CN100532538C - Lichen bacillus strain (B-0A12) and preparation thereof - Google Patents
Lichen bacillus strain (B-0A12) and preparation thereof Download PDFInfo
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- CN100532538C CN100532538C CNB2003101155355A CN200310115535A CN100532538C CN 100532538 C CN100532538 C CN 100532538C CN B2003101155355 A CNB2003101155355 A CN B2003101155355A CN 200310115535 A CN200310115535 A CN 200310115535A CN 100532538 C CN100532538 C CN 100532538C
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
One strain for preventing and controlling root rot caused by soybean cytocyst nematosis and fusarium is named as Bucillus licheniformis CGMCC No. 1035. The liquid bacteria preparation for preventing and controlling root rot caused by soybean cytocyst nematosis and fusarium is prepared through inoculation with the said strain, fermentation and addition of additive. The fermentation process has temperature of 27-35 deg.c and pH 6.8-8.0; and the bacteria content in finishing fermentation is 700 million/ml. Corresponding solid preparation and seed coating preparation may be also prepared. The preparation for preventing and controlling soybean cytocyst nematosis and fusarium can also regulate microbial ecologic system around root system, promote plant growth and increase yield.
Description
Technical field
The present invention relates to microorganism field, or rather, relate to and be used to prevent and treat the plant root disease, especially for the lichem bacillus strain and the preparation thereof of soybean cyst nematode and root rot control.
Technical background
Soybean originates from China, and along with the expansion of cultivated area, the meeting next batch of soybean problem is more and more serious.Wherein the onset area of soybean cyst nematode and root rot accounts for more than 70% of soybean acreage, and with a toll of 10-40%, the serious plot of falling ill is emerged irregular, plant is short and small, and yellow is commonly called as " flue seedling ", even causing total crop failure, onset area has increase trend in recent years.
Produce at present to go up soybean Cyst nematode and root rot effective chemical medicine are still adopted furans pellet and formulation of Duofu mixture, the chemical pesticide life-time service causes the pollution of environment, and huge malicious agricultural chemicals-furans pellet is banned use of by country.Therefore utilize biological method control root disease more and more to receive publicity.
Domestic and international application Nematophagous fungi and parasitics bacterium control plant nematode research report more (Lin Maosong, 1990; Liu Xingzhong etc., 1991,1992), the fungi Pochonia chlamydosporum of control soybean Cyst nematode, Paecilomyceslilacinus has entered field experiment (Liu Xingzhong, the patent No.: ZL95121114.5; Pan Cangsang, the patent No.: CN1149393, CN1149394; Liu Weizhi, the patent No.: CN1225236), but since the characteristics of fungi itself make its application be restricted as: 1) the general fermentation period of fungi preparation is long, the liquid bacterial agent fermentation time needs 5-7 days usually, if adopt the liquid-solid two-phase fermentation, time will be increased to 10-20 days (Pan Cangsang), and cost is increased.2) lack storage period, though liquid bacterial agent carries out seed treatment when being beneficial to northeast big area sowing, storage time is short, has only 3-6 month, is unfavorable for commercialization.3) liquid-solid two-phase fermentation, though can make the extended pot life to 18 month of preparation, the liquid-solid two-phase fermentation time prolongs greatly.The liquid-solid two-phase fermentation is generally open tray and cultivates, and floor space is bigger, pollutes easily.Fungi is not obtained important breakthrough as yet at aspects such as formulation, mass production at present.
Though and that the parasitics bacterium Pasteuria penetrans of control nematode studies is more, but successful control nematode infection (Stirling and Wachtel, 1980), but be obligatory parasite because of it, being difficult to artificial culture, the field application is restricted.Rhizosphere is first line that root defence cause of disease infects, so rhizosphere bacteria is strong except having with root avidity, can grow surely outside the characteristics of plant rhizosphere, also have strong resistance short of money and be easy to characteristics such as application, rhizosphere bacteria can prevent and treat pathogenetic bacteria and fungi, promotes plant-growth (Weller, 1988; Li Changsong, 1992; Guo Rongjun, 1996), this is inspired people, and rhizosphere bacteria control plant nematode is explored, and becomes one of focus of current research.
