CN101637186A - Bactericide for preventing and curing crop bacterial diseasess as well as preparation method and application thereof - Google Patents
Bactericide for preventing and curing crop bacterial diseasess as well as preparation method and application thereof Download PDFInfo
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- CN101637186A CN101637186A CN200910042262A CN200910042262A CN101637186A CN 101637186 A CN101637186 A CN 101637186A CN 200910042262 A CN200910042262 A CN 200910042262A CN 200910042262 A CN200910042262 A CN 200910042262A CN 101637186 A CN101637186 A CN 101637186A
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Abstract
The invention discloses a bactericide for preventing and curing crop bacterial diseasess as well as a preparation method and application thereof. In the preparation method, oligosaccharide is added into bdellovibrio bacteriovorus solution to be evenly mixed to obtain the bactericide for preventing and curing crop bacterial diseasess. The invention applies bdellovibrio bacteriovorus solution for preventing and curing crop bacterial diseases for the first time, fills the vacancy of researching and developing bdellovibrio bacteriovorus at home and abroad and adds new species for biopesticide. Asbdellovibrio bacteriovorus has the characteristics of broad cracking pattern and quick growth and has the outstanding capability of eliminating various pathogenicbacteria and bacteria, after combinedwith oligosaccharide, the bdellovibrio bacteriovorus strengthens crop resistance, greatly improves pesticide effect and enlarges the preventing and curing spectrum of bacterial diseases. The bactericide prepared by the invention has no toxicity or pollution, is safe to natural enemies of crop diseases and pests, pathogenic bacteria have small possibility of producing resistance, so that the invention overcomes 3R problems brought by abusing antibiotics and avoids the defects of serious chemical pesticide pollution and ecological balance damage at present.
Description
Technical field
The invention belongs to biological technical field, the particularly a kind of microbial inoculum of preventing and treating bacterial diseases of crops and preparation method thereof and application.
Background technology
In recent years, China's agricultural develops rapidly, and along with the raising of intensive cropping degree, some common bacterial diseases become more popular and stubbornness, the present poor effect of effective way of past, even so-called in the past sensitive medicaments is also failed to respond to any medical treatment.Bacterial disease is by the disease due to the bacterium infection process, as bacterial blight of rice, soft rot, canker, bacterial wilt etc., has caused the heavy losses of agricultural production.Particularly bacterial blight of rice is one of three big diseases of paddy rice, and China area takes place throughout the year more than 1,000 ten thousand mu, the general underproduction 10%～20%, and the severe patient underproduction reaches 50%.Since cause bacterial diseases of crops the pathogen kind many, host range is wide, and is strong to the adaptive faculty of environment, the survival mode is various, so difficulty of prevention and cure is big.At present, pesticidal chemical kind with respect to the control fungal disease, the pesticide variety of control bacterial diseases of crops is also fewer, existing pesticide species is also poor as the control efficiency of agricultural streptomycin, bismerthlazol and inorganic copper agent medicaments such as (Kocide SD, copper sulphate, copper oxychloride, cuprous oxide), resistance produces fast, and seriously polluted, brought severe " 3R " problem, agricultural product security is difficult to be guaranteed.Therefore, bacterial disease has become one of agricultural sustainable development problem demanding prompt solution.
Biopesticide is the effective ways of control bacterial diseases of crops, by handle management crops rhizosphere soil micro-ecological environment, eliminate pathogen, form the rhizosphere soil ecosystem stable, balance, through exploring for many years, have been found that the biopesticide that is used to prevent and treat corps diseases, for example utilized Trichoderma to prevent and treat bakanae disease, bacillary glume blight and bacillary damping off; Antibiotic jinggangmeisu control wheat and the rice sheath blight disease of utilizing streptomycete to produce; Utilize bacillus subtilis to prevent and treat rice sheath blight disease, cladosporium leaf and fruit mould of tomato, root rot, the mould worry of apple, cotton seedling blight and cotton wilt etc.But biopesticide is used for preventing and treating fungal disease more at present, and only germ is had bacteriostasis, and does not have bactericidal action, and disease is disappeared.
Bdellovibrio with parasitic character is a kind of effective tool that is used to eliminate the environment pathogen of Recent study, it is the host with the bacterium, can cracking eliminate pathogen, and fragmentation pattern is extensive, the field planting ability is strong, breeding is fast, utilizes it as the natural biological modifying agent in the environment and " living antibiotics ", has vast potential for future development in the control bacterial diseases of crops.Up to the present, developed Bdellovibrio and be applied to the product that pathogen is eliminated in the aquaculture, but do not seen that as yet Bdellovibrio is used to prevent and treat the effective ways of bacterial diseases of crops.
Summary of the invention
In order to overcome the weak point of utilizing Bdellovibrio effectively to prevent and treat bacterial diseases of crops is not arranged in the prior art, primary and foremost purpose of the present invention is to provide a kind of microbial inoculum of control bacterial diseases of crops of efficient, no side effects.
Another object of the present invention is to provide the preparation method of above-mentioned microbial inoculum.
A further object of the present invention is to provide the application of above-mentioned microbial inoculum.
Purpose of the present invention is achieved through the following technical solutions: a kind of microbial inoculum of preventing and treating bacterial diseases of crops comprises Bdellovibrio and oligosaccharides.
