CN102972192B - Method for improving pathogen-inhibiting and disease-controlling pesticide effect of rapeseed meal by applying myrosinase preparation in field - Google Patents
Method for improving pathogen-inhibiting and disease-controlling pesticide effect of rapeseed meal by applying myrosinase preparation in field Download PDFInfo
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Abstract
The invention relates to a method for improving the pathogen-inhibiting and disease-controlling pesticide effect of rapeseed meal by applying a myrosinase preparation in the field. Glucosinolates in the rapeseed meal is rapidly degraded by adopting the myrosinase preparation for releasing butyl isosulfocyanate and further develop the pathogen-inhibiting and disease-controlling pesticide effect. The method is characterized by comprising the steps of: removing residue generated by previous crops in the field at a soil temperature of above 15 DEG C, carrying out rotary tillage for 20-25cm after a base fertilizer is applied to soil, uniformly spreading the rapeseed meal into the field, wherein the consumption of the rapeseed meal is 200-400kg/mu; uniformly spreading the myrosinase preparation in the field, wherein the consumption of the myrosinase preparation is 1-1.5kg/mu; carrying out rotary tillage and watering on the soil to ensure that the content of the water in the soil is 60-70 percent of the maximum water-holding capacity in the field, covering a plastic film, and coating for 15-20 days at a soil temperature of higher than 25 DEG C, coating for 20-30 days at a soil temperature of lower than 25 DEG C, and then removing the film for ventilating for 2-3 days so that the pathogen-inhibiting and disease-controlling pesticide effect of the rapeseed meal is improved.
Description
Technical field
The present invention relates to a kind of method that myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect of using in field.Belong to agricultural wastes comprehensive utilization field.
Background technology
Rape is the large oil crop in third place in the world, and vegetable seed output is only second to soybean and cottonseed, is one of most important oil crop.Since 1978, oneself is sure to occupy No. 1 in the world China's rapeseed cultivation area and output, reaches 26% of 7,000,000 hectares of ，Zhan world rape seed areas, can produce the dish dregs of rice of approximately 1,000 ten thousand tons.In the dish dregs of rice, protein content is up to 40-45%, it is a kind of good protein raw materials, there is the application potential as feed protein exploitation, but its adding proportion generally can not be higher than 15-20%, because the Secondary Metabolism of Plant product glucosinolate (GSLs) containing in the dish dregs of rice can decompose noxious material and other antinutritional factor such as producing cyanogen, oxazolidine thioketones (OZT), isothiocyanates (ITCs) under the effect of myrosase, affect growing of animal, greatly affected the feeding value of rapeseed dregs.The dish dregs of rice can be feeding after detoxification, but detoxification process is complicated, and cost is higher, has " three wastes " to pollute, and Technique Popularizing still faces very large difficulty.Therefore, the current feeding dish dregs of rice are less than 1/3rd of total amount.
The dish dregs of rice are except containing high-quality nitrogen, and the content of phosphorus, potassium and sulphur also reaches 1.2-1.5% respectively, are a kind of fertilizers of high-quality.The dish dregs of rice are used and have long history as organic manure.A large amount of data show, use the dish dregs of rice can improve soil organic matter content and soil fertility in the new soil of opening up wasteland, and Study on Fertilizing soil, improves output; And the arable land of worsening at soil quality is used dish dregs of rice class fertilizer can significantly improve the physico-chemical and biological properties of soil, play rehabilitating soil and the effect that overcomes soil constraint factor, the consumption that simultaneously reduces chemical fertilizer and agricultural chemicals, improves quality of agricultural product.
Although the application effect of dish dregs of rice as fertilizer sources is generally approved, yet another function that the dish dregs of rice have-" drug effect " is rarely known by the people, in soil, role is also brought into play far away.At the dish dregs of rice, during as feed applications, the main catabolite isothiocyanates of the glucosinolate containing in the dish dregs of rice has toxic action to animal, is to limit its feeding disadvantage factor; Yet, the dish dregs of rice are applied in soil, glucosinolate decomposes the isothiocyanates producing and but has broad-spectrum sterilization activity, the pests such as the nematode in soil, bacterium, fungi are had to the very strong effect of killing livestock, can be used for the control of crops soil-borne disease insect pest, so the dish dregs of rice are a kind of good " the fertile economic benefits and social benefits of medicine " living resources.But after the dish dregs of rice are manured into soil, glucosinolate decomposes generation isothiocyanates and causal organism is played to lethal effect, must possess certain application technique, meets corresponding soil management condition.