At present the research report bacterial species that is used to prevent and treat plant nematode has Bacillus spp., Pseudomonas spp., Streptomyces spp., Clostridium spp., Serratia sp. and Agrobacterium sp. (Becker, etal., 1988; Oostendorp ﹠amp; Sikora, 1989; Sikora, 1988; Racke ﹠amp; Sikora, 1992; McInnis, 1990; Tian Honglin etc., 1995), Gluconobacter sp., Sporolactobacillus sp. (Guo Rongjun etc., 1998), Acinebacter sp. (Tian Honglin etc., 1995).Use many (the Siddiqui and Malhmood of research report of plant nematode-root knot nematodes such as pseudomonas control tomato, cucumber, beet, cotton, peanut, 1999), some bacterial strain preventive effect in the greenhouse experiment can surpass 60% (Sikora, 1988), even reach 80% (Tian Honglin etc., 1995), but effect is poor in non-sterilization soil, and the preventive effect to root knot nematode reaches 75% in the field to have only Oostendorp and Sikora (1989) report pseudomonas Pseudomonas fluorescensA-59.Use the pseudomonas control nematode patent applied for (patent No.: EP0171381).Pseudomonas is very potential bacterial strain, but when being applied to produce, the using method of most convenient economy is to carry out seed treatment.Pseudomonas non-refractory, microbial inoculum period of storage weak point are so limited its application.
Genus bacillus is relatively good because of the tolerance to poor environment, is convenient to be processed into various formulations and is used widely.Use the subtilis of bacillus, bacillus megaterium, bacillus cereus, more (the Siddiqui and Malhmood of research of Bacillus licheniformis control root knot nematode, 1999), but the plant difference of different sorts nematode infection, the antagonistic bacterium kind that obtains from different plant rhizospheres separation is also different, and these antagonism bacterium are to the reaction of physiological feature (Kloepper etc. also inequality, 1992), therefore each bacterial isolates may be different to the antagonism mechanism of cause of disease, relate to following one or more aspect: kill the line material as producing, produce hydrolase, change root secretion and nematode interaction mode (Oostendorp and Sikora, 1990), produce system induction resistance (Hasky-Gunther etc., 1998) etc.The rhizosphere bacteria of specific rhizosphere can consume or change the secreted form of root secretion, thereby influences the growth (Sikora andHoffmann-Hergarten, 1993) of root secretion dependent form plant nematode.Based on above principle, should be at different plants, Different Kinds of Pathogens screening biocontrol strain.And for most of plants, microbial inoculum and plant itself and be associated with factor such as edatope may be undesirable to another kind of cause of disease effect to the effective bacteria agent of a kind of cause of disease, and this also is the difficult point of Plant diseases biological and ecological methods to prevent plant disease, pests, and erosion.The report of using Bacillus licheniformis control root knot nematode is many, crops such as root knot nematode main harm tomato, cucumber, cotton, the nematode of harm soybean mainly is the soybean Cyst nematode, and Cyst nematode is different with the life history of root knot nematode, is two kinds of Different Kinds of Pathogens.Therefore also need be at soybean Cyst nematode screening soybean rhizosphere bacteria.The inventor once reported the antagonistic action (Guo Rongjun etc., 1998) of different rhizosphere bacteria bacterial strains to root rot and soybean cyst nematode.Do not see at present and use Bacillus licheniformis control soybean cyst nematode and the microbial STUDY ON ROOT ROT DISEASE OF SOYBEAN report of reaping hook.
Summary of the invention
The objective of the invention is based on the microecology principle, a kind of bacterial strain of preventing and treating soybean cyst nematode and root rot is provided.The present invention also provides the bacterial preparation process of this bacterial strain control soybean cyst nematode of a kind of application and root rot.
