CN1314325C - Biological prepn. for preventing and controlling soft rot of konjak, and its application - Google Patents
Biological prepn. for preventing and controlling soft rot of konjak, and its application Download PDFInfo
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- CN1314325C CN1314325C CNB200510048755XA CN200510048755A CN1314325C CN 1314325 C CN1314325 C CN 1314325C CN B200510048755X A CNB200510048755X A CN B200510048755XA CN 200510048755 A CN200510048755 A CN 200510048755A CN 1314325 C CN1314325 C CN 1314325C
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Abstract
The present invention relates to a biological agent for controlling konjak bacterial soft rot and an application thereof, which belongs to the technical field of the biological pesticide. The production strain of the present invention is bacillus amyloliquefaciens C3 of which the preservation number is CGMCC No. 1553. The strain of the present invention has the following distinctive characteristics that 1, colony presents an irregular round shape, and the edge of the colony presents a cracking shape; the colony is viscous in the initial stage, and wrinkles and is milky and nontransparent in the late stage; the mycelium is difficult to disperse in liquid, generates spore, and presents a rod shape; 2, the strain is colonized so as to have strong bacteriostatic capacity, promote the konjak to grow and has the functions of disease prevention and yield increase. The present invention obtains liquor or wettable powder which are prepared by adding wetting agent, spreader, auxiliary materials, etc. to bacteria mycelium culture by the liquid fermentation production of the strain, and has the advantages of obvious stable control efficiency for konjak bacterial soft rot, good integral character and easy commercial process.
Description
Technical field:
The present invention relates to a kind of biologic product and application thereof that prevents and treats the konjaku bacterial slimy soft rot, belong to biological pesticide technical field.
Background technology:
Konjaku is rich in glucomannans, is one of only several plants that contain natural glucomannans of natural world, the plant that also be that unique distribution is wide, adaptability is strong, can extract glucomannans in a large number.Be a kind of medical-care health food and the raw material of industry of great exploitation potential for its.Along with the adjustment with the structure of rural undertaking continually developed of konjaku product, konjaku has become a kind of main economic crops in mountain area, China Midwest, is peasant programme, revitalizes one of effective way of local economy.According to incompletely statistics, 2005, Yunnan Province's cultivated area reached 150,000 mu, and the cultivated area in the whole nation is about 800,000 mu.No matter being external or domestic, is first limiting factor of konjaku industry development by bacterial soft rot.Often several years ago growth momentum is better for konjaku, but prolongation along with the plantation time limit, the expansion of area under cultivation, the soft rot popular cycle is shorter and shorter, the state of an illness increases the weight of year by year, some areas soft rot cause total crop failure, seriously hinder the raising of konjaku yield and quality, cause cultivated area to fall sharply at last.Serious contusion the enthusiasm of peasant planting konjaku, hindered the development of local economy.
The konjaku bacterial slimy soft rot is that a kind of production has the worldwide plant disease of crushing harm to konjaku, studies confirm that mainly through us to be caused by carrot soft rot Erwinia carrot subspecies Erwinia carotovora subsp.carotovora, secondly is chrysanthemum Erwinia E.chrysanthermi.According to incompletely statistics, only in Yunnan Province, the annual comprehensive incidence of disease of konjak soft rot reaches more than 35%, and the konjaku percent of loss reaches 21.5%, and every mu is caused 774 yuan of economic losses, and the annual because soft rot in the whole county endangers the konjaku that causes with a toll of more than 1,200 ten thousand yuan.For the konjaku bacterial slimy soft rot, at present domestic mainly with agricultural measures and heavy dose of prophylactico-therapeutic measuress such as chemical pesticide such as crop rotations, but under the situation that China has a large population and a few land, the feasibility that the employing crop rotation prevents and treats the konjaku bacterial slimy soft rot is more and more lower; Chemical control is cost height, anti-heterodyne not only, and contaminated environment, influence quality, and some pathogenetic bacterias show pesticide resistance to producing the agricultural streptomycin of using always, makes control efficiency poorer.In addition, because chemical pesticide uses and persticide residue surpasses international standard, influenced the growth of its export abroad.