The microbial inoculum of described control bacterial diseases of crops contains 10 in preferred every liter of solution
6～10
12Bdellovibrio and the 10～30g oligosaccharides of pfu (plaque-forming units, plaque-forming unit);
Described Bdellovibrio is preferably the typical culture collection center preservation in China, and preservation date is that Bdellovibrio (Bdellovibrio sp.) BDS01, preservation date that August 7, deposit number in 2009 are CCTCC NO:M 209169 are that August 7, deposit number in 2009 Bdellovibrio (Bdellovibrio sp.) BDS02 that is CCTCC NO:M 209170 or preservation date are at least one strain among August 7, deposit number in 2009 Bdellovibrio (Bdellovibrio sp.) BDSM08 that is CCTCCNO:M 209171;
Described Bdellovibrio more preferably Bdellovibrio (Bdellovibrio sp.) BDS01 mixed than 1: 1 with bacterial population with Bdellovibrio (Bdellovibriosp.) BDSM08;
Described oligosaccharides is preferably at least a in chitosan oligosaccharide, agaropectin oligose, ligoglucoside, algin oligosaccharide or the oligomeric maltose;
The preparation method of the microbial inoculum of described control bacterial diseases of crops may further comprise the steps: is that to adjust concentration be 10 for 7.2～7.6 phosphate buffer with Bdellovibrio with DNB medium, water, physiological saline or 0.2mol/L pH value
6～10
12Add oligosaccharides behind the pfu/L, the addition of oligosaccharides is 10～30g/L, and mixing obtains preventing and treating the microbial inoculum of bacterial diseases of crops;
Described Bdellovibrio is preferably at least one strain among Bdellovibrio BDS01, Bdellovibrio BDS02 or the Bdellovibrio BDSM08;
Described Bdellovibrio is preferably by application number " 200710031166.X ", and name is called the fermentation process of " the fermented and cultured technology of high density Bdellovibrio telotroch " disclosed high density Bdellovibrio of national inventing patent application telotroch and cultivates;
Described Bdellovibrio is preferably by Bdellovibrio BDS01 and Bdellovibrio BDSM08 respectively according to application number " 200710031166.X ", the fermentation process that name is called " the fermented and cultured technology of high density Bdellovibrio telotroch " disclosed high density Bdellovibrio of national inventing patent application telotroch carries out fermented and cultured, and zymotic fluid is pressed bacterial population mixing in 1: 1 and got;
Described oligosaccharides is at least a in chitosan oligosaccharide, agaropectin oligose, ligoglucoside, algin oligosaccharide or the oligomeric maltose;
The application of the microbial inoculum of described control bacterial diseases of crops is the microbial inoculum of described control bacterial diseases of crops to be sprayed stem, leaf, fringe, the fruit of plant or irritates root drench root;
The content of Bdellovibrio is at least 10 in the microbial inoculum of described control bacterial diseases of crops
3Pfu/mL, preferred 10
5～10
9Pfu/mL;
The microbial inoculum of described control bacterial diseases of crops sprays stem, leaf, fringe or the fruit of plant, and consumption is minimum to be 80mL/m
2, preferable amount is 100～200mL/m
2
The microbial inoculum of described control bacterial diseases of crops is used to irritate root and drenches root, and consumption is minimum to be the 100mL/ strain, and preferable amount is 150～250mL/ strain;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat soft rot of crucifers, black rot, root rot, and using method is drenched root or sprayed cauline leaf for irritating root;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat cucumber and leguminous plant bacterial angular leaf spot, and using method is drenched root for irritating root, sprayed stem, leaf or fruit;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat solanaceous vegetable bacterial wilt, soft rot, canker, and using method is drenched root for irritating root, sprayed stem, leaf or fruit;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat gramineae farm crop bacterial stripe, bacterial leaf-blight, and using method is for spraying stem, leaf or fringe.
Principle of the present invention: Bdellovibrio is the host with the bacterium, can cracking eliminate pathogen, and fragmentation pattern is extensive, and the field planting ability is strong, and breeding is fast.Oligosaccharides can combine with the crop plant signal transducers, is the acquired disease-resistant derivant of plant, makes plant obtain the resistance of system, permanent, wide spectrum.The present invention is when using Bdellovibrio to eliminate pathogen, and interpolation can improve the oligosaccharides of crops acquired resistance, makes preventive effect stable more lasting, has solved problems such as the residual and resistance of the medicine that adopts antibiotic control bacterial disease to bring in the past.
The present invention has following advantage and effect with respect to prior art:
1. the present invention is applied to Bdellovibrio the control of bacterial diseases of crops first, fills up the blank of domestic and international Bdellovibrio research and development, has increased the new kind of biopesticide.It is wide that Bdellovibrio has fragmentation pattern, and the characteristics rapidly of growing have excellent elimination ability to the various bacteria pathogen.Oligosaccharides is with it after the coupling, strengthen the crops resistance, drug effect significantly improves, and has enlarged the control spectrum of bacterial disease, thus diseases such as high-efficiency prevention and control Solanaceae, Cruciferae, pulse family and unifacial leaf crops bacterial stripe, angular leaf spot, canker, soft rot, bacterial leaf-blight.Test shows, is used to prevent and treat angular leaf spot of cucumber, dispenser after 10 days preventive effect reach 87.5%, be better than the gloomy copper preventive effect of 20% thiophene (as shown in Figure 3).
2. the prepared microbial inoculum of the present invention is nontoxic, pollution-free, and to the diseases and pests of agronomic crop natural enemies security, and pathogen substantially produces resistance to it.As biologic product, do not produce poisoning, overcome the antibiotic abuse and brought " 3R " problem, also avoided current chemical pesticide seriously polluted, destroy the ecological balance, the drawback of harm natural enemy.The prepared microbial inoculum of the present invention is behind the intact host bacteria of cracking, Bdellovibrio can be withered away automatically because of hungry, therefore humans and animals is had no side effect, improve quality of agricultural product and safety, meet the requirement of the people the demand and the sustainable development of agricultural production of food health green.