The separation of bacterial strain:
I gather soil from Su County, Anhui Province soybean root rot and Cyst nematode grave illness ground healthy plant rhizosphere, the chamber planting soybean, when soybean grows 2 compound leaves, take out seedling, shake off the peripheral friable soil of rhizosphere, keep the soil of being close to the root table through 80 ℃ of heating in water bath 10 minutes, dilution is coated on the beef-protein medium then, the bacterium colony of picking different shape is stored in behind the purifying on the beef extract-peptone inclined-plane.Pine root fungus and Cyst nematode are two different cause of diseases, and the two is to soybean infection mechanism difference, and rhizosphere bacteria is also different because of bacterial strain to the antagonism mechanism of soybean Cyst nematode and pine root fungus.The bacterial strain SY-NS that in the greenhouse experiment soybean Cyst nematode is done very well does not show antagonistic action (Guo Rongjun etc., 1998) to root rot.This bacterial strain finds that this bacterial strain has certain restraining effect to big bean curd germ growth when dull and stereotyped antagonism is measured and the body outer suppressioning experiment of nematode detected, antibacterial band about 3mm, to soybean Cyst nematode second instar larvae lethality rate 76.3%.Gramstaining and microscopic examination to this bacterial strain show that this bacterial strain produces gemma, are very potential bacterial strains.
The feature of this bacterial strain: on beef-protein medium, form the oyster white bacterium colony, G dyeing is positive, and thalline is shaft-like, size 0.8 * 2.1-0.9 * 3.9, produce gemma, the gemma multiterminal are given birth to, not obvious expanding, ellipse or column, anaerobic growth, the catalase positive becomes nitrite, utilizes propionic salt at 7% NaCl growth, PH5.7 growth, the V.P positive, hydrolyzed starch, reduction nitrate, therefore is accredited as Bacillus licheniformis (Bacillus licheniformis).This bacterial strain has been preserved in Institute of Microorganism, Academia Sinica's preservation, and preserving number is: CGMCC No.1035.
The inventor uses the Bacillus licheniformis that is separated to and handles seed through fermentation and interpolation recombiner according to little ecological principle, from regulating the little ecology of plant rhizosphere, improves many-sided soybean cyst nematode and root rot of preventing and treating such as the disease resistance of plant own.The microbial inoculum of using the development of this bacterial strain reaches 67.2% to the preventive effect of soybean cyst nematode, to the root rot preventive effect 54.9%.
Thereby the invention provides the soybean rhizosphere bacteria of strain control soybean cyst nematode and root rot.Its name is called Bacillus licheniformis CGMCC No.1035.
Lichem bacillus strain of the present invention is used for a large amount of cultivations, and after adding composite additive, is used for the large area control of soybean cyst nematode and root rot.The present invention also provides a kind of liquid preparation that is used to prevent and treat soybean cyst nematode and root rot.It is characterized in that making by three grade fermemtation technology.It is characterized in that,
The bacterial strain that is used to inoculate is Bacillus licheniformis CGMCC No.1035;
The temperature of liquid fermenting is controlled at 27-35 ℃, is preferably 28-32 ℃; PH is controlled at 6.8-8.0, preferred pH7.0-7.4;
Said three grade fermemtation is to reach 7 * 10 when sporeformer content
8Individual/as can to finish fermentation during ml.
Above-mentioned three grade fermemtation technology is from the test tube original seed, through shaking the three grade fermemtation of bottle, seeding tank, fermentor tank.Inoculum size is 1-3%.Fermentation time is relevant with leavening temperature, air flow etc., and the fermentation time that shakes bottle and seeding tank was generally 36-48 hour, and the fermentation time of fermentor tank is 24-48h.
