CN102154157B - Bio-control strain 4AT8 capable of preventing and curing various bacterial diseases - Google Patents
Bio-control strain 4AT8 capable of preventing and curing various bacterial diseases Download PDFInfo
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- CN102154157B CN102154157B CN2010106086629A CN201010608662A CN102154157B CN 102154157 B CN102154157 B CN 102154157B CN 2010106086629 A CN2010106086629 A CN 2010106086629A CN 201010608662 A CN201010608662 A CN 201010608662A CN 102154157 B CN102154157 B CN 102154157B
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Abstract
The invention provides a bio-control strain 4AT8 capable of preventing and curing various bacterial diseases, belonging to the technical field of plant protection. Bacterium is fluorescent pseudomonas. The preparation method of the bio-control strain comprises the following steps of: vaccinating the bacterium in culture solution developed by an experiment; fermenting for 20-24h at 250rpm and under the temperature of 30 DEG C; centrifuging for 10min at 6000rpm; and preparing bactericide according to the proportion of wet bacterium to bacterium preservative fluid as 1:40 (g/ml), and the total concentration of the viable bacteria in the final product is 1*109-1*1012cfu/ml. The bio-control strain has 60-75% efficiency on various bacterial diseases among fields such as solanaceae crop bacterial wilt, canker, melon and fruit bacterial angular leaf spots, xanthomonas oryzae and the like; and the bactericide also has the functions of improving the soil microenvironment, and the like.
Description
One, technical field
The present invention relates to a kind of biocontrol strain 4AT8 that prevents and treats the various bacterial disease, belong to the farm crop field of preventing and curing diseases, be exclusively used in the non-environmental pollution control of vegetables and fruits class various bacterial disease.
Two, background technology
Bacterial disease is the another kind of Plant diseases that is different from fungal disease, on chamber vegetable crop, takes place very general.And almost be soil-borne disease entirely, should the strict comprehensive preventive health measures of carrying out soil-borne disease in control.But, being different from soil-borne fungus venereal disease evil again, in a single day plant catches an illness, and be mostly whole strain infection, and PD is rapid, can be very fast dead.What on plant of Solanaceae, take place usually has: bacterial wilt of tomato, canker of tomato, tomato soft rot, tomato shot hole, peppery (pimento) bacterial wilt, peppery (pimento) soft rot, peppery (pimento) shot hole, eggplant bacterial wilt etc.; The bacillary fruit blotch of cucumber etc. is normal one type of disease finding on the melon or fruit type crops; Paddy rice bacterial leaf spot, bacterial leaf streak of rice etc. also are the important diseases that influences the grain yield quality.
The solanaceous crops bacterial wilt that belongs to one of worldwide disease, be by
Ralstonia solanceariumThe bacterialo wilt disease that causes, this disease is many to begin morbidity at fruiting period, and their early stage is wilted daytime, and recover night, after 2-3 days, no longer recover, withered death, but plant still keeps green; The basal part of stem vascular bundle browning of disease plant with the hand extruding, has oyster white mucus to ooze out.This germ can endanger 300 various plants of section more than 50.Generally be injured heavier with cash crop such as solanaceous vegetables such as tomato, yam, sesame, peanut, soybean, capsicum and tobacco, bananas.Along with the discovery of new host, and temperature changes factor such as warm, and it is more and more general that bacterial wilt takes place, and be on the rise, and the annual financial loss that causes in the world is no less than 12,000,000,000 yuan.
Canker of tomato also is one of more common Micobial Disease, is mostly to put in order strain and catches an illness, and the upwards vertical volume of their early stage plant lower blade, wilting sagging; The wilting phenomenon appears in the part plant, and in the morbidity later stage, sick leaf leaf margin is withered and yellow; Flavescence between vein, full leaf browning is withered, but does not come off; And can cause stem long and narrow streak to occur, later stage scab cracking; Also can produce a large amount of aerial roots, stem marrow hollow is rotted, and gives out stink, and plant is withered when serious.Canker of tomato is a kind of destructive disease, from 1985 after Beijing finds, in succession in the Inner Mongol, province, autonomous regions such as Shanxi, Hebei, Heilungkiang, Jilin, Liaoning cause harm.The plot tomato underproduction of serious morbidity reaches 25% one 75%.China classifies it as Quarantine Objects to prevent and to control spreading of disease.
