CN113331213B - Soil bactericide for preventing and controlling apple or pear continuous cropping obstacle and application thereof - Google Patents
Soil bactericide for preventing and controlling apple or pear continuous cropping obstacle and application thereof Download PDFInfo
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- CN113331213B CN113331213B CN202110781677.3A CN202110781677A CN113331213B CN 113331213 B CN113331213 B CN 113331213B CN 202110781677 A CN202110781677 A CN 202110781677A CN 113331213 B CN113331213 B CN 113331213B
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- soil
- continuous cropping
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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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/34—Aspergillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
- A01M17/00—Apparatus for the destruction of vermin in soil or in foodstuffs
- A01M17/002—Injection of toxic gases or fluids into the soil
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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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
Abstract
The invention discloses a soil bactericide, which is prepared by the following steps: (1) Adding water into fresh earthworms for homogenate to prepare tissue homogenate, and sterilizing the tissue homogenate; (2) Inoculating the tissue homogenate sterilized in the step (1) into zymophyte liquid for fermentation to obtain the soil bactericide. The soil bactericide disclosed by the invention is green and environment-friendly, has a good bacteriostatic effect, and can be applied to prevention and control of continuous cropping obstacles of apples and pears, so that harmful fungi can be effectively inhibited and killed, the microbial environment of continuous cropping soil can be optimized, and the effect of prevention and control of continuous cropping obstacles can be achieved.
Description
Technical Field
The invention relates to the technical field of agricultural cultivation, in particular to a soil bactericide for preventing and controlling continuous cropping obstacles of apples or pears and application thereof.
Background
The continuous cropping obstacles of apple trees, pear trees and other fruit trees are represented by the phenomena of low survival rate, weak tree vigor, serious pest and disease damage, low yield, poor quality and great loss. Research results show that harmful microorganisms are the main factors causing continuous cropping obstacle of apples and pears.
The soil additive (bactericide) is one of effective measures for relieving continuous cropping obstacles of fruit trees, and the commonly used soil additives mainly comprise a chemical fumigant, a biological fumigant, bacterial manure and the like, but different soil additives have certain limitations. Common chemical fumigants such as methyl bromide, lime nitrogen, bitter chloride and the like have good effect of preventing and treating the stubble obstacle, but have the problems of high cost, environmental harm, human health and the like. Biological fumigants such as mustard seed meal, marigold and farmyard manure utilize gases with killing effect generated during self decomposition to inhibit or kill microorganisms in continuous cropping soil, so that the harm of harmful microorganisms to plants is reduced. The bacterial manure is another common soil additive, is developed by matching beneficial microorganisms with antagonistic and killing effects on harmful fungi in continuous cropping obstacles with a carrier through culture and fermentation, can effectively inhibit the growth of the harmful microorganisms in a green manner, improves the continuous cropping soil environment, reduces the harm caused by the continuous cropping obstacles, and has the defects of unstable effect and the like. Therefore, the soil bactericide which is green, stable, effective, economical and easy to popularize has important significance for preventing and controlling continuous cropping obstacles of fruit trees.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a soil bactericide for preventing and controlling continuous cropping obstacles of apples or pears and application thereof.
In order to realize the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a soil fungicide is provided, which is prepared by the following steps:
(1) Adding water into fresh earthworms for homogenate to prepare tissue homogenate, and sterilizing the tissue homogenate;
(2) Inoculating the tissue homogenate sterilized in the step (1) into zymophyte liquid for fermentation to obtain the soil bactericide.
Preferably, in the step (1), the mass ratio of the fresh earthworms to the water is (3-6): 1.
Preferably, in step (1), the homogenization is carried out in a tissue homogenizer.
Preferably, in the step (1), the sterilization is pasteurization, the sterilization temperature is 70 ℃, and the sterilization time is 30min.
Preferably, in the step (2), the zymocyte liquid is bacillus subtilis and aspergillus niger, and the adding mass ratio of the zymocyte liquid to the tissue homogenate is 1.
Preferably, the mass ratio of the bacillus subtilis to the aspergillus niger is 1.
Preferably, the effective viable count of Bacillus subtilis is 1X 10 9 CFU/ml, spore concentration of Aspergillus niger of 1.2X 10 9 CFU/ml。
Preferably, in the step (2), the fermentation is aerobic fermentation, the fermentation temperature is 35-38 ℃, and the fermentation time is 8-12 days.
