WO2012081420A1 - カンキツグリーニング病の治療液及びこれを用いた治療方法 - Google Patents
カンキツグリーニング病の治療液及びこれを用いた治療方法 Download PDFInfo
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- WO2012081420A1 WO2012081420A1 PCT/JP2011/077868 JP2011077868W WO2012081420A1 WO 2012081420 A1 WO2012081420 A1 WO 2012081420A1 JP 2011077868 W JP2011077868 W JP 2011077868W WO 2012081420 A1 WO2012081420 A1 WO 2012081420A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
Definitions
- the present invention relates to a treatment liquid for citrus greening disease and a treatment method using the same.
- Citrus greening disease (Huanglongbing: hereinafter also referred to as HLB disease) is one of the most important diseases of citrus fruits. HLB disease is a plant disease that occurs when HLB bacteria infect trees such as citrus fruits.
- HLB disease The fruit of citrus fruits suffering from HLB disease is small and most of it remains green, but its taste is quite bitter. Therefore, there is little commercial value of citrus fruits suffering from HLB disease. In addition, trees suffering from HLB disease fall and eventually die. For this reason, HLB disease is a serious disease causing a great blow to horticultural agriculture.
- Patent Document 1 discloses a method for detecting citrus greening disease and a detection kit.
- a tree suffering from HLB disease can be easily detected, but a tree suffering from HLB disease has to be cut down.
- the present invention has been made in view of the above-mentioned matters, and an object of the present invention is to provide a therapeutic solution for citrus greening disease capable of treating citrus trees suffering from citrus greening disease and a treatment method using the same. It is to provide.
- the treatment solution for citrus greening disease according to the first aspect of the present invention contains Fe ions, and at least a part of the Fe ions is Fe 2+ ions.
- the total Fe ion concentration is preferably 10 mg / L to 100 mg / L.
- the therapeutic solution further contains an acid in addition to the total Fe ions.
- the acid is preferably an organic acid.
- the organic acid preferably has at least one of a carboxyl group and a hydroxyl group, and the total of the carboxyl group and the hydroxyl group is preferably 2 or more.
- the organic acid is preferably at least one of citric acid, malic acid, tartaric acid and ascorbic acid.
- the plant to which the therapeutic liquid is applied is a citrus plant.
- the plant to which the treatment liquid is applied is rough lemon, tankan, or seek shark.
- a method for treating citrus greening disease according to the second aspect of the present invention provides a leaf solution, a rhizosphere, or a leaf surface and a rhizosphere of a citrus plant infected with the citrus greening disease using any of the above treatment solutions. It is applied to both to treat citrus greening disease by reducing or eliminating pathogenic bacteria in the citrus plant.
- the citrus greening disease treatment solution according to the present invention is applied to a tree suffering from citrus greening disease, so that the citrus greening disease can be treated and there is no need to cut down the tree.
- (A)-(d) is a figure which shows the PCR diagnostic result of the sample which applied the therapeutic liquid. It is a figure which shows the PCR diagnostic result of the test substance which applied the treatment liquid. It is a figure which shows the expansion
- (A) (b) is a figure which shows the PCR diagnostic result of the sample which applied the therapeutic liquid. It is a figure which shows the PCR diagnostic result of the test substance which applied the treatment liquid. It is a figure which shows the measurement result of the active oxygen by luminol reaction. It is a figure which shows the measurement result of the active oxygen by luminol reaction. (A) (b) is a figure which shows the measurement result of the active oxygen by luminol reaction.
- treatment solution for citrus greening disease contains Fe 2+ ions. This treatment solution holds Fe 2+ ions stably.
- the treatment liquid according to the present embodiment can be obtained by dissolving an iron compound capable of supplying Fe 2+ ions in water.
- the iron compound which can supply a Fe 2+ ion, as long as it can contain the Fe 2+ ions in aqueous solution is not particularly limited.
- a divalent iron compound such as FeO or FeSO 4 can be used.
- the powder is an iron compound containing trivalent iron such as iron citrate, it may contain Fe 2+ ions in an aqueous solution due to the equilibrium between Fe 3+ ions and Fe 2+ ions when dissolved in water. Anything is possible.
- the concentration of total Fe ions contained in the treatment liquid according to the present embodiment is preferably 10 mg / L to 100 mg / L. More preferably, it is 12 mg / L to 50 mg / L. More preferably, it is 15 mg / L to 30 mg / L. When it is lower than 10 mg / L, a sufficient therapeutic effect for HLB disease cannot be obtained. Moreover, when higher than 100 mg / L, there exists a possibility of damaging the tree itself which suffered from HLB disease. Normally, when iron is applied as a nutrient component to plants, it is used in a concentration range of 1 to 1.5 mg / L. On the other hand, the treatment solution according to the present embodiment has an excellent effect in treating HLB disease by containing total Fe ions at a high concentration of 10 to 100 mg / L.
- total Fe ions includes divalent iron ions (Fe 2+ ions) and trivalent iron ions (Fe 3+ ions).
