A method for disinfecting pollen using a chelating agent that enhances the effectiveness of disinfectants.

Abstract

This invention provides a novel disinfection method applicable to solution pollination. [Solution] A disinfectant for fruit tree pollen comprising an aqueous solution containing a disinfecting component and a chelating agent, characterized in that the disinfecting component is hydrogen peroxide or a polyphosphate.

Description

[Technical Field]

[0001] The present invention relates to a disinfectant for fruit tree pollen and a method for disinfecting fruit tree pollen. The disinfectant and the disinfection method of the present invention are effective for controlling kiwifruit bacterial canker. [Background Art]

[0002] Kiwifruit is a crop for which artificial pollination is indispensable: the greater the number of seeds contained therein, the better the fruit enlargement, and kiwifruit is dioecious. As methods of artificial pollination, there are two types: "solution pollination", in which dried pollen is suspended in a liquid bulking agent and sprayed onto female flowers with a hand sprayer, and "powder pollination", in which powdered dried pollen is directly applied using a tool such as a梵天 brush or pollen duster. These methods are used depending on the kiwifruit cultivar, but "solution pollination" is currently the mainstream.

[0003] In recent years, a pandemic of the highly pathogenic strain Psa3 of Pseudomonas syringae pv. actinidiae (hereinafter referred to as Psa), the pathogen that causes kiwifruit bacterial canker, has occurred. Since this Psa3 strain is transmitted by contamination in pollen, it has spread throughout the world through the import and export of pollen. Japan also relied on imports for most of its artificial pollination pollen for kiwifruit, so the pathogen invaded in 2014, causing devastating damage. Currently, Japan imposes quarantine on all imported pollen, and implements measures to allow only clean pollen into the country. However, since Psa3 is pandemic globally, the quarantine-approved volume has decreased sharply, and the domestic price in 2023 has jumped to more than 3.3 times that before the outbreak of Psa3.

[0004] As a countermeasure, domestic production of pollen is being strengthened. However, pollen is produced in orchards where it is unclear whether they are clean (for fear of reputational damage, it has not been disclosed which domestic orchards are contaminated and which are clean); furthermore, there is no quarantine for domestic pollen, and it remains freely distributable. Therefore, even for domestic pollen, the risk of Psa3 contamination in pollen still remains.

[0005] Currently, most domestically produced pollen is consumed within the production area, but as domestic production increases, it is expected that distribution outside the area will also become more active. However, there is a risk that pollen of unknown cleanliness may be distributed domestically, and since solution pollination is the mainstream method of pollination, there is a high demand from producers for sterilization technologies applicable to solution pollination.

[0006] Prior to this, there was knowledge that contaminated pollen could be disinfected by immersing it in (1) alcohols, (2) pesticides (antibiotics), or (3) sodium hypochlorite solution. While all of these methods showed sufficient sterilization effect, they had the drawback of also killing the pollen. To solve these problems, the inventors searched for a disinfectant that had a sterilization effect without affecting pollen germination. As a result, they found that by using (1) hydrogen peroxide, (2) peracetic acid, or (3) hypochlorous acid with a pH of 3.0 or higher to treat the pollen, it is possible to achieve both maintenance of the pollen germination rate and disinfection effect (Patent Document 1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-131669 [Overview of the project] [Problems that the invention aims to solve]

[0008] When hydrogen peroxide, peracetic acid, or sodium percarbonate were used on actual kiwi fruit pollen contaminated with bacteria, even sodium percarbonate, which was the most effective, left approximately 2.5% of the bacteria untreated. This invention was made against this backdrop and aims to provide a new disinfection method applicable to solution pollination. [Means for solving the problem]

[0009] The inventors of the present invention conducted extensive research to solve the above problems and found that the antibacterial effect of aqueous solutions containing hydrogen peroxide and polyphosphates is enhanced by the addition of a chelating agent. The inventors also found that if pollen and antibacterial components are not added to a solution pollination expander in a specific order, the pollen germination rate decreases. The present invention was completed based on these findings. That is, the present invention provides the following [1] to

[10] .

[0010] [1] A disinfectant for fruit tree pollen comprising an aqueous solution containing a disinfectant component and a chelating agent, characterized in that the disinfectant component is hydrogen peroxide or a polyphosphate.

[0011] [2] The disinfectant for fruit tree pollen according to [1], characterized in that the disinfectant component is hydrogen peroxide.

[0012] [3] The disinfectant for fruit tree pollen according to [1], characterized in that the disinfectant component is a polyphosphate.

[0013] [4] The fruit tree pollen disinfectant according to [3], characterized in that the polyphosphate is sodium polyphosphate.

