CN1347981A - Pseudomonas stutzeri Zh9944 and its screening method and bacterial wilt preventing and controlling microbial prepn - Google Patents
Pseudomonas stutzeri Zh9944 and its screening method and bacterial wilt preventing and controlling microbial prepn Download PDFInfo
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to one kind of bacteria and one kind of bacterial wilt preventing and controlling microbial preparation with the bacterium as main component. The said bacterium is pseudomonas stutzeri Zh9944. The said bacterial wilt preventing and controlling microbial preparation has pseudomonas stutzeri Zh9944 as active component, graminesous plant seed coat powder or stem powder as carrier and a water content of 8-15 %, and the number of live bacteria in each gram carrier is greater than 50 billion CFU. The microbial preparation is one kind of new pesticide with high efficiency, no toxicity, high safety and no residue and has powerful inhibition effect on bacterial wilt of tobacco, tomato, peanut, eggplant, chilli, etc. caused by pseudomonas.
Description
The present invention relates to the microorganism formulation of the bacterial wilt of a kind of bacterium and screening technique thereof and the control crop take this bacterium as main component such as tobacco, tomato etc.
Fallen ill in tobacco, tomato, peanut, eggplant, capsicum etc. crop by the bacterial wilt that the bacterial wilt pseudomonad causes, not yet find up to now effective agricultural chemicals and prevent and treat method.
The purpose of this invention is to provide a kind of Pseudomonas stutzeri and screening technique thereof and the microorganism formulation of preventing and treating bacterial wilt take this bacterium as main component.
Realize that the technical scheme that a kind of Pseudomonas stutzeri is provided in the object of the invention is: Pseudomonas stutzeri (Pseudomonas stutzeri) Zh9944 (being designated hereinafter simply as the PSZh9944 bacterial strain), separation screening occurs in the collected specimens in area and get from Greater, Changzhou, China Jiangsu Province bacterial wilt of tomato in March, 1999, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 26th, 2000, it is referred to as CGMCC, and deposit number is NO.0494.
The separation screening of above-mentioned Pseudomonas stutzeri and the preparation method of bacterial strain are, the method that adopts the mutual antagonism screening model of plate to combine with directed screening, its technology path is: (1) gathers root, stem and the rhizosphere soil of morbidity strain and healthy strain in the field in the bacterial wilts such as tobacco, tomato generation area; (2) isolated strains from collected specimens; (3) by the mutual antagonistic effect of plate between isolated strains and the pathogenicity of isolated strains, distinguish antagonistic strain and pathogenic strains two classes; (4) choose antagonistic strain with correlation between the analysis of the biological parameters such as antagonistic rate, anti-antagonistic rate Antagonistic Fungi, pathogen and other classes and at division bacteria genus false pseudomonas bacillus (Pseudomonas spp.) or bacillus (Bacills spp.); (5) antagonistic strain of selecting is not less than 60% by residential quarter, field multiple spot test take field efficacy and selects effective strain as benchmark.
The PSZh9944 bacterial strain has following character:
1, morphological feature
With light microscope and electron microscope observation bacterial strain. The result shows: bacterial strain is single rhabdocyte, and size is 0.8 * 1.5～3.8 microns, and the Gram’s staining thalline is redness, is negative, do not form gemma, move with single utmost point hair of giving birth to, produce the verdigris color water colo(u)r at nutrient agar, do not produce fluorchrome and pyocyanin.
On nutrient agar, bacterium colony is circular, smooth surface, and glossy, the edge is more neat, and powder blue is translucent, cultivates 48 hours for 30 ℃, and colony diameter is in 0.5～2.0 millimeter.