Raising along with science and technology development and living standards of the people, people are to healthy and environmental problem growing interest, development high yield, high-quality, konjaku production have efficiently become the key issue that improves taro district peasant economy income, particularly organic in recent years konjaku or nuisanceless konjaku production have become the dominant direction of konjaku industry development, more need aspect the disease agricultural and biological control etc. not to cause the residual prophylactic measures with environmental pollution of farming preventing and treating.Therefore, the biologic product of a kind of efficient, nontoxic, safe noresidue, control konjaku bacterial slimy soft rot easy to use is developed in screening, to alleviating disease hazard, improve quality, strengthen konjaku field ecosystem stability, ensureing the sound development of konjaku industry, make the peasant increase production, increase income and have important practical significance and wide application prospect.
Summary of the invention:
The objective of the invention is to overcome the deficiency of existing method, the biologic product of a kind of efficient, nontoxic, safety, noresidue, control konjaku bacterial slimy soft rot easy to use is provided.
The bacterium that the present invention adopts is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on December 05th, 2005; The numbering CGMCC No.1553 that preservation is registered on the books.
Production bacterial strain bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) C
3, separate from Yunnan natural resources konjaku rhizosphere soil, to measure through morphology, cultivation proterties, conventional Physiology and biochemistry and the full-automatic identification systems of Biolog, this biocontrol bacteria is a new bacterial strain (Bacillus amyloliquefaciens) C of bacillus amyloliquefaciens
3This bacterial strain has following feature: (1) bacterium colony irregular cycle, and colony edge splits laminated, bacterium colony initial stage thickness, the later stage is wrinkling, and milky is opaque, and thalline is difficult for disperseing in liquid, does not produce pigment, produces gemma; Examine under a microscope: thalline direct rod shape, size 0.7～0.8 * 2.0～2.4 μ m; (2) this bacterial strain colonization ability is strong, bacteriostasis strong, can promote the konjaku growth, has the effect of sterilization, diseases prevention, volume increase.This bacterial strain can at the rhizosphere of konjaku and body is decided at the higher level but not officially announced be grown.The main pathogenic fungi Erwinia carotovora on this crop there is remarkable inhibitory action, antibacterial circle diameter average out to 14.0-20.0mm.(3) has following physiological and biochemical property: Gram-positive, can utilize glycogen, polysorbate40, N-acetyl-D-aminoglucose, ribitol, L-arabinose, the D-arabite, the L-fucose, the D-galactose, gentiobiose, the meso-inositol, milk ketose, D-mannitol, the D-psicose, the D-raffinose, the L-rhamnose, the D-sorbierite, the methyl-prop ketone acid, acetate, citric acid, formic acid, in the D-galactonic acid, the D-gluconic acid, the A-glycolic acid, the Y-glycolic acid, itaconic acid, the a-batanone acid, the a-ketovaleric acid, propionic acid, succinic acid, the D-alanine, the L-alanine, L-alanyl-glycine, the L-aspartic acid, L-glutamic acid, the L-leucine, the L-serine, the L-threonine, the DL-carnitine, y-aminobutyric acid, uridylic acid, trophicardyl, uridine, thymidine, phenyl ethylamine, butanediamine, the 2-ethylaminoethanol, Robison ester; Can not utilize cyclodextrin, dextrin, N-acetyl-D-galactose, cellobiose, D-fructose, a-D-glucose, a-D-lactose, maltose, sucrose, D-trehalose, xylitol, malonic acid, L-proline, D-serine, glycerine.
The present invention is achieved in that the test tube cultivation of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C3 bacterial strain, the shaking table enlarged culture, fermented and cultured is prepared as biologic product, and it is applied on the konjaku, measures its control efficiency and output to the konjaku bacterial slimy soft rot.
