JP2010200628A - Method for sterilizing seeds and seeds - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a method for sterilizing seeds by which an incidence with pathogenic microorganisms on the whole seeds can be further reduced compared with conventional methods. <P>SOLUTION: The method for sterilizing the seeds includes alternately soaking the seeds in warm water and cold water to steriliz the seeds, wherein the seeds are soaked in the warm water at least twice. It is preferable that the temperatures of the warm water and the cold water are 40 to 75°C and 0 to 30°C, respectively. It is also preferable that the time for soaking the seeds in the warm water and the time for soaking the seeds in the cold water are 1 to 30 min and 1 to 10 min, respectively. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a seed disinfecting method used when disinfecting seeds such as vegetables and fruits, and a seed disinfected using this method.

  Fruit foul bacterial disease occurred in the United States in 1989-1995 and caused enormous damage. Therefore, Acidvorax avenae subsp. Citrulli (hereinafter referred to as “Aac”), which is a pathogenic bacterium, has been designated as a plant that requires cultivation site inspection in the Enforcement Regulations of the Plant Protection Act, and is wary of entering Japan. However, since it was first confirmed in Yamagata Prefecture in 1998, it has been sporadically occurring in watermelon production areas. In 2005, it has also been confirmed in melons, and there are cautions against further invasion and colonization of this disease (for example, Non-patent documents 1 and 2). Aac is known to infect and cause disease widely in cucurbitaceous plants, and since there is a problem of seed transmission in Japan, establishment of seed disinfection technology is urgently required.

  So far, as a method for disinfecting seeds, a mixed liquid immersion treatment and a peracetic acid solution immersion treatment of a copper wettable powder and an organic acid have been developed (for example, see Non-Patent Document 3). However, although this disinfection method is effective for watermelon and melon seeds, it has a low control effect on seeds of other crops such as pumpkin seeds. In particular, pumpkin seeds cannot be expected to have a sufficient control effect even by dry heat sterilization.

  In addition, a disinfection method using antibiotics such as oxytetracycline, oxytetracycline / streptomycin, oxytetracycline / streptomycin / copper wettable powder has been developed (for example, see Non-Patent Document 4), and has achieved certain results against Aac. Although listed, there is still room for improvement and it is unclear whether it is effective against many other pathogens.

Takashi Shirakawa (2005) Facility and Horticulture 131: 36-39. Takashi Shirakawa, Shigemi Kikuchi, Tomohiro Kato, Kazuo Abiko, Akira Kawai (2000) Nikkan Disease Report 66: 223-231. Kazuaki Hara, Ryoko Hikami, Kiichi Shimazu, Takashi Shirakawa (2006) Kansai Insect Research Bulletin (48): 57-60. Kazuaki Hara, Takashi Shirakawa, Ryoko Hikami, and Kiichi Shimazu, “Proceedings of the 2007 Annual Meeting of the Japanese Society of Plant Pathology”, March 12, 2007, p. 169

  The present invention has been made in view of the above points. A method for disinfecting seeds that can reduce the incidence of a wide variety of pathogens in general compared to conventional seeds, and a seed disinfected using the method are disclosed. It is intended to provide.

  The seed disinfection method according to claim 1 of the present invention is a seed disinfection method in which seeds are disinfected by alternately immersing seeds in warm water and cold water, wherein the seeds are immersed in warm water at least twice. is there.

  The invention according to claim 2 is characterized in that, in claim 1, the temperature of hot water is 40 to 75 ° C, and the temperature of cold water is 0 to 30 ° C.

  The invention according to claim 3 is characterized in that, in claim 1 or 2, the time of immersion in hot water is 1 to 30 minutes and the time of immersion in cold water is 1 to 10 minutes.

  The invention according to claim 4 is characterized in that, in any one of claims 1 to 3, both or one of hot water and cold water contains both or one of agrochemicals and organic acids.

  The seed according to claim 5 of the present invention is characterized in that it is disinfected using the method according to any one of claims 1 to 4.

  According to the method for disinfecting seeds according to claim 1 of the present invention, it is possible to reduce the incidence of a wide variety of pathogenic bacteria with respect to the whole seeds.

  According to the invention which concerns on Claim 2, a disease incidence rate can further be reduced.

  According to the invention of claim 3, the disease incidence can be further reduced.

  According to the invention of claim 4, in addition to causing physical damage to pathogenic bacteria by heating with hot water, both or one of hot water and cold water is used as a chemical solution, and the pathogenic bacteria are killed by chemically damaging the pesticide and organic acid. Thus, the disease incidence can be further reduced.

