Composition containing geraniol for control of plant-parasitic nematodes

Abstract

A composition for controlling nematodes contains plant essential oil, and more particularly, geraniol. The composition is derived from natural substances and is thus environmentally friendly, and exhibits superior nematocidal effects without causing phytotoxicity, and thus can be efficiently used as a nematicide.

Claims

1. A composition for controlling a plant-parasitic nematode, containing geraniol at a concentration of 100-500 ppm and a neem extract in a ratio of about 4:1 with the geraniol.

2. The composition of claim 1, wherein the nematode is at least one selected from the group consisting of a root-knot nematode, a root-lesion nematode, a spiral nematode and a cyst nematode.

3. A formulation for controlling a plant-parasitic nematode comprising the composition of claim 1 as an active ingredient.

4. A method of controlling a plant-parasitic nematode comprising controlling a nematode by treating the nematode with the composition of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows optical microscope images at 40× magnification of root-knot nematodes before and after treatment with geraniol according to an embodiment of the present invention;

(2) FIG. 2 is photographs showing the results of testing of phytotoxicity on tomato seedlings depending on the dilution factor of the nematocidal composition according to an embodiment of the present invention;

(3) FIG. 3 is photographs showing the pot test results of mixed solutions of geraniol and plant extracts according to an embodiment of the present invention; and

(4) FIG. 4 is microscope images showing the nematocidal effects of geraniol and palmarosa oil including the same amount of geraniol on root-knot nematodes.

BEST MODE

(5) The present invention is intended to provide a naturally derived material having nematocidal activity.

(6) In the present invention, six types of plant-derived materials and eight types of plant extracts were evaluated for nematocidal activity.

(7) In an embodiment of the present invention, nematocidal activity on root-knot nematodes (Meloidogyne spp.) was evaluated using geraniol, azadirachtin, clove oil, oregano oil, pyrethrin, oxymatrine, neem extract, Ostericum koreanum extract, clove extract, Myristica fragrans extract, Acorus gramineus extract, Kaempferia galanga extract and Amomum cardamomum L. extract. Based on the results thereof, when geraniol was used alone or in combination with a neem extract, clove extract or Ostericum koreanum extract, superior nematocidal activity was confirmed. In particular, when geraniol was used at 100 to 3,000 ppm, superior nematocidal effects were exhibited without phytotoxicity, but phytotoxicity was confirmed to occur upon treatment with geraniol at 4,000 ppm or more.

(8) Therefore, an aspect of the present invention pertains to a composition for controlling plant-parasitic nematodes containing geraniol at a concentration of 100 to 3,000 ppm.

(9) The geraniol is a monoterpene alcohol contained in plant essential oil, and may be used without limitation as to the source plant.

(10) In the present invention, if the amount of geraniol contained in the composition for controlling plant-parasitic nematodes is less than 100 ppm, the nematocidal activity may become insignificant. On the other hand, if the amount thereof exceeds 3,000 ppm, phytotoxicity may be caused in crops.

(11) The composition for controlling plant-parasitic nematodes according to the present invention may exhibit superior nematocidal activity even when containing geraniol alone, and in order to exhibit higher and more stable nematocidal efficiency, a plant extract selected from the group consisting of a neem extract, an Ostericum koreanum extract and a clove extract may be further included. Here, the volume ratio of the plant extract and the geraniol is preferably 3:1 to 5:1 based on the same concentration. If the volume ratio of the plant extract and the geraniol falls out of the above range, there is a concern in that phytotoxicity may occur.

(12) The plant extract may be obtained using a process selected from the group consisting of typically known extraction processes, such as alcohol extraction, hexane extraction, hot-water extraction, cold-water extraction, cold compression (expression), and steam distillation.

(13) In the present invention, the plant-parasitic nematodes may be exemplified by root-knot nematodes, root-lesion nematodes, spiral nematodes, cyst nematodes, and the like, and examples of the root-knot nematodes may include, but are not limited to, carrot root-knot nematodes, sweet-potato root-knot nematodes, Java root-knot nematodes, peanut root-knot nematodes, and the like.

(14) In the present invention, the composition for controlling plant-parasitic nematodes may be used alone, but is not particularly limited thereto, and may further include an appropriate diluent or excipient depending on the dosage form and/or use purposes of the composition. As the excipient, a typical material may be used depending on the dosage form, and when formulated, a filler, an extender, a wetting agent, a disintegrant or a surfactant may be used. Typical examples of the diluent or excipient may include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin and normal saline, and more preferably, secondary distilled water added with 0.1 ml of Triton X-100 per liter (L) is used.