The bacterial angular leaf spot of melon and fruit class also is that the greenhouse canopy is planted under the condition relatively one of severe diseases of morbidity, and this germ is a pseudomonas, all can take place in the whole growing of booth and open country cucumber.The main harm blade also endangers the climing and fruit of stem.When blade was injured, leaf front scab was filbert, and the back side receives the vein restriction to be polygon, the visible oyster white bacterial ooze in surface; Scab on the fruit can inwardly be expanded, and spreads to seed.At booth and open country, morbidity is all compared serious, between 30 ~ 50%, can reach 100% when serious.
The paddy rice bacterial leaf spot of serious harm paddy rice is the bacterial disease that is caused by xanthomonas.There is the yellow-green colour spot at plant initial stage blade tip and edge, form pale asphyxia, the rectangular spot of tawny along vein afterwards, become pearl at last and withered.The morbidity strain lodges easily, can have a strong impact on the spike of rice setting percentage.Generally can make the paddy rice underproduction 20 ~ 30%, can reach 50 ~ 60% when serious.。
At present, for bacterial disease, in chemical prevention, for a long time, how with agricultural streptomycin, can kill, first frost copper, DTM etc.But because this similar drug of life-time service, most bacterial diseases have produced very strong resistance to it, prevent and treat with this similar drug again, and weak effect has been difficult to control germ harm and development.Simultaneously, mostly bacterial disease is soil-borne disease, and it is wide to have host range; Strong stress resistance is difficult to characteristics such as control, aborning, lacks the effective chemical agricultural chemicals and prevents and treats; And chemical pesticide is understood polluted water, soil etc., destruction Soil structure, influence ecological environment; Directly the mikrobe of the non-target of harm is absolutely unsafe to people and animals, and these factors all make the use of chemical pesticide obtain serious limit.Therefore, property prophylactico-therapeutic measures has non-chemically become the core of control bacterial disease at present, and biological control then is the most promising method wherein.In the prior art according to this breadboard preservative fluid for thallus method, but thalline room temperature preservation 2～3 years.
Three, summary of the invention
Technical problem:The objective of the invention is to use chemical pesticide seriously polluted to current control plant various bacterial disease; Use cost is too high; And the problems such as biological prevention and control agent that the various bacterial disease do not had simultaneously higher preventive effect in the prior art; A kind of single biocontrol microorganisms bacterial strain and preparation of preventing and treating the various bacterial disease is provided, and the preventive effect of this microbial inoculum is more than 60%.
Technical scheme
A kind of biocontrol strain 4AT8 that prevents and treats the various bacterial disease, Pseudomonas fluorescens (
Pseudomonas fluorescens), being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 13rd, 2010, culture presevation number is CGMCC NO.3834.Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Biocontrol strain 4AT8 is used to prepare the method for biocontrol fungicide,
1) with this inoculation in the LB nutrient solution 30 ℃ cultivate the 180rpm shaking culture, after 12～16 hours in Bechtop the sampling OD value of surveying the 600nm place respectively, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2), will plant daughter bacteria liquid and add in the LB nutrient solution, 30 ℃ of 250rpm shaking culture 20～24 hours with the 1:100 volume ratio when being cultured to the OD value between 0.5～0.8 the time.The centrifugal 10min of 4000rpm then, every 1000ml nutrient solution obtain the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline.Get wet bacterium and preservative fluid for thallus and be made into microbial inoculum by 1 g:40ml, the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Cfu/ml.
Wherein, seed culture LB nutrient solution prescription: sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L, and regulating the pH value is 7.0; Preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, also contains the Tween20 that concentration is 0.001mol/L.
Said biocontrol fungicide can be used to prevent and treat bacterial diseases such as solanaceous crops bacterial wilt, Peptic Ulcers, melon and fruit bacterioid property angular leaf spot, paddy rice bacterial leaf spot.