In a second aspect of the invention, the application of the soil bactericide in preventing and controlling apple or pear continuous cropping obstacles is provided.
In a third aspect of the present invention, there is provided a method for preventing and controlling continuous cropping obstacles of apples or pears, comprising the following steps: digging planting furrows in the continuous cropping soil of the apples or the pears, digging planting holes in the planting furrows, applying the soil bactericide into the planting holes, and planting the young trees after 4-6 days.
Preferably, the soil bactericide is diluted by adding water and then applied to the planting holes, wherein the volume ratio of the soil bactericide to the water is 100ml: (8-9) L.
Preferably, the planting holes are 40cm square.
The invention has the beneficial effects that:
1. according to the invention, after the earthworms are homogenized, the earthworms are disinfected and inoculated with fermentation strains, the fermentation strains are fully fermented under specified conditions to form a fermentation product rich in a large amount of antibacterial peptides, the fermentation product is used as a soil bactericide and is applied to soil in a planting hole when a garden is built in continuous cropping, so that the effects of effectively inhibiting and killing harmful fungi, optimizing the microbial environment of the continuous cropping soil and preventing and controlling continuous cropping obstacles of apples and pears are achieved.
2. The soil bactericide is green and environment-friendly, has good bacteriostatic effect, and is a novel preparation for preventing and controlling continuous cropping obstacles of apples and pears, which is green, effective, simple and easy to popularize; the soil bactericide is rich in antibacterial small peptides and has a good antibacterial effect.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, currently, commonly used soil bactericides mainly comprise chemical fumigants, biological fumigants, bacterial fertilizers and the like, the commonly used chemical fumigants have a good effect of preventing and treating the continuous cropping obstacles, but have the problems of high cost, environmental harm, human health and the like, the biological fumigants are green and pollution-free green prevention and treatment technologies which do not cause damage to the environment, but generally have the defects of slow effect taking, incomplete effect and the like, and the bacterial fertilizers can effectively inhibit the growth of harmful microorganisms in a green manner, improve the continuous cropping soil environment, reduce the damage caused by the continuous cropping obstacles, but also have the defects of easy influence of environmental factors, unstable colonization of beneficial bacteria and the like. At present, no soil bactericide which is green and environment-friendly, has a good bacteriostatic effect and good stability exists.
Based on the above, the invention discloses a soil bactericide which is green and environment-friendly, has good bacteriostatic effect and good stability, earthworms are homogenized, sterilized and inoculated with fermentation strains, the fermentation strains are fully fermented under specified conditions to form a fermentation product rich in a large amount of antibacterial peptides, the fermentation product is used as the soil bactericide, and the soil bactericide is applied to soil of a fixed planting hole when a garden is built by continuous cropping, so that harmful fungi can be effectively inhibited and killed, the microbial environment of continuous cropping soil can be optimized, and the effect of preventing and controlling continuous cropping obstacles of apples and pears can be achieved.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example 1:
the preparation method of the soil bactericide comprises the following steps: (1) Adding water into fresh earthworms, and homogenizing in a tissue homogenizer to obtain tissue homogenate, wherein the mass ratio of the fresh earthworms to the water is 4; pasteurizing the homogenate;
(2) Inoculating the tissue homogenate sterilized in the step (1) into a zymophyte liquid for aerobic fermentation for 10 days at the temperature of 37 ℃, wherein the fermented product is the soil bactericide, the zymophyte liquid is bacillus subtilis and aspergillus niger, the adding mass ratio of the zymophyte liquid to the tissue homogenate is 1 9 CFU/ml, spore concentration of Aspergillus niger of 1.2X 10 9 CFU/ml。
Example 2:
the preparation method of the soil bactericide comprises the following steps: (1) Adding water into fresh earthworms, and homogenizing in a tissue homogenizer to obtain tissue homogenate, wherein the mass ratio of the fresh earthworms to the water is 3; pasteurizing the homogenate;
(2) Inoculating the tissue homogenate sterilized in the step (1) into a zymophyte liquid for aerobic fermentation for 8 days at the temperature of 38 ℃, wherein the fermented product is the soil bactericide, the zymophyte liquid is bacillus subtilis and aspergillus niger, the adding mass ratio of the zymophyte liquid to the tissue homogenate is 1 9 CFU/ml, aspergillus niger spore concentration of 1.2X 10 9 CFU/ml。
Example 3:
the preparation method of the soil bactericide comprises the following steps: (1) Adding water into fresh earthworms, and homogenizing in a tissue homogenizer to obtain tissue homogenate, wherein the mass ratio of the fresh earthworms to the water is 6; pasteurizing the homogenate;
(2) Inoculating the tissue homogenate sterilized in the step (1) into a zymophyte liquid for aerobic fermentation for 12 days at the temperature of 35 ℃, wherein the fermented product is the soil bactericide, the zymophyte liquid is bacillus subtilis and aspergillus niger, the adding mass ratio of the zymophyte liquid to the tissue homogenate is 1 9 CFU/ml, aspergillus niger spore concentration of 1.2X 10 9 CFU/ml。
The Bacillus subtilis (Bacillus subtilis) is purchased from China general microbiological culture collection center, and has the following numbering: 1.14985; aspergillus niger derived from China center for the Collection of common microorganisms, accession number: 3.15297.