(15) Another aspect of the present invention pertains to a formulation for controlling plant-parasitic nematodes including the composition for controlling plant-parasitic nematodes as an active ingredient and to a method of controlling plant-parasitic nematodes including controlling nematodes using the composition for controlling plant-parasitic nematodes.

(16) Here, the crops to be treated are not particularly limited, and may include one or more fruits and vegetables selected from the group consisting of all crops which are damaged or parasitized by nematodes, for example tomatoes, cucumbers, strawberries, melons, peppers, and watermelons.

Mode for Invention

(17) A better understanding of the present invention will be given through the following examples. These examples are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.

Example 1: Evaluation of Nematocidal Activity of Naturally Derived Material

(18) The nematodes used in the experiments were Meloidogyne spp. (hatched from eggs from infected roots of melons from farms in Seongju-gun, Gyeongsangbuk-do, Korea), and were propagated using tomato plants from Ecowin Co. Ltd. and used for experiments.

(19) The tomato roots infected with root-knot nematodes were washed with running water and were then cut to a size of about 1 cm, after which 50 g of the cut roots was placed in a 500 ml round-bottom flask containing 200 ml of 0.5% sodium hypochloride (NaOCl), stopped with a rubber stopper, and shaken vigorously for 3 min to sterilize the roots and at the same time burst the egg bag. Thereafter, the solution was passed through a sieve in which 200 mesh was superimposed on 500 mesh, and washed with distilled water, so the eggs of the root-knot nematodes from which the NaOCl solution was removed were recovered.

(20) Next, the eggs of the root-knot nematodes thus recovered were surface-sterilized with 0.5% NaOCl for 10 min, washed thoroughly with sterile distilled water, placed in a phosphate buffer (1%) containing sterilized normal saline, and hatched in an incubator at 25° C.

(21) Each of geraniol (available from Katyani Exports), azadirachtin (available from United Agro Industries), clove oil (available from Xi'Taima), oregano oil (available from Xi'Taima), pyrethrin (available from Xi'Taima) and oxymatrine (available from Kingbo, China) was mixed with an emulsifier (PLE9) at 1:1. Thereafter, each mixed solution was diluted to 2000 ppm, 2 to 3 ml thereof was poured into a Petri dish, after which 15 hatched root-knot nematodes were placed in the Petri dish, and nematocidal activity was measured and the results thereof are shown in Table 1 below.

(22) TABLE-US-00001 TABLE 1 Clove Oregano Classification Geraniol Azadirachtin oil oil Pyrethrin Oxymatrine 1 day  15.sup.a 7 2 1 5 7 2 days — 8 5 1 6 9 3 days — 10 7 3 11 10 Nematocidal 100 66.7 46.7 20.0 73.3 66.7 efficiency (%) .sup.aNumber of killed root-knot nematodes

(23) As is apparent from Table 1, the 2,000 ppm geraniol dilution solution exhibited 100% nematocidal efficiency on root-knot nematodes 1 day after treatment therewith, whereas the oregano oil killed 3 root-knot nematodes 3 days after treatment therewith and thus exhibited 20.0% nematocidal efficiency, which is evaluated to be the lowest nematocidal activity.

(24) Therefore, it was determined that geraniol, among the test samples, had the best nematocidal effect on root-knot nematodes, and nematocidal activity was measured depending on the geraniol dilution factor. The results thereof are shown in Table 2 below, and the images of root-knot nematodes before and after treatment with geraniol are shown in FIG. 1.

(25) TABLE-US-00002 TABLE 2 Geraniol (ppm) Duration 10,000 3,000 2,000 1,000 500 333 200 100 1 day  15.sup.a 15 15 10 5 5 5 5 2 days — — — 15 10 6 7 7 3 days — — — — 15 15 13 7 Nematocidal 100 100 100 100 100 100 86.7 46.7 efficiency (%) .sup.aThe number of root-knot nematodes used for the experiment on the number of killed root-knot nematodes was 15 at each dilution factor

(26) As is apparent from Table 2, the 10,000 ppm, 3,000 ppm and 2,000 ppm geraniol exhibited 100% nematocidal efficiency on root-knot nematodes 1 day after treatment therewith, and the 1,000 ppm geraniol exhibited 100% nematocidal efficiency 2 days after treatment therewith.

(27) Moreover, the 500 ppm and 333 ppm geraniol exhibited 100% nematocidal efficiency 3 days after treatment therewith, and the 200 ppm geraniol exhibited high nematocidal efficiency of 86.7% 3 days after treatment therewith, based on which the possibility of controlling root-knot nematodes thereby was confirmed.

