JP2018091849A - Method of detecting odorants - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detecting odorants.SOLUTION: A method of detecting odorants involves assaying taxis of a nematode using a substance under test and the nematode, and analyzing olfactory response of the nematode, where the nematode used herein is the pinewood nematode. A taxis index is measured, where a positive taxis index value indicates that the nematode is being attracted to an odorant and a negative taxis index value indicates that the nematode is being repelled from the odorant. Further, a method of trapping the pinewood nematode is provided, involving applying a substance that has been found to cause attractant action to the odorant by the above method to pine trees.SELECTED DRAWING: None

Description

  The present invention relates to a method for detecting an odor substance.

Pine wilt disease (PWD) is a serious disease (infectious disease) caused to Pinus species (pine) in East Asia and European countries, especially Japan, Portugal, China and Korea ( 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 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 not cause harm in the country of origin (Linit, 1988 (Non-patent Document 5) Reference 7)). However, pine wood nematode has pathogenicity against Asian or European pine, such as red pine, black pine, rhubarb pine, Thai red pine, Korean pine (Kwon et al., 2006); Mamiya, 1988 (Non-patent document 9); Niu et al., 2012 (non-patent document 10); Yun et al., 2012 (non-patent document 11)).
Although pine wilt causes fatal harm in pine forests, there is no effective tool to combat this disease.

  The chemotaxis plays an important role for pinewood nematodes to search for a host (Aikawa 2008 (Non-patent Document 12)). The pinewood nematode needs to be transferred from dying or dead trees to unharmed pine trees, which requires genus Monochamus. The dispersed third stage larvae of the pinewood nematode develop into the fourth stage larvae, gather around the pupae of the pinewood beetle, and enter the adult trachea through the air holes (Aikawa, 2008 (Non-patent Document 12) )). Past studies have shown that LIV formation in pinewood nematodes is induced by fatty acid ethyl esters and that the structure exists in the epidermis of pinewood beetle (Zhao et al., 2013 (Non-patent Document 13)). This suggests that the pinewood nematode finds pinewood beetle and uses chemotaxis to escape from dying or dead trees. In addition, it is reported that pine wood nematode is strongly attracted to α-pinene, β-pinene, and longifolene, which are pine compounds (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, 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. 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, 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. 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, 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.

However, the olfactory response to pinewood nematode non-pine composites has not been tested.
Then, an object of this invention is to provide the detection method of an odor substance.

  As a result of intensive studies to solve the above problems, the present inventor succeeded in detecting an odor substance having an incentive or a repellent effect on a linear animal by analyzing the olfactory response of the linear animal. The present invention has been completed.

That is, the present invention is as follows.
(1) A method for detecting an odorous substance, which comprises assaying the chemotaxis of a linear animal using a test substance and a linear animal and analyzing the olfactory response of the linear animal.
(2) The method according to (1), wherein the linear animal is a pinewood nematode.
(3) The method according to (1), wherein a chemotaxis index is measured.
(4) The method according to (3), wherein when the chemotaxis index is a positive value, it indicates attraction to an odor substance.
(5) The method according to (3), wherein when the chemotaxis index is a negative value, repelling to odor substances is indicated.
(6) A material of the pinewood nematode, which is detected by the method according to any one of (1) to (4) and which exhibits an attracting action against an odorous substance is applied to pine. Trap method.
(7) A method for extinguishing pinewood nematodes, characterized in that the pinewood nematodes trapped by the method according to (6) are extinguished.
(8) A substance, which is detected by the method according to any one of (1) to (3) and (5) and exhibits repellent behavior against an odor substance, is applied to a pine tree, Infection prevention method of pine wood nematode.
(9) A substance that exhibits repellent behavior against odorous substances, which is detected by the method according to any one of (1) to (3) and (5), is applied to the pine, and the pinewood nematode is removed from the pine. A method for extinguishing pinewood nematodes, characterized by repelling the pinewood nematodes that have been repelled in addition to the scabbard.
(10) A trap agent for pinewood nematodes, which contains a substance that has been detected by the method according to any one of (1) to (4) and that has shown an attracting action to an odorous substance.
(11) A pinewood nematode repellent, comprising a substance that has been detected by the method according to any one of (1) to (3) and (5) and exhibits repellent behavior against an odorous substance.