Description of drawings
Fig. 1 is that the microbial inoculum of embodiment 1 preparation is prevented and treated the bacterial blight of rice design sketch.
Fig. 2 is the microbial inoculum control paddy rice cecospora spot design sketch of embodiment 2 preparations.
Fig. 3 is that the microbial inoculum of embodiment 3 preparations is prevented and treated the angular leaf spot of cucumber design sketch.
Fig. 4 is that the microbial inoculum of embodiment 4 preparations is prevented and treated the canker of tomato design sketch.
Fig. 5 is the anti-soft rot of Chinese cabbage design sketch of the microbial inoculum of embodiment 5 preparations.
Fig. 6 is the anti-eggplant bacterial wilt design sketch of the microbial inoculum of embodiment 6 preparations.
Fig. 7 is the anti-Chinese cabbage black rot design sketch of the microbial inoculum of embodiment 7 preparations.
Fig. 8 is the anti-custard squash root rot design sketch of the microbial inoculum of embodiment 8 preparations.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
(1) preparation of Bdellovibrio bacterium liquid
The fermentation process of Bdellovibrio liquid adopts application number " 200710031166.X ", the fermentation process that name is called " the fermented and cultured technology of high density Bdellovibrio telotroch " disclosed high density Bdellovibrio of national inventing patent application telotroch carries out: inoculate Aeromonas hydrophila (Aeromonas hydrophila respectively in 3 conical flasks that 100mL LB (Luria-Bertani) liquid nutrient medium is housed, purchase in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172), 200rpm, 28 ℃ of shaking tables were cultivated 18 hours, culture fluid is respectively at 4 ℃, the centrifugal 10min of 8000rpm abandons supernatant.Respectively precipitation is joined 3 100mL DNB liquid nutrient medium (nutrient broth 0.8g is housed, caseinic acid hydrolysate 0.5g, yeast extract 0.1g, NaCl 30g, be dissolved in the 1000mL distilled water, the pH value is 7.2～7.6) conical flask in, insert Bdellovibrio BDS01 (choosing bacterial plaque from double-layer plate inserts), Bdellovibrio BDS02 (choosing bacterial plaque from double-layer plate inserts) and Bdellovibrio BDSM08 (choosing bacterial plaque from double-layer plate inserts) constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h more respectively.Culture fluid is got supernatant respectively at 4 ℃ of centrifugal 20min of 6000rpm, again with supernatant respectively at 4 ℃ of centrifugal 20min of 16000rpm, keep precipitation, add the aseptic DNB Bdellovibrio sediment that suspends again, making concentration is 10
9The Bdellovibrio liquid of pfu/mL.
Bdellovibrio BDS01 separates in the mud sample and obtains, and is preserved in Chinese typical culture collection center in the Chinese Wuhan City Wuhan University on August 7th, 2009, and deposit number is CCTCC NO:M 209169.It is carried out morphologic observation under electron microscope after the negative staining: Bdellovibrio BDS01 is unicellular, arc, and size is 0.9 * 0.25 μ m, and end is given birth to flagellum, and flagellum length is 4 μ m; It is cultivated the transparent circular plaque that can form diameter 1-2mm in four days with the double-layer plate method in 28 ℃.
Bdellovibrio BDS02 separates in the mud sample and obtains, and is preserved in Chinese typical culture collection center on August 7th, 2009, and deposit number is CCTCC NO:CCTCC M 209170.It is carried out morphologic observation under electron microscope after the negative staining: Bdellovibrio BDS02 is unicellular, arc, and size is 0.65 * 0.2 μ m, end is given birth to flagellum, flagellum length at least 1.8 μ m; It is cultivated the transparent circular plaque that can form diameter 1-2mm in four days with the double-layer plate method in 28 ℃.
Bdellovibrio BDSM08 carries out ultraviolet mutagenesis by Bdellovibrio BDS01 and obtains, and is preserved in Chinese typical culture collection center in the Chinese Wuhan City Wuhan University on August 7th, 2009, and deposit number is CCTCCNO:M 209171.It is carried out morphologic observation under electron microscope after the negative staining: Bdellovibrio BDSM08 is unicellular, arc, and size is 1.8 * 1.0 μ m, and end is given birth to flagellum, and flagellum length is 3.2 μ m; It is cultivated the transparent circular plaque that can form diameter 2-3mm in three days with the double-layer plate method in 28 ℃.
(2) preparation of the microbial inoculum of control bacterial diseases of crops
With the bacterium liquid of Bdellovibrio BDS01, Bdellovibrio BDS02 and Bdellovibrio BDSM08 respectively water be adjusted to 10
7Pfu/mL, adding chitosan oligosaccharide respectively, to make it concentration be 30g/L, shakes 30s with magnetic stirring apparatus, obtains preventing and treating microbial inoculum A, B and the C of bacterial diseases of crops.