2, the physiological and biochemical property of bacterial strain
Obligate is aerobic, under anaerobic can carry out denitrification, does not need growth factor, does not store up the Poly-β-hydroxybutyric Acid salt particle in cell, gelatin hydrolysate, and hydrolyzed starch does not produce levulan on SM. Other physiological and biochemical property is as follows:
Oxidizing ferment (+); Catalase (+);
O/F is determined as oxidized form and produces acid; H2S (-)；
Lysine decarboxylase (-); Arginine dihydrolase (-);
M.R. reaction (-); Tryptophan deaminase (-);
Indole test (+); V.P. reaction (-);
Urase (-); 41 ℃ of growths (+);
Arginine decarboxylase (+).
The G (-) because this bacterial strain gram test is negative, straight is shaft-like, move with single utmost point hair of giving birth to, oxidizing ferment (+), O/F are oxidized form, under anaerobic can carry out denitrification, catalase is positive, does not accumulate the Poly-β-hydroxybutyric Acid salt particle in cell, does not produce fluorchrome, 41 ℃ of features such as energy growth, can be classified as Pseudomonas stutzeri (Pseudomonas stutzeri).
Bacterial strain PSZh9944 and Pseudomonas stutzeri (Pseudomonas stutzeri) type strain relatively exists different as follows: on cultural characteristic, the growth-gen adhesion is on culture, all the time smooth, be powder blue, produce the verdigris color water colo(u)r at nutrient agar, at Physiology and biochemistry in nature, the PSZh9944 gelatin hydrolysate, hydrolyzed starch can not utilize maltose.
Realize that a kind of technical scheme of preventing and treating the microorganism formulation of bacterial wilt that provides in the object of the invention is: this microorganism formulation is pulvis, take grass kind skin powder, stem powder as carrier, take Pseudomonas stutzeri (Pseudomonas stutzeri) Zh9944 CGMCC NO.0494 as active component, every gram carrier contains more than several 50,000,000,000 CFU of active bacterium.
Above-mentioned carrier can be rice chaff powder, wheatfeed, rice straw powder, straw powder, kaoliang stalk powder or milled powders of cornstalk.
Preferred carrier is the rice chaff powder, and the granularity of rice chaff powder is being as the criterion by 20～60 mesh sieves, and the color of pulvis is that brown is to khaki.
The preferable range that above-mentioned carrier contains active bacterium number is every gram 1000～6,000 hundred million CFU.
Cultural method
Can send out the tank method pure with the microorganism formulation of routine and cultivate PSZh9944. The nutrient solution of gained mixes with the rice chaff powder in certain proportion, and material moisture should be reduced in 15%, is about 8%～15%, namely makes microorganism formulation of the present invention. Cultural method is Liquid Culture, and the bacterium suspension of producing bacterium is inoculated on the culture medium gas agitating that works of going forward side by side.
Nutrient source to culture medium there is no special regulation, and wherein carbon source is starch, glucose, should not use sucrose. Nitrogenous source can be peptone, soy meal, meat extract, corn steep liquor etc., and other nutrient source can suitably add such as phosphate, magnesium sulfate etc.
Temperature is limited between 29～31 ℃, the pH value can remain on disappear after 6.9～7.0, incubation time is 20～24 hours. Send out tank pure and cultivate stop after, in time nutrient solution is mixed to be fit to ratio with rice chaff, and under the condition of drying ventilation, making material moisture is in 8%～15% that the viable bacteria amount remains on more than every gram 50,000,000,000 CFU.
Microorganism formulation such as above gained has following physics and biological property:
1, shape: brown is to the khaki powder.
2, contain the viable bacteria amount: more than every gram 50,000,000,000 viable bacterias; Containing the viable bacteria amount in the following toxicity test is every gram 3,000 hundred million viable bacterias.
3, acute toxicity classification: rat oral LD50＞4640mg/kg, rat is through skin LD50＞2000mg/kg. Press GB-15670-1995 " agriculture chemical registration toxicology test method " Pesticides acute toxicity grading criteria, belong to the lower toxicity agricultural chemicals.
4, acute ocular skin irritatin classification: acute eye irritation score index is 2 (1 hours), and the acute skin irritation score value is 0 (1 hour). Press GB-15670-1995 " agriculture chemical registration toxicology test method " Pesticides acute skin eye irritation grade scale, to eyes, the equal nonirritant of skin.
