Agent for reducing nitrate ions and method for reducing nitrate ions

Abstract

To provide a nitrate ion reducing agent and a nitrate ion reducing method employing PGPB.SOLUTION: A nitrate ion reducing agent comprising a Pelomonas strain SPE11 strain (Pelomonas sp. SPE11) (NITE ABP-03793), and a method for reducing nitrate ions in which the SPE11 strain is supplied to the rhizosphere of a plant.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an agent for reducing nitrate ions contained in plants and a method for reducing the amount of nitrate ions contained in plants. [Background technology]

[0002] Nitrate ions are also approved as food additives, and the nitrate ion itself is not highly toxic. However, it has been pointed out that nitrate ions can be reduced in the body to produce nitrite ions, which cause methemoglobinemia, and can also be converted into nitroso compounds, which are carcinogenic. Therefore, it is considered desirable to consume a low amount of nitrate ions. For example, the Joint FAO / WHO Expert Committee on Food Additives estimates the acceptable daily intake of nitrates to be 0-5 mg per kg of body weight (0-3.7 mg as nitrate ions). Nitrogen is an essential nutrient for plants. Plants absorb nitrogen in the form of nitrate ions from their roots and accumulate it in their leaves. Therefore, it is said that most of the nitrate ions consumed by humans come from vegetables, and the European Food Safety Authority has established standard values ​​for the nitrate ion content of some vegetables to prevent excessive intake of nitrate ions (Non-Patent Document 1).

[0003] Given the above background, research is being conducted into cultivation methods that reduce the nitrate nitrogen content in vegetables, such as increasing the rate of nitrate ion reduction in vegetables and suppressing excessive absorption of nitrate ions. For example, Patent Document 1 reports a method in which vegetables that have reached harvest size are replaced with a nutrient solution that does not contain nitrate ions, and the vegetables are harvested after consuming the nitrate ions remaining in the vegetables. Patent Document 2 proposes a method for hydroponic cultivation of leafy vegetables under LED irradiation, in which the nitrate ion content is reduced and ascorbic acid is increased by simultaneously irradiating the leafy vegetables with green and purple light. Patent Document 3 reports a method for reducing the nitrate nitrogen content by using a culture solution containing glutathione. Furthermore, Non-Patent Document 2 reports that spraying Azospirillum brasilense onto the leaves of lettuce reduced the nitrate ion concentration. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-061570 [Patent Document 2] Japanese Patent Application Publication No. 2018-113934 [Patent Document 3] Japanese Patent Application Publication No. 2022-118839 [Non-patent literature]

[0005] [Non-Patent Document 1] COMMISSION REGULATION (EU) No 1258 / 2011 of 2 December 2011 amending Regulation (EC) No1881 / 2006 as regards maximum levels for nitrates in foodstuffs (Text with EEA relevance), Official Journal of the European Union, L 320 / 15-17 (2011) [Non-patent document 2] Moreria et al., Inoculation with Trichoderma harzianum and Azospirillum brasilense increases nutrition and yield of hydroponic lettuce, Arch Microbiol, 204(7):440, 2022. Summary of the Invention [Problem to be solved by the invention]

[0006] Plant growth-promoting bacteria (PGPB) exist in the natural environment. PGPB grow by utilizing carbon sources secreted from plant cells, and promote plant growth through various mechanisms, including nitrogen fixation, solubilization of insoluble phosphorus, iron chelation through siderophore production, promotion of plant hormone production, and suppression of plant pathogen growth. The present inventors have searched for PGPB that has the effect of reducing nitrate ions, and have completed the present invention. An object of the present invention is to provide a nitrate ion reducing agent and a nitrate ion reducing method that utilize PGPB. [Means for solving the problem]

[0007] The means for solving the problems of the present invention are as follows. 1. A nitrate ion reducer characterized by containing Pelomonas sp. SPE11 (NITE ABP-03793). 2. A method for reducing nitrate ions, comprising supplying Pelomonas sp. SPE11 (NITE ABP-03793) to the rhizosphere of a plant. 3. The method for reducing nitrate ions according to 2., wherein the plant is a leafy vegetable. [Effects of the Invention]

[0008] According to the present invention, the nitrate ion concentration in plants can be reduced, and therefore healthy, high-value-added vegetables can be produced. Because the Pelomonas bacterium SPE11 is a PGPB, its symbiosis with plants allows for reduced fertilizer and pesticide use, and it is expected to maintain and increase crop yields. Furthermore, because the Pelomonas bacterium SPE11 grows in the plant rhizosphere, it can be easily added to soil or nutrient solutions in hydroponic cultivation to reduce nitrate ion concentrations in plants. [Brief explanation of the drawings]