(3) microbial inoculum of the control bacterial diseases of crops of applying step (2) preparation is prevented and treated bacterial blight of rice
Using microbial inoculum prevents and treats bacterial blight of rice: the experimental field is positioned at Guangzhou Tianhe District Cen Cun paddy field, middle fertility, and testing used rice varieties is that late rice assists excellent 85.Paddy rice is in the shooting stage, and the experimental field piece is a regular incidence field piece.22.1～29.5 ℃ of duration of test daily mean temperatures, relative moisture 90～95%.Select the sub-district at random, each sub-district 2m
2, rice cultivation 20 caves, every sub-district.Clear water is established three sub-districts, and as blank, the contrast medicament is 500 times of liquid of the gloomy copper dilution of 20% thiophene.At the bacterial leaf-blight their early stage, by sub-district microbial inoculum A, B, C and the contrast medicament of the control bacterial diseases of crops of step of applying (3) preparation respectively, consumption was respectively 120mL/m when promptly fragmentary scab appearred in the rice field
2, using hand pressure sprayer spraying cauline leaf, blade face and blade back all evenly are sprayed onto, and each handles 2 replicated plots.Spray 1 time continuous 3 times later on every 10d.7d, 14d and 21d look into control efficiency after the 3rd dispenser.2 strain paddy rice are chosen in 5 samplings, each sample point, whenever select good strains in the field for seed and get boot leaf and following two leaves of boot leaf, carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
The preventive effect computing formula is:
Disease index=[∑ (the sick numbers of sheets at different levels * relative level numerical value)/(investigation sum * 9)] * 100
Control efficiency (%)=[(blank district disease index-treatment region disease index)/blank district disease index] * 100
Test shows that the control group bacterial blight of rice spreads rapidly, along the vein one or both sides or middle arteries and veins develop into undulatory yellowish green or celadon scab; Scab transfers canescence to behind 5～7d, and blade inwardly curls.Minority severe patient complete stool is withered behind the 14d.Use Bdellovibrio control paddy rice bacterial leaf spot effect remarkable, only a small amount of scab occurred at first day after using Bdellovibrio-oligosaccharides, behind the 2d, scab no longer enlarges, and the state of an illness is controlled, and later disease is no longer development almost.Bdellovibrio BDS01, Bdellovibrio BDS02 and Bdellovibrio BDSM08 respectively with chitosan oligosaccharide coupling spray medicine 21d after preventive effect be respectively 72.3%, 68.7%, 74.1%, all be better than the preventive effect (60.4%) of 500 times of liquid of the gloomy copper of 20% thiophene, control efficiency as shown in Figure 1.All are handled and all lose the poisoning generation, the tall and straight stalwartness of plant, and leaf is unfolded, and grows fine, and upgrowth situation is better than spraying 500 times of liquid of the gloomy copper of 20% thiophene.
Embodiment 2 control paddy rice cecospora spots
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
With Bdellovibrio BDS01, Bdellovibrio BDS02 and Bdellovibrio BDSM08 in twos (is BDS01 and BDS02 by bacterial population respectively at 1: 1, BDS01 and BDSM08, BDS02 and BDSM08) mix, and in every liter of mixed bacteria liquid, add 15g agaropectin oligose and 15g ligoglucoside respectively, shake 30s with magnetic stirring apparatus, obtain preventing and treating microbial inoculum A, B and the C of bacterial diseases of crops;
Bdellovibrio BDS01, Bdellovibrio BDS02 and Bdellovibrio BDSM08 are pressed bacterial population mixing in 1: 1: 1, in every liter of mixed bacteria liquid, add 15g agaropectin oligose and 15g ligoglucoside, shake 30s, obtain microbial inoculum D with magnetic stirring apparatus.
(3) the microbial inoculum control paddy rice cecospora spot of the control bacterial diseases of crops of applying step (2) preparation
Using microbial inoculum prevents and treats the paddy rice cecospora spot: the experimental field is positioned at Guangzhou Tianhe District Cen Cun paddy field, middle fertility, and testing used rice varieties is that late rice assists excellent 85.Paddy rice is in the shooting stage, and the experimental field piece is a regular incidence field piece.22.1～29.5 ℃ of duration of test daily mean temperatures, relative moisture 90～95%.Select the sub-district at random, each sub-district 2m
2, rice cultivation 20 caves, every sub-district.Clear water is established three sub-districts, as blank.The contrast medicament is 500 times of liquid of the gloomy copper of 20% thiophene, and at the cecospora spot their early stage, by sub-district microbial inoculum A, B, C, D and the contrast medicament of the control bacterial diseases of crops of step of applying (3) preparation respectively, consumption was respectively 120mL/m when promptly fragmentary scab appearred in the rice field
2, using hand pressure sprayer spraying cauline leaf, blade face and blade back all evenly are sprayed onto, and each handles 2 replicated plots.Spray 1 time continuous 3 times later on every 7d.7d, 14d and 21d look into control efficiency after the 3rd dispenser.2 strain paddy rice are chosen in 5 samplings, each sample point, whenever select good strains in the field for seed and get boot leaf and following two leaves of boot leaf, carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
Test shows that control group paddy rice cecospora spot disease spreads rapidly, and scab increases between vein, and the interior scab of 5-10d just is the water stain shape of dirty-green, after be yellowish-brown to bronzing, length expands 1cm to from 0.5mm.Bacterial ooze appears in the minority severe patient behind the 14d.Use Bdellovibrio-oligosaccharides control paddy rice cecospora spot effect remarkable, all processing only a small amount of scab occurred at first day, and behind the 2d, scab no longer enlarges, and the state of an illness is controlled, and later disease is no longer development almost.As shown in Figure 2, microbial inoculum D (is that three kinds of bacterium liquid of BDS01, BDS02 and BDSM08 mix at 1: 1: 1, and add agaropectin oligose, each 15g/L of glucan) preventive effect behind spray medicine 21d is 81.8%, be better than 20% thiophene, 500 times of liquid of gloomy copper (75.3%), microbial inoculum B (is that BDS01 mixes with BDSM08 bacterium liquid at 1: 1, and add agaropectin oligose, each 15g/L of glucan) preventive effect behind spray medicine 14d is 82.4%, preventive effect is 84.6% behind the spray medicine 21d, be better than other three kinds of microbial inoculums and 500 times of liquid of the gloomy copper of 20% thiophene, preventive effect is best.Microbial inoculum A, B, C, D all lose the poisoning generation to the processing of paddy rice, the tall and straight stalwartness of plant, and leaf is unfolded, and grows fine, and upgrowth situation is better than spraying 500 times of liquid of the gloomy copper of 20% thiophene.