Example 2: Experiment of Phytotoxicity of Geraniol on Crops

(28) Sterilized soil was uniformly mixed, 300 g thereof was then placed in each pot, and tomato seedlings not infected with root-knot nematodes were transplanted thereto. 50 ml of each of the geraniol dilution solutions (100, 200, 330, 500, 1,000, 2,000, 3,000, and 10,000 ppm) prepared in Example 1 was used for drenching treatment. Additionally, geraniol dilution solutions at 4,000 ppm, 5,000 ppm, and 20,000 ppm were further prepared, and then 50 ml of each was used for drenching treatment of each pot planted with tomato seedling. Here, the same amount of distilled water was used alone in an untreated group, and 1 week after treatment, the presence or absence of phytotoxicity was observed with the naked eye. The results thereof are shown in Table 3 below and in FIG. 2.

(29) TABLE-US-00003 TABLE 3 Geraniol Extent of concentration Test phytotoxicity Phytotoxicity (ppm) crop (0-5) symptom 100 Tomato 0 No 200 0 No 330 0 No 500 0 No 1,000 0 No 2,000 0 No 3,000 0 No 4,000 5 Withered 5,000 5 Withered 10,000 5 Withered 20,000 5 Withered

(30) As is apparent from Table 3 and FIG. 2, upon treatment with geraniol at different concentrations, there were no particular phytotoxicity symptoms before or after treatment in the concentration range of 100 ppm to 3,000 ppm. However, at 4000 ppm or more, it was observed that the bottoms of the stems dried up and all tomato seedlings collapsed and withered. Therefore, it was confirmed that geraniol had no effect on tomato seedlings at concentrations of 3,000 ppm or less, but geraniol at concentrations of 4,000 ppm or more caused phytotoxicity in tomato seedlings, making it impossible to use.

Example 3: Evaluation of Nematocidal Activity of Plant Extract

(31) Nematocidal activity on root-knot nematodes (treatment concentration: 2,000 ppm) was evaluated in the same manner as in Example 1, with the exception that Ostericum koreanum, clove, Myristica fragrans, Acorus gramineus, Kaempferia galanga and Amomum cardamomum L. extracts were used, in lieu of the naturally derived materials. The results thereof are shown in Table 4 below. Here, Ostericum koreanum, clove, Myristica fragrans, Acorus gramineus, Kaempferia galanga and Amomum cardamomum L. were purchased from a medicinal plant retailer located in the Daegu area and then extracted using ethanol according to a typical method. Specifically, each medicine was finely crushed, mixed with ethanol at a ratio of 1:5, and extracted at 180 rpm at room temperature for 48 hr, after which as much ethanol as possible was concentrated using a concentrator.

(32) TABLE-US-00004 TABLE 4 Type of plant extract Ostericum Myristica Acorus Kaempferia Amomum Duration koreanum Clove fragrans gramineus galanga cardamomum L. 1 day 5 4 4 1 2 2 2 days 14 11 6 5 2 5 3 days 15 15 6 6 2 8 Nematocidal 100 100 40 40 13.3 53.3 efficiency (%) a: The number of inoculated root-knot nematodes used for the experiment on the number of killed root-knot nematodes was 15

(33) As is apparent from Table 4, the Ostericum koreanum and clove extracts killed more than half of the root-knot nematodes 2 days after treatment therewith, and exhibited 100% mortality on the 3.sup.rd day, but Myristica fragrans, Acorus gramineus, Kaempferia galanga and Amomum cardamomum L. exhibited mortality rates equal to or less than about 50% even on the 3r.sup.d day after treatment therewith.

Example 4: Evaluation of Nematocidal Activity of Neem Extract

(34) Nematocidal activity on root-knot nematodes was evaluated in the same manner as in Example 1, with the exception that a neem extract was used in lieu of the naturally derived materials. The results thereof are shown in Table 5 below.

(35) TABLE-US-00005 TABLE 5 Neem extract (ppm) Duration 20,000 8,000 4,000 2,000 1,000 1 day  10.sup.a 7 5 2 1 2 days 13 9 10 4 2 3 days 15 15 15 9 5 Nematocidal efficiency (%) 100  100 100 60 33.3 .sup.aNumber of killed root-knot nematodes

(36) As is apparent from Table 5, when the dilution factor of the neem extract was 20,000 ppm, 8,000 ppm and 4,000 ppm, 15 root-knot nematodes were killed 3 days after treatment therewith, thus exhibiting 100% nematocidal efficiency, and at 2,000 ppm, 60% nematocidal efficiency was manifested. Therefore, the possibility of controlling root-knot nematodes using the neem extract at 20,000 to 4,000 ppm was confirmed.