[0009] [Figure 1] Graph showing the growth of 21 bacterial strains isolated from the rhizosphere of duckweed under lettuce cultivation conditions. [Figure 2] Graph showing the wet leaf weight (per plant) of lettuce after cultivation in Experiment 2. [Figure 3] Graph showing the nitrate ion concentration in lettuce after cultivation in Experiment 2. [Figure 4] Graph showing the nitrate ion concentration in lettuce after cultivation in Experiment 3. DETAILED DESCRIPTION OF THE INVENTION

[0010] SPE11 strain Pelomonas sp. SPE11 (hereinafter also referred to as "SPE11 strain") was isolated from the rhizosphere of the aquatic plant, duckweed (Spirodela polyrhiza). Properties of SPE strains Morphological characteristics: Shape: rod-shaped, size: 2-3 μm, motility: yes Physiological properties Gram staining: negative

[0011] The SPE11 strain was internationally deposited on December 7, 2022, with the National Institute of Technology and Evaluation's Patent Microorganism Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818)) under the accession number NITE BP-03793. The SPE11 strain is a PGPB that inhabits the rhizosphere of plants, promoting plant growth and reducing the nitrate ion concentration in plants.

[0012] Nitrate ion reducer The nitrate ion-reducing agent of the present invention includes the SPE11 strain. Note that in the nitrate ion-reducing agent of the present invention, the SPE11 strain may be dormant as long as it is active after being supplied to the rhizosphere of a plant. The nitrate ion reducing agent of the present invention may contain the SPE11 strain, and may be, for example, a culture containing the SPE11 strain, an isolate obtained by separating the SPE11 strain from the culture and washing it, or a formulation of the culture or isolate together with a carrier known in the field of pesticide formulations.

[0013] The concentration of the SPE11 strain contained in the nitrate ion reducing agent of the present invention is not particularly limited as long as it is within the range in which the effects of the present invention are exhibited. For example, in the case of a liquid agent, 3 ~1×10 12 cfu / ml. The dosage form of the nitrate ion reducing agent of the present invention is not particularly limited, and can be any formulation such as a dust, granule, liquid, emulsion, suspension, wettable powder, etc. Furthermore, when the agent is formulated, additives used in the field of pesticide formulations, such as organic fertilizer components, inorganic fertilizer components, nutrients, and even PGPB other than the SPE11 strain, can be included, as long as they do not impair the effects of the present invention.

[0014] Nitrate ion reduction method The method for reducing nitrate ions of the present invention involves supplying the SPE11 strain to the rhizosphere of a plant. The SPE11 strain can be supplied as a bacterial cell or as the above-mentioned nitrate ion reducing agent. The plant from which the SPE11 strain is supplied is not particularly limited, but is preferably a leafy vegetable (leafy vegetable) because nitrate nitrogen accumulates in the leaves of the plant. Examples of leafy vegetables include Asteraceae plants such as lettuce, sunny lettuce, romaine lettuce, frilled lettuce, lettuce, and salad greens, and Garland chrysanthemum plants such as Garland chrysanthemum; Amaranthaceae plants such as spinach; and Brassica plants such as cabbage, Chinese cabbage, komatsuna, and bok choy; with Asteraceae plants being more preferred, and Lactuca being even more preferred.

[0015] In the method for reducing nitrate ions of the present invention, the SPE11 strain can be supplied to the rhizosphere of plants grown either in soil or hydroponics, but hydroponics is preferred because it is easier to control the rhizosphere environment. Note that in soil cultivation, since many other microorganisms grow in the soil, the SPE11 strain is supplied after sterilization as necessary.

[0016] When the SPE11 strain is supplied to the rhizosphere in the form of a nitrate ion reducer, it can be applied directly as is, or diluted with water or the like before application. The method of application of the nitrate ion reducer is not particularly limited, and examples include adding it to soil or nutrient solution, attaching it to crops or seeds by spraying or immersing them, or adding it to water to be added to crops or soil. Among these, the SPE11 strain used in the present invention is a bacterium that interacts with plant roots, and in order to give it priority over other microorganisms, it is preferred to add it to soil or nutrient solution at the start of plant cultivation, or to attach it by immersing at least the roots in soil or nutrient solution before transplanting. When added to nutrient solution for hydroponic cultivation, the OD 600 It is preferable to add it so that the difference is 0.03 to 0.1. [Example]

[0017] "Experiment 1" Microorganism Selection Test strains Twenty-one strains of bacteria isolated from the rhizosphere of duckweed were used. The names of the strains and their growth-promoting effects on duckweed are shown in Table 1. [Table 1] Culture medium For bacterial preculture, 1 / 10 TSB (Difco) was used.

[0018] Lettuce cultivation Lettuce was grown using MK1 and MK2 manufactured by M-type Hydroponics Research Institute Co., Ltd. The composition and concentration are shown in Table 2. [Table 2]

[0019] Measurement of growth rate of rhizosphere microorganisms under lettuce cultivation conditions Among 21 strains of duckweed rhizosphere bacteria, we screened for strains suitable for lettuce cultivation. 250 mg / L glucose was added as a carbon source to the lettuce cultivation solution, which was then sterilized with a 0.22 μm filter. 600 The bacteria were inoculated so that OD = 0.01, and cultured at 20°C and 150 rpm for 7 days. After 4 and 7 days of culture, the OD 600 The results are shown in Figure 1.