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
Bdellovibrio BDS01 bacterium liquid and Bdellovibrio BDSM08 bacterium liquid are pressed bacterial population mixing in 1: 1, and with the physiological saline dilution, concentration is respectively 10
3Pfu/mL, 10
5Pfu/mL, 10
7Pfu/mL and 10
9Four concentration scales of pfu/mL, then respectively in the Bdellovibrio solution of variable concentrations, every liter of mixed bacteria liquid adds 10g chitosan oligosaccharide and 20g ligoglucoside, shakes 30s with magnetic stirring apparatus, obtains preventing and treating microbial inoculum A, B, C and the D of bacterial diseases of crops; Bdellovibrio BDS01, BDS02 and BDSM08 bacterium liquid are pressed bacterial population mixing in 1: 1: 1, and with the physiological saline dilution, concentration is respectively 10
3Pfu/mL, 10
5Pfu/mL, 10
7Pfu/mL and 10
9Four concentration scales of pfu/mL then respectively in the Bdellovibrio solution of variable concentrations, add 10g agaropectin oligose and 20g algin oligosaccharide in every liter of mixed bacteria liquid, shake 30s with magnetic stirring apparatus, obtain preventing and treating microbial inoculum E, F, G and the H of bacterial diseases of crops.
(3) microbial inoculum of the control bacterial diseases of crops of applying step (2) preparation is prevented and treated angular leaf spot of cucumber
Use microbial inoculum cucumber angular leaf spot in seedling stage is prevented and treated test: test is carried out in Baiyun District good farmland, Guangzhou town booth, and experimental cultivar is No. five strong yellow melons of precious poplar.Select the sub-district at random, each sub-district 3m
2, 10 strains of every sub-district plantation cucumber.When cucumber grew to 3～4 true leaves, on the occasion of rainy weather, the canopy temperature high humility was big, experimental period 22.8～26.3 ℃ of mean temperatures, relative moisture is 97～100%, cucumber seedling begins to have fragmentary angular leaf spot to occur in the booth.Clear water is established three sub-districts, as blank.The contrast medicament is 200 times of liquid of the gloomy copper of 20% thiophene, at the angular leaf spot their early stage, by sub-district microbial inoculum A, B, C, D, E, F, G, H and the contrast medicament of the control bacterial diseases of crops of step of applying (3) preparation respectively, consumption is respectively 120mL/m when fragmentary scab promptly occurring
2, using hand pressure sprayer spraying cauline leaf, blade face and blade back all evenly are sprayed onto, and each handles 2 replicated plots.Irritate every 10d later on and execute continuous 3 times 1 time.5d, 10d and 15d " Invest, Then Investigate " preventive effect behind the 3rd the spray medicine.A strain cucumber seedling is chosen in 5 samplings, each sample point, every strain cucumber chooses, in two leaves, carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
After the dispenser, the control group incidence of disease is lasting to rise, and scab increases and enlarges, and spreads along vein to be hexagon, and bacterial ooze appears in blade back portion, the perforation of severe patient blade, and drying up comes off plant death.And after using Bdellovibrio mixed liquor 2d the 2nd time, use and mix Bdellovibrio liquid group scab and no longer enlarge, it is few that new scab produces, and cucumber seedling grows fine, and only is low concentration (Bdellovibrio content 10
3Pfu/mL) mixed liquor produces many relatively new scabs, but the scab slower development, the state of an illness is not serious.And Bdellovibrio concentration is 10
7After using twice, do not see the high concentration mixed liquor of pfu/mL that poisoning produces.Test shows, the effect optimum be microbial inoculum C, preventive effect was respectively 87.5% and 85.3% after microbial inoculum C used 10d and 15d, preventive effect is a little more than microbial inoculum G and 200 times of liquid of 20% Thiodiazole-copper, as shown in Figure 3.
Embodiment 4 prevents and treats canker of tomato
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
Bdellovibrio BDS01 bacterium liquid and Bdellovibrio BDSM08 bacterium liquid are pressed bacterial population mixing in 1: 2, and adjusting bacterial concentration with phosphate buffer (0.2mol/L, pH7.2～7.6) is 10
7Pfu/mL adds 12g chitosan oligosaccharide and 18g algin oligosaccharide in every liter of mixed bacteria liquid, shake 30s with magnetic stirring apparatus, obtains preventing and treating the microbial inoculum A of bacterial diseases of crops; Bdellovibrio BDS01, BDS02 and BDSM08 bacterium liquid are pressed bacterial population mixing in 1: 1: 2, and adjusting bacterial concentration with phosphate buffer is 10
7Pfu/mL adds 12g agaropectin oligose and 18g ligoglucoside in every liter of mixed bacteria liquid, shake 30s with magnetic stirring apparatus, obtains preventing and treating the microbial inoculum B of bacterial diseases of crops.
(3) microbial inoculum of the control bacterial diseases of crops of applying step (2) preparation is prevented and treated canker of tomato
Cultivate canker of tomato pathogen Michigan rod shape bacillus (Clavibacter michiganensis) (available from Chinese common micro-organisms culture presevation administrative center CGMCC with Jin Shi B (KB) solid culture medium, bacterial strain CGMCC 1.1909), behind 27 ℃ of cultivation 24h lawn is scraped, being diluted to concentration is 10
7～10
8The bacteria suspension of cfu/mL unit, stand-by.