Example 5: Evaluation of Nematocidal Activity of Mixed Solution of Geraniol and Plant Extract

(37) Synergistic effects were evaluated by mixing each of the neem extract, clove extract and Ostericum koreanum extract with geraniol, which showed excellent nematocidal activity.

(38) Nematocidal activity on root-knot nematodes was evaluated in the same manner as in Example 1, with the exception that the plant extract and geraniol were mixed at a volume ratio of 4:1 (a mixed solution of geraniol and natural substance: 2500 ppm) and used as a sample. The results thereof are shown in Table 6 below.

(39) TABLE-US-00006 TABLE 6 Ostericum koreanum Neem extract + Clove extract + extract + Duration geraniol (4:1) geraniol (4:1) geraniol (4:1) 1 day  9.sup.a 5 8 2 days 13 11 13 3 days 15 15 15 Nematocidal 100  100 100 efficiency (%) .sup.aThe number of inoculated root-knot nematodes used for the experiment on the number of killed root-knot nematodes was 15

(40) As is apparent from Table 6, all of the mixed solution of neem extract and geraniol, the mixed solution of clove extract and geraniol, and the mixed solution of Ostericum koreanum extract and geraniol killed all 15 root-knot nematodes 3 days after treatment therewith, thus exhibiting 100% nematocidal efficiency.

(41) Thereby, all three were judged to have nematocidal effects on root-knot nematodes, and pot experiments and phytotoxicity experiments were thus conducted thereon.

Example 6: Experiment on Phytotoxicity of Mixed Solution of Geraniol and Plant Extract

(42) Sterilized soil was uniformly mixed, 300 g thereof was then placed in each pot, and tomato seedlings not infected with root-knot nematodes were transplanted thereto. The sample prepared in Example 4 was diluted to a standard amount or a multiple thereof for each treatment group, and then 50 ml thereof was used for drenching treatment. Here, the same amount of distilled water was used alone in an untreated group, and 1 week after treatment, the presence or absence of phytotoxicity was observed with the naked eye. The results thereof are shown in Table 7 below and in FIG. 3.

(43) TABLE-US-00007 TABLE 7 Extent of phytotoxicity (0-5) Test Standard Multiple Phytotoxicity Test drug crop amount amount symptom Neem extract + Tomato 0 0 No geraniol Clove extract + Tomato 0 0 No geraniol Ostericum Tomato 0 0 No koreanum extract + geraniol

(44) As is apparent from Table 7 and FIG. 3, there were no particular phytotoxicity symptoms before or after treatment with the mixed solution of geraniol and plant extract. Therefore, it was determined that the mixed solution of geraniol and plant extract did not cause any harm to the growth of the tomato seedlings.

Example 7: Pot Experiment on Mixed Solution of Geraniol and Plant Extract

(45) Soil infected with root-knot nematodes was uniformly mixed, the initial root-knot nematode density was measured (number of nematodes/300 g), and 300 g of the soil infected with root-knot nematodes was placed in each pot and then treated with 50 ml of the sample prepared in Example 4. In order to prevent moisture evaporation, the pot was covered with vinyl film and then treated two times with the sample at intervals of 1 week, and after 2 weeks, the density of root-knot nematodes was measured. The results thereof are shown in Table 8 below. Here, 50 ml of distilled water was used alone in an untreated group, and the experiment was repeated three times.

(46) TABLE-US-00008 TABLE 8 Ostericum Neem Clove koreanum extract + extract + extract + Initial geraniol geraniol geraniol density Control (4:1) (4:1) (4:1) Density 3,367 2,127 550 967 750 (number of nematodes/ 300 g) Efficiency of — 36.83 83.66 71.28 77.72 controlling nematodes (%)

(47) As is apparent from Table 8, the initial density of root-knot nematodes in the soil was 3,367 nematodes/300 g. Based on the results of treatment of the soil infected with root-knot nematodes with each sample, the control showed a density of 2,127 nematodes/300 g, and the neem extract+geraniol (4:1), the clove extract+geraniol (4:1) and the Ostericum koreanum extract 30 geraniol (4:1) showed densities of 550 nematodes/300 g, 967 nematodes/300 g and 750 nematodes/300 g, respectively.