Beneficial effect
Advantage of the present invention and positively effect show: the present invention is specially to the biological prevention and control agent of various bacterial diseases such as the bacillary fruit blotch of bacterial wilt, canker of tomato, cucumber of plant of Solanaceae, paddy rice bacterial leaf spot exploitation.Owing to be biotechnological formulation; There are not a series of problems that use brought fully because of chemical pesticide; Thereby help the nuisanceless production of farm crop such as vegetables and fruits class and paddy rice; The peasant can or reduce the consumption of other chemical pesticides, and this not only can be the peasant and reduces expenses, and helps the export trade of related prods.
Biocontrol microorganisms 4AT8 thalline and supernatant show in dull and stereotyped antagonistic effect has good inhibitory effect to bacterial wilt of tomato, canker of tomato, cucumber bacterial stripe, the former bacterium of bacterial blight of rice; Greenhouse test shows that this microbial inoculum all has remarkable prevention effect to bacterial wilt of tomato, canker of tomato, cucumber bacterial stripe, bacterial blight of rice; The warmhouse booth test shows that this microbial inoculum has preventive effect preferably to bacterial wilt of tomato, canker of tomato, cucumber bacterial stripe, bacterial blight of rice, and preventive effect is 60 ~ 75%.
This microbial inoculum is made into microbial inoculum with wet bacterium and this breadboard preservative fluid for thallus by 1:40 (g/ml), but room temperature preservation 2～3 years.This microbial inoculum is irritated except can transplant the time root uses again, and can also before transplanting, be mixed and made into compost with fertilizer, buries in the soil in whole ground process with fertilizer then and uses.
Four description of drawings
Fig. 1 biological and ecological methods to prevent plant disease, pests, and erosion Pseudomonas fluorescens 4AT8 thalline and the antagonism result of fermentation supernatant to the bacterial wilt pathogenic bacteria;
Fig. 2 biological and ecological methods to prevent plant disease, pests, and erosion Pseudomonas fluorescens 4AT8 thalline and the antagonism result of fermentation supernatant to the canker of tomato pathogenic bacteria;
Fig. 3 biological and ecological methods to prevent plant disease, pests, and erosion Pseudomonas fluorescens 4AT8 thalline and the antagonism result of fermentation supernatant to the former bacterium of bacterial blight of rice.
Five embodiments
A kind of biocontrol strain 4AT8 that prevents and treats the various bacterial disease, Pseudomonas fluorescens (
Pseudomonas fluorescens), from the wheat rhizosphere soil of Hebei, separate obtaining.
Biocontrol strain 4AT8 is used to prepare the method for biocontrol fungicide,
1) with this inoculation in the LB nutrient solution 30 ℃ cultivate the 180rpm shaking culture, after 12～16 hours in Bechtop the sampling OD value of surveying the 600nm place respectively, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time, will plant daughter bacteria liquid and add in the LB nutrient solution with the 1:100 volume ratio, 30 ℃ of 250rpm shaking culture 20～24 hours.The centrifugal 10min of 4000rpm then, every 1000ml nutrient solution obtain the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline.Get wet bacterium and preservative fluid for thallus and be made into microbial inoculum by 1 g:40ml, the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Cfu/ml.Wherein, seed culture LB nutrient solution prescription: sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L, and regulating the pH value is 7.0; Preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, also contains the Tween20 that concentration is 0.001mol/L.
Above-mentioned biocontrol fungicide is used to prevent and treat bacterial diseases such as solanaceous crops bacterial wilt, Peptic Ulcers, melon and fruit bacterioid property angular leaf spot, paddy rice bacterial leaf spot.
Implementation method: the dull and stereotyped antagonistic effect of (1) Pseudomonas fluorescens 4AT8 thalline and supernatant
Selection confession examination pathogenic bacteria tomato Ralstonia solanacearum ZJ3721 (
Ralstonia solanacearum, JG Li et al., 2010, PopW of Ralstonia solanacearum, a new two-domain harpin targeting the plant cell wall), tomato ulcer ACCC 01233 (
Clavibacter michiganense, buy in Chinese agriculture microbial strains preservation center), bacterial leaf spot ACCC 03530 (
Xanthomonas oryzae, buy in Chinese agriculture microbial strains preservation center).With pathogenic bacteria tomato ulcer bacterium, bacterial leaf spot at NA nutrient solution (beef extract 3g/L, peptone 5g/L, yeast powder 1g/L; Sucrose 10g/L, pH=7.0), with pathogenic bacteria tomato Ralstonia solanacearum at YGPA (glucose 10g/L; Yeast soaks powder 5g/L; Peptone 5g/L,, pH=7.0) middle cultivation 16-24 h to CFU is 10
8/ ml), add fusing and be cooled in the corresponding solid medium about 40 ℃, volume ratio 1:200,1:200 adds 10%TTC (chlorinated triphenyl tetrazole) dyestuff again.Every 20mL pours in the flat board of 9 cm diameters with substratum.After to be cooled the solidifying, toothpick inoculation biocontrol microorganisms 4AT8 or punching add 4AT8 biocontrol microorganisms fermentation supernatant 100ul.Record inhibition zone ring footpath behind 24 h.