Biocontrol bacterial strain bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) C
3Cultural method (below be weight percentage):
1, the test tube kind is cultivated
The test tube slant culture medium prescription is: beef soaks 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7-2.0%, pH6.8-7.0.
With Bacillus amyloliquefaciens C
3Move on on the test tube slant medium,, obtain the test tube kind at 28 ℃ of constant temperature culture 1-2 days.
2, liquid fermentation and culture
The liquid fermentation medium prescription is: analysis for soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH
2PO
40.1%, peptone 0.2% (NH
4)
2SO
40.05%, MgSO
47H
2O 0.03%, CaCO
30.1%, peanut oil 0.1%, defoamer 0.01%, pH 7.5-8.0.
The test tube kind is inoculated in the 500ml triangular flask liquid nutrient medium, every bottled 200ml, in 28 ℃ of constant-temperature shaking culture 14 hours, rotating speed was 150-200rpm, the gained culture fluid promptly can be preparation, also switchablely carries out fermenting and producing to fermentation tank, obtains liquid preparation.
Liquid fermentation condition
The jar temperature: the jar Wen Jun of fermentation tank is controlled at 25-32 ℃, by inserting the thermometer measure of medium, regulates with the method that feeds cooling water or hot water in the interlayer.
Tank pressure: the tank pressure of fermentation tank is controlled at 0.5/ square centimeter, regulates by filtrated air inlet and Waste gas outlet.
Stir: the mixing speed of fermentation tank is 200 rev/mins.
Sampling check and condition of culture are regulated: per 2 hours from probe tube sampling 1 time, measure pH, and smear with violet staining microscopy thalli morphology, have or not the pollution of assorted bacterium, observe thalline and whether produce gemma etc., treat that microscopy thalline gemma number accounts for 5% when above of whole thalline, about reducing ventilation and reducing cultivation temperature to 22 ℃, other condition is constant, continue to cultivate and sampling check, treat that microscopy thalline gemma number accounts for 85% when above of whole thalline, stop to cultivate and putting jar, microscopy counting when fermentation tank is put jar, and carry out the counting of viable bacteria with the culture medium flat plate method.
Cultivation cycle: the cultivation cycle of fermentation tank is about 36-48 hour.
The liquid fermentation preparation: measure bacteria containing amount through plate count, bacteria containing amount reaches 18,000,000,000 CFU/ml, uses for the field control disease.
3, solid fermentation is cultivated
At first carry out the liquid fermentation and culture (the same) of bacterium.Through adding filler, plate-frame filtering, making beating, drying, pulverizing, make wetting powder then.Detailed process is as follows:
The concrete filler that adds: zymotic fluid is pressed into basin, adds diatomite (precipitated calcium carbonate) as filler, add the back and stirred 30 minutes according to following computing formula.
Remnant (kilogram) in the addition (kilogram) of filler diatomite (precipitated calcium carbonate)=" zymotic fluid bacterium number (hundred million/milliliter) * put tank volume (liter) * yield "/finished product bacterium number-zymotic fluid.
Plate-frame filtering: the pressure with 2 kg/cm, material is pressed into sheet frame by basin, filter by No. 7 filter clothes, pressure should be regulated at any time, makes the bacteria containing amount in the filtrate be no more than 0.2 hundred million/milliliter.
Making beating: filter cake is discharged into the making beating jar, presses 8% of adding calcium carbonate quantity and add dense newborn No. 100, or, add the even stirring of an amount of filtrate 30 minutes by 3% adding SDS.
Mixing: the bacterium diatomite that contains after will pulling an oar is pulverized, dries in the shade, and again by 5 times of volume dilution, stirring and evenly mixing.
Dry: aeration-drying is to water content 5%-8% in the baking room of temperature below 60 ℃.