The dish dregs of rice are general designations of the rape dregs of rice and the leaf mustard dregs of rice.Although China is at research and extension double-low rapeseed energetically, but to so far, due to the poor-performing of the adverse circumstances such as double-low rapeseed disease and insect resistance, low temperature, arid, normal rapeseed plantation still be take as main in western part and the middle part of the ， China of some areas, Ye Jin middle and lower reach of Yangtze River popularization at present; Simultaneously, it is that the plantation of object leaf mustard is extensively risen that the fast development of world wide endogenous substance energy industry makes to manufacture biological material oil, a large amount of leaf mustard dregs of rice will produce thereupon, and in the leaf mustard dregs of rice content of glucosinolate far above the rape dregs of rice, feeding grade of fit is less, and it is disposed and utilizes and will face a larger difficult problem.Therefore the predicament that, the most dish dregs of rice are still faced with rationally, efficiently utilize within a very long time in future.On the other hand, China's arable area constantly dwindles, and fast development and the improving constantly of living standards of the people of society make people increasingly increase the demand of agricultural product quantity and kind, the advantage of high-efficiency agriculture advances and makes modern plant high speed development in China, the large loose water of facility cultivation and high cropping index make soil all the year round in high loaded process state, the physics of soil, chemistry and biology proterties sharply worsens, soil quality significantly declines, soil-borne disease insect pest generating plane is long-pending constantly to be increased with degree, seriously restricted greatly developing of modern plant, active demand is new, eco-friendly soil melioration and reparation product and technology.Therefore, not only there is good effect of fertilizer but also can multiple soil-borne disease bacterium had and be killed or inhibition, the Cruciferae brassica plant that can effectively prevent and kill off soil-borne disease and grouts thereof become that association area is paid close attention to and the focus of research again.The countries such as America and Europe have been developed as business-like pesticide-fertilizer product by leaf mustard green manure and the leaf mustard dregs of rice, in the soil-borne disease controls such as root-knot nematode, epidemic disease and blight of the crops such as vegetables, flowers, fruit tree, obtain good result, show huge application potential.
About coordinate as fertilizer sources to use the patent report of prevention and elimination of disease and pests to have with other compositions with the dish dregs of rice or the dish dregs of rice: application (patent) number: 93115650.5, by capsicum resin, rare earth chloride, colza cake or cottonseed cake, zinc sulphate, hydrochloric acid, made according to a certain ratio.The present invention has the function of desinsection, sterilization and promotion plant production simultaneously.Application (patent) number: 200610128495.1: the invention discloses a kind of composite organic modifier of preventing and treating root knot nematode disease and preparation method thereof, after specifically colza cake, loose sawdust wherein in the above colza cake, loose sawdust, three kinds of compositions of castor bean leaf fully being become thoroughly decomposed, dried and crushed is standby; Application (patent) number: 201010130961.6, a kind of composite microbiological fertilizer and preparation method thereof.It is characterized in that it is prepared from according to a certain percentage by rape colza cake, crops straw, mycothallus, bacterium thalline and water, the present invention, by 2 solid fermentation methods, mixes fungus and bacterium.Other be take the dish dregs of rice as primary raw material or are the patent of batching and the composite production fertilizer of other fertilizer in addition.
Comprehensively above material can be found out, all publications are all the fertilizer that the dish dregs of rice and chemical fertilizer, organic manure or the composite acquisition of other nutrient components have growth promotion, the effect of diseases prevention worm.Although also studies have reported that and use the generation that the dish dregs of rice or its composite fertilizer can alleviate various crop damage by disease and insect, but the fertilizer efficiency that is finally all summed up as the dish dregs of rice has promoted plant growth and has improved the disease resistance of crop itself, and all relate to, does not utilize the glucosinolate containing in the dish dregs of rice to decompose the isothiocyanates producing and play the prevention and control effect to disease.
