CN102972192A - 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 the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect of using in the 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 the No. 1 in the world China's rapeseed cultivation area and output, reaches 7,000,000 hectares, accounts for 26% of world's rape seed area, can produce about 1,000 ten thousand tons dish dregs of rice.Protein content is up to 40-45% in the dish dregs of rice, it is a kind of good protein raw materials, application potential as the feed protein exploitation is arranged, but its adding proportion generally can not be higher than 15-20%, because the Secondary Metabolism of Plant product glucosinolate (GSLs) that contains 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.Can be feeding after the detoxification of dish dregs of rice process, but detoxification process is complicated, cost is higher, has " three wastes " to pollute, and Technique Popularizing still faces very large difficulty.Therefore, the present 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 use as organic manure long history.A large amount of data show, use the dish dregs of rice in the new soil of opening up wasteland and can improve soil organic matter content and soil fertility, and Study on Fertilizing soil improves output; And use dish dregs of rice class fertilizer can significantly improve the physico-chemical and biological properties of soil in the arable land that soil quality worsens, play rehabilitating soil and the effect that overcomes soil constraint factor, reduce simultaneously the consumption of chemical fertilizer and agricultural chemicals, improve 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, role is also brought into play far away in soil.During as feed applications, the main catabolite isothiocyanates of the glucosinolate that contains in the dish dregs of rice has toxic action to animal, is to limit its feeding disadvantage factor at the dish dregs of rice; Yet, the dish dregs of rice are applied in the soil, glucosinolate decomposes the isothiocyanates that produces and but has the broad-spectrum sterilization activity, the pests such as the nematode in the soil, bacterium, fungi had 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 to decompose the generation isothiocyanates and causal organism is played lethal effect by rear glucosinolate so that the dish dregs of rice are manured into soil, must possess certain application technique, satisfy 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, because the poor-performing of the adverse circumstances such as double-low rapeseed disease and insect resistance, low temperature, arid, at present also only in the popularization of some areas, the middle and lower reach of Yangtze River, in western part of China and middle part still take the normal rapeseed plantation as main; Simultaneously, the fast development of world wide endogenous substance energy industry is to make biological material oil as extensively rise of purpose leaf mustard plantation, 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 to utilize and will face a larger difficult problem.Therefore, the most dish dregs of rice within a very long time in future, still be faced with rationally, the efficient predicament that utilizes.On the other hand, China's arable area constantly dwindles, and the improving constantly so that people increase increasingly to the demand of agricultural product quantity and kind of the fast development of society and living standards of the people, the advantage of high-efficiency agriculture advances so that modern plant high speed development in China, the large loose water of facility cultivation and high cropping index are so that soil is in the high loaded process state all the year round, the physics of soil, the chemistry and biology proterties sharply worsens, soil quality significantly descends, 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 having had good effect of fertilizer but also can have multiple soil-borne disease bacterium kills or inhibition, the Cruciferae brassica plant that can effectively prevent and kill off soil-borne disease and grouts thereof become again that association area is paid close attention to and the focus of research.The countries such as America and Europe have been developed as business-like pesticide-fertilizer product with leaf mustard green manure and the leaf mustard dregs of rice, obtain good result in the soil-borne diseases such as root-knot nematode, epidemic disease and the blight control of the crops such as vegetables, flowers, fruit tree, show huge application potential.
About cooperate 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, made according to a certain ratio by capsicum resin, rare earth chloride, colza cake or cottonseed cake, zinc sulphate, hydrochloric acid.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 wherein colza cake, loose sawdust in the above colza cake, loose sawdust, three kinds of compositions of castor bean leaf fully being become thoroughly decomposed, dried and crushed is for subsequent use; 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 mixes fungus and bacterium by 2 solid fermentation methods.Also have other take the dish dregs of rice as primary raw material or be the patent of batching with the composite production fertilizer of other fertilizer.