[0014] [5] The fruit tree pollen disinfectant according to [1], characterized in that the aqueous solution containing the disinfectant component and the chelating agent is an aqueous solution containing hydrogen peroxide, a chelating agent and sodium carbonate.

[0015] [6] The fruit tree pollen disinfectant according to [5], characterized in that the aqueous solution containing hydrogen peroxide, a chelating agent, and sodium carbonate is an aqueous solution obtained by dissolving sodium percarbonate and a chelating agent in water.

[0016] [7] The fruit tree pollen disinfectant according to [1], characterized in that the chelating agent is EDTA or sodium metaphosphate.

[0017] [8] The fruit tree pollen disinfectant according to [1], characterized in that the fruit tree pollen is kiwi fruit pollen.

[0018] [9] The bactericidal component and chelating agent-containing aqueous solution is an aqueous solution containing hydrogen peroxide, a chelating agent and sodium carbonate, the chelating agent is EDTA or sodium metaphosphate, and the fruit tree pollen is kiwifruit pollen. The bactericidal agent for fruit tree pollen according to [1], characterized in that.

[0019]

[10] A method for sterilizing fruit tree pollen, characterized in that step (2) is performed after the following step (1), (1) a step of adding a liquid extender for solution pollination and pollen to a container and mixing them, (2) a step of adding the bactericidal agent for fruit tree pollen according to any one of [1] to [9] to the container and shaking the container.

Effects of the Invention

[0020] The present invention provides a novel bactericidal agent for fruit tree pollen. This bactericidal agent is effective for controlling bacterial canker in kiwifruit.

Brief Description of Drawings

[0021] [Figure 1] Photograph after culture of a plate medium coated with a pollen suspension. [Figure 2] Figure showing the influence of the order of adding pollen and bactericidal agent on germination rate (n=6, bars indicate standard deviation).

Mode for Carrying Out the Invention

[0022] Hereinafter, the present invention will be described in detail. (A) Bactericidal agent The present invention is characterized by comprising an aqueous solution containing hydrogen peroxide and a chelating agent, or an aqueous solution containing a polyphosphate and a chelating agent. The aqueous solution containing hydrogen peroxide and a chelating agent preferably contains sodium carbonate in addition to hydrogen peroxide and a chelating agent, and may further contain other components. The aqueous solution containing hydrogen peroxide, a chelating agent and sodium carbonate can be prepared, for example, by dissolving sodium percarbonate and a chelating agent in water. Here, "sodium percarbonate" is also called "sodium carbonate hydrogen peroxide" or "sodium percarbonate," and is a compound in which sodium carbonate and hydrogen peroxide are mixed in a ratio (molar ratio) of 2:3.

[0023] The chelating agent used is not particularly limited as long as it is effective in disinfecting and does not significantly reduce the pollen germination rate. Examples include etidronic acid, tartaric acid, phytic acid, gluconic acid, sodium gluconate, citric acid monohydrate, trisodium citrate dihydrate, disodium EDTA calcium (disodium ethylenediaminetetraacetic acid calcium), EDTA (ethylenediaminetetraacetic acid), sodium metaphosphate, sodium hexametaphosphate, or lactoferrin. Among these, EDTA or sodium metaphosphate are preferred.

[0024] The concentration of the chelating agent used during disinfection is not particularly limited, as long as it is a concentration that can disinfect without significantly reducing the pollen germination rate. When EDTA is used as the chelating agent, its concentration is preferably 0.1 to 5 mM, more preferably 0.5 to 2.5 mM, and even more preferably 0.7 to 2 mM. When sodium metaphosphate is used as the chelating agent, its concentration is preferably 0.01 to 0.2% by weight, more preferably 0.03 to 0.15% by weight, and even more preferably 0.05 to 0.1% by weight. When using a chelating agent other than EDTA or sodium metaphosphate, its concentration can be appropriately determined depending on the type of chelating agent, but it can also be the same concentration as that of EDTA or sodium metaphosphate mentioned above.

[0025] The concentrations of hydrogen peroxide and sodium carbonate used for disinfection are not particularly limited, as long as they are effective in disinfecting and do not significantly reduce the pollen germination rate. When an aqueous solution containing only hydrogen peroxide and no sodium carbonate is used, the concentration of hydrogen peroxide is preferably 0.01 to 1% by weight, more preferably 0.05 to 0.5% by weight, and even more preferably 0.1 to 0.3% by weight. As described above, an aqueous solution containing both sodium carbonate and hydrogen peroxide can be prepared by dissolving sodium percarbonate in water. In this case, the concentration of sodium percarbonate is preferably 0.005 to 0.5% by weight, more preferably 0.01 to 0.1% by weight, and even more preferably 0.03 to 0.07% by weight.