  According to the seed according to claim 5 of the present invention, it is possible to supply a highly safe crop because the incidence of a wide range of pathogenic bacteria is lower than the conventional one.

It shows the relationship between the number of microspores in the melon vine split fungus microspore suspension (vertical axis) and the accumulated time (horizontal axis) of holding the melon vine split microbe microspore suspension at 47.5 ° C. It is a graph. It is a graph which shows the relationship between the number of bacteria in a cucumber spot bacterial disease bacterial suspension (vertical axis) and the integration time (horizontal axis) for holding the cucumber spot bacterial disease bacterial suspension at 45 ° C. It is a graph which shows the relationship between the number of bacteria in the cruciferous black rot bacterial suspension (vertical axis) and the accumulated time (horizontal axis) which holds the cruciferous black rot bacterial suspension at 45 ° C. . A graph showing the relationship between the number of bacteria in the suspension of Yugao brown spot bacterial disease (vertical axis) and the cumulative time (horizontal axis) for holding the suspension of Yugao brown spot bacterial disease at 42.5 ° C. It is. It is a graph which shows the relationship between the number of bacteria in a tomato scab bacterial suspension (vertical axis) and the integration time (horizontal axis) which keeps the tomato scab bacterial suspension at 45 degreeC. It is a graph which shows the relationship between the bacterial count (vertical axis) in fruit stain bacterial pathogen (Aac) suspension and the integration time (horizontal axis) which keeps Aac suspension at 50 degreeC. It is a graph which shows the relationship between the disease incidence (vertical axis) of the disinfected artificially contaminated seed and the accumulated time (horizontal axis) in which the artificially contaminated seed is immersed in hot water at 50 ° C. It is a bar graph which shows the prevention | control value of Examples 3-9 and Comparative Examples 3-9.

  Embodiments of the present invention will be described below.

  The seeds to be disinfected in the present invention are not particularly limited. For example, cucurbitaceae vegetables such as watermelon, melon, cucumber, pumpkin, yugao, and gangan, and solanaceae vegetables such as tomato, pepper and eggplant. Examples include cruciferous vegetables such as radish, Chinese cabbage, cabbage, and other crops such as rice, wheat and barley. Furthermore, sprouts, seeds of vegetable vegetables such as alfalfa and silkworm radish can be mentioned.

  The pathogenic bacteria that can be killed by the present invention are not particularly limited, and examples thereof include filamentous fungi, bacteria, viruses, and the like, and nematodes and the like can also be killed by the present invention. Among these, examples include filamentous fungi, wilt disease fungus, black spot fungus, anthracnose fungus, rice blast fungus, sesame leaf blight fungus, stupid seedling fungus, maggot smut fungus, naked smut fungus, leaf spot fungus, etc. Can do. Examples of the bacteria include fruit stain bacterial pathogen (Aac), scab pathogen, spotted bacterial pathogen, black rot pathogen, brown spot bacterial pathogen, and rice wilt bacterial pathogen.

  In the seed disinfection method according to the present invention, the seed contaminated with the above-mentioned pathogenic bacteria is heated with warm water (water such as distilled water heated to a predetermined temperature) and cold water (water such as distilled water with a predetermined temperature). It is disinfected by alternately dipping in a cooled one), but in particular, the seed is immersed in warm water at least twice. For example, seeds are immersed in the order of hot water → cold water → hot water, and this is the most basic disinfection method, but as long as the part of `` hot water → cold water → hot water '' is included, the first and last immersion is Either immersion in hot water or immersion in cold water may be used. The seed after the last soaking is preferably air-dried and then stored at 0 to 20 ° C., but is not limited to this storage temperature.

  Conventionally, as a physical method for seed disinfection, a hot water soaking method and a cold hot water soaking method are known. Both of these methods immerse seeds in warm water only once. However, a one-time immersion in warm water cannot completely kill a wider range of pathogenic bacteria, and the disease incidence cannot be sufficiently reduced. Therefore, it is conceivable to immerse the seeds in warm water for a long time. However, if the time for continuous soaking becomes longer, the pathogens will adapt to the surrounding environment and become heat resistant, or the seeds themselves may become longer. There is a risk of damage by continuous heating. In contrast, in the present invention, since the seeds are immersed in warm water at least twice or more, a wide range of pathogenic bacteria can be completely killed, and the incidence rate is reduced and the germination rate is improved. It is something that can be done. Further, in the present invention, the seeds are not immersed continuously in hot water for a long time, but the seeds are alternately immersed in hot water and cold water for a short time, so the environment where the seeds are placed is thus By changing frequently, stress can be applied to a wide range of pathogens that contaminate the seeds, preventing them from acquiring the ability to adapt to the environment, such as heat resistance, and preventing the seeds themselves from being damaged. Is something that can be done. Furthermore, since the present invention does not require any particularly complicated treatment other than immersing the seeds in warm water at least twice or more, the seed disinfection method according to the present invention can be applied to all seeds contaminated with a wide range of pathogenic bacteria. It can be done. In addition, although the frequency | count of immersing in warm water changes with the temperature and immersion time of warm water mentioned later, the upper limit is about 20 times, for example.