Pulverize: drop temperature can not surpass 60 ℃ during pulverizing, to prevent the thalline inactivation.
The end product quality index determining: bacteria containing amount, water content, outstanding prisoner lead, the mensuration of fineness is all carried out with reference to national company standard (Q/KWL02-2003), bacteria containing amount 1,000,000,000/gram, and all the other every indexs all meet standard.
Biologic product of the present invention is prevented and treated soft rot field control effectiveness test and volume increase test
1, preparation biologic product: by aforementioned Bacillus amyloliquefaciens C
3Cultural method preparation test preparation.
2, biocontrol microorganisms C
3Liquid preparation is to konjak soft rot field efficacy and volume increase test
2.1 reagent agent and processing:
A, biocontrol microorganisms C
3250 times of dilutions of liquid preparation: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
B, biocontrol microorganisms C
3500 times of dilutions of liquid preparation: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
C, biocontrol microorganisms C
31000 times of dilutions of liquid preparation: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
D, 500 times of times of dilutions of contrast fungicide chlorothalonil: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
E, blank: use clear water, adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed and used for the first time, uses 7 days at interval altogether 2 times.
2.2, test method: for to study thing is elephant-foot yam (Fuyuan County landrace), controlling object is the konjaku bacterial slimy soft rot, and test site is bamboo plantation town, Fuyuan County, Yunnan Province.
5 processing are established in test, 3 repetitions, totally 15 sub-districts.The sub-district area is not less than 20 square metres, randomized arrangement.
Establish guard row all around, every sub-district is surveyed and is produced during results.
2.3, investigation method: in the konjaku morbidity later stage, disease survey is carried out in the experimental plot. according to konjak soft rot acrial part symptom feature and PD trend, we are divided into circulating type and longitudinal type with scab, the former scab is around petiole, PD is fast. and can cause the compound leaf lodging very soon, harmfulness is big; How latter's scab is vertically expanded along vascular bundle only in petiole one side, and the later stage causes whole petiole to rot after horizontal expansion.
The grade scale of konjak soft rot (state of an illness is divided into 5 grades):
0 grade: asymptomatic; 1 grade: petiole one side has the following small-sized water soaking mode scab of 2cm, or scab length accounts for below 1/10 of petiole total length, the slight yellow of homonymy blade; 2 grades: petiole one side has the above scab of 2cm, or scab length accounts for the 1/10--5/10 of petiole total length, the yellow of homonymy blade; 3 grades: scab is circulating type, or longitudinal type scab length accounts for more than 5/10 of petiole length overall, and most of yellow of blade or part are withered; 4 grades: full leaf is withered and yellow or lodging is rotten.
If whole compound leaf begins yellow from the top, there is soft rotten symptom petiole and stem tuber junction, are the stem tuber morbidity, and the state of an illness is included into 3 grades.
3, biocontrol microorganisms C
3Wetting powder is to konjak soft rot field efficacy and volume increase test
1, reagent agent and processing:
A, biocontrol microorganisms C
3250 times of dilutions of wetting powder: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
B, biocontrol microorganisms C
3500 times of dilutions of wetting powder: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
C, biocontrol microorganisms C
31000 times of dilutions of wetting powder: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
D, 500 times of times of dilutions of contrast fungicide chlorothalonil: adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed medication for the first time, and dispenser is 2 times altogether, 7 days at interval.
E, blank: use clear water, adopt and water the root method, every strain konjaku root waters 50 milliliters of soups, and their early stage is executed and used for the first time, uses 7 days at interval altogether 2 times.
2, test method: for studying thing is elephant-foot yam (Fuyuan County landrace), and controlling object is the konjaku bacterial slimy soft rot, and test site is bamboo plantation town, Fuyuan County, Yunnan Province.
5 processing are established in test, 3 repetitions, totally 15 sub-districts.The sub-district area is not less than 20 square metres, randomized arrangement.
Establish guard row all around, every sub-district is surveyed and is produced during results.