(48) As for the efficiency of controlling root-knot nematodes using each drug, the neem extract+geraniol (4:1) exhibited the highest nematocidal effect of 83.66% compared to the initial density thereof, and then the Ostericum koreanum extract+geraniol (4:1) and the clove extract +geraniol (4:1) exhibited nematocidal effects of 77.72% and 71.28%, respectively. The nematocidal effect of the control was 36.83%.

Example 8: Nematocidal Effects of Geraniol on Other Plant-Parasitic Nematodes

(49) 2-3 ml of a 2,000 ppm dilution solution of geraniol (available from Katyani Exports) was poured into Petri dishes, and 15 root-lesion nematodes, 15 spiral nematodes, and 15 cyst nematodes, which are different kinds of plant-parasitic nematodes, were placed in respective

(50) Petri dishes, followed by measurement of nematocidal activity. The results thereof are shown in Table 9 below.

(51) TABLE-US-00009 TABLE 9 Root-lesion Spiral Cyst Classification nematodes nematodes nematodes 1 day  14.sup.a 7 8 2 days 15 9 11 3 days — 13 12 Nematocidal efficiency 100  86.7 80 .sup.aThe number of nematodes used for the experiment on the number of killed root-knot nematodes was 15.

(52) As is apparent from Table 9, nematocidal efficiency of 80% or more was exhibited on all of root-lesion nematodes, spiral nematodes and cyst nematodes.

Comparative Example 1: Nematocidal Effect of Palmarosa Oil on Root-Knot Nematodes

(53) In order to compare the nematocidal effects of palmarosa oil and geraniol, as disclosed in Korean Patent Application Publication No. 2009-0011029, a composition including 93% canola, 2% palmarosa oil (made by AOS Products Private Limited, geraniol content: about 68.5%) and 20.5% polysorbate, which were mixed together, was prepared.

(54) The composition thus prepared was diluted to a concentration of 5% (v/v) with tap water, after which the diluted solution (geraniol concentration: 685 ppm) was vigorously stirred for 1 hr using a high-speed magnetic stirrer and allowed to stand for 30 min so that layer separation occurred, after which only the solution (A) in the lower water layer, rather than the layer formed stably on the surface, was taken, and the nematocidal activity thereof on root-knot nematodes was evaluated. The results thereof are shown in Table 10 below. Here, the solution obtained by mixing geraniol with an emulsifier (PLE9) at 1:1 as in Example 1 and performing dilution to a geraniol concentration of 685 ppm was used as a control.

(55) TABLE-US-00010 TABLE 10 Nematocidal efficiency on root-knot nematodes (%) 1 day 2 days 3 days Untreated — — — Palmarosa oil 17.6 17.6 23.5 composition dilution solution (A) Geraniol 685 ppm 72.5 93.6 100.0

(56) As is apparent from Table 10, even when the geraniol content was the same, the diluted solution of the geraniol-containing palmarosa oil composition exhibited nematocidal efficiency of 17.6% on 1-2 days and 23.5% on the 3.sup.rd day, whereas when using geraniol, the nematocidal efficiency was 72.5% on the 1.sup.st day, 93.6% on the 2.sup.nd day, and 100% on the 3.sup.rd day, indicative of very high nematocidal effects of geraniol compared to the palmarosa oil. This is deemed to be due to the antagonistic effects of materials that make up the palmarosa oil.

(57) Specifically, in Korean Patent Application Publication No. 2009-0011029, the nematocidal activity of palmarosa oil, which is a complex, was confirmed, but there is no description of what compounds exhibit substantial nematocidal activity, among compounds such as dihydrotagetone, thiophene, ocimene, linalool, thymol, estragole, geraniol and the like, constituting palmarosa oil. However, it was confirmed in the present invention that the material that shows substantial nematocidal activity among the compounds constituting palmarosa oil is geraniol, and the use of geraniol alone has a superior nematocidal effect about 4.2 times as high as that of palmarosa oil containing the same amount of geraniol. Furthermore, the present invention is significant in that the concentration of geraniol that can exhibit the maximum nematocidal effect without causing phytotoxicity was found to be 100 to 3,000 ppm.

(58) Although specific embodiments of the present invention have been disclosed in detail as described above, it will be obvious to those skilled in the art that the description is merely of preferable exemplary embodiments and is not to be construed to limit the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

(59) According to the present invention, a composition for controlling plant-parasitic nematodes containing geraniol at a concentration of 100 to 3,000 ppm is derived from natural substances and is thus environmentally friendly, and can exhibit superior nematocidal effects without causing phytotoxicity, and can thus be efficiently used as a nematicide.