  Here, it is preferable that the temperature of warm water is 40-75 degreeC, and it is preferable that the temperature of cold water is 0-30 degreeC. Thus, the disease incidence can be further reduced by setting the temperature of hot water and cold water. However, if the hot water temperature is lower than 40 ° C., pathogenic bacteria with low heat resistance can be killed, but pathogenic bacteria with high heat resistance may remain without being completely killed. On the other hand, if the hot water temperature is higher than 75 ° C., even if a wide variety of pathogenic bacteria can be completely killed, the seed itself may be damaged by heat and not germinate. If the cold water temperature is lower than 0 ° C., germination damage may occur. On the other hand, if the cold water temperature is higher than 30 ° C., the temperature difference from the hot water becomes small, and there is a possibility that the seeds are not different from the case where the seeds are continuously immersed in the hot water for a long time.

  Moreover, it is preferable that the time immersed in warm water is 1 to 30 minutes, and it is preferable that the time immersed in cold water is 1 to 10 minutes. Thus, the disease incidence can be further reduced by setting the time for immersion in hot water and cold water. However, if the hot water immersion time is shorter than 1 minute, the seed may not be heated to the temperature of the hot water, or the number of hot water immersions may increase. Conversely, if the warm water immersion time is longer than 30 minutes, even if a wide variety of pathogenic bacteria can be completely killed, the seed itself may be damaged by heat and not germinate. In addition, when the cold water immersion time is shorter than 1 minute, the seed cannot be cooled to the temperature of the cold water, and there is a possibility that the seed is not different from the case where the seed is continuously immersed in the hot water for a long time. Conversely, if the cold water immersion time is longer than 10 minutes, the total time required for seed disinfection may be increased.

  Moreover, it is preferable that both or one of an agrochemical and an organic acid is contained as a chemical | medical agent in both or one of warm water and cold water. In addition to causing physical damage to pathogens by heating with warm water, both pathogenic bacteria are killed by chemically damaging them with pesticides and organic acids using both hot and cold water as chemicals. Can be further reduced. In particular, when a chemical solution is prepared using a plurality of agricultural chemicals or organic acids, the disease incidence can be further reduced by a synergistic effect thereof.

  Here, as an agrochemical, a copper wettable powder etc. can be used, for example. When using a copper wettable powder, the dilution factor with water is preferably 50 to 2000 times. Thus, the disease rate can be further reduced by containing the copper wettable powder in hot water or cold water. However, if the dilution factor of the copper wettable powder is larger than 2000 times, the effect of reducing the disease rate by the copper wettable powder may not be sufficiently obtained. Conversely, the dilution factor of the copper wettable powder is more than 50 times. If it is small, germination damage may occur, and the cost of the drug at the time of disinfection may increase.

  On the other hand, as an organic acid, ascorbic acid, acetic acid, peracetic acid, malic acid, lactic acid, etc. can be used, for example. When using ascorbic acid, the content is preferably 0.01 to 0.5M. Thus, ascorbic acid is contained in warm water or cold water, the onset rate can be further reduced. However, if the ascorbic acid content is less than 0.01M, the effect of reducing the incidence of ascorbic acid may not be sufficiently obtained. Conversely, if the ascorbic acid content is more than 0.5M, germination There is a possibility that a failure may occur, and the cost of the medicine at the time of disinfection may increase. Moreover, when using acetic acid, it is preferable that the content is 0.01-0.5M. Thus, acetic acid is contained in warm water or cold water, so that the disease incidence can be further reduced. However, if the content of acetic acid is less than 0.01M, there is a possibility that the effect of reducing the disease rate by acetic acid cannot be sufficiently obtained. Conversely, if the content of acetic acid is more than 0.5M, germination damage occurs. In addition, there is a risk that the cost of the medicine at the time of disinfection increases. Moreover, when using peracetic acid, it is preferable that the content is 800-3200 microgram / ml. As described above, peracetic acid is contained in warm water or cold water, so that the disease incidence can be further reduced. However, if the content of peracetic acid is less than 800 μg / ml, the effect of reducing the incidence of peracetic acid may not be sufficiently obtained. Conversely, if the content of peracetic acid is more than 3200 μg / ml, germination There is a possibility that a failure may occur, and the cost of the medicine at the time of disinfection may increase.