5, to the toxicity safety evaluatio of birds, fish, honeybee, silkworm:
To birds, inject gavage with the direct per os of the disposable dosage of solution that is higher than one times of the highest working concentration, without the phenomena of mortality and other poisoning symptoms, illustrate that quail is to the tolerance dose LD of pulvis through 10 days experimental observations0＞100 hundred million CFU/kg, therefore, normally using this biologic product is safe to birds.
To fish, zebra fish is in the solution that is higher than one times of the highest working concentration, and through all experimental observations, without the phenomena of mortality and other poisoning symptoms, zebra fish is to the maximal tolerable concentration LC of pulvis0＞300 times of dilutions (10,000 hundred million CFU/ liter). Therefore, normally using this biologic product is safe to fish.
To honeybee, with the liquid of field usual amounts and twice field usual amounts honeybee is carried out stomach virus killing property and the toxicity test of tagging, tested honeybee poisoning symptom and the phenomena of mortality all do not occur at experimental observation in the phase. This biologic product is to the honeybee low toxicity, when normally using to honeybee safety.
To silkworm, feed with the impregnated mulberry leaf of liquid of field usual amounts and twice field usual amounts and to play silkworm tested two ages, change after 24 hours to feed and do not soak the medicine mulberry leaf, tested silkworm is at 24 hours, 48 hours, rises to three ages in observation period of silkworm poisoning symptom and death not to occur. Continue to observe and rise to four ages, growth is normal. Above result shows that this biologic product normally uses silkworm safety the silkworm low toxicity.
The present invention has positive effect: (1) can be controlled without any agricultural chemicals up to now because the bacterial wilt that causes because of Pseudomonas solanacearum is on the rise (comprising rising and the expansion in susceptible degree, crop and area). The bacterial isolates that can be effective to prevent and treat bacterial wilt provided by the present invention produces microbial pesticide take PSZh9944 as main component, is the novel microbial agricultural chemicals of a kind of efficient, nontoxic, safety, noresidue. (2) PSZh9944 can be the former bacterium of susceptible crop inside and outside antagonism bacterial wilt. This bacterial strain has the ability of breeding in susceptible crop rhizosphere soils, and assembles and enter root tissue inside to crop root, upwards is transferred to stem. And be colonizated between the phloem parenchyma cells of root and stem, consist of so-called " antagonism defence line ", and form intergrowth relation with susceptible crop. PSZh9944 becomes corresponding relation to the size of Pseudomonas solanacearum antagonistic ability with bacterium amount to Field inoculation PSZh9944. (3) microorganism formulation of the present invention has very strong inhibitory action to the bacterial wilt of the plants such as the tobacco that causes because of Pseudomonas solanacearum, tomato, peanut, eggplant, capsicum, and field efficacy reaches about 60～80%. (4) PSZh9944 of the present invention is that main microorganism formulation not only has therapeutic action to the bacterial wilt evil that Pseudomonas solanacearum causes, and makes and applied thing and have prevention effect to the bacterial wilt evil, can also stimulate plant growth. (5) when making described microorganism formulation, with fiber substance, especially more suitable take the rice chaff powder as carrier adsorption, can not or mix use with other fungicide compoundings.
Below with regard to manufacturing of the present invention, the test example illustrate.
Production Example
With glucose 1.5%, starch 2.0%, dusty yeast 1.5%, peptone 0.4%, MgSO4Be 0.05%, KH2PO
4Be 0.05%, CaCO3Be that 0.6% ratio is made culture medium, pH value is 7.2～7.4 before the sterilization, at 70 milliliters of culture mediums of 250 milliliters of triangular flasks placements, PSZh9944 is inoculated in this culture medium. Shaken cultivation 48 hours is as seed liquor under 30 ℃, 220r.p.m, gets the 1000ml seed liquor in 1 ton is sent out tank pure, the culture medium of the above-mentioned same ratio that interior Sheng is 600 liters. At 30 ℃, to fermentation tank ventilate, stir culture 20～24 hours. After cultivating termination, in time be that the rice chaff of 40 mesh sieves mixes with 1: 1～1: 3 ratio with nutrient solution and granularity, aeration-drying to material moisture is 8～15%, and the viable bacteria amount reaches 1000～6,000 hundred million CFU/g, and color and luster is that khaki is to brown.