Comprehensive above material can find out that all publications all are 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 being arranged, research report uses the generation that the dish dregs of rice or its composite fertilizer can alleviate the various crop damage by disease and insect, but the fertilizer efficiency that all is summed up as at last the dish dregs of rice promoted plant growth and improved the disease resistance of crop itself, do not utilize the isothiocyanates that the glucosinolate decomposition that contains in the dish dregs of rice produces and plays prevention and control effect to disease and all relate to.
Recently research finds that the percent hydrolysis of glucosinolate in the dish dregs of rice (GSLs) is low to be to cause the main cause that isothiocyanates in the soil (ITCs) concentration is low, affect bactericidal effect.The factor that affects the GSLs hydrolysis has dish dregs of rice degree of crushing, myrosase activity, soil temperature, moisture etc.Morra strengthens the material fragmentation degree and increases soil water content under laboratory condition, so that the percent hydrolysis of GSLs brings up to 30% by 15%, and under the state of field, the GSLs percent hydrolysis still is lower than 15%.Gimsing etc. are under the state of 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 the detection soil, discovery was through 12 days, the percent hydrolysis of bi-material GSLs still only has 26% and 56%, and most GSLs still is not hydrolyzed, but the activity of myrosase is proportionate in the percent hydrolysis of research discovery GSLs and the soil.The plant tissue (comprising the dish dregs of rice) that contains GSLs 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 the myrosase that the dish dregs of rice itself contain is found in a large amount of research owing to be subjected to the impact of the factors such as its content, activity can not satisfy the demand that GSLs is hydrolyzed at short notice in a large number, can significantly promote GSLs to be hydrolyzed in a large number at short notice, to discharge ITCs by external source interpolation myrosase and strengthen the Biofumigation effect.Omirou etc. are under indoor condition of culture, (contain the 1.5g dish dregs of rice) adding active unit in 1kg soil is the plant resource myrosase preparation of 3U, so that the percent hydrolysis in 3 days of the GSLs in the dish dregs of rice reaches 100%, the concentration of ITCs is 5-6 times that contrasts in the soil, and contrast GSLs percent hydrolysis after 8 days still is 40%.As seen, add the degradation rate that myrosase can significantly improve GSLs by external source and strengthen bactericidal effect.More than the myrosase that uses of experiment is the plant resource myrosase, and experimentation only limits in the laboratory, all the practical problem used of unresolved field.
At present commercially available myrosase all extracts from the Cruciferae brassica plant and obtains, and extraction step is complicated, and cost is higher, causes myrosase expensive.At first, plant material source difficulty, in the countries and regions that arable area is nervous, intensive degree is high, it is very large that 2-3 month kind plant of cost is used for extracting the myrosase difficulty; Secondly, the factors such as plant growth region, season and weather directly affect the growing amount of its body endoenzyme, so that output and quality that enzyme extracts are all unstable; In addition, the offcuts that produce behind complicated extraction process and the extraction myrosase are disposed the important bottleneck that difficulty also is restriction the method.Can satisfy any scale, demand any time and utilize microbial fermentation to produce myrosase, productive rate is high, steady quality, has huge advantage.
About the microbial source myrosase, carried out the Aspergillus niger strain that following research: Ohtsuru screening obtains to produce myrosase in the born of the same parents abroad, and carried out separation and purification of enzyme, enzymatic property and affect the factor research of enzymic activity, but not specifically application; 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) promote GSL to decompose generation methyl ITC by producing myrosase.Rakariyatham successively screens the many strains of acquisition and produces the aspergillus of myrosases and the fungi that Fusarium oxysporum belongs to, and obtains the superior strain of 3.5 times of myrosase output increaseds by ultraviolet mutagenesis.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 generation benzyl ITCs with GSLs, thereby effectively prevention and control are by Pythium ultimum
( Pythium ultimum) generation of the pea soil-borne disease that causes.Domestic only have the research such as Wu Hongwei to be separated to myrosase from the aspergillus oryzae culture, but further research report not.The applicant has submitted to name to be called to produce myrosase dark green trichoderma strain from March 15th, 2012 to State Intellectual Property Office 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, also do not utilize the myrosase of dark green trichoderma production to be applied to the report that dish dregs of rice class fertilizer bacteriostatic and disease prevention function is improved in the field both at home and abroad.