  Further, in the present invention, seeds are sterilized by alternately immersing seeds in hot water and cold water. If each immersion time is short, the temperature of the surface portion of the seed is equal to the temperature of hot water or cold water in a short time. Even if it can, the temperature inside the seed may not be equal to the temperature of hot water or cold water in a short time. Therefore, before immersing the seed in hot water or cold water for the first time, the seed is immersed for 10 to 120 minutes in water such as distilled water set to 5 to 35 ° C. or the aforementioned chemical solution set to 5 to 35 ° C. Is preferred. As described above, when the seed is pretreated with water or a chemical solution, the water or the chemical solution penetrates into the inside of the seed and heat is easily transmitted, and the temperature inside the seed is quickly changed to the temperature of hot water or cold water. It can be made equal. In particular, in the case of pretreatment using a chemical solution, in addition to the above-described effects, the effect that most of the pathogenic bacteria existing on the seed surface and inside can be preliminarily killed by the chemical solution can be obtained. In addition, when the immersion time of pretreatment is shorter than 10 minutes, there exists a possibility that water or a chemical | medical solution cannot be penetrate | infiltrated to the inside of a seed. On the other hand, if the pretreatment soaking time is longer than 120 minutes, the seed storage quality may be adversely affected.

  As described above, in the seed sterilized by using the seed disinfection method according to the present invention, the disease incidence caused by a wide range of pathogenic bacteria is reduced as compared with the conventional method, so that a highly safe crop is supplied. Is something that can be done.

  Although the technical field is different from the present invention, it is considered that the present invention can be applied to disinfection of milk, beer, etc. in the food field, and disinfection of plasma, etc. in the medical field.

  Hereinafter, the present invention will be specifically described by way of examples.

[Disinfection of spore suspension of melon vine split fungus]
Melon vine split fungus race 1.2y (Me102065) was cultured with shaking in PS medium in a flask for 4 days. Next, this was filtered with gauze and then centrifuged at 3500 rpm for 20 minutes. Then, after discarding the supernatant and suspending in phosphate buffer, count the number of microspores using a Thoma hemocytometer, and add phosphate buffer so that the number of microspores is 10 ⁶ cfu / mL. By this, a melon vine split fungus microspore suspension was prepared.

  Next, the PCR microtube to which 100 μL of the melon vine split microbe spore suspension prepared as described above was added was attached to a DNA amplification apparatus (manufactured by Bio-Rad Laboratories Co., Ltd.). Sterilized by method 3. Note that the DNA amplification device was used for temperature control, not for DNA amplification. The same applies to the following.

  In the first method, the treatment of holding a melon vine split fungus microspore suspension at 47.5 ° C. for 10 minutes and then holding at 4 ° C. for 2 minutes was repeated 6 times as one cycle.

  Further, in the second method, the treatment of holding the melon vine split microspore suspension at 47.5 ° C. for 5 minutes and then holding at 4 ° C. for 2 minutes was repeated 12 times as one cycle.

  Furthermore, in the third method, the suspension of melon vine split fungus microspores was held at 47.5 ° C. for 60 minutes.

  And in the said 1st-3rd method, the number of living microspores in each melon vine split fungus microspore suspension was measured by the plate dilution method using a PSA culture medium. The result is shown in FIG. In addition, the horizontal axis of the graph of FIG. 1 is the integration time which hold | maintains the melon vine split fungus microspore suspension at 47.5 degreeC.

  As shown in FIG. 1, at the time when the accumulated time of holding the melon vine split fungus microspore suspension at 47.5 ° C. reaches 60 minutes, the first and second methods are compared with the third method. In this method, it was confirmed that the number of microspores was significantly reduced. Moreover, in the second method, when the accumulated time of holding the melon vine split fungus microspore suspension at 47.5 ° C. reaches 40 minutes, the melon vine split fungus microspore is completely killed. It was confirmed that

[Disinfection of bacterial suspension of cucumber spotted bacterial disease]
Cucumber spotted bacterial disease bacterium (MAFF730011) was stirred and cultured at 25 ° C. for 24 hours in YP medium in a vial. Next, after centrifuging at 3500 rpm for 20 minutes, the supernatant was discarded and suspended in a phosphate buffer of 20 times the amount of YP medium to prepare a cucumber spotted bacterial disease bacterial suspension.

  Next, the PCR microtube to which 100 μL of the cucumber spot bacterial disease bacterial suspension prepared as described above was added was mounted on a DNA amplification apparatus (Bio-Rad Laboratories Co., Ltd.). It was disinfected by the method.