- the Fe 2+ ion concentration can be measured by an existing method using, for example, o-phenanthroline. is o- phenanthroline to form a selectively complex with Fe 2+ ions by measuring the absorbance of the complex, it is possible to selectively quantify Fe 2+ ions. Further, the total amount of Fe ions can be quantified by reducing Fe 3+ ions contained in the solution in advance to convert all Fe ions to divalent iron and then quantifying them using the o-phenanthroline method.
- Fe 2+ ions are likely to be oxidized to Fe 3+ ions, but the treatment liquid according to the present embodiment can preferably hold Fe 2+ ions stably by containing an acid.
- the acid contained in the therapeutic solution may be an organic acid or an inorganic acid as long as it can stably hold Fe 2+ ions.
- Organic acids are preferred because they can hold Fe 2+ ions more stably.
- the organic acid is an acid having a carboxyl group and / or a hydroxyl group and having a total of two or more carboxyl groups and hydroxyl groups, and forms a chelate with Fe 2+ ions generated from the above iron compound in water. Thereby, Fe ⁇ 2+> ion exists stably in water.
- the organic acid having a carboxyl group include citric acid (anhydrous citric acid), malic acid, tartaric acid, and oxalic acid.
- Examples of the organic acid having a hydroxyl group include ascorbic acid.
- citric acid, malic acid, tartaric acid etc. are mentioned as an organic acid provided with both a carboxyl group and a hydroxyl group. These may use only 1 type or may use 2 or more types together.
- citric acid, malic acid, tartaric acid, and ascorbic acid are preferable because the stability of Fe 2+ ions in the treatment solution is excellent. Furthermore, when an aqueous solution containing these organic acid and iron compound is prepared, since the Fe 2+ ion concentration is high with respect to the organic acid concentration, citric acid and tartaric acid are preferable. Among these, when an aqueous solution containing an organic acid and an iron compound is prepared, citric acid is most preferable because the Fe 2+ ion concentration with respect to the organic acid concentration is particularly high. Moreover, since citric acid is also an organic acid which citrus itself produces
- the water used for this treatment liquid is not particularly limited, and various waters can be used. Highly purified water such as pure water and ion-exchanged water may be used, and water usually used such as tap water, industrial water, agricultural water, and groundwater may be used.
- the treatment liquid may contain other nutritional components such as magnesium and calcium.
- the treatment liquid according to the present embodiment can be obtained by the following manufacturing method.
- An organic acid powder and an iron compound powder capable of supplying Fe 2+ ions can be obtained by heating and dissolving in water.
- ferrous organic acid ferrous powder such as ferrous citrate can be added to water and heated.
- the organic acid powder, the iron compound powder that supplies Fe 2+ ions, and water may be mixed without heating.
- the treatment solution according to the present embodiment can be applied to citrus trees suffering from HLB disease to treat HLB disease.
- Citrus fruits include, for example, rough lemon (Citrus verrucosa), tankan (Citrus tankan), Sikhwasher (Citrus depressa Hayata), and Citrus unshiu.
- rough lemon Citrus verrucosa
- tankan Citrus tankan
- shikwasha Citrus depressa Hayata
- HLB bacteria are phloem-localized and difficult-to-cultivate diversity Gram-negative bacteria-like microorganisms, which are classified into three strains called Asian, African, and American types, respectively, Candidatus Liberobacter asiaticus, Candidatus Liberobacter africanus, and , Candidatus Liberobacter americanus is classified into three systems.
- the therapeutic solution according to the present embodiment can be used for infection by any strain of HLB bacteria.
- the treatment liquid may be applied, for example, by spraying on the leaf surface of the tree or by irrigating the root of the tree, that is, the rhizosphere.
- the treatment mechanism of citrus greening disease is not clear, it is presumed that the hydroxy radical generated by the treatment liquid applied to the tree is affected, as will be described in the following examples.
- active oxygen is involved in the resistance of cells to invaders such as pathogens. This active oxygen has a function to protect the cell from pathogenic stress in the cell, and the concentration of active oxygen in the cell is Fenton in which hydrogen peroxide generated in the cell reacts with divalent iron to generate a hydroxy radical. Controlled through reaction.
- the treatment liquid according to the present embodiment stably holds Fe 2+ ions and can continuously generate hydroxy radicals as shown in the examples described later.
- citrus trees suffering from citrus greening disease have been found to have reduced iron content compared to normal trees.
- plants reduce Fe 3+ ions from the roots and ingest Fe 2+ ions.
- the treatment liquid according to the present embodiment is an aqueous solution in which Fe 2+ ions exist stably, Fe 2+ Ions can be taken as they are.
- this therapeutic solution contains a large amount of Fe 2+ ions, it is considered that citrus trees can directly take in a large amount of Fe 2+ ions, supplement the lack of iron, and enhance the therapeutic effect.
- Fe aqueous solutions for the following tests were prepared respectively. 1.
- Fe-EDTA aqueous solution Treatment solution A (aqueous solution) 3.
- Treatment solution B aqueous solution 4).