[0026] The polyphosphate used is not particularly limited as long as it is antibacterial and does not significantly reduce the pollen germination rate; for example, sodium polyphosphate.

[0027] The concentration of polyphosphate used for disinfection is not particularly limited, as long as it is a concentration that can disinfect and does not significantly reduce the pollen germination rate. When sodium polyphosphate is used as the polyphosphate, its concentration is preferably 0.005 to 0.5% by weight, more preferably 0.01 to 0.1% by weight, and even more preferably 0.03 to 0.07% by weight. When a polyphosphate other than sodium polyphosphate is used, its concentration can be appropriately determined depending on the type of polyphosphate, but it can also be the same concentration as that of sodium polyphosphate as described above.

[0028] The molar ratio of hydrogen peroxide to chelating agent is not particularly limited as long as it is a molar ratio that can disinfect and does not significantly reduce the pollen germination rate. However, the molar ratio (hydrogen peroxide:chelating agent) is preferably 1:30 to 1:100, more preferably 1:40 to 1:80, and even more preferably 1:50 to 1:70. The molar ratio of sodium carbonate to hydrogen peroxide (sodium carbonate:hydrogen peroxide) is 2:3 when sodium percarbonate is used, but a molar ratio other than 2:3 may be used if sodium percarbonate is not used.

[0029] The weight percentage ratio of polyphosphate to chelating agent is not particularly limited as long as it is a weight percentage ratio that can eliminate bacteria and does not significantly reduce the pollen germination rate. However, the weight percentage ratio (polyphosphate:chelating agent) is preferably 0.01:0.01 to 0.1:0.05, more preferably 0.03:0.01 to 0.07:0.03, and even more preferably 0.05:0.01 to 0.05:0.02.

[0030] The fruit tree pollen targeted for disinfection is primarily kiwi fruit pollen, but other fruit tree pollens, such as those from Rosaceae fruit trees (apples, peaches, pears, plums, cherries, etc.), may also be targeted for disinfection.

[0031] The plant pathogen targeted for elimination is primarily the kiwifruit canker fungus (Pseudomonas syringae pv. actinidiae), but other plant pathogens, such as apple blight fungus (Erwinia amylovora), pear branch blight bacterial disease (Erwinia amylovora, biovar 4), and pear flower blight bacterial disease (Pseudomonas syringae pv. syringae), may also be targeted for elimination.

[0032] The disinfectant effect of the disinfectant of the present invention is thought to be due to hydroxyl radicals generated from hydrogen peroxide, the active ingredient. Furthermore, the increased disinfectant effect with chelating agents is thought to be partly because hydroxyl radicals are highly reactive and disappear in a very short time, but the addition of chelating agents chelates catalytic heavy metals, thereby stabilizing the hydroxyl radicals. In addition, Gram-negative bacteria, to which the kiwifruit canker fungus belongs, have an outer membrane made of lipopolysaccharides, etc., and it is thought that this outer membrane hinders the passage of substances into the cell ("Effects of two or three surfactants on succinate dehydrogenase in Escherichia coli suspension" (1981), Tsutsumi et al., Kyushu University Journal of Agriculture 35:89-95). In the pharmaceutical field, there is evidence that treating Gram-negative bacteria with EDTA alters the permeability of the outer membrane, allowing drugs that normally have difficulty penetrating the outer membrane to reach their target sites on the cell membrane ("Permeability of Gram-negative bacterial outer membrane and affinity for penicillin-binding protein of Ceftazidime (SN401)" (1983), Yokota & Sekiguchi, CHEMOTHERAPY 31:17-21). Although the mechanism by which the combined use of hydrogen peroxide and chelating agents enhances the antibacterial effect on pollen is not yet fully understood, it is suspected that the stabilization of hydroxyl radicals by the chelating agent and the alteration of the outer membrane permeability of the kiwifruit canker fungus are involved in enhancing the effectiveness of the antibacterial agent.

[0033] (B) Sterilization method The present invention provides a method for sterilizing fruit tree pollen, characterized by performing step (2) after step (1) described below.

[0034] In step (1), the liquid extender for solution pollination and pollen are added to the container and mixed.

[0035] Liquid fillers for solution pollination are commercially available, so commercially available products, such as "Hana Mirai" (manufactured by Shiraishi Calcium Co., Ltd.), can be used. Also, since liquid fillers for solution pollination are usually made by dissolving sucrose, agar, and a coloring agent in water, an aqueous solution containing sucrose or an aqueous solution containing sucrose and agar may be used instead of a liquid filler for solution pollination. The concentrations of sucrose and agar in these aqueous solutions are not particularly limited, but the sucrose concentration can be 3 to 10% by weight, preferably 6 to 8% by weight, and the agar concentration can be 0.03 to 0.2% by weight, preferably 0.05 to 0.15% by weight.