  In the first method, the treatment of holding the cucumber spot bacterial disease bacterial suspension at 45 ° C. for 10 minutes and then holding at 20 ° C. for 2 minutes was repeated four times as one cycle.

  In the second method, the treatment of holding the cucumber spot bacterial disease bacterial suspension at 45 ° C. for 5 minutes and then holding at 20 ° C. for 2 minutes was repeated 8 times as one cycle.

  Furthermore, in the third method, the cucumber spotted bacterial disease bacterial suspension was kept at 45 ° C. for 40 minutes.

  And in the said 1st-3rd method, the number of living bacteria in each cucumber spot bacterial disease bacterial suspension was measured by the plate dilution method using a YPA culture medium. The result is shown in FIG. In addition, the horizontal axis of the graph of FIG. 2 is the integration time which hold | maintains the cucumber spot bacterial disease bacterial suspension at 45 degreeC.

  As shown in FIG. 2, when the accumulated time of holding the cucumber spot bacterial disease bacterial suspension at 45 ° C. has reached 40 minutes, the first and second methods completely eliminate the cucumber spot bacterial disease bacteria. In the third method, it was confirmed that cucumber spotted bacterial diseases still remained. Moreover, in the second method, the number of bacteria becomes 0 at the time when the cumulative time during which the cucumber spotted bacterial disease bacterial suspension is maintained at 45 ° C. reaches 30 minutes, which is earlier than in the first method. It was confirmed that the cucumber spotted bacterial disease was completely killed.

[Disinfection of Brassicaceae black rot bacterial suspension]
Cruciferous black rot bacteria (Xcc9501) were stirred and cultured at 25 ° C. for 24 hours in YP medium in a vial. Next, after centrifuging at 3500 rpm for 20 minutes, the supernatant was discarded and suspended in a phosphate buffer of 20 times the amount of YP medium to prepare a cruciferous black rot bacterial suspension.

  Next, a PCR microtube to which 100 μL of the cruciferous black rot bacterial suspension prepared as described above was added was attached to a DNA amplification apparatus (Bio-Rad Laboratories Co., Ltd.). Sterilized by method 3.

  In the first method, the treatment of holding the cruciferous black rot bacterial suspension at 45 ° C. for 10 minutes and then at 20 ° C. for 2 minutes was repeated four times as one cycle.

  In the second method, the treatment of holding the Brassicaceae black rot bacterial suspension at 45 ° C. for 5 minutes and then at 20 ° C. for 2 minutes was repeated 8 times as one cycle.

  Furthermore, in the third method, the Brassicaceae black rot bacterial suspension was kept at 45 ° C. for 40 minutes.

  In the first to third methods, the number of surviving bacteria in each cruciferous black rot bacterial suspension was measured by a plate dilution method using a YPA medium. The result is shown in FIG. In addition, the horizontal axis of the graph of FIG. 3 is the integration time which hold | maintains the Brassicaceae black rot bacterial suspension at 45 degreeC.

  As shown in FIG. 3, the first and second methods are compared to the third method at the time when the accumulated time of holding the Brassicaceae black rot bacterial suspension at 45 ° C. reaches 40 minutes. Then, it was confirmed that the number of bacteria decreased remarkably. Moreover, in the second method, the cruciferous black rot bacteria are completely killed when the cumulative time of holding the cruciferous black rot bacterial suspension at 45 ° C. reaches 40 minutes. Was confirmed.

[Disinfection of Bacterial suspension of Yugao brown spot bacterial disease]
The stagnation fungus (MAFF301081) was stirred and cultured at 25 ° C. for 24 hours in YP medium in a vial. Next, after centrifuging at 3500 rpm for 20 minutes, the supernatant was discarded and suspended in a phosphate buffer of 20 times the amount of YP medium to prepare a suspension of Yugao brown spot bacterial disease.

  Next, the PCR microtube to which 100 μL of the suspension of Yugao brown spot bacterial disease prepared as described above was attached to a DNA amplification apparatus (manufactured by Bio-Rad Laboratories Co., Ltd.). Sterilized by method 3.

  In the first method, the treatment of holding the suspension of the flyfish brown spot bacterial disease at 42.5 ° C. for 10 minutes and then holding at 20 ° C. for 2 minutes was repeated four times as one cycle.

  Moreover, in the 2nd method, the process of hold | maintaining 2 minutes at 20 degreeC after hold | maintaining the suspension of a stagnation brown spot bacterial disease for 5 minutes at 42.5 degreeC was repeated 8 times.