- Iron sulfate aqueous solution Iron sulfate aqueous solution
- Fe-EDTA aqueous solution (1. Fe-EDTA aqueous solution)
- the Fe-EDTA aqueous solution was prepared by dissolving Fe-EDTA (manufactured by Sigma Aldrich Japan, trade name: sodium ethylenediaminetetraacetate (III)) with demineralized distilled water so that the total Fe ion concentration was 15 mg / L. .
- Treatment solution A (aqueous solution)
- the therapeutic solution A is 14 g of citric acid per 100 mL of water, and Fe per 100 mL of water is 40 mass parts of the therapeutic liquid A stock solution when the citric acid content is 100 mass parts. It adjusted by diluting with demineralized distilled water so that a density
- Treatment solution B (aqueous solution)
- Treatment liquid B has 14 g of citric acid per 100 mL of water, and Fe per 100 mL of water is 13 parts by mass of treatment liquid B stock solution when the citric acid content is 100 parts by mass. It adjusted by diluting with demineralized distilled water so that a density
- the aqueous iron citrate solution was prepared by dissolving iron citrate (Showa Kako Co., Ltd.) with demineralized distilled water so that the total Fe ion concentration was 15 mg / L.
- the aqueous iron sulfate solution was prepared by dissolving iron sulfate with demineralized distilled water so that the total Fe ion concentration was 15 mg / L.
- the Fe 2+ ion concentration in each Fe aqueous solution was measured in order to confirm Fe 2+ ions contained in these aqueous solutions. In addition, this measurement was performed using Fe aqueous solution adjusted so that total Fe ion concentration might be about 50 mg / L.
- the therapeutic solution A aqueous solution was prepared by diluting the therapeutic solution A stock solution with ion-exchanged water so that the total Fe ion concentration was about 50 mg / L.
- the Fe 2+ ions and total Fe ions contained in the obtained aqueous solution are measured. did.
- the measurement was performed according to the protocol attached to the reflective quaternary iron ion test paper. The amount obtained by subtracting the Fe 2+ ion amount from the total Fe ion amount was converted as the Fe 3+ ion amount. In this measurement, the work was always performed in a room where direct sunlight was not inserted.
- the Fe 2+ ion concentration was 10.4 mg / L
- the Fe 3+ ion concentration was 36.6 mg / L
- the total of Fe 2+ ions and Fe 3+ ions was 100% by mass.
- the 2+ ion was 22% by mass.
- the treatment solution B, iron citrate, Fe-EDTA, and iron sulfate were adjusted so that the total Fe ion concentration was about 50 mg / L, and the Fe 2+ ion concentration and the total Fe ion concentration in each aqueous solution, The proportion of Fe 2+ ions in the total Fe ions was measured. The results are shown in Table 1.
- the therapeutic solution A aqueous solution, the therapeutic solution B aqueous solution, the iron citrate aqueous solution, and the iron sulfate aqueous solution all contain Fe 2+ ions at a predetermined ratio.
- the Fe—EDTA aqueous solution stably held Fe 3+ ions.
- cultivation was carried out in a gross cabinet. The test was performed under the conditions of a daytime temperature of 32 ° C and a nighttime temperature of 28 ° C. Nutrients were applied to the soil every 10 days. The applied nutrient was an aqueous solution containing 10 mM calcium nitrate, 2.5 mM monopotassium dihydrogen phosphate, 2.5 mM magnesium sulfate heptahydrate, and 1 mM potassium sulfate, which was given 50 mL / pot per pot.
- the above-mentioned Fe-EDTA aqueous solution and distilled water were applied by spraying on the leaves of the specimen and irrigating the root of the specimen. Application to the leaves and irrigation to the roots were performed once every 5 days.
- the amount of Fe-EDTA aqueous solution and distilled water sprayed on the leaves is 50 mL at a time.
- the amount of the Fe-EDTA aqueous solution and distilled water irrigated to the root is 50 mL at a time.
- PCR diagnosis On the 60th day after the start of the Fe-EDTA aqueous solution treatment (nurturing), about 3 to 5 leaves were collected from each specimen, DNA was extracted, amplified by PCR, and diagnosed for citrus greening disease (hereinafter referred to as “citrus greening disease”). , Referred to as PCR diagnosis). Specifically, it was performed as follows.
- CTAB solution 10% CTAB, 0.7 M NaCl
- An equal amount of CTAB precipitation 1% CTAB, 50 mM Tris-HCl, pH 8.0, 0.10 mM EDTA
- the nucleic acid in the supernatant binds to CTAB and precipitates at a low concentration (less than 0.35M NaCl). It was allowed to stand overnight, settled, centrifuged at 1800 rpm ⁇ 15 minutes, and the supernatant containing starch was discarded.
- a precipitation lysate (1 M NaCl, 50 mM Tris-HCl, 10 mM EDTA) was added to separate the nucleic acid / CTAB complex and dissolve the nucleic acid.
- An equal amount of isoamyl alcohol was slowly added to the nucleic acid solution to precipitate the nucleic acid. Centrifugation was performed at 1800 rpm ⁇ 10 minutes, and the supernatant containing CTAB was discarded.
- the precipitate and the CTAB on the side of the centrifuge tube were washed with 70% ethanol, and centrifuged in the same manner to remove CTAB.