Recently research finds, the percent hydrolysis of glucosinolate in the dish dregs of rice (GSLs) is low is to cause the main cause that isothiocyanates in soil (ITCs) concentration is low, affect bactericidal effect.The factor that affects GSLs hydrolysis has dish dregs of rice degree of crushing, myrosase activity, soil temperature, moisture etc.Morra strengthens material fragmentation degree and increases soil water content under laboratory condition, and the percent hydrolysis of GSLs is brought up under the state of 30%，Er field by 15%, and GSLs percent hydrolysis is still lower than 15%.Under the state of Gimsing Deng field, adopt the bi-material of high and low GSLs content, optimize material fragmentation degree and soil water content, the concentration of GSLs and ITCs in detection soil, discovery was through 12 days, the percent hydrolysis of bi-material GSLs still only has 26% and 56%, and most GSLs is not still hydrolyzed, but in the percent hydrolysis of research discovery GSLs and soil, the activity of myrosase is proportionate.The plant tissue that contains GSLs (comprising the dish dregs of rice) contains myrosase simultaneously, in complete plant cell, the two is distributed in the different parts of cell, and when plant is subject to attack of insect, mechanical damage or pathogen infection, myrosase will be released, and catalysis GSLs hydrolysis produces living killing compound.And a large amount of research finds that myrosase that the dish dregs of rice itself contain is owing to being subject to the impact of the factors such as its content, activity can not meet the demand that GSLs is hydrolyzed at short notice in a large number, by external source, add myrosase and can significantly promote GSLs to be hydrolyzed in a large number at short notice, to discharge ITCs to strengthen Biofumigation effect.Omirou etc. are under indoor condition of culture, in 1kg soil, (contain the 1.5g dish dregs of rice) and add the plant resource myrosase preparation that active unit is 3U, make the GSLs percent hydrolysis in 3 days in the dish dregs of rice reach 100%, in soil, the concentration of ITCs is 5-6 times that contrasts, and contrast GSLs percent hydrolysis after 8 days is still 40%.Visible, by external source, add the degradation rate that myrosase can significantly improve GSLs and strengthen bactericidal effect.The myrosase that more than experiment is used is plant resource myrosase, and experimentation only limits in laboratory, all the practical problem of unresolved field application.
At present commercially available myrosase all extracts and obtains from Cruciferae brassica plant, and extraction step is complicated, and cost is higher, causes myrosase expensive.First, plant material source difficulty,, intensive degree Gao countries and regions nervous at arable area, it is very large that 2-3 month kind plant of cost is used for extracting myrosase difficulty; Secondly, the factors such as plant growth region, season and weather directly affect the growing amount of its body endoenzyme, and output and quality that enzyme is extracted are all unstable; In addition, the offcuts that produce after complicated extraction process and extraction myrosase are disposed the important bottleneck that difficulty is also restriction the method.And utilizing microorganism fermenting and producing myrosase can meet any scale, demand any time, productive rate is high, steady quality, has huge advantage.
About microbial source myrosase, carried out following research: Ohtsuru screening abroad and obtained the Aspergillus niger strain that can produce myrosase in born of the same parents, and carried out separation and purification, the enzymatic property of enzyme and affect the factor research of enzymic activity, but specifically do not applied; Tani to enterobacter cloacae (
enterobacter cloacae) myrosase that produces carried out separation and purification and enzymatic property research.Palop report quick lactic acid bacteria (
lactobacillus agilis) by producing myrosase, promote GSL to decompose generation methyl ITC.Rakariyatham successively screens and obtains the aspergillus of many strains product myrosases and the fungi that Fusarium oxysporum belongs to, and by ultraviolet mutagenesis, obtains the superior strain that myrosase output improves 3.5 times.Ishimoto is separated to a strain sickle-like bacteria bacterial strain Ls-F-in-4-1(
fusariumspp), the myrosase that this bacterial strain produces decomposes GSLs to produce benzyl ITCs, thereby effectively prevention and control are by Pythium ultimum
( pythium ultimum) generation of the pea soil-borne disease that causes.Domestic only have the researchs such as Wu Hongwei from aspergillus oryzae culture, to be separated to myrosase, but further do not study report.Applicant in March, 2012 15Xiang State Intellectual Property Office submitted to name to be called and to produce the dark green trichoderma strain of myrosase and the application for a patent for invention of application thereof, application number is 201210068201.6, mention first by dark green trichoderma strain and extract myrosase, at present, the myrosase that does not also utilize dark green trichoderma to produce is both at home and abroad applied to the report that dish dregs of rice class fertilizer bacteriostatic and disease prevention function is improved in field.
summary of the invention
The object of the invention is to: on the one hand, in the dish dregs of rice of annual millions of tons, 1/3 the dish dregs of rice of only having an appointment add in feed with lower ratio after treatment, a large amount of dish dregs of rice dish dregs of rice that especially glucosinolate content is high do not have that technology can operate, the economically viable approach that utilizes so far, most of dish dregs of rice still apply in soil as common fertilizer is extensive, are far from bringing into play its due effect.Under the state of ，Cai Po field, GSLs percent hydrolysis is still low on the other hand, does not give full play to its series of problems such as effect of killing livestock to pest in soil, has proposed a kind of method that improves dish dregs of rice bacteriostatic and disease prevention drug effect in field by myrosase preparation.