Nutrient media components is:
Medium component content
Zulkovsky starch 1-2% (W/V, down together)
Soybean cake powder 1-2%
Extractum carnis 0.5-1.0%
Glucose 0.1-0.5%
NaNO
3 0.01-0.03％
Mg
2SO
4·7H
2O 0.01-0.03％
CaCO
3 0.1-0.3％
After fermentation ends, add some trace elements that soybeans they grow needs as: ZnSO
450-100ppm, H
3BO
45-20ppm, NaMoO
4.2H
2O 2-10ppm.Chitin can be used as the additive of soil and microbial inoculum and prevents and treats soil-borne disease, but chitin needs the long period could be by microbiological degradation in soil, therefore in additive of the present invention, not only add 0.1%-0.3% (W/V) chitin, also added 0.01-0.02% (W/V) chitosan.The chitosan molecule amount is less, can be utilized very soon by microorganism in the soil, promote the breeding of antagonistic microbe flora, improve the immunizing power of plant itself simultaneously, chitin decomposes to discharge and kills line material antagonism pine root fungus and nematode subsequently, decomposes the growth that small-molecule substance that the back produces can also promote microorganism and plant itself in the soil.In this microbial inoculum, also added 0.5-1 ‰ (W/V) sodium humate, sodium humate be added with the physiological status that helps regulate plant itself, promote the growth of plant.In bacterium liquid, add 0.1-0.2% (W/V) carboxymethyl cellulose in addition and do bonding agent, more help bacterium liquid dressing on soybean seeds, do not influence the breathing of seed simultaneously.
The using method of liquid preparation of the present invention is: the liquid bacterial agent that every mu of soybean seeds (5-6Kg) adds 50-100ml mixes, and waits to dry the back sowing.Preferred version: the thalline after the filtration of fermented liquid process is centrifugal adsorbs with diatomite or other carriers, and drying is made pulvis.When using, be used for seed treatment with bonding agent is mixed after evenly in proportion.The advantage of this scheme is that technology is simple, is convenient to storage and packing, transportation, prolongs the storage period of bacterium.
Fermented liquid can be processed into pulvis through spraying drying, also can adopt carrier absorption, makes solid preparation or is processed into granule.The bacteria suspension of ferment product is rarer, so among the present invention the ratio of absorption carrier is improved, is beneficial to the drying of microbial inoculum.The ratio of bacterium liquid and absorption carrier is 1:1-3:1 (W/W).Absorption carrier can be selected for use: diatomite, the peat composed of rotten mosses, kaolin or other do not influence the mixture of active absorption carrier of bacterial strain and various carriers.This kind preparation can keep the viable bacteria content and the metabolite activity of thalline simultaneously with thalline and fermentating metabolism product absorption.
Compare with the chemical pesticide or the similar biological pesticide of prior art, the present invention has enlarged the range of application that bacillus is prevented and treated nematodiasiss, and the advantage of this microbial inoculum is: 1. the sporeformer fermentation period is short, reduces production costs save energy.Nontoxic, pollution-free.2. gemma is high temperature resistant and dry, is beneficial to be processed into various formulations, is convenient to store and transportation.3. can be at the soybean rhizosphere colonization, antagonism soybean Cyst nematode and pine root fungus.Improve rhizosphere environment simultaneously, promote the growth of soybean plant strain.4. the additive mixture can promote seed germination, improves the physiological status of plant, promotes soybeans they grow, improves the immunizing power of plant, the organic nematicide material that also produces in decomposition course in the mixture.
Specific embodiment 1:
The Bacillus licheniformis CGMCC No.1035 that employing is cultivated on the beef-protein medium inclined-plane cultivates by the three grade fermemtation technology of shaking bottle → seeding tank → fermentor tank as the seed inclined-plane again, and inoculum size is 2%, and the culture medium prescription of employing is:
Medium component content
Zulkovsky starch 1% (W/V, down together)
Soybean cake powder 1%
Extractum carnis 0.5%
Glucose 0.3%
NaNO
3 0.01％
Mg
2SO
4·7H2O 0.02％
CaCO
3 0.2％
PH 7.0-7.2
The control leavening temperature is at 28-30 ℃, pH7.0-7.4, and the second order fermentation time is decided to be 20-24h, is decided to be that bacterial number reaches 7.0 * 10 in the fermented liquid three grade fermemtation time
8Individual/ml, whole fermentation time is 60-72h.In this fermented liquid, add the micro-mother liquor (ZnSO of 0.5% (V/V)
41% (W/V), H
3BO
40.1% (W/V), NaMoO
4.2H
2O 0.05% (W/V), 0.01% (W/V) chitosan, 0.3% (W/V) chitin, 0.5 ‰ (W/V) sodium humate, 0.2% (W/V) carboxymethyl cellulose mixes the back and mixes seed dressing by every 100ml preparation with 5 kilograms of soybean seeds, carries out the diseases prevention experiment.Do contrast with the soybean that is left intact.4 compound leaf phases are investigated the disease index of soybean cyst nematode and root rot, the results are shown in subordinate list-1.