Use microbial inoculum canker of tomato is prevented and treated test: test is carried out in Baiyun District good farmland, Guangzhou town booth, and experimental cultivar is good red No. 4.Experimental period 22.8～26.3 ℃ of mean temperatures, relative moisture is 100%.Select the sub-district at random, each sub-district 3m
2, 10 strains of every sub-district plantation tomato.The contrast medicament is 300 times of liquid of 20% Thiodiazole-copper, and clear water is established three sub-districts, is blank.The tomato phase of bearing fruit entangles the intact young fruit of 0.5～1cm with freshness protection package, sack is fastened with cord.When treating that tomato grows to diameter 2～3cm, untie freshness protection package.Spray plant Michigan bacillus bacteria suspension 100mL/m with hand pressure sprayer
2, making whole plant surface wettability, microbial inoculum A, B and the contrast medicament of the control bacterial diseases of crops that after drying naturally step (3) is prepared evenly spray plant by the sub-district with hand pressure sprayer, and consumption is respectively 180mL/m
2Sprayed medicine more once every 8 days, connect and spray 2 times.7d, 14d and 21d investigation diseased fruit rate and occurring degree behind the 2nd the spray medicine carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
Inoculation 1d-2d, new canker scab all appears in each group, but processed group obviously is less than control group, and behind the 5d, the control group state of an illness spreads and increases the weight of, and bacterial ooze is overflowed in sick portion on the young fruit, and scab is to the young fruit internal extended, and the sick fruit of minority is caducous.Use microbial inoculum group premunition and 200 times of liquid phases of 20% Thiodiazole-copper and work as, the minority scab is only arranged, scab no longer enlarges behind the 2d, and rare scab endangers pulp in flakes.Experimental result shows that the 14d preventive effect is 71.4% after the microbial inoculum A dispenser for the second time, and is close with 200 times of liquid preventive effects of 20% Thiodiazole-copper, and does not see that poisoning takes place.Continue to use microbial inoculum in the tomato growth process, disease seldom takes place, and best in quality when ripe, color and luster is dark green smooth, little scab.The mean fruit weight amount of using microbial inoculum overweights the clear water control group, than clear water control group volume increase 32.5%.Experiment simultaneously shows that microbial inoculum B also has good control efficiency to canker of tomato, and concrete preventive effect as shown in Figure 4.
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
Bdellovibrio BDS01 bacterium liquid is mixed by bacterial population with Bdellovibrio BDSM08 bacterium liquid at 1: 3.In every liter of mixed bacteria liquid, add 10g chitosan oligosaccharide, 10g agar-agar glycan and 10g oligomeric maltose, shake 30s, obtain preventing and treating the microbial inoculum A of bacterial diseases of crops with magnetic stirring apparatus; In every liter of mixed bacteria liquid, add 3.3g chitosan oligosaccharide, 3.3g agar-agar glycan and 3.3g oligomeric maltose, shake 30s, obtain preventing and treating the microbial inoculum B of bacterial diseases of crops with magnetic stirring apparatus.
(3) the microbial inoculum control soft rot of Chinese cabbage of the control bacterial diseases of crops of applying step (2) preparation
Contain the preparation of Erwinia soil: with common nutrient broth (NB) medium, 160rpm cultivates 12～16h for 28 ℃, it is 105 that propagation Erwinia (Erwinia sp.) (purchasing in Chinese common micro-organisms culture presevation administrative center CGMCC, bacterial strain CGMCC1.1215) makes bacterial concentration
cFu/mL, NB suspends, and is 1: 4 ratio mixing by weight in bacteria suspension and aseptic soil.
Soft rot of Chinese cabbage is prevented and treated: test is carried out at Guangzhou Tianhe District Cen Cun, and the duration of test mean temperature is that 22.8～26.3 ℃, relative moisture are 100%.Testing used Chinese cabbage cultivar is No. 2, Shandong.The contrast medicament is 500 times of liquid of 75% tpn wetting powder, and clear water is done blank.Adopt aseptic soil greenhouse pot culture Chinese cabbage seedling, treat when Chinese cabbage is cultivated to 5～6 leaves Chinese cabbage moved in the above-mentioned soil that contains Erwinia and test every sub-district 0.5m
2, 20 strains of every sub-district.Spray microbial inoculum A, B and the contrast medicament of the control bacterial diseases of crops of step (3) preparation by the sub-district with hand pressure sprayer immediately after the transplanting, each handles 2 replicated plots, and consumption is distinguished 120mL/m
2Repeat dispenser more once behind the 10d.Observe incidence behind the 2nd dispenser 7d, 14d and the 21d, carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
After the dispenser, begin to occur the soft rot illness behind the control group 3d, base portion is morbidity earlier, the little yellow scab of water stain shape occurs, and fester in petiole base or rhizome place subsequently, flows out mucus, the severe patient perish.The incidence of disease of processed group significantly is lower than control group, and this group Chinese cabbage morbidity is few, and cauline leaf is healthy and strong, and color and luster is dark green, grows fine.Experimental result shows, the best results of microbial inoculum A reaches 82.6% behind the 2nd the dispenser 21d, as shown in Figure 5.The effect of microbial inoculum B is inferior slightly, but continues to use microbial inoculum B in the Chinese cabbage process of growth, and disease seldom takes place, and is best in quality when ripe, and on average individual plant weight overweights control group and Bdellovibrio use group separately, compares average single weightening finish 23.7% with control group.