3, investigation method: in the konjaku morbidity later stage, disease survey is carried out in the experimental plot. according to konjak soft rot acrial part symptom feature and PD trend, we are divided into circulating type and longitudinal type with scab, the former scab is around petiole, PD can cause the compound leaf lodging soon very soon, and harmfulness is big; How latter's scab is vertically expanded along vascular bundle only in petiole one side, and the later stage causes whole petiole to rot after horizontal expansion.
The grade scale of konjak soft rot (state of an illness is divided into 5 grades):
0 grade: asymptomatic; 1 grade: petiole one side has the following small-sized water soaking mode scab of 2cm, or scab length accounts for below 1/10 of petiole total length, the slight yellow of homonymy blade; 2 grades: petiole one side has the above scab of 2cm, or scab length accounts for the 1/10--5/10 of petiole total length, the yellow of homonymy blade; 3 grades: scab is circulating type, or longitudinal type scab length accounts for more than 5/10 of petiole length overall, and most of yellow of blade or part are withered; 4 grades: full leaf is withered and yellow or lodging is rotten.If whole compound leaf begins yellow from the top, there is soft rotten symptom petiole and stem tuber junction, are the stem tuber morbidity, and the state of an illness is included into 3 grades.
4, result of the test
4.1 biocontrol microorganisms C
3Liquid preparation is to konjak soft rot field efficacy and volume increase test
Table 1 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3Liquid preparation is to the konjak soft rot field control effect
| Handle | Disease index | Total strain number (strain) | Average diseased plant rate (%) | Average disease refers to | Control efficiency (%) | ||
| Repeat I | Repeat II | Repeat III | |||||
| 500 times of 250 times of dilutions of CK dilution | 19.17 3.33 4.67 | 21.67 5.92 9.33 | 17.17 5.83 13.08 | 900 941 953 | 12 3.59 3.22 | 19.34 4.26 9.03 | - 77.9 53.3 |
| Dilute 1000 times | 6.33 | 11.75 | 12.83 | 952 | 3.68 | 10.30 | 46.7 |
| 500 times of tpns | 10.17 | 15.08 | 13.67 | 927 | 5.23 | 12.97 | 32.9 |
Table 2 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3Liquid preparation is to the effect of increasing production of konjaku
| Handle | Per mu yield (kg) | Units increased in production (kg) | Rate of growth (%) |
| 1000 times of 500 times of dilutions of 250 times of dilutions of CK dilution | 1600.8 2267.8 2134.4 2134.4 | - 667 533.6 533.6 | - 41.67 33.33 33.33 |
| 500 times of tpns | 1734.2 | 133.4 | 8.33 |
From table 1 and table 2 biocontrol microorganisms C as can be seen
3Liquid preparation is obvious to konjak soft rot control and effect of increasing production, it is best wherein to dilute 250 times of processing controls and effect of increasing production, control efficiency reaches the processing that 77.9%. irritates 500 times of roots and 1000 times, konjak soft rot is controlled effect be respectively 53.3% and 46.7%.Compare with 500 times of tpns of dilution, use biocontrol microorganisms C
3(250 times of each processing of liquid preparation, 500 times, 1000 times) diseased plant rate lower by 1.64%, 2.01% than it respectively, 1.55%, disease index is lower by 8.71,3.94,2.67 than tpn, and control efficiency is higher by 45.00%, 20.40% than it, 13.80%, shown that this biological prevention and control agent has preventive effect preferably; Compare with 500 times of tpns of dilution simultaneously, use biocontrol microorganisms C
3Each processing (250 times, 500 times, 1000 times) rate of growth higher by 33.34%, 25.00%, 25.00% than it respectively, show that this product has certain short life, production-increasing function, produce the primary condition that goes up large-scale popularization so this product has possessed.