- nucleic acid was dissolved with 1/10 TE solution (10 mM Tris-HCl, 1 mM EDTA). The purity of the nucleic acid solution was evaluated with a spectrophotometer at 260 nm / 230 nm for starch contamination and 260 nm / 280 nm for protein contamination. Both values were 1.8 or higher for the next experiment. Further, a polymer nucleic acid solution of ⁇ DNA (47.5 kb) or more was used in the next experiment by agarose electrophoresis. The amount of DNA was measured with a fluorescence spectrophotometer.
- PCR reaction solution 20 ⁇ L 48 mM MgCl 2 1 ⁇ L, Takara Premix tag 2 ⁇ PCR solution 10 ⁇ L (Takara, Bio Inc., Shiga, Japan), 90 ng / ⁇ L forward primer MHO035 1 ⁇ L, 90 ng / ⁇ L reverse primer MHO0354 1 ⁇ L, extracted above
- a DNA sample 2 ⁇ L (20 ng) and sterilized water 5 ⁇ L were mixed to prepare a PCR reaction solution 20 ⁇ L.
- This PCR reaction solution was set in DNA Thermal Cycler PTC-1148 (Bio-Rad Laboratories, Inc.), and DNA was amplified under the following conditions.
- Primer Forward primer (MHO0353): 5'-CACCGAAGATATGGACAACA-3 '(SEQ ID NO: 1)
- Reverse primer (MHO0354): 5'-CAGGTTCTTGTGGTTTTTCTG-3 '(SEQ ID NO: 2) PCR conditions 90 ° C 3 minutes 1 cycle ⁇ 94 ° C 1 minute, 68.5 ° C 1 minute, 72 ° C 3 minutes ⁇ 35 cycles 72 ° C 3 minutes 1 cycle Store at 4 ° C until electrophoresis
- PC a DNA sample extracted from a leaf collected from a pathogenic tree infected with the pathogenic strain Ishi-1 by the above-described DNA extraction method was used.
- FIG. 1 and Table 2 show the PCR diagnosis results of the sample applied with the Fe-EDTA aqueous solution and the PCR diagnosis results of the sample applied with distilled water.
- m indicates a lane in which a molecular weight marker is passed.
- a positive band was detected at a predetermined position (indicated by an arrow in the figure) in the positive control (PC).
- PC positive control
- Table 2 shows the band intensity of each specimen when the intensity of the positive band of PC is 100%.
- the band intensity of the electrophoresis result was analyzed using ImageJ (image processing program, NIH). First, the PC band part (white area in the figure) was selected, and the luminance (darkness) of the selected area was digitized. Regarding the band portion of each sample, the density in the region having the same area as the region selected in the PC band was quantified.
- Treatment Solution A The above specimens were continued to be grown as they were after the 61st day of growth. From the 61st day, instead of the Fe-EDTA aqueous solution, the treatment solution A was applied to 5 samples (sample A to sample E) at a total Fe ion concentration of 15 mg / L.
- Treatment liquid A was applied by spraying the leaves of the specimen and irrigating the root of the specimen in the same manner as the application of the Fe-EDTA aqueous solution. Application to the leaves and irrigation to the roots were performed once every 5 days. The treatment solution sprayed on the leaves is 50 mL at a time. Further, the amount of the treatment liquid A irrigated to the root is 50 mL at a time.
- the treatment solution A used was prepared by diluting the treatment solution A stock solution with demineralized distilled water so that the total Fe ion concentration was 15 mg / L as described above.
- sample F For the other 5 samples (sample F to sample J), distilled water was applied under the same conditions as described above continuously from the 61st day.
- FIG. 2 and Table 3 show the PCR diagnosis results of the sample applied with the treatment liquid A and the sample applied with distilled water. Table 3 shows the band intensity of each specimen when the intensity of the positive band of PC is 100%. In addition, when the intensity
- each specimen was nurtured under the same conditions after the 115th day.
- day 252 day 192 after applying the treatment solution
- about 5 leaves were collected from each of the upper, middle and lower leaves of each specimen, and PCR diagnosis was performed.
- the results of PCR diagnosis of the sample applied with the therapeutic solution and the sample applied with distilled water are shown in FIGS. 3 (a) to 3 (d) and Tables 4 (a) to (d).
- sample F to sample J In samples (sample F to sample J) that continued to be applied with distilled water, only sample F did not show a band at the same position as PC, but in other samples G to J, all of them were the same position as PC ( A positive band appeared at (indicated by an arrow in the figure).
- each sample was grown under the same conditions from day 253 onward. Then, about 3 to 5 leaves were collected from each specimen after 1 year and 9 months of growth (1 year and 7 months after applying the treatment solution), and PCR diagnosis was performed in the same manner as described above.
- FIG. 4 shows the PCR diagnosis result of the sample to which treatment solution A or distilled water was applied.
- Table 5 shows the band intensity of each specimen when the intensity of the positive band of PC is 100%.