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 the 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 so far that technology can operate, the economically viable approach that utilizes, most of dish dregs of rice still apply in the soil as common fertilizer is extensive, are far from bringing into play its due effect.On the other hand, the dish dregs of rice are under the state of field, and the GSLs percent hydrolysis is still low, do not give full play to it to the series of problems such as the effect of killing livestock of pest in the soil, proposed a kind of method that improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field by the 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 the field by the myrosase preparation, to promote glucosinolate fast degradation in the dish dregs of rice by the 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 ℃, remove the residual body that preceding crop in the field produces clean, soil has been executed behind the base fertilizer rotary tillage 20-25 centimetre, the dish dregs of rice evenly are spilled into the field, dish dregs of rice consumption is 200-400 kg/acre, again the myrosase preparation evenly is spilled into the field, myrosase preparation consumption is 1-1.5 kg/acre, again with soil rotary tillage, water, so that soil water content is the 60-70% of maxmun field capacity, and covered with plastic film, when soil temperature is higher than 25 ℃, the overlay film duration is 15-20 days, and when soil temperature was lower than 25 ℃, the overlay film duration was 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 used dish dregs of rice is at least 40 μ mol/g, and the myrosase activity is 100-200U/g in the myrosase preparation.
In the present invention, described myrosase preparation is cultivated by dark green trichoderma liquid pure and is obtained, concrete grammar is for to carry out pure culture to dark green trichoderma, obtain the liquid pure culture, the liquid pure culture is crossed four layers of gauze, collect the mycelia on the gauze, with mycelia behind ultrasonic disruption, leave standstill, centrifugal, collect supernatant and namely obtain myrosase liquid, with myrosase liquid with carrier adsorption after, naturally dry or 30 ℃ of oven dry after be the myrosase preparation.
In the present invention, to carry out pure culture to dark green trichoderma, obtaining the liquid pure culture refers to: dark green trichoderma is inoculated into carries out activation culture in the test tube slant medium, again the capacity of the cultured inoculation in inclined-plane to correspondence is 250ml and is equipped with in the triangular flask of 80ml liquid seed culture medium, triangular flask is placed rotating speed 160-180r/min, temperature is to cultivate 24-36 hour on 28 ± 2 ℃ the shaking table, and then be linked into respectively capacity as 2000ml and be equipped with in the large triangular flask of 800ml liquid production medium take the inoculum concentration of volume ratio 5-10%, large triangular flask is placed rotating speed 180r/min, temperature is to cultivate 72-96 hour on 28 ± 2 ℃ the shaking table, when liquid culture just begins yellow color to occur, finish to cultivate, obtain dark green trichoderma liquid pure culture;
Described test tube slant medium is the PDA medium, and compound method is: potato 200 grams, glucose or sucrose 20 grams, agar 15-20 gram, 1000 milliliters in water, pH nature.Peeling potatoes is cut into piece and boils half an hour, then uses 2 layers of filtered through gauze, and again sugaring and agar are supplied water to 1000 milliliter, 121 ℃ of autoclaving 20min after dissolving;
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, with mycelia behind ultrasonic disruption, leave standstill, centrifugal, the collection supernatant namely obtains myrosase liquid and refers to: will evenly mix with weight ratio 1:5 with the phosphate buffer of PH7.0 from the mycelia that gauze is collected, it is in the 200W ultrasonic disruption instrument broken 10 minutes at power, then the mycelia liquid that fragmentation is good places 4 ℃ of refrigerators to place 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 are dissolved in 1000 ml waters;
In the present invention, be the peat composed of rotten mosses powder of 60 mesh sieves with myrosase liquid with the used carrier of carrier adsorption, this peat composed of rotten mosses powder is through 121 ℃, sterilize and use 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 namely obtain the myrosase preparation.