This application method adopts and waters the root method, finds in the research, and the konjaku plant of root irrigation, the probability that pathogen is invaded from basal part of stem significantly reduces, but still finds some field acrial parts plant of rotting, and mainly invades from overground parts such as blades.Therefore, on application method, use from now on and water Gen Fa and the foliar spray method is used in combination, reach the purpose of effective control konjak soft rot.
4.2 biocontrol microorganisms C
3Wetting powder is to konjak soft rot field efficacy and volume increase test
Table 3 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3Wetting powder is to the konjak soft rot field control effect
| Handle | Disease index | Total strain number (strain) | Average diseased plant rate (%) | Average disease refers to | Control efficiency (%) | ||
| Repeat I | Repeat II | Repeat III | |||||
| 500 times of 250 times of dilutions of CK dilution | 19.17 4.00 7.05 | 21.67 4.50 9.30 | 17.17 3.53 10.14 | 900 950 959 | 12 3.20 3.32 | 19.34 4.01 8.83 | - 79.3 54.3 |
| Dilute 1000 times | 8.23 | 10.11 | 9.05 | 960 | 3.57 | 9.13 | 52.8 |
| 500 times of tpns | 10.17 | 15.08 | 13.67 | 927 | 5.23 | 12.97 | 32.9 |
Table 4 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3Wetting powder is to the effect of increasing production of konjaku
| Handle | Per mu yield (kg) | Units increased in production (kg) | Rate of growth (%) |
| 1000 times of 500 times of dilutions of 250 times of dilutions of CK dilution | 1600.8 2301.3 2190.2 2103.4 | - 700.5 589.4 502.6 | - 43.76 36.82 31.40 |
| 500 times of tpns | 1734.2 | 133.4 | 8.33 |
From table 3 and table 4 biocontrol microorganisms C as can be seen
3Wetting powder is obvious to konjak soft rot control and effect of increasing production, it is best wherein to dilute 250 times of processing controls and effect of increasing production, control efficiency reaches the processing that 79.3%. irritates 500 times of roots and 1000 times, konjak soft rot is controlled effect be respectively 54.3% and 52.8%.Compare with 500 times of tpns of dilution, use biocontrol microorganisms C
3(250 times of each processing of wetting powder, 500 times, 1000 times) diseased plant rate lower by 2.03%, 1.91% than it respectively, 1.66%, disease index is lower by 8.96,4.14,3.84 than tpn, and control efficiency is higher by 46.40%, 21.40% than it, 19.90%, shown that this biological prevention and control agent has preventive effect preferably; Compare with 500 times of tpns of dilution simultaneously, use biocontrol microorganisms C
3Each processing (250 times, 500 times, 1000 times) rate of growth higher by 35.43%, 28.49%, 23.07% than it respectively, show that this product has certain short life, production-increasing function, produce the primary condition that goes up large-scale popularization so this product has possessed.
Total result shows that biologic product of the present invention (no matter being liquid preparation or wetting powder) control konjaku bacterial slimy soft rot effect is remarkable, and has the short effect of giving birth to, increasing production concurrently.
The main controlling object of preparation of the present invention is a konjak soft rot.This preparation can be used by seed soaking, the modes such as root, spraying of irritating.Preparation of the present invention has carried out sub-district, field efficiency test in Fuyuan County, Qujing, Yunnan Province, prove this biological prevention and control agent preventive effect significantly, stable, can solve effectively the farming of long-term puzzlement taro the konjaku bacterial slimy soft rot prevent and treat a difficult problem.Konjaku bacterial slimy soft rot preventive effect is reached 46.7-79.30%.
Embodiment:
This biological prevention and control agent can with other kill bacterial pesticide, plant growth regulator mix uses, but must assurance mixed medicament to bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C of the present invention's employing
3Have no side effect.