- sample F to specimen J In specimens (sample F to specimen J) that continued to be applied with distilled water, 4 specimens (sample G to specimen J) had a band at the same position as the PC (indicated by the arrow in the figure), and the medical condition was improved. I never did. In addition, the positive band disappeared in one sample (Sample F). The cause is unknown, but in citrus greening disease, it is known that there are individuals that rarely detect bacteria, and this individual is also considered to be one of them.
- citrus greening disease can be completely cured by applying the treatment liquid A containing Fe 2+ ions to the tree of rough lemon suffering from citrus greening disease.
- FIG. 5 shows the length of the branches extended for 67 days.
- the extension of the branch is about 25 cm, whereas when the treatment solution A is applied, the extension of the branch is 35 cm or more and is not affected. It was found to be as good as or better than healthy tree branch elongation. From the above results, it was proved that the application of the treatment liquid A containing Fe 2+ ions also has an effect of promoting branch elongation.
- the treatment liquid according to the present embodiment has a surprising effect that citrus greening disease can be treated without impairing the growth of citrus trees.
- the treatment solution B having a total Fe ion concentration of 30 mg / L was applied to these affected specimens 1 to 3.
- treatment solution A having a total Fe ion concentration of 30 mg / L was applied.
- Each treatment solution was applied by spraying treatment solution B or treatment solution A on the leaves of the specimen.
- the leaves were sprayed once every 7 days.
- the amount of the treatment solution sprayed on the leaves is 1.5 L per specimen.
- the used treatment solution B was prepared by diluting the treatment solution B stock solution with demineralized distilled water so that the total Fe ion concentration was 30 mg / L.
- Treatment solution A was prepared by diluting treatment solution A stock solution with demineralized distilled water so that the total Fe ion concentration was 30 mg / L.
- A indicates an old leaf and B indicates a new leaf.
- No band was detected at the same position as PC (shown as Con in the figure) in all of the samples to which the treatment solution was applied (samples 1 to 3 and 5), and citrus greening disease was completely cured in these samples 1 to 3 and 5. I found out. Therefore, it was proved that the treatment liquid B and the treatment liquid A containing Fe 2+ ions are effective in treating citrus greening disease even in tankan.
- an Fe-EDTA aqueous solution having a total Fe ion concentration of 15 mg / L, a therapeutic solution B, an iron citrate aqueous solution, an iron sulfate aqueous solution, and distilled water were applied. Each Fe aqueous solution was evaluated using 2 to 3 specimens.
- Fe aqueous solution was performed by spraying on the leaves of the specimen and irrigating the root of the specimen. Application to the leaves and irrigation to the roots were performed once every 5 days.
- the amount of the Fe aqueous solution or the like sprayed on the leaves is 50 mL at a time. Further, the amount of the Fe aqueous solution or the like irrigated to the root is 50 mL at a time.
- Table 8 shows the average value of the band intensity of each specimen when the intensity of the positive band of PC is 100%.
- the treatment liquid according to the present embodiment contains Fe 2+ ions. Therefore, it is considered that when a therapeutic solution is applied to trees, it reacts with hydrogen peroxide generated in the cells to generate active oxygen. Therefore, the amount of active oxygen generated by the various Fe aqueous solutions and the stability thereof were evaluated.
- the active oxygen generated when each of the Fe aqueous solutions prepared in the above-described test Fe aqueous solution was added to distilled water was measured by the luminol reaction.
- active oxygen is generated by adding an aqueous solution containing Fe 2+ ions.
- FIG. 8 100 ⁇ L of each of the above aqueous solutions was taken, 50 ⁇ L of distilled water was added, 50 ⁇ L of luminol solution was added after 0, 60, 180 and 360 minutes, and the amount of chemiluminescence was measured.
- the Fe-EDTA aqueous solution as shown in FIG. 8, almost no active oxygen was detected.
- the treatment liquid A, the treatment liquid B, the iron citrate aqueous solution, and the iron sulfate aqueous solution were added to distilled water, strong luminescence intensity was detected, indicating that active oxygen was generated.
- the treatment liquid according to the present embodiment contains Fe 2+ ions, it was confirmed that the stably hold the Fe 2+ ions.
- the treatment liquid according to the present embodiment contains Fe 2+ ions and is surprisingly applied to citrus trees suffering from citrus greening disease, thereby completely curing citrus greening disease. It became clear that it had a power effect.
- citrus fruits with citrus greening disease can be treated by using the citrus greening disease treatment solution according to the present invention. Therefore, utilization in the agricultural field where citrus fruits are cultivated is expected.