The object of the present invention is achieved like this: a kind of method that improves dish dregs of rice bacteriostatic and disease prevention drug effect in field by myrosase preparation, to promote glucosinolate fast degradation in the dish dregs of rice by myrosase preparation, discharge the drug effect of isothiocyanates and then performance bacteriostatic and disease prevention, it is characterized in that: soil temperature is more than 15 ℃, the residual body that preceding crop in field is produced is removed clean, soil has been executed rotary tillage 20-25 centimetre after base fertilizer, the dish dregs of rice are evenly spilled into field, dish dregs of rice consumption is 200-400 kg/acre, again myrosase preparation is evenly spilled into field, myrosase preparation consumption is 1-1.5 kg/acre, again by soil rotary tillage, water, making soil water content is the 60-70% of maxmun field capacity, and covered with plastic film, soil temperature is during higher than 25 ℃, the overlay film duration is 15-20 days, soil temperature is during lower than 25 ℃, the overlay film duration is 20-30 days, then take off film ventilation 2-3 days, can improve dish dregs of rice bacteriostatic and disease prevention drug effect.
In the present invention, the glucosinolate content of the dish dregs of rice used is at least 40 μ mol/g, and in myrosase preparation, myrosase activity is 100-200U/g.
In the present invention, described myrosase preparation is cultivated and is obtained by dark green trichoderma liquid pure, concrete grammar is for to carry out pure culture to dark green trichoderma, obtain liquid pure culture, liquid pure culture is crossed to four layers of gauze, collect the mycelia on gauze, mycelia is standing after ultrasonic disruption, centrifugal, collect supernatant and obtain myrosase liquid, by myrosase liquid with after carrier adsorption, naturally dry or 30 ℃ of oven dry after be myrosase preparation.
In the present invention, to carry out pure culture to dark green trichoderma, obtaining liquid pure culture refers to: dark green trichoderma is inoculated into and in test tube slant medium, carries out activation culture, the cultured inoculation in Zai Jiang inclined-plane is 250ml and is equipped with in the triangular flask of 80ml liquid seed culture medium to corresponding capacity, triangular flask is placed in to rotating speed 160-180r/min, temperature is to cultivate 24-36 hour on the shaking table of 28 ± 2 ℃, and then take the inoculum concentration of volume ratio 5-10% and be linked into respectively capacity as 2000ml and be equipped with in the large triangular flask of 800ml liquid production medium, large triangular flask is placed in to rotating speed 180r/min, temperature is to cultivate 72-96 hour on the shaking table of 28 ± 2 ℃, when just starting to occur yellow color, finishes liquid culture to cultivate, obtain dark green trichoderma liquid pure culture,
Described test tube slant medium is PDA medium, and compound method is: 200 grams of potatos, 20 grams of glucose or sucrose, agar 15-20 gram, 1000 milliliters, water, pH nature.Peeling potatoes, is cut into piece and boils half an hour, then uses 2 layers of filtered through gauze, then sugaring and agar, after dissolving, supplies water to 1000 milliliter, 121 ℃ of autoclaving 20min;
Described liquid seed culture medium and liquid production medium are: oat 20 g, FeSO
47H
2o 0.001g, MnCl
24H
2o 0.001g, ZnSO
47H
2o 0.001g, is dissolved in 1000 ml waters, 121 ℃ of autoclaving 25min.
In the present invention, mycelia is standing after ultrasonic disruption, centrifugal, collection supernatant obtains myrosase liquid and refers to: the mycelia of collecting from gauze is evenly mixed with weight ratio 1:5 with the phosphate buffer of PH7.0, at power, it is in 200W ultrasonic disruption instrument broken 10 minutes, then the good mycelia liquid of fragmentation being placed in to 4 ℃ of refrigerators places 1 hour, then at 4 ℃, centrifugal 15 min of 4000 r/min, get centrifugal rear supernatant and are myrosase liquid;
The phosphate buffer of PH7.0 refers to: sodium chloride 8g, and potassium chloride 0.2g, potassium dihydrogen phosphate 0.24g, sodium hydrogen phosphate 3.58g is dissolved in 1000 ml waters;
In the present invention, by myrosase liquid, with carrier adsorption carrier used, be the peat composed of rotten mosses powder of 60 mesh sieves, this peat composed of rotten mosses powder is through 121 ℃, sterilizing was used after 40 minutes, the mixed proportion of myrosase liquid and peat composed of rotten mosses powder is volume ratio 1:8-1:10, mixed mixture natural air drying or 30 ℃ of rear mistake 40 mesh sieves of oven dry, obtain myrosase preparation.