The invention has the advantages that: at first, the present invention is take the body material of the dish dregs of rice as using common, easy acquisition, add myrosase and adopt simple method regulation and control soil condition by external source, the glucosinolate fast degradation discharges the environmental condition of the gas of killing livestock in the creation promotion dish dregs of rice, reaches the purpose of killing or suppressing pest in the 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 through soil covering and suitable moisturizing process, 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 also 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 the pathogen growth.Whole application is simple, easy operating; Can significantly improve soil quality, balance soil nutrient after using and enrich the population structure of edaphon; performance is to the effect of 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 to the growth inhibition effect of frequently seen plants pathogen with to the control effect of corps diseases and studied, research finds that the dish dregs of rice more than 90% 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 the dish dregs of rice are used, myrosase content, the loosing soil situation, soil water content, soil temperature and soil covering situation etc. all have certain requirement.
Test shows that after the dish dregs of rice were manured into soil, external source was added myrosase and regulated soil to suitable moisture, and then covered with plastic film is kept soil moisture, can significantly improve the dish dregs of rice to the growth inhibitory effect of pathogen.Not only opened up new way for the efficient utilization of a large amount of dish dregs of rice, realized the high-end recycling of agricultural wastes, 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 that adopts 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 have 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: potato 200 grams, glucose or sucrose 20 grams, agar 15-20 gram, 1000 milliliters in water, pH nature.Peeling potatoes, be cut into piece and boil half an hour, then use 2 layers of filtered through gauze, again sugaring and agar, heating is boiled to agar and is dissolved, supply volume to 1000 milliliter, suitably be cooled to about 60 ℃ minute to install in the 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, the pendulum inclined-plane is for subsequent use to cooling off after taking out while hot, 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 by weighing 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, for subsequent use after the cooling.
Embodiment 2:
The preparation of myrosase preparation
Dark green trichoderma preservation of bacteria strain among the embodiment 1 is inoculated into carries out the test tube slant activation in the test tube slant medium, then, to activate good bacterial classification inoculation in the 250ml triangular flask that the 80ml liquid seed culture medium is housed with oese, place shaking table to cultivate 24 hours triangular flask, the rotating speed 160r/min of shaking table, cultivation temperature is 28 ± 2 ℃; Be equipped with in the 2000ml triangular flask of 800ml liquid production medium with 5% inoculum concentration (V/V) access after finishing, place shaking table to cultivate triangular flask, the rotating speed 180r/min of shaking table, cultivation temperature is 28 ± 2 ℃, cultivates and can finish in 3-4 days to cultivate when liquid culture begins flavescence; With liquid culture through four layers of filtered through gauze, collect the mycelia on the 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, with myrosase liquid and cross 60 mesh sieves and the went out peat composed of rotten mosses powder of bacterium of 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, namely obtain the myrosase preparation, the myrosase vigor is 100-200U/g in the preparation.
Embodiment 3
The dish dregs of rice are to the growth inhibition effect of plant pathogenic fungi mycelia and spore or sclerotium
All disease funguses of participating in test can obtain from Chinese Academy of Sciences microorganism fungus kind preservation center.All disease funguses of participating in test are inoculated in the PDA flat board, cultivate after 5 days for subsequent use with the mycelia piece of 4 millimeters of card punch punching acquisition diameters for 28-30 ℃.