(2) in the greenhouse test, sow tomato in the culture medium, cucumber treats that tomato, cucumber grew to for 4 leaf phases, selects the consistent plantlet of transplant of growing way in plastic cup, to make an experiment.With 4AT8 microbial inoculum weaker concn to 1x10
8Irritating root behind the CFU/ml and use, after 7 days, is 1x10 with the pathogenic bacteria weaker concn
7CFU/ml irritates root and uses, and wherein cucumber bacterial angular leaf spot uses the soil of morbidity field piece.
(3) warmhouse booth test
Field test is established three processing: A, biocontrol fungicide 4AT8 altogether; B, chemical agent (the withered peaceful 400 times of liquid of 70% agricultural streptomycin SP1000 times liquid and 25% leaf); C, blank.Each sub-district 16m
2, the triplex row isolated area is established in the minizone, and each is handled by completely random district group and arranges, and repeats 4 times, and complete test is totally 12 sub-districts.Divided the sub-district by testing program before the test, held isolating trenches successfully, every mu of usage quantity is 1000 milliliter 10
9The biocontrol fungicide 4AT8 of CFU/ml.Add up the disease severity of various diseases respectively, and calculate preventive effect.
Test-results:
(1) biocontrol microorganisms 4AT8 antagonism pathogenic bacteria test-results: biological and ecological methods to prevent plant disease, pests, and erosion Pseudomonas fluorescens 4AT8 thalline shows the dull and stereotyped preferably effect that suppresses with the fermentation supernatant to bacterial wilt (Fig. 1), Peptic Ulcers (Fig. 2) and paddy rice bacterial leaf spot (Fig. 3).
(2) greenhouse test shows, biocontrol microorganisms 4AT8 in the greenhouse to the preventive effect of bacterial wilt (table 1), Peptic Ulcers (table 2) and cucumber bacterial angular leaf spot (table 3) more than 80%, pathogenic bacteria has been played the better controlled effect.
(3) field test shows, biocontrol microorganisms 4AT8 has preferably disease resisting effect to bacterial wilt (table 4), Peptic Ulcers (table 5), cucumber bacterial angular leaf spot (table 6) and paddy rice bacterial leaf spot (table 7) to biocontrol microorganisms 4AT8 in the greenhouse.
Table 1: biocontrol fungicide 4AT8 is to the greenhouse control effect of bacterial wilt of tomato
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 12.75 | 85.61 |
| Agricultural streptomycin | 25.71 | 70.98 |
| Blank | 88.59 | —— |
Table 2: biocontrol fungicide 4AT8 is to the greenhouse control effect of canker of tomato
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 17.76 | 78.33 |
| Agricultural streptomycin | 43.33 | 47.13 |
| Blank | 81.95 | —— |
Table 3: biocontrol fungicide 4AT8 is to the greenhouse control effect of cucumber bacterial angular leaf spot
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 23.33 | 71.59 |
| Agricultural streptomycin | 35．71 | 56.53 |
| Blank | 82.14 | —— |
Table 4: biocontrol fungicide 4AT8 is to the field control effect of bacterial wilt of tomato
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 23.75 | 78.82 |
| Agricultural streptomycin | 49.78 | 45.13 |
| Blank | 90.73 | —— |
Table 5: biocontrol fungicide 4AT8 is to the field control effect of canker of tomato
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 26.67 | 69.67 |
| Agricultural streptomycin | 49.19 | 44.06 |
| Blank | 87.93 | —— |
Table 6: biocontrol fungicide 4AT8 is to the field control effect of cucumber bacterial angular leaf spot
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 29.17 | 63.16 |
| Agricultural streptomycin | 42.86 | 45.86 |
| Blank | 79.17 | —— |
Table 7: biocontrol fungicide 4AT8 is to the field control effect of paddy rice bacterial leaf spot
| The medicament title | Disease severity % | Control effect % |
| Preparation 4AT8 | 26.67 | 63.63 |
| Leaf is withered peaceful | 40.00 | 45.46 |
| Blank | 73.33 | —— |
Claims (5)
1. biocontrol strain 4AT8 who prevents and treats the various bacterial disease; Be Pseudomonas fluorescens (Pseudomonas fluorescens); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 13rd, 2010, culture presevation number is CGMCC NO.3834.