The invention has the advantages that: first, that the present invention be take is common, the dish dregs of rice of easy acquisition are the body material using, by external source, add myrosase and adopt simple method regulation and control soil condition, in the creation promotion dish dregs of rice, glucosinolate fast degradation discharges the environmental condition of the gas of killing livestock, and reaches the object of killing or suppressing pest in soil; In addition, the method has not only improved sterilization, the diseases prevention effect of the dish dregs of rice, given full play to " drug effect " of the dish dregs of rice, and cover and suitable moisturizing process through soil, also the mineralization process of organic macromolecule material in the dish dregs of rice, degraded and the Nutrient Transformation of the promotion dish dregs of rice have been accelerated, improve its fertilizer validity, the fertility that strengthens soil has also increased quantity, activity and the bio-diversity of Soil Microorganism simultaneously, has also indirectly played the effect that suppresses pathogen growth.Whole application is simple, easy operating; After using, can significantly improve soil quality, balance soil nutrient and enrich the population structure of edaphon; the effect of performance to promoting crop growth, diseases prevention; thereby reduce the usage amount of chemical fertilizer, agricultural chemicals, for new thinking is opened up in the safety in production of resource circulation utilization, environmental protection and agricultural product.
We are to from Jiangsu, Shandong, Anhui, Jiangxi, Hubei, Sichuan, Shaanxi, the dish dregs of rice of more than ten province such as Gansu, carried out the growth inhibition effect of frequently seen plants pathogen and the control effect research to corps diseases, research finds that the more than 90% dish dregs of rice have the growth inhibition effect of 30-100% to the mycelia of pathogen and conidium or sclerotium, to capsicum, the control effect of the soil-borne diseases such as tomato can reach 60-100%, but make its performance diseases prevention effect, the dish dregs of rice particle size when dish dregs of rice are used, myrosase content, loosing soil situation, soil water content, soil temperature and soil covering situation etc. all there are certain requirements.
Test shows, after the dish dregs of rice are manured into soil, external source is added myrosase and also regulated soil to arrive suitable moisture, and then covered with plastic film maintains soil moisture, can significantly improve the growth inhibitory effect of the dish dregs of rice to pathogen.Not only opened up new way for the efficient utilization of a large amount of dish dregs of rice, realized the high-end of agricultural wastes and recycled, and the improvement of facility cultivation soil property, soil-borne disease prevention and control and the sustainable use of facility soil and the sustainable development of industrialized agriculture have been significant
Embodiment
Embodiment 1
The dark green trichoderma adopting in the present embodiment is that this case applicant is at the dark green trichoderma of Chinese microorganism strain preservation center registration preservation, its preserving number is: CGMCC NO.5609, this dark green trichoderma has been to public by 201210068201.6 Chinese invention patent application case.
The preparation of dark green trichoderma medium
The preparation of test tube slant medium: 200 grams of potatos, 20 grams of glucose or sucrose, agar 15-20 gram, 1000 milliliters, water, pH nature.Peeling potatoes, be cut into piece and boil half an hour, then use 2 layers of filtered through gauze, then sugaring and agar, heating is boiled to agar and is dissolved, supply volume to 1000 milliliter, be suitably cooled to 60 ℃ of left and right to divide and install in test tube, loading amount is the 1/5-1/6 of test tube height, with the good test tube mouth of test tube plug plug, 121 ℃ of autoclaving 25min, take out Hou Bai inclined-plane while hot to cooling standby, and bevel altitude is no more than 1/3 of test tube height.
The preparation of liquid seed culture medium and liquid production medium: take respectively oat 20 g, FeSO
47H
2o 0.001g, MnCl
24H
2o 0.001g, ZnSO
47H
2o 0.001g, is dissolved in 1000 ml waters, then packing, and liquid amount is 80ml/250ml, 800ml/2000ml triangular flask, with the good bottleneck of tampon plug, 121 ℃ of autoclaving 25min, cooling rear standby.
Embodiment 2:
The preparation of myrosase preparation
Dark green trichoderma preservation of bacteria strain in embodiment 1 is inoculated into and in test tube slant medium, carries out test tube slant activation, then, with oese, the bacterial classification having activated is inoculated in the 250ml triangular flask that 80ml liquid seed culture medium is housed, triangular flask is placed in to shaking table to be cultivated 24 hours, the rotating speed 160r/min of shaking table, cultivation temperature is 28 ± 2 ℃; After end, with 5% inoculum concentration (V/V) access, be equipped with in the 2000ml triangular flask of 800ml liquid production medium, triangular flask is placed in to shaking table to be cultivated, the rotating speed 180r/min of shaking table, cultivation temperature is 28 ± 2 ℃, cultivates and within 3-4 days, when liquid culture starts flavescence, can finish to cultivate; By liquid culture through four layers of filtered through gauze, collect the mycelia on gauze, mycelia is evenly mixed by 200W power ultrasonic fragmentation 10 minutes with the phosphate buffer one weight ratio 1:5 of PH7, place after 1 hour again at 4 ℃ for 4 ℃, centrifugal 15 minutes of 4000 r/min, collect supernatant and be myrosase liquid, the peat composed of rotten mosses powder that myrosase liquid is sterilized with crossing 60 mesh sieves and high temperature mixes with volume ratio 1:8-1:10, mixed mixture natural air drying or 30 ℃ of rear mistake 40 mesh sieves of oven dry, obtain myrosase preparation, in preparation, myrosase vigor is 100-200U/g.