The test and application example
According to the nosogenesis of bacterial wilt, for the control of this type of disease, can received method can only be main prevention method in order to biological control, reach to stop with this and hide in ralstonia solanacearum invaded plants root and develop into disease. Therefore, use biologic product of the present invention opportunity should sow, heel in, after field planting and the field planting 20～60 days, 4～5 times altogether, total consumption is 2～2.5kg (300,000,000,000 CFU/g), in use take water as " solvent ", thalline is discharged, preventive effect can reach 60～80%. Concrete test example sees Table 1, application examples sees Table 2. Table 1
Table 2
| Test number | The crop title | Test site | The test time | For examination area (mu) | Processing method | Process | Contrast | Preventive effect % | ||
| Diseased plant rate % | Disease index | Diseased plant rate % | Disease index | |||||||
| 1 | Tomato | The changzhou | 2000 | 1.0 | Seed soaking+seedbed is sprinkled and is watered, altogether once. 150g/ mu | 10.13 | 10.13 | 14.96 | 16.51 | 38.6 |
| 2 | Tomato | The changzhou | 2000 | 1.0 | Seed soaking+seedbed is sprinkled and is watered, and heels in and waters root, twice totally. 11kg/ mu. | 40 | 64 | 37.5 | ||
| 3 | Tomato | The changzhou | 2000 | 1.0 | Seed soaking+seedbed is sprinkled and is watered, and heels in and waters root, and root is watered in field planting, totally three times. 1.5kg/ mu | 40.0 | 87.0 | 54.0 | ||
| 4 | Tomato | The Nanchang | 1999 | 73 | Seed soaking+seedbed is sprinkled and is watered, and heels in and waters root, and root is watered in field planting, waters root after the field planting, totally four times. 2kg/ mu | 8.7 | 4.8 | 33 | 19.7 | 75.6 |
| Test number | The crop title | Test site | The test time | For examination area (mu) | Processing method | Process | Contrast | Preventive effect % | ||
| Diseased plant rate % | Disease index | Diseased plant rate % | Disease index | |||||||
| 1 | Tomato | The changzhou | 1999 | 20 | Seed soaking+seedbed is sprinkled and is watered, and heels in and waters root, and root is watered in field planting, waters root after the field planting, totally four times. 2kg/ mu. | 4.2 | 40.7 | 89.7 | ||
| 2 | Tomato | The Nanchang | 2000 | 50 | The same | 18.1 | 89.2 | 79.4 | ||
| 3 | Tobacco | Chenzhou, Hunan Province | 1999 | 10 | The same | 44.67 | 22.57 | 77.28 | 51.01 | 55.05 |
Claims (6)
1, a kind of Pseudomonas stutzeri is characterized in that: pseudomonas stutzeri Zh 9944, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 26th, 2000, and it is referred to as CGMCC, and deposit number is NO.0494.
2, the preparation method of the screening of a kind of Pseudomonas stutzeri and bacterial strain is, the method that adopts the mutual antagonism screening model of plate to combine with directed screening, its technology path is: (1) gathers root, stem and the rhizosphere soil of morbidity strain and healthy strain in the field in the bacterial wilts such as tobacco, tomato generation area; (2) isolated strains from collected specimens; (3) by the mutual antagonistic effect of plate between isolated strains and the pathogenicity of isolated strains, distinguish antagonistic strain and pathogenic strains two classes; (4) choose antagonistic strain with correlation between the analysis of the biological parameters such as antagonistic rate, anti-antagonistic rate Antagonistic Fungi, pathogen and other classes and division bacteria genus pseudomonas or bacillus; (5) antagonistic strain of selecting is not less than 60% by residential quarter, field multiple spot test take field efficacy and selects effective strain as benchmark.