  Furthermore, in the third method, the suspension of Yugao brown spot bacterial disease was kept at 42.5 ° C. for 40 minutes.

  And in the said 1st-3rd method, the number of living bacteria in each Yugao brown spot bacterial disease bacterial suspension was measured by the plate dilution method using a YPA culture medium. The result is shown in FIG. In addition, the horizontal axis of the graph of FIG. 4 is the integration time which hold | maintains Yugao brown spot bacterial disease bacterial suspension at 42.5 degreeC.

  As shown in FIG. 4, at the time when the accumulated time of holding the suspension of Yugao brown spot bacterial disease at 42.5 ° C. has reached 40 minutes, the first and second methods are compared with the third method. In this method, it was confirmed that the number of bacteria was remarkably reduced. Moreover, in the second method, at the time when the accumulated time of holding the suspension of the fungus brown spot bacterial disease at 42.5 ° C. reaches 40 minutes, the fly fungus brown spot bacterial disease bacteria are completely killed. It was confirmed that

[Disinfection of bacterial suspension of tomato wilt disease]
Tomato scab disease bacterium (MAFF730041) was stirred and cultured at 25 ° C. for 48 hours in YP medium in a vial. Next, after centrifuging at 3500 rpm for 20 minutes, the supernatant was discarded and suspended in a phosphate buffer of 20 times the amount of YP medium to prepare a tomato scab bacterial suspension.

  Next, the PCR microtube to which 100 μL of the tomato scab bacterial suspension prepared as described above was added was attached to a DNA amplification apparatus (Bio-Rad Laboratories Co., Ltd.), and the following first to third Disinfected by the method.

  In the first method, the tomato scab disease bacterial suspension was held at 45 ° C. for 10 minutes and then held at 20 ° C. for 2 minutes, and was repeated four times as one cycle.

  In the second method, the treatment of holding the tomato scab bacterial suspension for 5 minutes at 45 ° C. and then for 2 minutes at 20 ° C. was repeated 8 times as one cycle.

  Further, in the third method, the tomato scab bacterial suspension was kept at 45 ° C. for 40 minutes.

  And in the said 1st-3rd method, the number of living bacteria in each tomato scab disease suspension was measured by the plate dilution method using the YPA culture medium for 5g of sucrose per liter. The result is shown in FIG. In addition, the horizontal axis of the graph of FIG. 5 is the integration time which has maintained the tomato scab bacterial suspension at 45 degreeC.

  As shown in FIG. 5, compared to the third method, the first and second methods at the time when the accumulated time of maintaining the tomato scab bacterial suspension at 45 ° C. reached 40 minutes, It was confirmed that the number of bacteria decreased.

[Disinfection of suspension of fruit stain bacteria (Aac)]
Aac (watermelon isolate: Aac9801) was stirred and cultured at 36 ° C. for 24 hours in YP medium in a vial. Next, this was centrifuged at 3500 rpm for 20 minutes, and then the supernatant was discarded and suspended in a phosphate buffer. Then, after measuring the absorbance of the suspension at a wavelength of 660 nm using a spectrophotometer, a phosphate buffer solution is added using a calibration curve so that the bacterial density is 10 ⁸ cfu / mL, thereby suspending the Aac suspension. A liquid was prepared.

  Next, the PCR microtube to which 100 μL of the Aac suspension prepared as described above was added was attached to the DNA amplification apparatus, and sterilized by the following first to third methods.

  That is, in the first method, the treatment of holding the Aac suspension at 50 ° C. for 10 minutes and then holding at 20 ° C. for 2 minutes was repeated four times as one cycle.

  In the second method, the treatment of holding the Aac suspension at 50 ° C. for 5 minutes and then holding at 20 ° C. for 2 minutes was repeated 8 times as one cycle.

  Furthermore, in the third method, the Aac suspension was held at 50 ° C. for 40 minutes.

  In the first to third methods, the number of living bacteria in each Aac suspension was measured by a plate dilution method using an Aac selective medium AacSM basal medium. The result is shown in FIG. In addition, the horizontal axis of the graph of FIG. 6 is the integration time during which the Aac suspension is maintained at 50 ° C.

  As shown in FIG. 6, when the accumulated time of maintaining the Aac suspension at 50 ° C. reaches 40 minutes, the first and second methods show that Aac is completely dead. In the third method, it was confirmed that Aac still remained.

  As described above, melon vine split fungus microspore suspension, cucumber spotted bacterial disease bacterial suspension, cruciferous black rot bacterial suspension, Yugao brown spot bacterial disease bacterial suspension, tomato scab bacterial suspension The liquid and Aac suspension were alternately contaminated with various pathogens contained in these suspensions because it was confirmed that they could be disinfected if they were held alternately at high and low temperatures and kept at high temperatures at least twice. Similarly, it is considered that seeds can be sterilized by alternately immersing seeds in warm water and cold water and immersing them in warm water at least twice.