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
BDS01 Bdellovibrio liquid and BDSM08 Bdellovibrio liquid are pressed bacterial population mixing in 2: 3.In every liter of mixed bacteria liquid, add 5g chitosan oligosaccharide, 10g agaropectin oligose and 15g algin oligosaccharide, shake 30s, obtain preventing and treating the microbial inoculum of bacterial diseases of crops with magnetic stirring apparatus.
(3) the microbial inoculum control eggplant bacterial wilt of the control bacterial diseases of crops of applying step (2) preparation
Using microbial inoculum prevents and treats the eggplant bacterial wilt: test is carried out at Guangzhou Tianhe District Cen Cun, used eggplant kind is the Xinlong purple long eggplant, the duration of test mean temperature is 22.8～26.3 ℃, relative moisture is 100%, the contrast medicament is 500 times of liquid of 75% tpn wetting powder, and clear water is a blank.Adopt aseptic soil greenhouse pot culture eggplant seedling, treat to move into when eggplant is educated to 5～6 leaves in the booth each sub-district 1m
2, 20 strains of plantation eggplant.Use the microbial inoculum and the contrast medicament of the control bacterial diseases of crops of step (3) preparation to irritate root respectively by the sub-district immediately after the transplanting, every strain consumption 150ml, each handles 2 sub-districts.Repeat dispenser more once every 10d.The 2nd dispenser 7d, 14d and the every sub-district of 21d " Invest, Then Investigate " diseased plant number.Carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
After the dispenser, begin to occur the soft rot illness behind the control group 3d, the disease plant blade present wither vertical, after expand to gradually on the whole strain branch.Just be light green, browning is shrivelled, and sick leaf comes off or remains on the branch.Stem's cortex strip off xylem is brown.The severe patient brown stain extends to the xylem of top branch.The marrow of branch the inside rots hollow mostly.The incidence of disease of processed group significantly is lower than control group, and this group eggplant seedling morbidity is few, and cauline leaf is healthy and strong, and color and luster is dark green, grows fine.Experimental result shows that the 2nd dispenser 21d preventive effect of microbial inoculum reaches 73.5%, as shown in Figure 6.Continue to use microbial inoculum in the eggplant process of growth, disease seldom takes place, and best in quality when ripe, average single weight overweights control group and Bdellovibrio use group separately, compares average single weightening finish 10.8% with control group.
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
BDS01 Bdellovibrio liquid and BDSM08 Bdellovibrio liquid are pressed bacterial population mixing in 1: 4.In every liter of mixed bacteria liquid, add 5g chitosan oligosaccharide, 5g agaropectin oligose, 5g algin oligosaccharide and 5g oligomeric maltose, shake 30s, obtain preventing and treating the microbial inoculum A of bacterial diseases of crops with magnetic stirring apparatus; In every liter of mixed bacteria liquid, add 5g chitosan oligosaccharide, 5g agaropectin oligose, 5g ligoglucoside, 5g oligomeric maltose, shake 30s, obtain preventing and treating the microbial inoculum B of bacterial diseases of crops with magnetic stirring apparatus.
(3) the microbial inoculum control Chinese cabbage black rot of the control bacterial diseases of crops of applying step (2) preparation
Using microbial inoculum Chinese cabbage black rot prevents and treats: this test is located at the heavier Guangzhou Tianhe District Cen Cun of morbidity over the years, and breeds of Chinese cabbage is No. 2, Shandong.The soil types of test region is a sandy loam, and soil property is fertile.Experimental period 21.5～25.3 ℃ of mean temperatures, relative moisture is 91～95%.The contrast medicament is 4000 times of liquid of 72% agricultural streptomycin, and clear water is a blank.Every sub-district 1m is established in test
2, 15 strains of plantation Chinese cabbage.Spray microbial inoculum A, B and the contrast medicament of the control bacterial diseases of crops of step (3) preparation, consumption 120mL/m by the sub-district with hand pressure sprayer respectively during the Chinese cabbage field planting
2Every 10 days 1 time, prevent and treat continuously 2 times afterwards.Observe incidence behind 5d, 10d and the 15d behind the 2nd medicine, carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
Carry out pilot survey and calculate preventive effect with reference to the pesticide field efficacy medicine test criterion.
Experiment shows that the clear water group vegetative period occurs the symptom of black rot Chinese cabbage, and the cotyledon of catching an illness is water soaking mode, and morbidity back 2-5d tikka is inwardly expanded from leaf margin, and root marrow blackening behind the 7d finds behind the 10d that seedling is withered.The incidence of disease of processed group significantly is lower than control group, and this group Chinese cabbage has only fragmentary morbidity, and the state of an illness is not serious, no withered seedling.Experimental result shows that the plant color and luster of using microbial inoculum A is dark green, grows fine.Preventive effect reaches 79.2% behind the 2nd dispenser 15d of microbial inoculum A.Continue to use Bdellovibrio-oligosaccharides in the Chinese cabbage process of growth, disease seldom takes place, and is best in quality when ripe, and average individual plant weight overweights control group and 4000 times of liquid groups of agricultural streptomycin, compares average single weightening finish 15.1% with control group.
Embodiment 8 control custard squash root rot
(1) preparation of Bdellovibrio bacterium liquid is with embodiment 1 step (1).