Subordinate list-1 microbial inoculum is handled pathogen of soybean root rot and soybean Cyst nematode prevention effect
The disease index of root rot is 23.8%, and the disease index of blank is 52.8%, and preventive effect is 54.9%.The disease index of soybean cyst nematode is reduced to 12.6%, and the contrast disease index is 38.4%, and preventive effect is 67.2%.
Claims (3)
1, a kind of microorganism strains that is used to prevent and treat soybean cyst nematode and root rot is characterized in that, it is Bacillus licheniformis CGMCC No.1035.
2, a kind of preparation that is used to prevent and treat soybean cyst nematode and root rot is through fermentation culture and the liquid preparation of making after adding mixture by Bacillus licheniformis CGMCC No.1035 bacterial strain.
3, preparation as claimed in claim 2 is characterized in that, the culture medium prescription that uses in the Bacillus licheniformis CGMCC No.1035 fermentation culture process is:
Nutrient media components content, volume ratio by weight
Zulkovsky starch 1-2%
Soybean cake powder 1-2%
Extractum carnis 0.5-1.0%
Glucose 0.1-0.5%
NaNO
3 0.01-0.03％
Mg
2SO
4·7H
2O 0.01-0.03％
CaCO
3 0.1-0.3％。
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| CN101531971B (en) * | 2008-03-14 | 2012-11-28 | 天津现代职业技术学院 | Bacillus subtilis and application thereof in prevention and treatment of continuous cropping obstacle of greenhouse vegetable |
| CN101779678B (en) * | 2009-01-20 | 2013-03-27 | 北京嘉博文生物科技有限公司 | Biological sweet sorghum seed pelleting agent and preparation method thereof |
| CN102308853A (en) * | 2011-03-02 | 2012-01-11 | 沈阳农业大学 | The biological seed coating agent of control soybean SCN |
| UY36334A (en) * | 2014-12-29 | 2017-04-28 | Fmc Corp | COMPOSITIONS OF BACILLUS LICHENIFORMIS RT1184 AND USES METHODS TO BENEFIT THE GROWTH OF PLANTS |
| CR20190001A (en) * | 2016-05-27 | 2019-07-04 | Agro Res International Llc | Composición biopesticida para el uso en la prevención y reducción al mínimo de la enfermedad de la planta |
| CN106719844B (en) * | 2016-12-26 | 2019-08-09 | 山西省农业科学院蔬菜研究所 | It is a kind of for preventing and treating the biological seed coating agent and preparation method thereof of cucurbita pepo root rot |
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| CN1090130A (en) * | 1993-01-20 | 1994-08-03 | 河南华能科技发展公司 | Produce the method for biopesticide with bacillus licheniformis |
| CN1095892A (en) * | 1994-03-28 | 1994-12-07 | 北京大学 | Efficient peanut yield increasing agent and preparation method thereof |
| US5589381A (en) * | 1994-06-30 | 1996-12-31 | Rutgers, The State University Of New Jersey | Bacillus licheniformis producing antifungal agents and uses thereof for control of phytopathogenic fungi |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1090130A (en) * | 1993-01-20 | 1994-08-03 | 河南华能科技发展公司 | Produce the method for biopesticide with bacillus licheniformis |
| CN1095892A (en) * | 1994-03-28 | 1994-12-07 | 北京大学 | Efficient peanut yield increasing agent and preparation method thereof |
| US5589381A (en) * | 1994-06-30 | 1996-12-31 | Rutgers, The State University Of New Jersey | Bacillus licheniformis producing antifungal agents and uses thereof for control of phytopathogenic fungi |
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