Bacterium biological prevention and control agent of the present invention is generally used in crop sowing or the disease phase of starting.Before method is sowing, adopts fermentation preparation and plant ball and soak after (mixing) plant, during airing is manured into soil, or in the disease phase of starting this preparation is applied konjaku basal part of stem or spray method by irritating root, dilution 300-500 doubly uses.Serious area is taken place the konjaku bacterial slimy soft rot can impose 2-3 time in the konjaku process of growth.
The present invention have to the konjaku bacterial slimy soft rot prevent, to control effect obvious, is applicable to the konjaku whole growing, has characteristics such as cost is low, easy to use, the term of validity is long.
Be described in further detail the present invention with embodiment below, but content of the present invention is not limited thereto.
Embodiment one:
With bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3Forward on the test tube slant medium, the test tube slant culture medium prescription is: beef soaks 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7%, pH:7.0.
28 ℃ of constant temperature culture 2 days, obtain the test tube kind.
The liquid fermentation medium prescription is: analysis for soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH
2PO
40.1%, peptone 0.2% (NH
4)
2SO
40.05%, MgSO
47H
2O 0.03%, CaCO
30.1%, peanut oil 0.1%, defoamer 0.01%, pH 7.5;
The test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, and culture medium prescription is: beef soaks 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7.In 28 ℃ of constant-temperature shaking culture 14 hours, rotating speed was 150rpm, and the gained culture fluid is biologic product of the present invention.
Embodiment two:
At first obtain test tube kind (with embodiment one), the test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, culture medium prescription is: beef soaks 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 2.0%.In 28 ℃ of constant-temperature shaking culture 14 hours, rotating speed was 200rpm, and the gained culture fluid is forwarded to and carries out fermenting and producing in the fermentation tank, obtained liquid preparation.
The liquid fermentation medium prescription is: analysis for soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH
2PO
40.1%, peptone 0.2% (NH
4)
2SO
40.05%, MgSO
47H
2O 0.03%, CaCO
30.1%, peanut oil 0.1%, defoamer 0.01%, pH 8.0.
Liquid fermentation condition
The jar temperature: the jar Wen Jun of fermentation tank is controlled at 25 ℃, by inserting the thermometer measure of medium, regulates with the method that feeds cooling water or hot water in the interlayer.
Tank pressure: the tank pressure of fermentation tank is controlled at 0.5/ square centimeter, regulates by filtrated air inlet and Waste gas outlet.
Stir: the mixing speed of fermentation tank is 200 rev/mins.
Sampling check and condition of culture are regulated: per 2 hours from probe tube sampling 1 time, measure pH, and smear with violet staining microscopy thalli morphology, have or not the pollution of assorted bacterium, observe thalline and whether produce gemma etc., treat that microscopy thalline gemma number accounts for 5% when above of whole thalline, about reducing ventilation and reducing cultivation temperature to 22 ℃, other condition is constant, continue to cultivate and sampling check, treat that microscopy thalline gemma number accounts for 85% when above of whole thalline, stop to cultivate and putting jar, microscopy counting when fermentation tank is put jar, and carry out the counting of viable bacteria with the culture medium flat plate method.
Cultivation cycle: the cultivation cycle of fermentation tank is about 36 hours.
The liquid fermentation preparation: measure bacteria containing amount through plate count, bacteria containing amount reaches 18,000,000,000 CFU/ml, uses for the field control disease.
Promptly get biologic product of the present invention.
Embodiment three:
At first carry out the liquid fermentation and culture (with embodiment two) of bacterium.Through adding filler, plate-frame filtering, making beating, drying, pulverizing, make wetting powder then.Detailed process is as follows:
The concrete filler that adds: zymotic fluid is pressed into basin, and 10% adding filler diatomite (precipitated calcium carbonate) by putting tank volume adds the back and stirred 30 minutes.
Plate-frame filtering: the pressure with 2 kg/cm, material is pressed into sheet frame by basin, filter by No. 7 filter clothes, pressure should be regulated at any time, makes the bacteria containing amount in the filtrate be no more than 0.2 hundred million/milliliter.