[0036] The containers used are not particularly limited; for example, centrifuge tubes, PET bottles (1.5-2L), etc., can be used.

[0037] The amount of liquid pollination liquid extender and pollen to be placed in the container is not particularly limited, but 0.01 to 1 g, preferably 0.05 to 0.5 g, of pollen can be added per 20 ml of extender.

[0038] The means of mixing are not particularly limited; for example, the container may be shaken continuously or intermittently. The time for this mixing operation is not particularly limited, but can be 0.5 to 15 minutes, preferably 1 to 10 minutes.

[0039] In step (2), the fruit tree pollen disinfectant of the present invention is added to the container and shaken. If the pollen is brought into contact with the fruit tree pollen disinfectant of the present invention without going through step (1) above, the pollen germination rate will decrease, so step (2) must be performed after step (1).

[0040] The amount of the fruit tree pollen disinfectant of the present invention added is not particularly limited, but it is preferable to add it so that the components in the disinfectant (chelating agent, hydrogen peroxide, sodium carbonate) reach the concentrations described above.

[0041] The time for shaking the container is not particularly limited, but can be 5 to 90 seconds, preferably 10 to 60 seconds. [Examples]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0043] [Comparative Example] We investigated the disinfecting effect against contaminated pollen using hydrogen peroxide, peracetic acid, or sodium percarbonate as disinfectants.

[0044] Experimental method 1. Hydrogen peroxide treatment (1) 20 ml of a commercially available liquid pollination extender ("Hana Mirai", Shiraishi Calcium Co., Ltd.) was placed in a 50 ml Corning tube. (2) 0.114 ml of 35% hydrogen peroxide solution (Fujifilm Wako) was added to (1) and mixed (final hydrogen peroxide concentration 0.2%). (3) Add 0.1g of contaminated pollen to (2) and shake vigorously by hand for 1 minute to thoroughly mix the pollen with "Hana Mirai". Note that the contaminated pollen was a bacterial solution (concentration 10) of the kiwi fruit canker fungus (Pseudomonas syringae pv. actinidiae). 9 It was prepared by mixing cfu / ml with kiwi fruit pollen (the same contaminated pollen was used in the following experiments as well). (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0045] 2. Peracetic acid treatment (1) 20 ml of a commercially available liquid pollination extender ("Hana Mirai", Shiraishi Calcium Co., Ltd.) was placed in a 50 ml Corning tube. (2) 1.33 μl of 15% peracetic acid solution (Persan MP2-J, Kanto Chemical) was added to (1) and mixed (final peracetic acid concentration 10 ppm). (3) Add 0.1g of contaminated pollen to (2) and shake vigorously by hand for 1 minute to thoroughly mix the pollen with "Hana Mirai". (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0046] 3. Sodium percarbonate treatment (1) 20 ml of a commercially available liquid pollination extender ("Hana Mirai", Shiraishi Calcium Co., Ltd.) was placed in a 50 ml Corning tube. (2) 0.03 g of sodium percarbonate powder (Fujifilm Wako) was added to (1) and mixed (final concentration of sodium percarbonate 0.15%). (3) Add 0.1g of contaminated pollen to (2) and shake vigorously by hand for 1 minute to thoroughly mix the pollen with "Hana Mirai". (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0047] 4. Dilution plate Each of the pollen suspensions treated as described above was serially diluted 10-fold with sterilized water. 0.1 ml of each suspension was then spread onto YP (0.5% yeast extract, 0.5% peptone, 0.5% sodium chloride, 1.5% agar) plates containing 50 ppm rifampicin (Fujifilm Wako) and 200 ppm tebuconazole (Bayer CropScience), and incubated at 23°C for 4 days to observe the appearance of colonies.

[0048] Experimental results Table 1 shows the disinfection efficiency of each disinfectant. [Table 1]

[0049] As shown in the table, the disinfectants used did not provide a practical level of disinfection against actual contaminated pollen. Even with sodium percarbonate, which was the most efficient, about 2.5% of bacteria remained.

[0050] [Example 1] The disinfecting effect of a mixed treatment with EDTA and sodium percarbonate was investigated. For comparison, the disinfecting effects of EDTA alone and sodium carbonate alone were also investigated.