  According to the present invention, it is possible to detect an odor substance having an incentive or a repellent effect on a linear animal. Substances that pinewood nematodes have shown repellent behavior can be used as pine defensive agents, i.e. pinewood nematode repellents, while substances that have attracted odorants can be used to trap pinewood nematodes. Can be used as a trapping agent

It is a figure of the agar plate used for behavior analysis of a nematode. The odorant was placed on one end of the 9 cm assay plate, and the washed nematode was placed near the center of the plate. Sodium azide (NaN3) was used to paralyze nematodes. One hour later, the number of nematodes in the region A, B or C was counted, and the chemotaxis index was calculated by the following formula. (A-B) / (A + B + C) The chemotaxis index is between +1.0 and -1.0. Positive values indicate attraction to odorants, negative values indicate avoidance. It is a figure which shows the behavioral response of Shimabara with respect to an attractive odor. The assay was performed as shown in FIG. 1, with 1 μl of undiluted attractant (geraniol) or diluted attractant (other) present. As a control, ethanol used as a solvent was spotted on one end of the plate. All odorants showed a chemotaxis 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 action response of the nematode with respect to an unpleasant smell. The assay was performed as shown in FIG. 1 using diluted odorants (10 ⁻² ). As a control, ethanol used as a solvent was spotted on one end of the plate. All odorants show a chemotaxis index significantly different from the control (P <0.05, Dunnett's test, N = 3-4). Error bars indicate SEM. It is a figure in which Shimabara and S10 show different behavioral responses to odorants. The white bar indicates S10 and the black bar indicates Shimabara. Two pinewood 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 embodiment is an example for explaining the present invention, and is not intended to limit the present invention only to this embodiment. The present invention can be implemented in various forms without departing from the gist thereof.

The present inventor analyzed the olfactory response to 53 odorants in the isolated pine wood nematode strains Shimbara and S10. As a result, the present inventor has found that Shimabara is attracted to 14 odor substances. Of these 14 odorants, 1-octanol strongly attracted Shimabara. S10, on the other hand, did not attract 53 odorants, but showed strong repellent behavior against isoamyl isovalerate. Guaiacol and 2-nonanone also made S10 uncomfortable.
These results show the difference in odor selectivity among the pinewood nematode isolates.

As mentioned above, pine wilt disease severely damages pine trees in East Asia and European countries. Although pine wilt disease is caused by pine wood nematode, there is no effective tool to prevent the disease. Past studies have shown that chemotaxis is important for pinewood nematodes to look for hosts. Here, olfactory responses to 53 odorants were tested using two pine wood nematode strains (Shimabara and S10) isolated by the present inventors.
As a result, Shimabara was attracted to 14 odorants, of which 1-octanol was most strongly attracted. On the other hand, S10 attracted 53 odorants, but S10 showed strong repelling to isoamyl isovalerate, guaiacol, and 2-nonanone.
These results suggest that there are genetic variations in odorant preferences among isolated pine wood nematodes.
However, nematodes applicable to the present invention are not limited to the above-mentioned two pine wood nematode strains (Shimabara and S10), and can be applied to other linear animals. Examples of other linear animals include agricultural pests such as soybean cyst nematode, neptune nematode, and root-knot nematode.

  In the present invention, a substance that has attracted an odor substance can be used as a trap for controlling pinewood nematodes. For example, a substance showing an incentive action for an odor substance is applied to a pine trunk or buried in the soil, and pine wood nematode is attracted to the substance. The pine wood nematode is killed by disinfecting it when the pine wood nematode gathers. Thereby, the infection to the pine of a pinewood nematode can be prevented. 1-octanol is preferable as the substance showing the inducing behavior. Further, examples of the extermination treatment include treatment with an insecticide and incineration.

On the other hand, the substance which showed the repelling action with respect to an odor substance can be used as an infection prevention agent to the pine wood nematode pine. For example, a substance exhibiting repellent behavior against odorous substances is applied to the trunk of a pine or buried in the soil to prevent the pinewood nematode from infecting the pine. Alternatively, a substance that exhibits repellent behavior against odorous substances is injected into the trunk of the pine. By capturing or exterminating the pinewood nematode that had been in the trunk of the pine, the pinewood nematode can be prevented from infecting the pine, and the pinewood nematode can be removed. be able to. Examples of substances exhibiting repellent behavior against odorous substances include isoamyl isovalerate, guaiacol, 2-nonanone and the like.
The usage, dosage, etc. when applying a substance that has shown an incentive action to a odor substance or a substance that has a repellent action to a odor substance to a pine can be appropriately set, and are not particularly limited. .