3, a kind of microorganism formulation of preventing and treating bacterial wilt, this microorganism formulation is pulvis, it is characterized in that: take grass kind skin powder, stem powder as carrier, take pseudomonas stutzeri Zh 9944 CGMCC NO.0494 as active component, every gram carrier contains more than several 50,000,000,000 CFU of active bacterium; Water content is 8～15%.
4, the microorganism formulation of preventing and treating bacterial wilt according to claim 3 is characterized in that: carrier is rice chaff powder, wheatfeed, rice straw powder, straw powder, kaoliang stalk powder or milled powders of cornstalk.
5, the microorganism formulation of preventing and treating bacterial wilt according to claim 4 is characterized in that: carrier is the rice chaff powder, and the granularity of rice chaff powder is being as the criterion by 20～60 mesh sieves, and the color of pulvis is that brown is to khaki.
6, according to claim 3 to one of the 5 described microorganism formulations of preventing and treating bacterial wilt, it is characterized in that: every gram carrier contains several 1000～6,000 hundred million CFU of active bacterium.
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| CNB001190180A CN1159434C (en) | 2000-10-11 | 2000-10-11 | Pseudomonas stutzeri Zh9944 and its screening method and bacterial wilt preventing and controlling microbial prepn |
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| CNA200310124465XA Division CN1515666A (en) | 2000-10-11 | 2000-10-11 | Method for screening pseudomonads for controlling blight |
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| CN1347981A true CN1347981A (en) | 2002-05-08 |
| CN1159434C CN1159434C (en) | 2004-07-28 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101712941B (en) * | 2009-11-30 | 2012-05-30 | 浙江大学 | Paenibacillus polymyxa and application thereof |
| CN102604857A (en) * | 2011-12-22 | 2012-07-25 | 中国农业科学院烟草研究所 | Biological control pseudomonas monteilii strain against tobacco mosaic virus (TMV) |
| CN108570433A (en) * | 2018-05-09 | 2018-09-25 | 中国农业科学院烟草研究所 | One plant of acid proof defence pseudomonad CLP-6 and its application |
| CN109207398A (en) * | 2018-09-20 | 2019-01-15 | 宁国市百立德生物科技有限公司 | One plant of Pseudomonas stutzeri and its application |
-
2000
- 2000-10-11 CN CNB001190180A patent/CN1159434C/en not_active Expired - Lifetime
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101712941B (en) * | 2009-11-30 | 2012-05-30 | 浙江大学 | Paenibacillus polymyxa and application thereof |
| CN102604857A (en) * | 2011-12-22 | 2012-07-25 | 中国农业科学院烟草研究所 | Biological control pseudomonas monteilii strain against tobacco mosaic virus (TMV) |
| CN102604857B (en) * | 2011-12-22 | 2013-09-04 | 中国农业科学院烟草研究所 | Biological control pseudomonas monteilii strain against tobacco mosaic virus (TMV) |
| CN108570433A (en) * | 2018-05-09 | 2018-09-25 | 中国农业科学院烟草研究所 | One plant of acid proof defence pseudomonad CLP-6 and its application |
| CN108570433B (en) * | 2018-05-09 | 2020-07-31 | 中国农业科学院烟草研究所 | Acid-resistant pseudomonas defensins C L P-6 and application thereof |
| CN109207398A (en) * | 2018-09-20 | 2019-01-15 | 宁国市百立德生物科技有限公司 | One plant of Pseudomonas stutzeri and its application |
| CN109207398B (en) * | 2018-09-20 | 2020-10-09 | 宁国市百立德生物科技有限公司 | Pseudomonas stutzeri and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1159434C (en) | 2004-07-28 |
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