[Disinfection of Pumpkin Seeds Contaminated with Fruit Bacteria Bacteria (Aac)]
(Creation of artificially contaminated seeds)
Aac was cultured in an Erlenmeyer flask with stirring at 36 ° C. for 24 hours. Next, this was centrifuged at 3500 rpm for 20 minutes, and then the supernatant was discarded and suspended in a phosphate buffer. Then, after measuring the absorbance of the suspension at a wavelength of 660 nm using a spectrophotometer, a phosphate buffer solution is added using a calibration curve so that the bacterial density is 10 ⁸ cfu / mL, thereby suspending the Aac suspension. 2 L of the liquid was prepared.

  Then, 3L of pumpkin 'Aqua Shintosa' (about 6000 grains) is immersed in the Aac suspension prepared as described above, decompressed with an aspirator for 30 minutes, and then air-dried to pumpkin seeds contaminated with Aac. It was created.

(Disinfection of artificially contaminated seeds 1)
<Example 1>
First, artificially contaminated seeds were immersed in distilled water set at 20 ° C. for 60 minutes.

  Next, 1 L beakers containing distilled water were installed in two water baths, respectively, the temperature of distilled water in one beaker was adjusted to 50 ° C., and the temperature of distilled water in the other beaker was adjusted to 15 ° C. Then, the artificially contaminated seeds pretreated as described above were immersed in 50 ° C. warm water for 10 minutes and then immersed in 15 ° C. cold water for 5 minutes. .

<Example 2>
As a chemical solution, copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and Alcorbine An acid containing 0.5M was prepared.

  First, artificially contaminated seeds were immersed in a chemical solution set at 20 ° C. for 60 minutes.

  Next, the same treatment as in Example 1 was repeated four times as one cycle, except that chemical solutions having the same composition as above were used as hot water and cold water, and then air-dried.

<Comparative example 1>
The artificially contaminated seeds were immersed in distilled water set at 20 ° C. for 60 minutes and then air-dried.

<Comparative example 2>
First, artificially contaminated seeds were immersed in distilled water set at 20 ° C. for 60 minutes.

  Next, the artificially contaminated seeds pretreated as described above were continuously immersed in hot water at 50 ° C. for 40 minutes, and then air-dried.

(Test of control effect)
A test tube having an inner diameter of 14 mm and a length of 100 mm was filled with sphagnum containing water, and the sterilized artificially contaminated seeds of Examples 1 and 2 and Comparative Example 2 were sown one by one. After these artificially contaminated seeds germinated, they were irrigated and placed under 25-35 ° C., 12 hours of temperature change, 12 hours of day length to promote disease. Then, for 10 days from 1 week, the affected individuals were examined using the latex agglutination method to determine whether the disease was caused by Aac, and the disease incidence was determined. The results are shown in Table 1 and FIG.

  As shown in Table 1 and FIG. 7, at the time when the accumulated time in which artificially contaminated seeds were immersed in warm water of 50 ° C. reached 40 minutes, in Examples 1 and 2, the disease incidence was 0%. On the other hand, in Comparative Example 2, it was confirmed that the disease incidence did not become 0%. Moreover, in Example 2, when the accumulated time in which artificially contaminated seeds are immersed in hot water at 50 ° C. reaches 20 minutes, the disease incidence becomes 0%, and the disease incidence is 0 earlier than in Example 1. % Was confirmed.

(Disinfection of artificially contaminated seeds 2)
<Comparative Example 3>
As a chemical solution, a copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and acetic acid is used. Containing 0.2M was prepared.

  First, artificially contaminated seeds were immersed in a chemical solution set at 20 ° C. for 60 minutes.

  Thereafter, the artificially contaminated seeds pretreated as described above were continuously immersed in a chemical solution at 55 ° C. (the same composition as that used in the pretreatment) for 40 minutes, and then air-dried.

<Comparative example 4>
The same treatment as in Comparative Example 3 was performed except that a chemical solution containing 0.2 M of ascorbic acid in distilled water was used.

<Comparative Example 5>
The same treatment as in Comparative Example 3 was performed except that a chemical solution containing 0.5 M ascorbic acid in distilled water was used.

<Comparative Example 6>
As a chemical solution, copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and ascorbine The same treatment as in Comparative Example 3 was performed, except that an acid containing 0.2M was used.