the bacillus subtilis widely exists in nature and is a nontoxic, harmless and pollution-free strain; meanwhile, active substances such as subtilin, polymyxin, gramicidin and the like generated by the bacillus subtilis in the growth process can effectively inhibit the activity of pathogenic bacteria, and the bacillus subtilis can synthesize various enzymes such as alpha-amylase, protease, cellulase and the like, thereby playing a key role in fermenting earthworm tissue homogenate. Aspergillus niger is a commonly used strain in fermentation, and can produce amylase, acid protease, cellulase, pectinase and glucose oxidase.
1. Determination of effective components of soil bactericide:
and (3) filtering and centrifuging the fermented product, further purifying, and detecting by mass spectrometry to find that the earthworm fermented product is rich in a large amount of earthworm antibacterial small peptides, wherein the main component of the earthworm antibacterial small peptides is earthworm antibacterial peptides.
The experimental steps are as follows:
(1) And (3) taking 2000mL of earthworm fermentation product, centrifuging for 10min at a speed of 5000 r/min, and taking supernatant to obtain crude extract of the earthworm fermentation product antibacterial peptide.
(2) And (3) activation: add 200. Mu.L of ACN, centrifuge for 2min at 3000rpm and repeat the procedure once.
(3) Balancing: adding 200. Mu.L of 50-% ACN-50-0.1-% TFA, centrifuging at 3000rpm for 2min, and repeating the operation once. Add 200. Mu.L of 0.1% TFA solution, centrifuge at 3000rpm for 2min, and repeat the procedure once.
(4) Loading: transferring the sample solution to a molecular sieve chromatography column (Sephadex G-25, eluent: 0.033M acetic acid), and collecting the eluent
(5) Desalting: add 200. Mu.L of 0.1% TFA solution and centrifuge at 3000rpm for 2min.
(6) And (3) elution: adding 100 μ L of 80-vol ACN-20-vol TFA solution, centrifuging at 3000rpm for 2min, and collecting the supernatant to be tested.
(7) Mass spectrometry analysis (column information: 300um i.d.x. 5mm, packed with Acclaiim PepMap RSLC C18,5um,
NanoViper Acclaim PepMap 75um X150mm, C18,3um,100A; a mobile phase A:0.1% formic acid; mobile phase B:0.1% formic acid, 80% acn; flow rate: 300 nL/min), the tested solution contains a plurality of amino acid fragments with different sequences, and some of the amino acid fragments have good bactericidal action.
2. Indoor bacteriostasis verification of the soil bactericide:
(1) The soil fungicide obtained in example 1, the homogenate obtained in example 1, and sterile water were added to the PDA medium that had not solidified after sterilization, and the mixture was mixed well, poured into a petri dish (dish diameter 9 cm), and allowed to stand for solidification.
(2) And (3) beating the four cultured Fusarium oxysporum strains of the continuous cropping obstacle of apple under test, namely the four cultured Begonia reticulata, the rotten skin, the conidia and the stratified Fusarium oxysporum strains with the diameter of 0.5cm by using a puncher with the diameter of 0.5cm, inoculating the fungus cakes into the culture medium, and culturing at the constant temperature of 28 ℃.
(3) After 7 days the colony diameter was measured and the inhibition of harmful bacteria by different treatment conditions was observed and recorded.