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Abstract
Description
本実施の形態に係るカンキツグリーニング病の治療液(以下、単に治療液ともいう)は、Fe2+イオンを含有する。この治療液は、Fe2+イオンを安定に保持している。
Fe2+イオン濃度は、例えばo-フェナントロリンを用いた既存の方法によって測定することができる。o-フェナントロリンはFe2+イオンと選択的に錯体を形成するため、この錯体の吸光度を測定することにより、Fe2+イオンを選択的に定量することができる。また、溶液中に含まれるFe3+イオンをあらかじめ還元して全Feイオンを二価鉄とした後にo-フェナントロリン法を用いて定量することにより、総Feイオン量を定量することができる。
有機酸粉末とFe2+イオンを供給することができる鉄化合物粉末とを水に加熱溶解して得ることができる。
一般に、病原体等の侵入物に対する細胞の耐性には活性酸素が関与していることが知られている。この活性酸素は細胞内において病原性ストレスから細胞を保護する働きを有し、細胞内の活性酸素濃度は、細胞内で発生した過酸化水素が二価鉄と反応してヒドロキシラジカルを生成するフェントン反応を介して制御される。
本実施の形態に係る治療液は、Fe2+イオンを安定に保持し、後述の実施例に示すようにヒドロキシラジカルを持続的に発生することができる。そのため、この治療液を樹木に施用すると、柑橘類の細胞内で反応性の高いヒドロキシラジカルを生じ、このヒドロキシラジカルが直接的にHLB菌を死滅させる、或いは、細胞内で何らかの反応性を促して、二次的作用でHLB菌を死滅させることにより、カンキツグリーニング病を治療することができると考えられる。
以下の試験用のFe水溶液をそれぞれ調整した。
1.Fe-EDTA水溶液
2.治療液A(水溶液)
3.治療液B(水溶液)
4.クエン酸鉄水溶液
5.硫酸鉄水溶液
Fe-EDTA水溶液は、Fe-EDTA(シグマアルドリッチジャパン製、商品名:エチレンジアミン四酢酸(III)ナトリウム)を総Feイオン濃度が15mg/Lになるように脱塩蒸留水で溶解することにより調整した。
治療液Aは、水100mLあたりのクエン酸が14gであり、水100mLあたりのFeが、クエン酸の含有量を100質量部とした場合に、40質量部である治療液A原液を総Feイオン濃度が15mg/Lになるよう脱塩蒸留水で希釈することにより調整した。
治療液A原液を希釈した水溶液は、各々Fe2+イオンとFe3+イオンとを含有し、Fe2+イオンとFe3+イオンとの合計量を100質量%とした場合に、Fe2+イオンが20~40質量%である。但し、この各イオン濃度は、後述する測定方法により測定された値である。
治療液Bは、水100mLあたりのクエン酸が14gであり、水100mLあたりのFeが、クエン酸の含有量を100質量部とした場合に、13質量部である治療液B原液を総Feイオン濃度が15mg/Lになるよう脱塩蒸留水で希釈することにより調整した。
治療液B原液を希釈した水溶液は、各々Fe2+イオンとFe3+イオンとを含有し、Fe2+イオンとFe3+イオンとの合計量を100質量%とした場合に、Fe2+イオンが50~90質量%である。但し、この各イオン濃度は、後述する測定方法により測定された値である。
クエン酸鉄水溶液は、クエン酸鉄(昭和化工株式会社)を総Feイオン濃度が15mg/Lとなるように脱塩蒸留水で溶解することにより調整した。
硫酸鉄水溶液は、硫酸鉄を総Feイオン濃度が15mg/Lとなるように脱塩蒸留水で溶解することにより調整した。
治療液A、治療液B、クエン酸鉄水溶液、Fe-EDTA水溶液及び硫酸鉄水溶液について、これらの水溶液中に含まれるFe2+イオンを確認するため、各Fe水溶液におけるFe2+イオン濃度を測定した。なお、本測定は、総Feイオン濃度が約50mg/Lになるように調整されたFe水溶液を用いて行った。
まず、上記と同様にして、治療液A原液を総Feイオン濃度が約50mg/Lとなるようにイオン交換水で希釈して治療液A水溶液を調整した。その後、すぐにRQflex多項目水質検査器(Merck社製)とリフレクトクァント鉄イオン試験紙(Merck社製)を用いて、得られた水溶液に含有されるFe2+イオンと総Feイオンとを測定した。測定は、リフレクトクァント鉄イオン試験紙に添付のプロトコールに従って行った。また、総Feイオン量からFe2+イオン量を差し引いた量をFe3+イオン量として換算した。尚、この測定では常に直射日光の差し込まない室内において作業を行った。
カンキツグリーニング病に感染した柑橘類の樹木に、上記のFe水溶液を施用し、Fe2+イオンを含有する治療液のカンキツグリーニング病に対する治療効果について検証した。
検体としてラフレモン(Citrus verrucosa Lush.)の樹木を用いた。まず、ラフレモンの種子を発芽させ、約1Lのポット植、野菜育苗用培土(タキイ種苗株式会社製)で育成した。育成1年後に、接ぎ木によって病原木から病原菌を摂取させて、カンキツグリーニング病を感染させた。病原木は、石垣島から採取され「Ishi-1」と命名された病原菌株を感染させたものである。感染後、更に1年育成した検体を試験に供した。同様にして、カンキツグリーニング病を感染させた検体を10検体(検体A~検体J)準備した。