(2) preparation of the microbial inoculum of control bacterial diseases of crops
BDS01 Bdellovibrio liquid and BDSM08 Bdellovibrio liquid are pressed bacterial population mixing in 1: 1, regulate thalline number to 10 with phosphate buffer (0.2mol/L, pH7.2～7.6)
7Pfu/mL adds 5g chitosan oligosaccharide, 5g agaropectin oligose, 5g ligoglucoside, 5g algin oligosaccharide and 5g oligomeric maltose in every liter of mixed bacteria liquid, shake 30s with magnetic stirring apparatus, obtains preventing and treating the microbial inoculum of bacterial diseases of crops.
(3) the microbial inoculum control custard squash root rot of the control bacterial diseases of crops of applying step (2) preparation
Using microbial inoculum prevents and treats the custard squash root rot: this test is located at the heavier Guangzhou Tianhe District Cen Cun of morbidity over the years, and the soil types of test region is a sandy loam, and soil property is fertile.Experimental period mean temperature 21.5-25.3 ℃, relative moisture is 91%-95%.Pumpkin variety is for cold beautiful.Every sub-district 3m is established in test
2, 12 strains of plantation custard squash.The contrast medicament is 500 times of liquid of 75% tpn wetting powder, and clear water is established three sub-districts, as blank.Evenly irritate in the plant root with above Bdellovibrio oligosaccharides mixed liquor and tpn solution by the sub-district respectively during the land for growing field crops field planting, 150mL is irritated in every strain, and each handles three replicated plots.Irritate every 15d later on and execute continuous 2 times 1 time.The every sub-district of statistics diseased plant number behind 5d, 10d and the 15d carries out pilot survey and calculates preventive effect with reference to the pesticide field efficacy medicine test criterion after the 2nd dispenser.
After the field planting, the land for growing field crops custard squash begins to take place root rot.In this test, clear water group morbidity is heavier, and disease plant presents water soaking mode at first, and stem dwindles withered, and sick place begins that putrefactive phenomenon is arranged behind the 8d, the vascular bundle browning, and serious wilting of later stage is withered.The processed group custard squash grows fine, and rarely catches an illness.Morbidity strain scab does not spread, and the sign that fades away.Experiment shows, uses that preventive effect is 82.0% behind the 15d for the 2nd time, is better than 500 times of liquid of 75% tpn wetting powder.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1, a kind of microbial inoculum of preventing and treating bacterial diseases of crops, it is characterized in that: described microbial inoculum comprises Bdellovibrio and oligosaccharides.
2, the microbial inoculum of control bacterial diseases of crops according to claim 1 is characterized in that: contain 10 in every liter of described microbial inoculum
6～10
12The Bdellovibrio of pfu and 10～30g oligosaccharides.
3, the microbial inoculum of control bacterial diseases of crops according to claim 2 is characterized in that: described Bdellovibrio is at least one strain among Bdellovibrio (Bdellovibrio sp.) BDS01, Bdellovibrio (Bdellovibriosp.) BDS02 or Bdellovibrio (Bdellovibrio sp.) BDSM08.
4, the microbial inoculum of control bacterial diseases of crops according to claim 2 is characterized in that: described Bdellovibrio is that Bdellovibrio BDS01 mixed than 1: 1 with bacterial population with Bdellovibrio BDSM08.
5, the microbial inoculum of control bacterial diseases of crops according to claim 2 is characterized in that: described oligosaccharides is at least a in chitosan oligosaccharide, agaropectin oligose, ligoglucoside, algin oligosaccharide or the oligomeric maltose.
6, the preparation method of the microbial inoculum of each described control bacterial diseases of crops of claim 2～5 is characterized in that: is that to be adjusted to bacterial concentration be 10 for 7.2～7.6 phosphate buffer with Bdellovibrio with DNB medium, water, physiological saline or 0.2mol/L pH value
6～10
12Add oligosaccharides behind the pfu/L, the addition of oligosaccharides is 10～30g/L, and mixing obtains preventing and treating the microbial inoculum of bacterial diseases of crops.
7, the application of the microbial inoculum of each described control bacterial diseases of crops of claim 1～5 is characterized in that: the microbial inoculum of described control bacterial diseases of crops is sprayed stem, leaf, fringe, the fruit of plant or irritates root drench root; The content of Bdellovibrio is at least 10 in the microbial inoculum of described control bacterial diseases of crops
3Pfu/mL.
8, according to the application of the microbial inoculum of the described control bacterial diseases of crops of claim 7, it is characterized in that: the microbial inoculum of described control bacterial diseases of crops sprays stem, leaf, fringe or the fruit of plant; The microbial inoculum of perhaps described control bacterial diseases of crops is used to irritate root and drenches root.
9, the application of the microbial inoculum of described according to Claim 8 control bacterial diseases of crops is characterized in that: the microbial inoculum of described control bacterial diseases of crops sprays stem, leaf, fringe or the fruit of plant, and consumption is minimum to be 80mL/m
2
The microbial inoculum of described control bacterial diseases of crops is used to irritate root and drenches root, and consumption is minimum to be the 100mL/ strain.
10, according to the application of the microbial inoculum of the described control bacterial diseases of crops of claim 7, it is characterized in that:
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat soft rot of crucifers, black rot, root rot, and using method is drenched root or sprayed stem or leaf for irritating root;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat cucumber and leguminous plant bacterial angular leaf spot, and using method is drenched root for irritating root, sprayed stem, leaf or fruit;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat solanaceous vegetable bacterial wilt, soft rot, canker, and using method is drenched root for irritating root, sprayed stem, leaf or fruit;
The microbial inoculum of described control bacterial diseases of crops is used to prevent and treat gramineae farm crop bacterial stripe, bacterial leaf-blight, and using method is for spraying stem, leaf or fringe.
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