(4) When the culture dish was covered with the control group pathogenic bacteria colonies to which sterile water was added, the diameters of the pathogenic bacteria colonies of the two treatment groups of the soil bactericide (fermentation product in table 1) and the tissue homogenate were measured and recorded, and the inhibition ratios (%) = [ (control colony diameter-treatment colony diameter)/control colony diameter ] × 100% were calculated, respectively.
TABLE 1 inhibition of the fermentation products against four harmful fungi with continuous cropping obstacles
As can be seen from Table 1, the inhibition rates of the earthworm fermentation products on the four main harmful fungi of conidium, layer emergence, rotten skin and beading in the apple continuous cropping obstacle reach 83.81%, 84.03%, 83.79% and 86.02% respectively.
3. Field verification of the soil bactericide:
when the apple orchard is renewed and rebuilt, a planting groove is dug on the continuous cropping soil of the apples, a planting hole with the square of 40cm is dug in the planting groove, 300ml of the soil bactericide (fermentation product in table 2) prepared in the embodiment 1 is diluted by adding 50 jin of water and then is applied to the planting hole, and apple saplings are planted after 5 days to serve as an experimental group. Meanwhile, a control group (CK) and sterilized soil treatment are set, sterile water with the same amount as the soil bactericide is applied to the control group (CK) in the planting holes, and other conditions are the same as those of the experimental group. The results are shown in tables 2 and 3.
As can be seen from Table 2, after one year of normal water and fertilizer management, the plant height, diameter and branch development of the young apple trees in the experimental group are respectively improved by 18.5%, 18.0% and 74.1% compared with those in the control group.
TABLE 2 Effect of fermentation products on growth of apple saplings under continuous cropping conditions
TABLE 3 Effect of fermentation products on harmful fungi
As can be seen from Table 3, after qPCR detection, the number of four harmful bacteria, namely beading, rotten skin, conidia and stratifying, in the continuous cropping soil is reduced by 49.8%, 56.9%, 42.4% and 56.9% compared with the control group after the soil bactericide is applied.
Therefore, the antibacterial peptide in the soil bactericide can inhibit the growth of harmful fungi in continuous cropping soil, improve the microbial environment of the continuous cropping soil and reduce the harm of continuous cropping to apple saplings.
Application example
From 3 months in 2017, field verification was performed on Dashaling village (37.40 degrees N120.09 degrees E), feng Maozilancura (37.41 degrees N120.07 degrees E) and gulf head village (37.09 degrees N119.80 degrees E) in Laizhou city, a Yantai city, shandong province, and the textures of the three soils were sandy loam, loam and clay loam respectively. The apple seedlings for the test are 2-year-old grafted seedlings, the combination of the stock and the scion is 3/T337 rich in tobacco, the stem thickness of the grafted seedlings is about 10mm, the stem is set to 1.4m, and the row spacing of the planted seedlings is 1.5m multiplied by 4m. During garden construction, planting furrows are dug in the apple continuous cropping soil, planting holes of 40cm square are dug in the planting furrows, 300ml of earthworm fermentation products (soil bactericide prepared in example 1) are added with 50 jin of water to be diluted and then applied to the planting holes, apple saplings are planted after 5 days, the apple saplings are used as experimental groups, and 300ml of sterile water is applied to 50 jin of water as a control group. And (5) performing normal field water and fertilizer management, and periodically measuring the growth vigor of the apple saplings. The results are shown in Table 4.
The results show that: the continuous cropping obstacle control effect of the method in the continuous cropping orchard with three different soil textures of Shandong tobacco platforms is proved, the method for controlling the continuous cropping obstacle of the apples can effectively improve the survival rate of young apple trees under the continuous cropping condition, obviously improve tree vigor, improve yield and effectively overcome the continuous cropping obstacle of the apples.
TABLE 4 growth vigor of fruit trees after fermentation product application
In Table 4, CK is the control group and FP is the experimental group.