[0051] Experimental method 1. Untreated (1) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (2) 20 ml of a commercially available liquid pollination extender ("Hana Mirai", Shiraishi Calcium Co., Ltd.) was added to (1). (3) Shake by hand for 5 minutes to thoroughly mix the "Hanamirai" with the pollen. (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0052] 2.1 mM EDTA monotherapy (1) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (2) Prepare another Corning tube, add 20 ml of "Hanamirai", and then add 40 μl of 0.5 M EDTA (Nacalai Tesque) (final EDTA concentration 1 mM) and mix. (3) Add (2) to (1) and shake by hand for 5 minutes to thoroughly mix everything together. (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0053] 3. 0.05% sodium percarbonate monotherapy (1) A 5% sodium percarbonate (Fujifilm Wako) aqueous solution was prepared in advance. (2) 0.1g of contaminated pollen was placed in a 50ml Corning tube, and then 20ml of "Hana Mirai" was added and shaken by hand for 5 minutes to allow it to mix thoroughly. (3) Add 0.2 ml of (1) to (2) and shake by hand for 30 seconds (final concentration of sodium percarbonate 0.05%). (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0054] 4.1 mM EDTA + 0.05% sodium percarbonate mixed treatment (1) A 5% sodium percarbonate solution was prepared in advance. (2) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (3) Prepare another Corning tube, add 20 ml of "Hanamirai", and add 40 μl of 0.5 M EDTA (Nacalai Tesque) (final EDTA concentration 1 mM). (4) Add (3) to (2) and shake by hand for 5 minutes to thoroughly mix the EDTA-added "Hanamirai" with the pollen. (5) Add 0.2 ml of (1) to (4) (final sodium percarbonate concentration 0.05%) and shake by hand for 30 seconds. (6) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0055] 5. Dilution Plate Each of the pollen suspensions treated as described above was serially diluted 10-fold with sterilized water. 0.1 ml of each suspension was then spread onto YP (0.5% yeast extract, 0.5% peptone, 0.5% sodium chloride, 1.5% agar) plates containing 50 ppm rifampicin (Fujifilm Wako) and 200 ppm tebuconazole (Bayer CropScience), and incubated at 23°C for 4 days to observe the appearance of colonies.

[0056] Experimental results Figure 1 shows a photograph of agar plates coated with pollen suspension after incubation. Table 2 shows the number of colonies formed. Furthermore, Table 3 shows the sterilization efficiency when treated with a mixture of EDTA and sodium percarbonate. [Table 2] [Table 3]

[0057] As shown in Figure 1 and Table 2, numerous Psa3 colonies were produced on YP agar plates after EDTA monotherapy and sodium percarbonate monotherapy. On the other hand, no colonies were produced when EDTA and sodium percarbonate were used in combination, confirming that EDTA enhances the antibacterial effect of sodium percarbonate. Furthermore, as shown in Table 3, the antibacterial efficiency was higher when 1.0 mM EDTA was used than when 0.7 mM EDTA was used.

[0058] [Example 2] The relationship between EDTA concentration and pollen germination rate in a mixed treatment with EDTA and sodium percarbonate was investigated.

[0059] Experimental method 1. Untreated (1) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (2) Add 5 ml of a commercially available liquid pollination extender ("Hanamirai", Shiraishi Calcium Co., Ltd.) to (1) and shake by hand for 5 minutes to thoroughly mix the contents. (3) Add 15 ml of "Hanamirai" to (2) and shake by hand for 5 minutes to mix thoroughly. (4) 10 μl of pollen suspension was spread on a germination bed and germination was tested.

[0060] Treatment with 2.0.5 mM EDTA + 0.15% sodium percarbonate (1) A 5% sodium percarbonate (Fujifilm Wako) aqueous solution was prepared. (2) Put 20 ml of "Hanamirai" into a Corning tube, add 0.6 ml of (1) and mix (final sodium percarbonate concentration 0.15%), then add 20 μl of 0.5 M EDTA (Nacalai Tesque) and mix. (3) Prepare another 50 ml Corning tube and put 0.1 g of fresh pollen into it. (4) Add 5 ml of (2) to (3) and shake by hand for 5 minutes to thoroughly mix everything together. (5) Add the remaining 15 ml from (2) to (4) and shake by hand to thoroughly mix the contents. (6) 10 μl of (5) was spread on the germination bed and a germination test was performed.

[0061] Treatment with 3.1 mM EDTA + 0.15% sodium percarbonate (1) A 5% sodium percarbonate (Fujifilm Wako) aqueous solution was prepared. (2) Put 20 ml of "Hanamirai" into a Corning tube, add 0.6 ml of (1) and mix (final sodium percarbonate concentration 0.15%), then add 40 μl of 0.5 M EDTA (Nacalai Tesque) and mix. (3) Prepare another 50 ml Corning tube and put 0.1 g of fresh pollen into it. (4) Add 5 ml of (2) to (3) and shake by hand for 5 minutes to thoroughly mix everything together. (5) Add the remaining 15 ml from (2) to (4) and shake by hand to thoroughly mix the contents. (6) 10 μl of (5) was spread on the germination bed and a germination test was performed.