2. the biocontrol fungicide for preparing with the said biocontrol strain 4AT8 of claim 1.
3. the application of the said biocontrol fungicide of claim 2 in control solanaceous crops bacterial wilt, Peptic Ulcers, melon and fruit bacterioid property angular leaf spot, paddy rice bacterial leaf spot bacterial disease.
4. the preparation method of the said biocontrol fungicide of claim 2 comprises:
1) the said biocontrol strain 4AT8 of claim 1 is inoculated into 30 ℃ of cultivation 180rpm shaking culture in the LB nutrient solution, in Bechtop, takes a sample respectively after 12～16 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time; To plant daughter bacteria liquid and add in the LB nutrient solution, 30 ℃ of 250rpm shaking culture 20～24 hours, the centrifugal 10min of 4000rpm then with 1: 100 volume ratio; Every 1000ml nutrient solution obtains the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline, get wet bacterium and preservative fluid for thallus by 1g: 40ml is made into microbial inoculum, and the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Cfu/ml; Wherein preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, also contains the Tween20 that concentration is 0.001mol/L.
5. method according to claim 4 is primarily characterized in that, used LB nutrient solution collocation method is: sodium-chlor 10g/L, and Tryptones 10g/L, yeast soak powder 5g/L, and regulating the pH value is 7.0.
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|---|---|---|---|---|
| CN104152380B (en) * | 2014-08-07 | 2017-03-15 | 领先生物农业股份有限公司 | One plant of ultraviolet mutagenesis type Pseudomonas fluorescens and application thereof |
| CN105543133A (en) * | 2015-12-31 | 2016-05-04 | 山东农业大学 | Separation and application of pseudomonas with significant antagonism to plant pathogenic bacteria |
| CN107646873A (en) * | 2017-10-12 | 2018-02-02 | 泰克美生物科技（喀左）有限公司 | A kind of microbial bacterial agent for preventing and treating bacterial plant disease and application |
| CN108192851B (en) * | 2018-03-13 | 2021-03-19 | 长沙艾格里生物肥料技术开发有限公司 | Oriental rhodobacter strain, oriental rhodobacter agent and application thereof |
| CN112522136A (en) * | 2020-11-19 | 2021-03-19 | 贵州省烟草科学研究院 | Pseudomonas and application thereof in controlling bacterial wilt of solanaceae crops |
| CN112608869B (en) * | 2020-12-23 | 2022-02-22 | 山东佐田氏生物科技有限公司 | Saline-alkali-tolerant Argentina pseudomonads as well as viable bacteria preparation and application thereof |
| CN113287454A (en) * | 2021-04-15 | 2021-08-24 | 冯传荣 | Method for improving root rot disease control effect of greenhouse tomatoes by means of frame-falling ridging |
| CN114854627B (en) * | 2022-04-29 | 2023-10-13 | 重庆西农植物保护科技开发有限公司 | Pseudomonas fluorescens for preventing and treating bacterial wilt and application thereof |
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| CN101054565A (en) * | 2007-04-10 | 2007-10-17 | 南京农业大学 | Biological control strain capable of preventing and curing root knot nematode disease for greenhouse vegetable |
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Non-Patent Citations (1)
| Title |
|---|
| JIAN-GANG LI et al.PopW of Ralstonia solanacearum, a new two-domain harpin targeting the plant cell wall.《Mol Plant Pathol.》.2010,第11卷(第3期),全文. * |
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