[0020] ·result Four strains, SP2, SP7, SPE11, and SPE14, showed excellent growth in the lettuce nutrient solution, and were therefore selected for use in lettuce cultivation.

[0021] "Experiment 2" Plant growth promotion effect of selected rhizosphere microorganisms (1) Experimental method ·Test plant Hydroponic lettuce ('Multi-leaf Frill Lettuce', Kaneko Seed Co., Ltd.) Lettuce cultivation method using rhizosphere microorganisms I. Seeding / raising seedlings Hydroponic lettuce was sown on a seedling mat immersed in a lettuce nutrient solution and kept dark for 2 days. After that, the light intensity was 100 μmol / m 2 The plants were grown for 12 days under conditions of 16 hours day length and 10 minutes day length, in a laboratory with a room temperature of 24°C and constant relative humidity. II. Planting and cultivation A submerged hydroponic cultivation system was used for cultivation, with a water temperature of 19°C and a light intensity of 250±1 μmol / m 2 ·s, 16-hour photoperiod. OD of bacteria into the bat 600 Five liters of lettuce nutrient solution inoculated to a pH of 0.03 was added, and ten seedlings of similar size were planted. The trays were placed in a hydroponic cultivation system and cultivated for three weeks. The pH of the nutrient solution was monitored during the cultivation period, and if it dropped to the pH 4 range, it was adjusted to the pH 6 range.

[0022] III. Measurement ·weight The above-ground parts were cut off and harvested, and immediately thereafter the weight was measured to calculate the wet leaf weight per plant. Measurement of nitrate ion concentration in lettuce Eight 7mm diameter leaf disks were cut from random locations on the lettuce leaves, weighed, crushed using a BioMasher II (Nippi), and dissolved in MilliQ to obtain an extract. The extract was filtered through a 0.45μm filter and diluted 10-fold with MilliQ, after which the nitrate ion concentration was measured by ion chromatography. The results are shown in Figures 2 and 3.

[0023] (2) Experimental results Inoculation with SP2, SP7, and SPE11 promoted lettuce growth, increasing lettuce weight by approximately two-fold compared to the uninoculated control. SPE14 did not promote lettuce growth. The nitrate ion concentration in the control was 1.39 mg / gFW (FW is fresh weight). Inoculation of SPE11 reduced the nitrate ion concentration to 82% of the control. In contrast, even when inoculated with SP2, SP7, and SPE14 strains, the nitrate ion concentration was almost the same as that of the control. Note that SPE14 strain did not promote lettuce growth, so the nitrate ion concentration was not measured. From the above, it was confirmed that supplying the SPE11 strain to the rhizosphere of plants can reduce nitrate ion concentrations and further promote plant growth.

[0024] "Experiment 3" Comparison with known strains (1) Experimental method Test strain Azospirillum brasilense Sp7 (ATCC29145, hereinafter referred to as Ab strain) was used. It has been reported that spraying Azospirillum brasilense on the leaves reduces the nitrate ion concentration in lettuce (Non-Patent Document 2).

[0025] Cultivation method Lettuce seeds were sown on a seedling mat soaked in a nutrient solution (M-type No. 1 + No. 2), kept in the dark for 2 days, and then grown for 11 days. 600 After immersing the seedlings for 24 hours in a culture solution containing a suspension of 0.1% of the bacteria, three seedlings were planted in 5 L of the culture solution without bacteria. The seedlings were cultivated for four weeks in a laboratory with a room temperature of 25°C, relative humidity, and a light intensity of 150 μmol / m² / S and a 16-hour day length. During the cultivation period, oxygen was supplied to the nutrient solution using a blower.

[0026] Nitrate ion concentrations were measured in the same manner as in Experiment 2 above. (2) Experimental results Even when the Ab strain was supplied to the rhizosphere, the nitrate ion concentration did not decrease. As described in Non-Patent Document 2, Azospirillum brasilense is a microorganism that reduces the nitrate ion concentration when sprayed on leaves, and it was confirmed that even when supplied to the rhizosphere, the nitrate ion concentration did not decrease.

Claims

1. A nitrate ion reducing agent comprising a bacterium of the genus Pelomonas, strain SPE11 (Pelomonas sp. SPE11) (NITE ABP-03793).

2. A method for reducing nitrate ions, comprising supplying a bacterium belonging to the genus Pelomonas, strain SPE11 (Pelomonas sp. SPE11) (NITE ABP-03793), to the rhizosphere of a plant.

3. 3. The method for reducing nitrate ions according to claim 2, wherein the plant is a leafy vegetable.

Images