[0062] 4.2.5 mM EDTA + 0.15% sodium percarbonate treatment (1) A 5% sodium percarbonate (Fujifilm Wako) aqueous solution was prepared. (2) Put 20 ml of "Hanamirai" into a Corning tube, add 0.6 ml of (1) and mix (final sodium percarbonate concentration 0.15%), then add 100 μl of 0.5 M EDTA (Nacalai Tesque) and mix. (3) Prepare another 50 ml Corning tube and put 0.1 g of fresh pollen into it. (4) Add 5 ml of (2) to (3) and shake by hand for 5 minutes to thoroughly mix everything together. (5) Add the remaining 15 ml from (2) to (4) and shake by hand to thoroughly mix the contents. (6) 10 μl of (5) was spread on the germination bed and a germination test was performed.

[0063] 5. Germination test (1) A germination bed (made by boiling 10% sucrose and 1% agar in water) was prepared in advance. (2) 8 ml of the melted germination bed was spread onto a microscope slide and allowed to solidify. (3) 10 μl of the sterilized pollen suspension was spotted into the center of the germination bed using a micropipette. (4) The spot was widened with a spatula or similar tool to a diameter of approximately 14 mm. (5) The germination beds, which had been spotted with pollen solution, were placed in petri dishes lined with moistened Kimwipes to prevent drying and incubated overnight at 28°C. (6) The following morning, an optical microscope (Leica DMR, 10x objective lens) was used to examine any one spot on the slide, and the number of germinated and ungerminated seeds per field of view was counted to calculate the germination rate.

[0064] Experimental results Table 4 shows the germination rates of pollen treated with each method. [Table 4]

[0065] As shown in Table 4, the germination rate decreased as the EDTA concentration increased. Therefore, it is thought that the germination rate can be improved by lowering the EDTA concentration. In addition, the overall pollen germination rate in this experiment was low, which is thought to be due to the high concentration of sodium percarbonate. Therefore, it is thought that the germination rate can also be improved by lowering the concentration of sodium percarbonate.

[0066] [Example 3] We investigated the effect of the procedure for adding pollen and disinfectant on germination rates.

[0067] Experimental method 1. Untreated (1) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (2) 20 ml of a commercially available liquid pollination extender ("Hana Mirai", Shiraishi Calcium Co., Ltd.) was added to (1). (3) Shake by hand for 5 minutes to thoroughly mix the contents. (4) 10 μl of (3) was spread on the germination bed and a germination test was performed.

[0068] 2. Inappropriate procedures (1) A 5% sodium percarbonate (Fujifilm Wako) aqueous solution was prepared in advance. (2) 20 ml of "Hanamirai" was placed in a 50 ml Corning tube, and 40 μl of 0.5 M EDTA (Nacalai Tesque) was added and mixed. (3) Add 0.2 ml of (1) to (2) and mix. (4) Add 0.1g of pollen to (3) and shake by hand for 5 minutes to ensure that everything is well combined. (5) 10 μl of (4) was spread on the germination bed and a germination test was performed.

[0069] 3. The correct procedure (1) A 5% sodium percarbonate solution was prepared in advance. (2) Put 0.1g of pollen into a 50ml Corning tube, then add 20ml of "Hana Mirai" and shake by hand for 5 minutes to thoroughly mix everything together. (3) Add 40 μl of 0.5 M EDTA and 0.2 ml of (1) to (2) and shake by hand for 30 seconds. (4) 10 μl of (3) was spread on the germination bed and a germination test was performed.

[0070] 4. Pollen germination test (1) A germination bed (made by boiling 10% sucrose and 1% agar in water) was prepared in advance. (2) 8 ml of the melted germination bed was spread onto a microscope slide and allowed to solidify. (3) 10 μl of the sterilized pollen suspension was spotted into the center of the germination bed using a micropipette. (4) The spot was widened with a spatula or similar tool to a diameter of approximately 14 mm. (5) The germination beds, which had been spotted with pollen solution, were placed in petri dishes lined with moistened Kimwipes to prevent drying and incubated overnight at 28°C. (6) The following morning, three random locations among the areas where the pollen had been spread were examined using an optical microscope (Leica DMR, 10x objective lens), and the number of germinated and ungerminated seeds per field of view were counted. (7) The germination rate was calculated by adding up the number of germinated and ungerminated seeds in the three fields of view.

[0071] Experimental results Figure 2 shows the germination rates under three conditions: no treatment, sterilization using an improper procedure, and sterilization using the correct procedure. Following the "correct procedure," it was confirmed that sterilization was achieved by first adding pollen to a liquid extender for solution pollination in a container and shaking, then adding sodium percarbonate and EDTA and shaking again, and that although pollen germination decreased slightly, it did not have a practical impact. On the other hand, following the "improper procedure," it was confirmed that sterilization was achieved by first adding sodium percarbonate and EDTA to a liquid extender for solution pollination in a container and mixing, and then suspending the pollen, but the germination rate decreased to a level that was impractical.