Volatile decomposition products is to the growth inhibition effect of plant pathogenic fungi mycelia in the dish dregs of rice: pour the PDA medium in culture dish (diameter 8.5cm, high 1.3cm), the inoculation diameter is 5 mm bacterium dish when putting to normal temperature; Add 0.2g toward culture dish ware lid central authorities, 0.4g, 0.6g, 0.8g, 1.0g, 1.2g cross the dish dregs of rice powder of 2mm sieve, add aqua sterilisa with the ratio of the dish dregs of rice: water=1:5 and fully mix, and contrast only adds aqua sterilisa 6mL, repeats 3 times.Then half culture dish that will inoculate microorganism tips upside down on the culture dish lid, with sealed membrane sealing, 28 ℃ of constant temperature culture.Measure respectively the rotten mould colony diameter of melon and fruit behind 12h, measure the Rhizoctonia solani Kuhn colony diameter behind the 24h, measure the colony diameter of all the other four kinds of disease funguses behind the 72h, every processing repeats 3 times.Calculate each compound to the inhibiting rate of mycelial growth, inhibiting rate (%)=[(the contrast bacterium colony increases diameter-processing bacterium colony and increases diameter)/(the contrast bacterium colony increases diameter)] * 100%.
Non-volatile catabolite is to the growth inhibition effect of plant pathogenic fungi mycelia in the dish dregs of rice: take by weighing dish dregs of rice powder and put into the 250mL triangular flask, add the 100mL sterile water.Add respectively 2g, 6g, 10g, the 14g dish dregs of rice.Only add sterile water in the test tube of contrast.45 ℃ of vibration 12h, the centrifugal 20min of 12000r/min, getting supernatant is 0. 22 μ m filtering with microporous membrane with the aperture on superclean bench.Get mixing in the PDA medium that 1mL filtrate adds 5mL40 ℃ of melting to, be tiled in the culture dish of diameter 5cm, the inoculation diameter is the bacterium piece of 5mm during to normal temperature, with the sealed membrane sealing, is inverted for 28 ℃ and cultivates.Measure the bacterial plaque diameter, calculate inhibiting rate, method is the same, and experimental result sees Table 1.
Table 1 dish dregs of rice catabolite is to the inhibitory action of the mycelial growth of plant pathogenic fungi
| Pathogen | EC 50V（g/L） | EC 50nv（g/L） |
| The 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 |
| The 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 |
Annotate: 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).There were significant differences (P=0. 05) between the different letter representation different disposal.
The 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, and is the most weak to the inhibitory action of phytophthora blight of pepper.And two large class catabolite all be the strongest to the inhibitory action of Sclerotinia sclerotiorum, secondly be to dosporium cucumerinumand its.By EC
50Value can find out that the volatile materials that the dish dregs of rice produce is maximum to the growth inhibition effect of Sclerotinia sclerotiorum, secondly is to dosporium cucumerinumand its.
During the embodiment 4 dish dregs of rice are manured into soil to the inhibition of capsicum pathogen mycelial growth
With 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 the 800g fresh vegetable garden mould, after mixing, add distilled water, keeping soil water content is 70% of maximum water-holding capacity.Being in the drier of 15cm with the vegetable garden soil bore of packing into, is that the hollow plastic tube distribution triangular in shape of 8mm is inserted in the soil with 3 diameters, supports the discoid platform in the drier.To inoculate respectively 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 the drier.The lid of drier and seam crossing are coated vaseline to be sealed.Two contrasts are set: blank, vegetable garden soil be not with the dish dregs of rice; Dazomet (i.e. 98% dazomet fine granule, consumption is 45kg/ha) is added in chemical fumigation contrast in the vegetable garden soil.Behind the good seal drier, it is positioned in 35 ℃ of incubators, opens drier behind the 3d, take out culture dish, measure the diameter of bacterium colony, calculate inhibiting rate, computing formula is with embodiment 3, and experimental result sees Table 2.
The table 2 different amounts dish dregs of rice are to the inhibitory action of phytopathogen mycelial growth
The result shows: after adding the dish dregs of rice in the soil capsicum pathogen mycelial growth is had 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 rates of all the other 5 kinds of pathogens is all reached 100%, reach identical effect with the dazomet chemical fumigation.But the inhibitory action to different pathogens under the identical dish dregs of rice consumption is different.