以上のように準備した10検体(検体A~検体J)を育成し、まず、育成60日目までは、5検体(検体A~検体E)に対して、総Feイオン濃度15mg/LのFe-EDTA水溶液を施用した。また、他の5検体(検体F~検体J)に対して、Fe-EDTA水溶液の代わりに蒸留水を施用した。
採取した各葉(3~5g)を蒸留水で洗浄して水分を除き、中肋を切り取った後、液体窒素を用いて凍結させた。これを蒸気滅菌・乾熱滅菌した乳鉢・乳棒でホモジナイズして、5mLの1×CTABバッファー(1%CTAB,50mMTris-HCl(pH8.0),0.7M NaCl,10mM EDTA)に溶解し、65℃で撹拌しながら30分間インキュベートした後、クロロホルム・イソアミルアルコール(24:1v/v)を5mL加え、30分間転倒混和し3000rpm×15分遠心分離し、上澄みをスポイドで新しい遠心チューブに移した。以上の除タンパク質処理を3回行った。上澄みの1/10量の10%CTAB溶液(10%CTAB、0.7M NaCl)を加え転倒混和し除タンパク質処理によって失われたCTABを補充した。その上澄みに等量のCTAB沈殿液(1%CTAB、50mM Tris-HCl,pH8.0、0.10mM EDTA)をゆっくり加え、静かに転倒混和した。上澄みにある核酸は低濃度(0.35M以下のNaCl)でCTABと結合し沈殿する。一晩静置、沈殿させ、1800rpm×15分で遠心分離し、デンプンを含む上澄みを捨てた。沈殿に1mLの沈殿溶解液(1M NaCl、50mM Tris-HCl、10mM EDTA)を加え、核酸・CTAB複合体を分離し、核酸を溶かした。この核酸溶液に等量のイソアミルアルコールをゆっくり加えて、核酸を沈殿させた。1800rpm×10分で遠心分離し、CTABを含む上澄みを捨てた。70%エタノールで沈殿及び遠心管側面のCTABを洗浄し、同様に遠心してCTABを除去した。最後に1/10TE溶液(10mM Tris-HCl、1mM EDTA)で核酸を溶解した。核酸溶液の純度は分光光度計で260nm/230nmでデンプン混入度を、260nm/280nmでタンパク質の混入度を評価し、両値とも1.8以上のものを次の実験に使用した。また、アガロース電気泳動によりλDNA(47.5kb)以上の高分子の核酸溶液を次の実験に使用した。DNA量は蛍光分光光度計で測定した。
PCR診断は、「Marjorie A.Hoy,Ayyamperumal Jeyaprakash,and Ru Nguyen(2001),Long PCR is a sensitive Method for Detecting Liberobacter asiaticum in Parasitoids Undergoing Risk Assessment in Quarantine. Biological Control 22,278-287」に記載の方法に従って行った。
具体的には、48mM MgCl2 1μL、Takara Premix tag2×PCR溶液10μL(Takara,Bio Inc.,Shiga,Japan)、90ng/μLフォワードプライマーMHO035 1μL、90ng/μLリバースプライマーMHO0354 1μL、上記にて抽出したDNA試料2μL(20ng)、及び滅菌水5μLを混合してPCR反応液20μLを調整した。このPCR反応液を、DNA Thermal Cycler PTC-1148(Bio-Rad Laboratories,Inc.)にセットし、以下の条件でDNAを増幅した。
プライマー
フォワードプライマー(MHO0353):
5’-CACCGAAGATATGGACAACA-3’ (配列番号1)
リバースプライマー(MHO0354):
5’-CAGGTTCTTGTGGTTTTTCTG-3’ (配列番号2)
PCR条件
90℃ 3分 1サイクル
{94℃ 1分、68.5℃ 1分、72℃ 3分}35サイクル
72℃ 3分 1サイクル
4℃で泳動まで保存
Fe-EDTA水溶液を施用した検体のPCR診断結果及び蒸留水を施用した検体のPCR診断結果を図1及び表2に示す。図中、mは分子量マーカーを流したレーンを示す。
電気泳動結果のバンド強度は、ImageJ(画像処理プログラム、NIH)を用いて解析した。まず、PCのバンド部分(図中、白い領域)を選択し、選択された領域の輝度(濃さ)を数値化した。各試料のバンド部分についても、PCのバンドで選択された領域と同じ面積を有する領域内の濃さを数値化した。次にブランクになっている黒い部分の濃さも同様にして数値化し、各バンドの濃さの数値から差し引いた値を各バンドの元の数値とした。表2には、ポジテイブコントロール(PC)の濃さを100%とした場合の各バンドの元の数値の相対値(%)を示している。
育成61日目以降も、上記の検体をそのまま育成し続けた。そして、61日目からはFe-EDTA水溶液に代えて、5検体(検体A~検体E)に治療液Aを総Feイオン濃度15mg/Lにて施用した。
以上の結果から、Fe2+イオンを含有する治療液Aの施用は、枝の伸長を促進する効果も有することが立証された。
このように、本実施の形態に係る治療液は、柑橘類樹木の成長を損なうことなく、カンキツグリーニング病を治療できるという驚くべき効果を有することが明らかとなった。