[0072] [Example 4] We investigated whether disinfection was possible by using hydrogen peroxide instead of sodium percarbonate, and by a mixed treatment of hydrogen peroxide and EDTA.

[0073] Experimental method 1. Untreated (1) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (2) 20 ml of a commercially available liquid pollination extender ("Hana Mirai", Shiraishi Calcium Co., Ltd.) was added to (1). (3) Shake by hand for 5 minutes to thoroughly mix the "Hanamirai" with the pollen. (4) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0074] 2.0.2% hydrogen peroxide treatment (1) 35% hydrogen peroxide solution (Fujifilm Wako) was diluted with distilled water beforehand to make 4% hydrogen peroxide solution. (2) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (3) Add 20 ml of "Hanamirai" to (2) and shake by hand for 5 minutes to thoroughly mix everything together. (4) Add 1 ml of (1) to (3) and shake by hand for 30 seconds (final hydrogen peroxide concentration 0.2%). (5) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0075] 3.1 mM EDTA + 0.2% hydrogen peroxide treatment (1) 35% hydrogen peroxide solution was diluted with distilled water beforehand to make 4% hydrogen peroxide solution. (2) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (3) Prepare another Corning tube, add 20 ml of "Hanamirai", and then add 40 μl of 0.5 M EDTA (Nacalai Tesque) and mix. (4) Add (3) to (2) and shake by hand for 5 minutes to thoroughly mix everything together. (5) Add 1 ml of (1) to (4) and shake by hand for 30 seconds (final hydrogen peroxide concentration 0.2%). (6) The solution was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0076] 4. Dilution plate Each of the pollen suspensions treated as described above was serially diluted 10-fold with sterilized water. 0.1 ml of each suspension was then spread onto YP (0.5% yeast extract, 0.5% peptone, 0.5% sodium chloride, 1.5% agar) plates containing 50 ppm rifampicin (Fujifilm Wako) and 200 ppm tebuconazole (Bayer CropScience), and incubated at room temperature around 23°C for 4 days to observe the appearance of colonies.

[0077] Experimental results Table 5 shows the number of colonies formed after each treatment. [Table 5]

[0078] As shown in Table 5, sufficient disinfection was not obtained with hydrogen peroxide solution alone, but the disinfection effect was increased by mixing hydrogen peroxide solution with EDTA.

[0079] [Example 5] We investigated whether the disinfecting effect of various chelating agents is increased when used in combination with sodium percarbonate.

[0080] Experimental method A commercially available liquid pollination extender ("Hana Mirai," Shiraishi Calcium Co., Ltd.) was mixed with the kiwifruit canker fungus (Pseudomonas syringae pv. actinidiae, Psa) to a concentration of 10%. 4 The solution was added to achieve a concentration of cfu / ml. The disinfectant effect was then examined by adding either the chelating agent alone or the chelating agent and sodium percarbonate.

[0081] Experimental results Table 6 shows the disinfectant effects of various chelating agents. In the table, ○ indicates "strong disinfectant effect," △ indicates "weak disinfectant effect," and × indicates "no disinfectant effect." [Table 6]

[0082] As shown in Table 6, the antibacterial effect of EDTA and sodium metaphosphate was enhanced by the addition of sodium percarbonate. In this experiment, no enhancement of antibacterial effect was observed with chelating agents other than EDTA and sodium metaphosphate, but it is possible that similar antibacterial enhancement effects can be obtained with these chelating agents by adjusting their concentrations.

[0083] [Example 6] Polyphosphates are known to exhibit bactericidal (antimicrobial) effects (Lee RM, Hartman PA, Stahr HM, Olson DG, Williams FD (1994) Antibacterial Mechanism of Long-Chain Polyphosphates in Staphylococcus aureus. Journal of Food Protection 57(4):289-294). This study investigated whether EDTA enhances the antimicrobial effect of this polyphosphate (sodium polyphosphate).

[0084] Experimental method Contaminated pollen was prepared by mixing healthy fresh pollen with canker bacteria. The amount of canker bacteria in the contaminated fresh pollen was 1 x 10⁶ per gram. 9 The goal was to achieve a CFU (container-free unit) result. Note that no untreated control group was included in this experiment.