Embodiment 3
The growth inhibition effect of the dish dregs of rice to plant pathogenic fungi mycelia and spore or sclerotium
The disease fungus of all participation tests can obtain from Chinese Academy of Sciences's microorganism fungus kind preservation center.The disease fungus of all participation tests is inoculated in PDA flat board, cultivates after 5 days standby with the mycelia piece of 4 millimeters of card punch punching acquisition diameters for 28-30 ℃.
The growth inhibition effect of volatile decomposition products to plant pathogenic fungi mycelia in the dish dregs of rice: pour PDA medium in culture dish (diameter 8.5cm, high 1.3cm), inoculating diameter while putting to normal temperature is 5 mm bacterium dish; Toward culture dish ware lid central authorities, add 0.2g, 0.4g, 0.6g, 0.8g, 1.0g, 1.2g crosses the dish dregs of rice powder of 2mm sieve, with the ratio of the dish dregs of rice: water=1:5, adds aqua sterilisa fully to mix, and contrast only adds aqua sterilisa 6mL, repeats 3 times.Then half culture dish of having inoculated microorganism is tipped upside down on culture dish lid, by sealed membrane sealing, 28 ℃ of constant temperature culture.After 12h, measure respectively the rotten mould colony diameter of melon and fruit, measure Rhizoctonia solani Kuhn colony diameter after 24h, measure the colony diameter of all the other four kinds of disease funguses after 72h, every processing repeats 3 times.Calculate the inhibiting rate of each compound to mycelial growth, inhibiting rate (%)=[(contrast bacterium colony increases diameter-processing bacterium colony and increases diameter)/(contrast bacterium colony increases diameter)] * 100%.
The growth inhibition effect of non-volatile catabolite to plant pathogenic fungi mycelia in the dish dregs of rice: take dish dregs of rice powder and put into 250mL triangular flask, add 100mL sterile water.Add respectively 2g, 6g, 10g, the 14g dish dregs of rice.In the test tube of contrast, only add sterile water.45 ℃ of vibration 12h, the centrifugal 20min of 12000r/min, getting supernatant is 0. 22 μ m filtering with microporous membrane in superclean bench Shang Yong aperture.Get in the PDA medium that 1mL filtrate adds 5mL40 ℃ of melting to and mix, be laid in the culture dish of diameter 5cm, during to normal temperature, inoculating diameter is the bacterium piece of 5mm, with sealed membrane sealing, is inverted for 28 ℃ and cultivates.Measure bacterial plaque diameter, calculate inhibiting rate, method is the same, and experimental result is in Table 1.
The inhibitory action of table 1 dish dregs of rice catabolite to the mycelial growth of plant pathogenic fungi
| Pathogen | EC 50V（g/L） | EC 50nv（g/L） |
| Cucumber Rhizoctonia solani Kuhn | 6.70±0.009 bc | 0.326±0.028 b |
| Withered germ of water-melon | 6.30±0.066 c | 0.145±0.005 b |
| Phytophthora blight of pepper | 7.63±0.316 ab | 7.171±0.261 a |
| Melon and fruit pythium spp | 8.78±0.720 a | 0.221±0.011 b |
| Dosporium cucumerinumand its | 4.76±0.357 d | 0.103±0.008 b |
| Sclerotinia sclerotiorum | 1.54±0.179 e | 0.068±0.007 b |
Note: EC
50Vdish dregs of rice volatile decomposition products suppresses the concentration of mycelial growth 50%, the g/L(of the unit space of volatilizing).EC
50 nvthe non-volatile catabolite of the dish dregs of rice suppresses the concentration of mycelial growth 50%, the g/L(of unit medium).Between different letter representation different disposal, there were significant differences (P=0. 05).
Result shows: the volatility of the dish dregs of rice and non-volatile catabolite are all inhibited to the mycelial growth of capsicum soil-borne pathogen, dish dregs of rice volatile decomposition products reaches significance level to the inhibitory action difference of 6 kinds of pathogens, but not volatile decomposition products is not remarkable to the inhibitory action difference of 6 kinds of pathogens, the most weak to the inhibitory action of phytophthora blight of pepper.And two large class catabolite be all the strongest to the inhibitory action of Sclerotinia sclerotiorum, be secondly to dosporium cucumerinumand its.By EC
50value can find out, the volatile materials that the dish dregs of rice produce is maximum to the growth inhibition effect of Sclerotinia sclerotiorum, is secondly to dosporium cucumerinumand its.