次に検体としてタンカン(Citrus tankan Hayata)の樹木を用いて評価を行った。まず、沖縄県恩納村の樹園地にて、複数のタンカンの樹木の中からカンキツグリーニング病に罹病している樹木を特定するため、各樹木の古葉を用いて上記のPCR診断を行った。その結果を図6(a)(b)、及び表6(a)(b)に示す。表6(a)(b)には、PCの陽性バンドの強度を100%としたときの各検体のバンド強度を示す。
従って、Fe2+イオンを含有する治療液B及び治療液Aは、タンカンにおいてもカンキツグリーニング病の治療に有効であることが立証された。
次に検体としてシークワーシャー(Citrus depressa Hayata)の樹木を用いて評価した。上記のラフレモンと同様に接ぎ木によって病原木から病原菌を接種させ、カンキツグリーニング病に感染した検体を準備した。
このように、本実施の形態に係る治療液は、シークワーシャーにおいてもカンキツグリーニング病の治療に有効であることが立証された。
上述したように、本実施の形態に係る治療液はFe2+イオンを含有する。そのため、治療液を樹木に施用すると、細胞内で発生した過酸化水素と反応して活性酸素を生じると考えられる。そこで、上記の各種Fe水溶液による活性酸素の発生量及びその安定性について評価した。
上述の試験用Fe水溶液の調整においてそれぞれ調整した各種Fe水溶液を蒸留水に添加した際に発生する活性酸素をルミノール反応により測定した。なお、蒸留水中には一定量の割合で過酸化水素が含まれるため、Fe2+イオンを含有する水溶液を添加することによって活性酸素が発生する。
各Fe水溶液を100μLずつとり、蒸留水50μL及びルミノール液を50μL添加して、ルミネッセンサー(アトー株式会社製)を用いて化学発光量を10秒間の積算にて測定した。各試料について3反復で測定した。溶液中の活性酸素の量が多いほど発光強度は高い値を示す。
さらに、活性酸素は蒸留水の添加後3時間以上継続して検出されたことから、これらFe2+イオンを含有する水溶液は、安定にFe2+イオンを保持できることが明らかとなった。
また、所定の濃度のクロロゲン酸存在下でも、Fe-EDTA水溶液以外のFe水溶液では発光強度がある程度維持されていた。従って、これらのFe水溶液は、ヒドロキシラジカルを捕捉、除去するような物質の存在下でも、安定に且つ持続的に活性酸素を供給できることが分かった。
Claims (9)
- Feイオンを含有し、該Feイオンの少なくとも一部がFe2+イオンであることを特徴とするカンキツグリーニング病の治療液。
- 総Feイオンの濃度が10mg/L~100mg/Lであることを特徴とする請求項1に記載のカンキツグリーニング病の治療液。
- 前記総Feイオンに加えさらに酸を含有することを特徴とする請求項2に記載のカンキツグリーニング病の治療液。
- 前記酸が有機酸であることを特徴とする請求項3に記載のカンキツグリーニング病の治療液。
- 前記有機酸がカルボキシル基及びヒドロキシル基のうち少なくとも一方を有し、該カルボキシル基及びヒドロキシル基の合計が2つ以上であることを特徴とする請求項4に記載のカンキツグリーニング病の治療液。
- 前記有機酸がクエン酸、リンゴ酸、酒石酸及びアスコルビン酸のうち少なくとも1種であることを特徴とする請求項4又は5に記載のカンキツグリーニング病の治療液。
- 施用する植物が、柑橘類植物であることを特徴とする請求項1乃至6のいずれか1項に記載のカンキツグリーニング病の治療液。
- 施用する植物が、ラフレモン、タンカン又はシークワーシャーであることを特徴とする請求項7に記載のカンキツグリーニング病の治療液。
- 請求項1乃至8のいずれか1項に記載の治療液をカンキツグリーニング病に感染した柑橘類植物の葉面、根圏、又は葉面及び根圏の両方に施用して、該柑橘類植物中の病原菌を減少又は消滅させることでカンキツグリーニング病を治療することを特徴とするカンキツグリーニング病の治療方法。
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JP2021134165A (ja) * | 2020-02-27 | 2021-09-13 | 国立研究開発法人農業・食品産業技術総合研究機構 | リベリバクター属細菌の増殖制御剤およびその制御方法 |
JP7381078B2 (ja) | 2020-02-27 | 2023-11-15 | 国立研究開発法人農業・食品産業技術総合研究機構 | リベリバクター属細菌の増殖制御剤およびその制御方法 |
WO2023176347A1 (ja) * | 2022-03-14 | 2023-09-21 | デンカ株式会社 | 柑橘類植物を栽培する方法及び液体組成物 |
WO2023176346A1 (ja) * | 2022-03-14 | 2023-09-21 | デンカ株式会社 | 柑橘類植物を栽培する方法及び柑橘類植物栽培用液体組成物 |
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TW201306744A (zh) | 2013-02-16 |
US8945631B2 (en) | 2015-02-03 |
US20130259954A1 (en) | 2013-10-03 |
JPWO2012081420A1 (ja) | 2014-05-22 |
TWI530254B (zh) | 2016-04-21 |
JP5920729B2 (ja) | 2016-05-18 |
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