By utilizing the product and the same technical method, the successive cropping obstacle prevention and control verification of pears in Laiyang Shandong, guangxian county, taian san and the like are respectively carried out, and good effects are also obtained.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (5)
1. The soil bactericide is characterized by being prepared by the following steps:
(1) Adding water into fresh earthworms for homogenate to prepare tissue homogenate, and sterilizing the tissue homogenate;
(2) Inoculating the tissue homogenate sterilized in the step (1) into zymophyte liquid for fermentation to prepare a soil bactericide;
in the step (1), the mass ratio of the fresh earthworms to the water is (3-6) to 1;
in the step (2), the zymocyte liquid is bacillus subtilis and aspergillus niger, and the adding mass ratio of the zymocyte liquid to the tissue homogenate is 1; the mass ratio of the bacillus subtilis to the aspergillus niger is 1; the bacillus subtilis is purchased from China general microbiological culture collection center, and is numbered as follows: 1.14985; the aspergillus niger is from China center for general microbiological preservation management, and the numbering is as follows: 3.15297;
in the step (2), the fermentation is aerobic fermentation, the fermentation temperature is 35-38 ℃, and the fermentation time is 8-12 days.
2. The soil fungicide according to claim 1, wherein: in the step (1), pasteurization is adopted for sterilization.
3. The soil fungicide according to claim 2, wherein: the effective viable count of Bacillus subtilis is 1 × 10 9 CFU/ml, aspergillus niger spore concentration of 1.2X 10 9 CFU/ml。
4. Use of a soil fungicide according to any one of claims 1 to 3 for the prevention and control of apple or pear continuous cropping disorders.
5. A method for preventing and controlling continuous cropping obstacles of apples or pears is characterized by comprising the following steps: digging a planting ditch on the continuous cropping soil of the apples or the pears, digging a planting hole in the planting ditch, applying the soil bactericide of any one of claims 1 to 3 into the planting hole, and planting the apples or the pears after 4 to 6 days;
diluting the soil bactericide with water and then applying the soil bactericide to the planting holes, wherein the volume ratio of the soil bactericide to the water is 100ml: (8-9) L.
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| CN104130078A (en) * | 2014-08-26 | 2014-11-05 | 姜汉军 | Bio-organic fertilizer for continuously cropping watermelons |
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| JP2019043827A (en) * | 2017-09-06 | 2019-03-22 | 日本製紙株式会社 | Micro-organism material and method of producing the same |
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| CN101870608B (en) * | 2010-06-21 | 2011-12-14 | 山东宝源生物有限公司 | Continuous cropping resisting biological organic and inorganic compound fertilizer and production method thereof |
| WO2012064096A2 (en) * | 2010-11-09 | 2012-05-18 | 한국생명공학연구원 | Novel strain of bacillus subtilis, and use thereof for preventing root rot disease in plants |
| CN102965314B (en) * | 2012-11-19 | 2014-03-05 | 山东农业大学 | Bacillus subtilis and preparation and application of microbial inoculum thereof |
| CN104447127A (en) * | 2014-12-17 | 2015-03-25 | 成都新朝阳作物科学有限公司 | Soil conditioner for resisting pseudo-ginseng continuous cropping diseases as well as preparation and application thereof |
| CN104788263A (en) * | 2015-04-16 | 2015-07-22 | 王龙潮 | Composite soil conditioner |
| CN106242829A (en) * | 2016-08-26 | 2016-12-21 | 尹求实 | A kind of overcome the soil remediation fertilizer of Radix Salviae Miltiorrhizae continuous cropping obstacle, preparation method and its usage |
| CN106278654A (en) * | 2016-08-26 | 2017-01-04 | 尹求实 | A kind of overcome the soil remediation fertilizer of Rhizoma Zingiberis Recens continuous cropping obstacle, preparation method and its usage |
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| CN110734875B (en) * | 2019-10-25 | 2021-08-20 | 山东康地恩生物科技有限公司 | Bacillus subtilis for preventing and treating plant diseases and application thereof |
| CN111758743A (en) * | 2020-07-10 | 2020-10-13 | 山东农业大学 | Soil bactericide for preventing and controlling apple or pear continuous cropping obstacle and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104130078A (en) * | 2014-08-26 | 2014-11-05 | 姜汉军 | Bio-organic fertilizer for continuously cropping watermelons |
| CN106614840A (en) * | 2016-12-11 | 2017-05-10 | 戴琪 | Method for preparing soil disinfector by using earthworm hydrolysate |
| JP2019043827A (en) * | 2017-09-06 | 2019-03-22 | 日本製紙株式会社 | Micro-organism material and method of producing the same |
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