<Comparative Example 7>
As a chemical solution, copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and ascorbine The same treatment as in Comparative Example 3 was performed except that an acid containing 0.5M was used.

<Comparative Example 8>
The same treatment as in Comparative Example 3 was performed except that a chemical solution containing 1600 μg / ml peracetic acid in distilled water was used.

<Comparative Example 9>
The artificially contaminated seeds were immersed in distilled water set at 20 ° C. for 60 minutes and then air-dried.

<Example 3>
As a chemical solution, a copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and acetic acid is used. Containing 0.2M was prepared.

  First, artificially contaminated seeds were immersed in a chemical solution set at 20 ° C. for 60 minutes.

  Thereafter, 1 L beakers containing the chemical solution having the same composition as above were installed in two water baths, respectively, the temperature of the chemical solution in one beaker was adjusted to 55 ° C., and the temperature of the chemical solution in the other beaker was adjusted to 15 ° C. . Then, the treatment of immersing the artificially contaminated seed in a chemical solution at 55 ° C. for 10 minutes and then immersing the artificial contaminated seed in a chemical solution at 15 ° C. for 10 minutes was repeated four times as one cycle, and then air-dried.

<Example 4>
The same treatment as in Example 3 was performed except that a chemical solution containing 0.2 M of ascorbic acid in distilled water was used.

<Example 5>
The same treatment as in Example 3 was performed except that a chemical solution containing 0.5 M ascorbic acid in distilled water was used.

<Example 6>
As a chemical solution, copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and ascorbine The same treatment as in Example 3 was performed, except that an acid containing 0.2M was used.

<Example 7>
As a chemical solution, copper wettable powder (made by Hokuko Chemical Co., Ltd., consisting of 84.1% by mass of basic copper chloride and 15.9% by mass of mineral fine powder) is diluted 500 times with distilled water, and ascorbine The same treatment as in Example 3 was performed, except that an acid containing 0.5M was used.

<Example 8>
The same treatment as in Example 3 was performed, except that a chemical solution containing 1600 μg / ml peracetic acid in distilled water was used.

<Example 9>
First, artificially contaminated seeds were immersed in distilled water set at 20 ° C. for 60 minutes.

  Thereafter, 1 L beakers containing distilled water were installed in two water baths, respectively, the temperature of distilled water in one beaker was adjusted to 55 ° C., and the temperature of distilled water in the other beaker was adjusted to 15 ° C. Then, the artificially contaminated seed was immersed in 55 ° C. distilled water for 10 minutes and then immersed in 15 ° C. distilled water for 10 minutes. The treatment was repeated four times as one cycle, and then air-dried.

(Test of control effect)
A test tube having an inner diameter of 14 mm and a length of 100 mm is filled with sphagnum containing water, and one sterilized artificially contaminated seed of Examples 3 to 9 and Comparative Examples 3 to 7 and one non-sterilized artificially contaminated seed of Comparative Example 9 are used. Seeded one by one. After these artificially contaminated seeds germinated, they were irrigated and placed under 25-35 ° C., 12 hours of temperature change, 12 hours of day length to promote disease. Then, for 10 days from 1 week, the affected individuals were examined using the latex agglutination method to determine whether the disease was caused by Aac, and the disease incidence was determined. The results are shown in Table 2 and FIG. In addition, the test of the control effect was performed 3 times (a total of 150 grains) for 50 artificially contaminated seeds.

  As shown in Table 2 and FIG. 8, Examples 3 to 8 are alternately immersed in a high-temperature chemical solution and a low-temperature chemical solution rather than Example 9 in which they are alternately immersed in high-temperature distilled water and low-temperature distilled water. It was confirmed that the control value was improved.

  Even in the case of using a chemical solution, Examples 3 to 8 are alternately immersed in a high-temperature chemical solution and a low-temperature chemical solution for a short time, instead of Comparative Examples 3 to 8 that are continuously immersed in a high-temperature chemical solution for a long time. However, it was confirmed that the control value was improved.

Claims (5)

  A seed disinfection method for disinfecting seeds by alternately immersing seeds in warm water and cold water, wherein the seeds are immersed in warm water at least twice.   The temperature of warm water is 40-75 degreeC, and the temperature of cold water is 0-30 degreeC, The seed disinfection method of Claim 1 characterized by the above-mentioned.   The method for disinfecting seeds according to claim 1 or 2, wherein the time for immersion in warm water is 1 to 30 minutes and the time for immersion in cold water is 1 to 10 minutes.   The seed disinfection method according to any one of claims 1 to 3, wherein both or one of hot water and cold water contains a pesticide and / or an organic acid.   Seeds that have been disinfected using the method according to any one of claims 1 to 4.

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