Inhibition to capsicum pathogen mycelial growth during the embodiment 4 dish dregs of rice are manured into soil
By the dish dregs of rice respectively with different amounts 0.2%(w/w), 0.25%(w/w), 0.3%(w/w), 0.4%(w/w) add in 800g fresh vegetable garden mould, after mixing, add distilled water, to keep soil water content be maximum water-holding capacity 70%.Vegetable garden soil is packed in the drier that bore is 15cm, and the hollow plastic tube that is 8mm by 3 diameters distribution triangular in shape is inserted in soil, supports the discoid platform in drier.Respectively by having inoculated the mycelia piece (5mm) of pathogen, PDA that diameter is 5.5cm dull and stereotyped (not covering culture dish ware lid) is placed on the platform in drier.The lid of drier and seam crossing are coated to vaseline to be sealed.Two contrasts are set: blank, vegetable garden soil is not with the dish dregs of rice; Chemical fumigation contrast, adds dazomet (i.e. 98% dazomet fine granule, consumption is 45kg/ha) in vegetable garden soil.After good seal drier, be positioned in 35 ℃ of incubators, open drier after 3d, take out culture dish, measure the diameter of bacterium colony, calculate inhibiting rate, computing formula is with embodiment 3, and experimental result is in Table 2.
The inhibitory action of the table 2 different amounts dish dregs of rice to phytopathogen mycelial growth
Result shows: after adding the dish dregs of rice in soil, capsicum pathogen mycelial growth is had to strong inhibitory action, and along with the increase of consumption, inhibition is more obvious.Under the condition of 400kg/ mu dish dregs of rice consumption, except being 78.18% to the rotten mould inhibiting rate of melon and fruit, the inhibiting rate of all the other 5 kinds of pathogens is all reached to 100%, reach identical effect with dazomet chemical fumigation.But different to the inhibitory action of different pathogens under identical dish dregs of rice consumption.
Embodiment 5
Under different soils moisture, use the growth inhibition effect of the dish dregs of rice to phytopathogen
In drier, simulate field state, the application process of the dish dregs of rice is with embodiment 4, dish dregs of rice consumption 0.25%(w/w), different soil moisture contents is set and is respectively 50%, 60%, 70%, 80%, 90% of maxmun field capacity, be positioned over after the incubator of 35 ℃ is cultivated 3d and measure colony diameter, calculate inhibiting rate, computing formula is with embodiment 3, and experimental result is in Table 3.
The impact of table 3 different soils moisture on dish dregs of rice bacteriostasis
The result of table 3 can draw, the comprehensive inhibition to 6 kinds of pathogens, can find out that the water capacity 70% delicacies of the season dregs of rice are the strongest to the inhibitory action of capsicum plant pathogen mycelial growth.Soil water content is lower than 70% time, and inhibitory action weakens along with the reduction of soil water content, and along with water capacity increases to 80%, 90%, the inhibitory action of mycelial growth is also reduced gradually.The suitable moisture of visible soil has larger synergistic effect for dish dregs of rice performance bacteria resistance function.
Embodiment uses in 6 fields myrosase preparation and the dish dregs of rice is suppressed to the enhancing effect of pathogen growth
Experiment is carried out in green house of vegetables, artificial infection Phytophthora capsici in plastic shed soil, and inoculum density is 1000 spores/gram soil, soil temperature 20-25 ℃, following processing is established in test: 1) contrast, do not use the dish dregs of rice and myrosase preparation; 2) only use myrosase preparation; 3) only use the dish dregs of rice; 4) the dish dregs of rice+mustard seed processing with enzyme preparation.400 kgs/acre of soil Chinese food dregs of rice consumptions, 1.5 kgs/acre of myrosase preparation (being provided by embodiment 2) consumptions, 20 square metres of each community areas, 3 repetitions of every processing, in experiment, the corresponding dish dregs of rice or myrosase preparation or the dish dregs of rice and myrosase preparation are spilled into field (control group does not spill), rotary tillage is watered, and making soil water content is 70% of maxmun field capacity.After soil covered with plastic film 20 days, take off film airing after 2 days, gather 30 centimetres of dark soil samples, measure the quantity of Phytophthora capsici in soil.Result is as table 4.
Table 4 adds myrosase preparation and the dish dregs of rice is suppressed to the enhancing effect of Phytophthora capsici
From table 4, in soil, add after myrosase preparation, the degradation rate of glucosinolate brings up to 86.2 from 30%, obviously; Add in the processing of myrosase, the quantity of pathogen be do not add that myrosase processes 1/3, showing that soil adds has significantly increased the growth inhibitory effect to Phytophthora capsici when having improved glucosinolate degradation rate after myrosase.