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

Culture- isolated pinewood nematodes were cultured and maintained on potato dextrose agar (PDA) at 25 ° C. Botrytis cinerea was used as food. Shimabara and S10 used in the present invention were obtained from National Forest Research Institute (Tsukuba, Japan).

Behavioral assay nematodes were placed on 6 cm PDA plates with gray mold and cultured for 7 days. Nematodes on the plate cover were collected with distilled water and 70-100 nematodes were spotted in the center of a 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. Odor substances were obtained from T. Hasegawa Co., Ltd.

Test odor substance
The 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 FIG. The number of nematodes in regions A, B, and C was visually counted 1 hour after the start of the test, and the chemotaxis index was calculated according to the following formula.
Taxis index = (AB) / (A + B + C)
A, B, and C represent the number of nematodes in the corresponding region, respectively.

Results and Discussion The inventor used two pine wood nematode isolates to test behavioral responses to 53 odorants including alcohol, phenol, and ketone. Shimabara was attracted to 14 odorants including alcohol, esters and aldehydes (Figure 2, Table 1). Shimabara was most strongly attracted to 1-octanol out of 14 odorants. S10 did not show any clear attraction to 53 odorants, but showed repellent guaniacol, 2-nonanone and isoamyl isovalerate (Fig. 3), and most strongly against isoamyl isovalerate among the 3 species. There are significant differences in olfactory responses to octanol, 3-methyl-1-butanol, octanal, benzaldehyde, and γ-nonalactone between Shimabara and S10 (Figure 4), with 53 common repellent or attractants tested. It was not present in the material.
These results indicate that the two isolates have different preferences for odorants.

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

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

  Caenorhabditis elegans shows strong preference for undiluted 3-methyl-1-butanol and repels undiluted benzaldehyde (Bargmann et al., 1993). However, pine wood nematode does not show selectivity for both of these substances (Table 1). C. elegans is neutral or repellent to Linalool, 1-octanol, ethyl hexanoate (Bargmann et al .. 1993), but Shimabara is attracted. However, Nonanon is repelled by both pinewood nematodes and C. elegans. Thus, the olfactory response to odor differs between pinewood nematode and C. elegans.

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

  In the present invention, first, the odor selectivity of different isolates of pinewood nematode to a panel of odorants was tested. Odor selectivity is thought to be related to PWD infection and spread. As a result of the present invention, a substance that pinewood nematode showed repellent behavior can be used as a pine defense agent, i.e., a pinewood nematode repellent, while a substance that showed attractant action against odorous substances is pinewood It shows 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 (11)

  A method for detecting an odorous substance, characterized by assaying the chemotaxis of a linear animal using a test substance and a linear animal and analyzing the olfactory response of the linear animal.   The method according to claim 1, wherein the linear animal is a pinewood nematode.   The method of claim 1, wherein the chemotaxis index is measured.   4. The method of claim 3, wherein when the chemotaxis index is a positive value, it indicates attraction to odorants.   4. The method of claim 3, wherein when the chemotaxis index is negative, it indicates repellent to odorants.   A method for trapping pine wood nematodes, characterized in that the substance detected by the method according to any one of claims 1 to 4 and exhibiting an attracting action against an odor substance is applied to a pine.   A method for extermination of pinewood nematodes trapped by the method according to claim 6.   A method for preventing infection of pine wood nematode, characterized by applying to a pine a substance that has been detected by the method according to any one of claims 1 to 3 and 5 and exhibits repellent behavior against an odorous substance. .   The substance detected by the method according to any one of claims 1 to 3 and 5, which exhibits repellent behavior against odorous substances, is applied to pine to repel pinewood nematodes from the pine and repel A method for extinguishing a pinewood nematode, comprising:   The trap agent of a pinewood nematode containing the substance which showed the attracting action with respect to the odorous substance detected by the method of any one of Claims 1-4.   A pinewood nematode repellent, comprising a substance that has been detected by the method according to any one of claims 1 to 3 and 5 and exhibits a repellent behavior against an odorous substance.