[0085] 1. EDTA processing (1) 0.1 g of contaminated pollen was placed in a 50 ml Corning tube. (2) 20 ml of a commercially available liquid bulking agent ("Hanamirai", Shiraishi Calcium Co., Ltd.) was added to (1). (3) Shake by hand for 5 minutes to thoroughly mix "Hanamirai" with the pollen. (4) Add 20 μl of 0.5 M EDTA (Nacalai Tesque) to (3) and shake by hand for 30 seconds (final EDTA concentration 0.5 mM). (5) The treated pollen suspension was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0086] 2. Treatment with 0.05% sodium polyphosphate (1) Sodium polyphosphate (Fujifilm Wako) was dissolved in distilled water beforehand to make a 5% aqueous solution. (2) 20 ml of "Hanamirai" was placed in a 50 ml Corning tube and mixed with 0.2 ml of the sodium polyphosphate aqueous solution from (1) (final concentration of sodium polyphosphate: 0.05%). (3) Add 0.1g of contaminated pollen to (2) and shake for 5 minutes. (4) The treated pollen suspension was serially diluted 10-fold with sterilized water, and 0.1 ml was spread onto a culture plate.

[0087] Treatment with 3.0.5 mM EDTA + 0.05% sodium polyphosphate (1) Sodium polyphosphate (Fujifilm Wako) was dissolved in distilled water beforehand to make a 5% aqueous solution. (2) 20 ml of "Hanamirai" was placed in a 50 ml Corning tube and mixed with 0.2 ml of the sodium polyphosphate aqueous solution from (1) (final concentration of sodium polyphosphate: 0.05%). (3) Add 0.1g of contaminated pollen to (2) and shake for 5 minutes. (4) Add 20 μl of 0.5 M EDTA (Nacalai Tesque) to (3) and shake by hand for 30 seconds (final EDTA concentration 0.5 mM). (5) The treated pollen suspension was serially diluted 10 times with sterilized water, and 0.1 ml was spread onto a culture plate.

[0088] 4. Dilution plate Each of the pollen suspensions treated as described above was serially diluted 10-fold with sterilized water. 0.1 ml of each suspension was then spread onto YP (0.5% yeast extract, 0.5% peptone, 0.5% sodium chloride, 1.5% agar) plates containing 50 ppm rifampicin (Fujifilm Wako) and 200 ppm tebuconazole (Bayer CropScience), and incubated at room temperature around 23°C for 4 days to observe the appearance of colonies.

[0089] Experimental results Table 7 shows the number of colonies that appeared after each treatment. Although no untreated group was included in this experiment, the estimated number of colonies that would have appeared is shown in the right-hand column. [Table 7]

[0090] As shown in Table 7, treatment with 0.5 mM EDTA alone did not produce a bactericidal effect, and while treatment with polyphosphate alone did show a bactericidal effect, it was weak. Combining EDTA and polyphosphate improved the bactericidal effect. [Industrial applicability]

[0091] This invention relates to a technology for disinfecting fruit tree pollen and can be used in industrial fields such as agriculture.

Claims

1. A disinfectant for fruit tree pollen comprising an aqueous solution containing a disinfecting component and a chelating agent, characterized in that the disinfecting component is hydrogen peroxide or a polyphosphate.

2. The disinfectant for fruit tree pollen according to claim 1, characterized in that the disinfecting component is hydrogen peroxide.

3. The disinfectant for fruit tree pollen according to claim 1, characterized in that the disinfecting component is a polyphosphate salt.

4. The fruit tree pollen disinfectant according to claim 3, characterized in that the polyphosphate is sodium polyphosphate.

5. The disinfectant for fruit tree pollen according to claim 1, characterized in that the aqueous solution containing the disinfectant component and the chelating agent is an aqueous solution containing hydrogen peroxide, a chelating agent, and sodium carbonate.

6. The fruit tree pollen disinfectant according to claim 5, characterized in that the aqueous solution containing hydrogen peroxide, a chelating agent, and sodium carbonate is an aqueous solution obtained by dissolving sodium percarbonate and a chelating agent in water.

7. The fruit tree pollen disinfectant according to claim 1, characterized in that the chelating agent is EDTA or sodium metaphosphate.

8. The disinfectant for fruit tree pollen according to claim 1, characterized in that the fruit tree pollen is kiwi fruit pollen.

9. The disinfectant for fruit tree pollen according to claim 1, characterized in that the aqueous solution containing the disinfectant component and the chelating agent is an aqueous solution containing hydrogen peroxide, a chelating agent and sodium carbonate, the chelating agent is EDTA or sodium metaphosphate, and the fruit tree pollen is kiwi fruit pollen.

10. A method for disinfecting fruit tree pollen, characterized by performing step (2) after step (1) below. (1) Add the liquid extender for solution pollination and pollen to a container and mix them together. (2) Adding the fruit tree pollen disinfectant described in any one of claims 1 to 9 to a container and shaking it.

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