Claims (6)
1.Yi Zhong uses in field the method that myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect, to promote glucosinolate fast degradation in the dish dregs of rice by myrosase preparation, discharge the drug effect of isothiocyanates and then performance bacteriostatic and disease prevention, it is characterized in that: soil temperature is more than 15 ℃, the residual body that preceding crop in field is produced is removed clean, soil has been executed rotary tillage 20-25 centimetre after base fertilizer, the dish dregs of rice are evenly spilled into field, dish dregs of rice consumption is 200-400 kg/acre, again myrosase preparation is evenly spilled into field, myrosase preparation consumption is 1-1.5 kg/acre, again by soil rotary tillage, water, making soil water content is the 60-70% of maxmun field capacity, and covered with plastic film, soil temperature is during higher than 25 ℃, overlay film, time is 15-20 days, soil temperature is during lower than 25 ℃, the overlay film time is 20-30 days, then take off film ventilation 2-3 days, can improve dish dregs of rice bacteriostatic and disease prevention drug effect.
2. the method for using myrosase preparation raising dish dregs of rice bacteriostatic and disease prevention drug effect in field according to claim 1, is characterized in that: the glucosinolate content of the dish dregs of rice used is at least 40 μ mol/g, and in myrosase preparation, myrosase activity is 100-200U/g.
3. according to claim 1 and 2ly in field, use the method that myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect, it is characterized in that: described myrosase preparation is cultivated and obtained by dark green trichoderma liquid pure, concrete grammar is for to carry out pure culture to dark green trichoderma, obtain liquid pure culture, liquid pure culture is crossed to four layers of gauze, collect the mycelia on gauze, mycelia is standing after ultrasonic disruption, centrifugal, collect supernatant and obtain myrosase liquid, by myrosase liquid with after carrier adsorption, naturally dry or 30 ℃ of oven dry after be myrosase preparation.
4. according to claim 3ly in field, use the method that myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect, it is characterized in that: dark green trichoderma is carried out to pure culture, obtaining liquid pure culture refers to: dark green trichoderma is inoculated into and in test tube slant medium, carries out activation culture, the cultured inoculation in Zai Jiang inclined-plane is 250ml and is equipped with in the triangular flask of 80ml liquid seed culture medium to corresponding capacity, triangular flask is placed in to rotating speed 160-180r/min, temperature is to cultivate 24-36 hour on the shaking table of 28 ± 2 ℃, and then take the inoculum concentration of volume ratio 5-10% and be linked into respectively capacity as 2000ml and be equipped with in the large triangular flask of 800ml liquid production medium, large triangular flask is placed in to rotating speed 180r/min, temperature is to cultivate 72-96 hour on the shaking table of 28 ± 2 ℃, when just starting to occur yellow color, finishes liquid culture to cultivate, obtain dark green trichoderma liquid pure culture,
Described test tube slant medium is PDA medium, compound method is: 200 grams of potatos, 20 grams of glucose or sucrose, agar 15-20 gram, 1000 milliliters, water, pH nature, peeling potatoes, is cut into piece and boils half an hour, then use 2 layers of filtered through gauze, sugaring and agar, supply water to 1000 milliliter after dissolving again, 121 ℃ of autoclaving 20min;
Described liquid seed culture medium and liquid production medium are: oat 20g, FeSO
47H
2o0.001g, MnCl
24H
2o0.001g, ZnSO
47H
2o0.001g, is dissolved in 1000 ml waters, 121 ℃ of autoclaving 25min.
5. according to claim 3ly in field, use the method that myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect, it is characterized in that: mycelia is standing after ultrasonic disruption, centrifugal, collection supernatant obtains myrosase liquid and refers to: the mycelia of collecting from gauze is evenly mixed with weight ratio 1:5 with the phosphate buffer of PH7.0, at power, it is in 200W ultrasonic disruption instrument broken 10 minutes, then the good mycelia liquid of fragmentation being placed in to 4 ℃ of refrigerators places 1 hour, then at 4 ℃, the centrifugal 15min of 4000r/min, the supernatant of getting after centrifugal is myrosase liquid;
The phosphate buffer of PH7.0 refers to: sodium chloride 8g, and potassium chloride 0.2g, potassium dihydrogen phosphate 0.24g, sodium hydrogen phosphate 3.58g is dissolved in 1000 ml waters.
6. according to claim 3ly in field, use the method that myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect, it is characterized in that: by myrosase liquid, with carrier adsorption carrier used, be the peat composed of rotten mosses powder of 60 mesh sieves, this peat composed of rotten mosses powder is through 121 ℃, sterilizing was used after 40 minutes, the mixed proportion of myrosase liquid and peat composed of rotten mosses powder is volume ratio 1:8-1:10, mixed mixture natural air drying or 30 ℃ of rear mistake 40 mesh sieves of oven dry, obtain myrosase preparation.
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