Embodiment 5
Under the different soils moisture, use the dish dregs of rice to the growth inhibition effect of phytopathogen
Simulation field state in drier, 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 is respectively 50%, 60%, 70%, 80%, 90% of maxmun field capacity, be positioned over and measure colony diameter after 35 ℃ incubator is cultivated 3d, calculate inhibiting rate, computing formula is with embodiment 3, and experimental result sees Table 3.
Table 3 different soils moisture is on the impact of dish dregs of rice bacteriostasis
The result of table 3 can draw, and comprehensively to the inhibition of 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.When soil water content was lower than 70%, inhibitory action weakened along with the reduction of soil water content, and along with water capacity increases to 80%, 90%, to the also gradually reduction of inhibitory action of mycelial growth.As seen the suitable moisture of soil has larger synergistic effect for dish dregs of rice performance bacteria resistance function.
Embodiment uses in 6 fields the myrosase preparation suppresses the pathogen growth to the dish dregs of rice enhancing effect
Experiment is carried out in green house of vegetables, and artificial infection Phytophthora capsici in the plastic shed soil, inoculum density are 1000 spores/gram soil, and soil temperature 20-25 ℃, following processing is established in test: 1) contrast, namely do not use the dish dregs of rice and myrosase preparation; 2) only use the myrosase preparation; 3) only use the dish dregs of rice; 4) the dish dregs of rice+mustard seed processing with enzyme preparation.400 kg/acres of soil Chinese food dregs of rice consumptions, 1.5 kg/acres of myrosase preparation (being provided by embodiment 2) consumptions, 20 square metres of each residential quarter areas, 3 repetitions of every processing, in the experiment dish dregs of rice of correspondence 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.Behind the soil covered with plastic film 20 days, take off the film airing after 2 days, gather 30 centimetres of dark soil samples, measure the quantity of Phytophthora capsici in the soil.Result such as table 4.
Table 4 adds the myrosase preparation suppresses Phytophthora capsici to the dish dregs of rice enhancing effect
By as seen from Table 4, in soil, add the myrosase preparation after, 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, show the growth inhibitory effect that soil adds significantly have been increased when having improved the glucosinolate degradation rate behind the myrosase Phytophthora capsici.
Claims (6)
1. use the method that the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field for one kind, to promote glucosinolate fast degradation in the dish dregs of rice by the 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 ℃, remove the residual body that preceding crop in the field produces clean, soil has been executed behind the base fertilizer rotary tillage 20-25 centimetre, the dish dregs of rice evenly are spilled into the field, dish dregs of rice consumption is 200-400 kg/acre, again the myrosase preparation evenly is spilled into the field, myrosase preparation consumption is 1-1.5 kg/acre, again with soil rotary tillage, water, so that soil water content is the 60-70% of maxmun field capacity, and covered with plastic film, when soil temperature is higher than 25 ℃, overlay film, the time is 15-20 days, when soil temperature is lower than 25 ℃, the overlay film time is 20-30 days, then takes off film ventilation 2-3 days, can improve dish dregs of rice bacteriostatic and disease prevention drug effect.
2. according to claim 1ly use the method that the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field, it is characterized in that: the glucosinolate content of the used dish dregs of rice is at least 40 μ mol/g, and the myrosase activity is 100-200U/g in the myrosase preparation.
3. according to claim 1 and 2ly use the method that the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field, it is characterized in that: described myrosase preparation is cultivated by dark green trichoderma liquid pure and is obtained, concrete grammar is for to carry out pure culture to dark green trichoderma, obtain the liquid pure culture, the liquid pure culture is crossed four layers of gauze, collect the mycelia on the gauze, with mycelia behind ultrasonic disruption, leave standstill, centrifugal, collect supernatant and namely obtain myrosase liquid, with myrosase liquid with carrier adsorption after, naturally dry or 30 ℃ of oven dry after be the myrosase preparation.
