold diluted with undiluted material and ethanol). However, in the present invention, the concentration of the odor response can be tested at other concentrations.

Caenorhabditis elegans show a strong preference for undiluted 3-methyl-1-butanol and a repellent to undiluted benzaldehyde (Bargmann et al., 1993). However, pine wood nematodes show no selectivity for both of these substances (Table 1). C. elegans are neutral or repellent to Linalool, 1-octanol, and ethyl hexanoate (Bargmann et al .. 1993), but Shimabara is attracted. However, nonanone is repelled by both pine wood nematodes and C. elegans. Therefore, the sensory response to odor differs between pine wood nematodes and C. elegans.

Past studies have shown that Korean pine wood nematodes have a strong preference for β-pinene, one of the pine tree products (Yun et al .. 2012). However, the inventor found that S10 and Shimabara were not attracted to β-pinene (Table 1). This indicates that the pine wood nematode changes its response to odorants depending on its habitat.

In the present invention, first, the odor selectivity of different isolated strains of pine wood nematode to a panel of odorants was tested. Smell selectivity is thought to be associated with PWD infection and spread. The results of the present invention show that substances that show repellent behavior to pine wood nematodes can be used as pine protective agents, that is, substances that show repellent behavior to pine wood nematodes, while substances that show attracting behavior to odorous substances are pine wood nematodes. It is shown that it can be used as a trapping agent for trapping nematodes.

<References>
Aikawa, T. (2008) Pine wilt disease transmission biology of Bursaphelenchus Xylophilus in relation to its insect vector, 123-124.
Akiba, M. (2006) Diversity of pathogenicity and virulence in the pinewood nematode, Bursaphelenchus Xylophilus. Journal of The Japanese Forestry Society 5, 383-91.
---. (2012) Virulence of Bursaphelenchus Xylophilus isolated from naturally infested pine forests to five resistant families of Pinus Thunbergii. Plant Disease 96, 249-52.
Bargmann, CI Hartwieg, E. and Horvitz, HR (1993) Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74, 515-27.
Dwinell, LD (1993) First report of pinewood nematode (Bursaphelenchus Xylophilus) in Mexico. Plant Disease, 846.
Dwinell, LD and Nickle WR (1989) An overview of the pine wood nematode ban in North America. United States Department of Agriculture (USDA): Washington, DC, 1989; USDA Forest Service General Technical Report SE-55
Iwahori, H. Tsuda K. Kanzaki, N. Izui, K. and Futai, K. (1998) PCR-RFLP and sequencing analysis of ribosomal DNA of Bursaphelenchus nematodes related to pine wilt disease. Fundamental and Applied Nematology 21, 655-66 ..
Jianghua, S. (2005) Forest invasive species: country report -people's republic of China. In the unwelcome guets, Proceeding of the Asia, 80-86.
Kiyohara, T. and Tokushige, Y. (1971) Inoculation experiments of a nematode, Bursaphelenchus Sp., onto pine trees. JOURNAL OF THE JAPANESE FORESTRY SOCIETY, 53
Kwon, TS Lim, JH Sim, SJ Kwon, YD Son, SK Lee, KY Kim, YT Park, JW Shin, CH Ryu, SB Lee, CK Shin, SC Chung, YJ and Park, YS (2006) Distribution patterns of Monochamus alternatus and M. Saltuarius (Coleoptera: Cerambycidae) in Korea. Journal of Korean Forest Society 95 (5), 543-50.
Linit, MJ (1988) Nematode-vector relationships in the pine wilt disease system. Journal of nematology 20 (2), 227-35.
Mamiya, Y. (1988) History of Pine Wilt Disease in Japan. Journal of nematology 20 (2), 219-26.
Manuel, MM Helen, B. Maria, AB Ana, CP Wolfgang, B. Kai, M. and Edmundo, S. (1999) First report of Bursaphelenchus Xylophilus in Portugal and in Europe. Nematology 1, 727-34.
Moon, YS Joung, AS and Chan, SJ (2013) Morphometric variation in pine wood nematodes, Bursaphelenchus Xylophilus and B. Mucronatus, isolated from multiple locations in South Korea. The Plant Pathology Journal 29 (3), 344-49.
Niu, H. Zhao, L. Lu, M. Zhang, S. and Sun, J (2012) The ratio and concentration of two monoterpenes mediate fecundity of the pinewood nematode and growth of its associated Fungi. PLoS ONE 7 (2), e31716
Yun, JE Kim, J. and Park, CG (2012) Rapid diagnosis of the infection of pine tree with pine wood nematode (Bursaphelenchus Xylophilus) by use of host-tree volatiles. Journal of Agricultural and Food Chemistry 60 (30), 7392 -97.
Zhao, L. Zhang, S. Wei, W. Hao, H. Zhang, B. Butcher, RA and Sun, J. (2013) Chemical signals synchronize the life cycles of a plant-parasitic nematode and its vector beetle. Current Biology 23, 2038-2043.

Claims (6)

Pine at least one of decanol, linalool, 1-octanol, benzaldehyde, cinnamaldehyde, octanal, trans-2-hexenal, methylamylketone, citronellol, geraniol, allyl hexaneate, benzyl acetate, ethyl hexaneate and methyl salicylate. A method of trapping the Shimabara strain of pine wood nematode, which is characterized by being applied to. A method for exterminating a Shimabara strain of a pine wood nematode, which comprises treating the Shimabara strain of the pine wood nematode trapped by the method according to claim 1. A method for preventing infection of S10 strain of pine wood nematode, which comprises applying at least one of gu ai acol, 2-nonanone and isoamyl isovalerate to pine. Apply at least one of gu ai acol, 2-nonanon and isoamyl isovalerate to pine to repel the S10 strain of pine wood nematode outside the pine and to exterminate the repelled S10 strain of pine wood nematode. A method for exterminating the S10 strain of pine wood nematode, which is characterized by this. Includes at least one of decanol, linalool, 1-octanol, benzaldehyde, cinnamaldehyde, octanal, trans-2-hexenal, methylamylketone, citronellol, geraniol, allyl hexaneate, benzyl acetate, ethyl hexaneate and methyl salicylate. A trapping agent for the Shimabara strain of Matsunozai nematode. A repellent for the S10 strain of pine wood nematode, comprising at least one of gu ai acol, 2-nonanone and isoamyl isovalerate.

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