Making beating: filter cake is discharged into the making beating jar, adds dense newborn No. 100, or, add 2% even the stirring 30 minutes of filtrate by 3% adding SDS by 8% of adding calcium carbonate quantity.
Mixing: the bacterium diatomite that contains after will pulling an oar is pulverized, dries in the shade, and again by 5 times of volume dilution, stirring and evenly mixing.
Dry: aeration-drying is to water content 8% in the baking room of temperature below 60 ℃.
Pulverize: drop temperature can not surpass 60 ℃ during pulverizing, to prevent the thalline inactivation.
The end product quality index determining: bacteria containing amount, water content, outstanding prisoner lead, the mensuration of fineness is all carried out with reference to national company standard (Q/KWL02-2003), bacteria containing amount 1,000,000,000/gram, and all the other every indexs all meet standard.
Promptly get biologic product of the present invention.
Claims (3)
1, a kind of biologic product of preventing and treating konjaku bacterial slimy soft rot disease is characterized in that: the production bacterial strain of said preparation is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C
3, preserving number CGMCC No.1553.
2, the described bacillus amyloliquefaciens of claim 1 (Bacillus amyloliquefaciens) C
3The cultural method of bacterial strain is characterized in that, described bacterial strain is to cultivate acquisition through conventional liquid and solid fermentation, and its liquid and solid fermentation culture medium prescription are as follows:
(1) liquid fermentation medium prescription: analysis for soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH
2PO
40.1%, peptone 0.2% (NH
4)
2SO
40.05%, MgSO
47H
2O 0.03%, CaCO
30.1%, peanut oil 0.1%, defoamer 0.01%, pH 7.5-8.0;
(2) solid fermentation culture medium prescription: beef soaks 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7-2.0%, pH6.8-7.0.
3, the described bacillus amyloliquefaciens of claim 1 (Bacillus amyloliquefaciens) C
3The application of bacterial strain in control konjaku bacterial slimy soft rot disease.
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| CNB200510048755XA CN1314325C (en) | 2005-12-27 | 2005-12-27 | Biological prepn. for preventing and controlling soft rot of konjak, and its application |
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| CN1314325C true CN1314325C (en) | 2007-05-09 |
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| CN101165171B (en) * | 2007-09-30 | 2010-05-19 | 华中农业大学 | Bacillus subtilis BSn5 with bacteriostasis active to carrot soft rot Erwinia and bacteriostasis protein APn5 |
| EP2179652B1 (en) * | 2008-09-10 | 2019-05-08 | ABiTEP GmbH Gesellschaft für AgroBioTechnische Entwicklung und Produktion | Use of an antibacterial agent for treating bacterial infection in crop plants |
| CN102174427B (en) * | 2010-12-24 | 2012-08-29 | 中南林业科技大学 | Endophytic antagonistic strain for controlling soft rot of camellia oleifera and application thereof |
| CN103131648A (en) * | 2011-11-30 | 2013-06-05 | 河北农业大学 | Bacillus amyloliquefaciens and application of bacillus amyloliquefaciens in peanut rot disease preventive treatment |
| CN102864100B (en) * | 2012-08-16 | 2015-01-28 | 北京雷力海洋生物新产业股份有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN103013854A (en) * | 2012-10-24 | 2013-04-03 | 昆明学院 | Biocontrol strain KMXU1 capable of preventing and treating blueberry blight and antibiological inoculant thereof |
| CN103274825B (en) * | 2013-06-09 | 2015-11-25 | 顾祥茂 | A kind of polynary microorganism special fertilizer preventing and treating celery soft rot and preparation method thereof |
| CN103451135B (en) * | 2013-09-09 | 2015-04-22 | 云南农业大学 | Bacillus subtilis M3 and application thereof |
| TWI649419B (en) * | 2017-07-13 | 2019-02-01 | 國立屏東科技大學 | Liquefied Bacillus starch and its application |
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