4. according to claim 3ly use the method that the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field, it is characterized in that: will carry out pure culture to dark green trichoderma, obtaining the liquid pure culture refers to: dark green trichoderma is inoculated into carries out activation culture in the test tube slant medium, again the capacity of the cultured inoculation in inclined-plane to correspondence is 250ml and is equipped with in the triangular flask of 80ml liquid seed culture medium, triangular flask is placed rotating speed 160-180r/min, temperature is to cultivate 24-36 hour on 28 ± 2 ℃ the shaking table, and then be linked into respectively capacity as 2000ml and be equipped with in the large triangular flask of 800ml liquid production medium take the inoculum concentration of volume ratio 5-10%, large triangular flask is placed rotating speed 180r/min, temperature is to cultivate 72-96 hour on 28 ± 2 ℃ the shaking table, when liquid culture just begins yellow color to occur, finish to cultivate, obtain dark green trichoderma liquid pure culture;
Described test tube slant medium is the PDA medium, compound method is: potato 200 grams, glucose or sucrose 20 grams, agar 15-20 gram, 1000 milliliters in water, the pH nature, peeling potatoes is cut into piece and boils half an hour, then use 2 layers of filtered through gauze, again sugaring and agar are supplied water to 1000 milliliter, 121 ℃ of autoclaving 20min after dissolving;
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.
5. according to claim 3ly use the method that the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field, it is characterized in that: mycelia is left standstill behind ultrasonic disruption, centrifugal, the collection supernatant namely obtains myrosase liquid and refers to: will evenly mix with weight ratio 1:5 with the phosphate buffer of PH7.0 from the mycelia that gauze is collected, it is in the 200W ultrasonic disruption instrument broken 10 minutes at power, then the mycelia liquid that fragmentation is good places 4 ℃ of refrigerators to place 1 hour, then at 4 ℃, centrifugal 15 min of 4000 r/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 use the method that the myrosase preparation improves dish dregs of rice bacteriostatic and disease prevention drug effect in the field, it is characterized in that: be the peat composed of rotten mosses powder of 60 mesh sieves with myrosase liquid with the used carrier of carrier adsorption, this peat composed of rotten mosses powder is through 121 ℃, sterilize and use 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 namely obtain the myrosase preparation.
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| WO2015013808A1 (en) * | 2013-08-02 | 2015-02-05 | Mpt Mustard Products & Technologies Inc. | Biopesticide compositions comprising water soluble polyols |
| CN105541508A (en) * | 2016-02-21 | 2016-05-04 | 宿林功 | Organic fertilizer capable of disinfecting soil |
| CN105993729A (en) * | 2016-05-25 | 2016-10-12 | 句容市天王镇戴庄有机农业专业合作社 | Organic paddy rice biological weeding method |
| CN110915339A (en) * | 2019-11-01 | 2020-03-27 | 山东省农业科学院植物保护研究所 | Method for preventing and treating soil-borne diseases by fumigating soil with plants |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015013808A1 (en) * | 2013-08-02 | 2015-02-05 | Mpt Mustard Products & Technologies Inc. | Biopesticide compositions comprising water soluble polyols |
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| CN105541508A (en) * | 2016-02-21 | 2016-05-04 | 宿林功 | Organic fertilizer capable of disinfecting soil |
| CN105993729A (en) * | 2016-05-25 | 2016-10-12 | 句容市天王镇戴庄有机农业专业合作社 | Organic paddy rice biological weeding method |
| CN110915339A (en) * | 2019-11-01 | 2020-03-27 | 山东省农业科学院植物保护研究所 | Method for preventing and treating soil-borne diseases by fumigating soil with plants |
| CN110915339B (en) * | 2019-11-01 | 2022-08-05 | 山东省农业科学院植物保护研究所 | Method for preventing and treating soil-borne diseases by fumigating soil with plants |
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