CN106754567B - A kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS of efficiently prevention various crop droop - Google Patents
A kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS of efficiently prevention various crop droop Download PDFInfo
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Abstract
The invention discloses a kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS of efficiently prevention various crop droop, it is made of bacillus JC65, Bacillus cercus CH01, Bacillus subtillis SM16.By 3 kinds of bacterium single doses according to each bacterium number of viable 1:1:1 is made into mix preparation, and viable bacteria total concentration is 1 × 10 in mixture8~1010CFU/ml.LAS mixture is to the prevention effect of crop droop up to more than 70%.To the growth-promoting effect of disease-free plant up to 20%, which needs to carry out sowing and transplant two links.
Description
Technical field
The present invention relates to a kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS of efficiently prevention various crop droop, belong to agro-ecology and prevent
Control technical field.
Background technology
Occupy very important status in the agricultural crops yield of watermelon, cucumber, muskmelon and pumpkin in the world, its
Middle wilt disease is one of Major Diseases for influencing 4 kinds of agricultural crops yield and qualities of appeal.Droop is by Fusarium oxysporum
(Fusarium oxysporum f.sp.) causes a kind of destructive soil-borne disease, is commonly called as stem wilt, dead seedling disease etc., alive
There is generation in the range of boundary.As the expansion of cultivated area and continuous cropping plantation have more aggravated the generation of droop.
At present, wilt disease prevents main measure and still uses chemical agent.Although chemical agent can be in certain journey
Droop is prevented on degree, but at the same time, many beneficial microorganisms can also be subject to different degrees of injury in soil.Some
The problem of use of chemical agent can cause soil pollution and chemical pesticide residual etc. serious.In addition, breeding resistant variety
Also have certain effect to controlling disease with enhancement of field management measure.But the selection and breeding cycle of disease-resistant variety is grown, and droop
Bacterium Variation of Virulence is big, and resistance easily disappears foster.
Relatively effective in agricultural measures is crop rotation, and especially rice field-upland field rotation, can effectively reduce bacterium in soil
Amount, reduces disease, but there are certain difficulty in practical applications.First, watermelon, cucumber, pumpkin and muskmelon belong to high economical
It is worth crop；Two be due to that Fusarium oxysporum bacterium can survive for more than ten to decades in the soil, and short ratation does not reach expected purpose；Three
That the most of non-host plants of plantation cannot reduce the quantity of Microsclerotia in the soil, and the root secretion of non-host remain to for
Microsclerotia survival provides nutrition.
Under this development, many sight begin to transfer to biological control means.Biological control and other methods phase
Than, have the characteristics that safely, effectively, it is lasting, be especially that of avoiding a series of problems that chemical prevention is brought.Biological prevention and control agent is to disease
Evil prevention effect is good, free from environmental pollution, noresidue nontoxic to people and animals；Killing high specificity to pest and disease damage, does not injure natural enemy,
Beneficial organism, can keep ecological balance；Raw materials for production and active ingredient are degradable for natural products, can go back to nature, and ensure
Sustainable development；Microorganism can be changed with the method for biotechnology and genetic engineering；Many factors and component play a role,
Pathogen is difficult to develop immunity to drugs.It is reported that the biocontrol fungi of droop mainly has, Trichoderma, viscous broom are mould, but above-mentioned biocontrol microorganisms
Prevention effect is not ideal enough, and main cause is that these biocontrol microorganisms are poor in host's rhizospere competition.Compare in droop biocontrol bacteria
More promising species is bacillus, bacillus subtilis, Pseudomonas fluorescens, bacillus etc..It is, in general, that a variety of lifes
It is fungi-proofing that to be used in mixed way its effect better than single biocontrol microorganisms.In addition, biocontrol agent and agricultural cultivation measure are used in combination with,
One good border of dwelling can be provided for biocontrol microorganisms, the prevention potential of itself can be played as far as possible.
The content of the invention
It is an object of the present invention to provide a kind of efficiently prevention microbial composite of various crop droop and prepared therefrom
Complex microbial inoculum.
It is a further object of the present invention to provide the application of the microbial composite and complex microbial inoculum prepared therefrom.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of microbial composite of efficiently prevention various crop droop, is made of following 3 kinds of bacteriums, is respectively：Bud
Born of the same parents bacillus JC65 (Bacillus sp.), it is general to be preserved in China Committee for Culture Collection of Microorganisms on December 15th, 2010
Logical microorganism center, culture presevation number is CGMCC No.4475；Bacillus cercus CH01 (Bacillus cereuse), in
On November 17th, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is
CGMCC NO.11677；Bacillus subtillis SM16 is (Bacillus subtilis), on 06 18th, 2009 are preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, culture presevation number are CGMCC No.3127.
Application of the microbial composite of the present invention in terms of prevention droop and promotion plant growth.
It is preferred that wet bacterium and the preservative fluid for thallus in this laboratory after described three kinds of bacterial strains JC65, CH01 and SM16 are fermented
(belong to this laboratory granted patent, patent No. ZL02112559.7, publication CN1358838, publication date：In July, 2002
17 days) press 1：40 (g/ml) are made into microbial inoculum, can room temperature preservation 2~3 years.The composite bacteria agent can be diluted when watermelon transplantation of seedlings
Pour and apply.
Microbial composite of the present invention is preparing prevention droop and is promoting the microbial composite bacteria of plant growth
Application in agent.
Complex microbial inoculum prepared by microbial composite of the present invention.
Complex microbial inoculum of the present invention, is preferably prepared by the following method to obtain：By the bacterial strain JC65,
CH01 and SM16 is inoculated into the flat lining outs of LB respectively, cultivates 14-16h in 28 DEG C of biochemical cultivation cases, after single bacterium colony is grown, chooses
In the test tube for taking LB nutrient solutions of the single bacterium colony access containing 5mL, 28 DEG C are placed in, is cultivated in the shaking table of 200r/min, seed is made
Liquid；Seed liquor is inoculated in the triangular flask of the LB nutrient solutions equipped with 500mL with 1% inoculum concentration, is placed in 28 DEG C, 200r/min
Shaking table in cultivate 48h, 3 kinds of bacterial concentrations are diluted to 5 × 107According to 1 after CFU/mL:1:1 ratio is uniformly mixed, and is
The complex microbial inoculum.
The preparation method of complex microbial inoculum of the present invention, including：By described bacterial strain JC65, CH01 and SM16
The flat lining outs of LB are inoculated into respectively, 14-16h is cultivated in 28 DEG C of biochemical cultivation cases, and after single bacterium colony is grown, picking single bacterium colony connects
In the test tube for entering the LB nutrient solutions containing 5mL, 28 DEG C are placed in, is cultivated in the shaking table of 200r/min, seed liquor is made；With 1%
Seed liquor is inoculated in the triangular flask of the LB nutrient solutions equipped with 500mL by inoculum concentration, is placed in 28 DEG C, is trained in the shaking table of 200r/min
48h is supported, 3 kinds of bacterial concentrations are diluted to 5 × 107According to 1 after CFU/mL:1:1 ratio is uniformly mixed.
Application of the complex microbial inoculum of the present invention in terms of prevention droop and promotion plant growth.
The application is preferably diluted pouring root when crop is transplanted and sows and uses.
Beneficial effect
The advantages and positive effects of the present invention are shown：The present invention is specific to the biological and ecological methods to prevent plant disease, pests, and erosion system of droop disease exploitation
Agent.Because it belongs to biological agent, completely not because of a series of problems caused by the use of chemical pesticide, beneficial to watermelon, cucumber,
The No-harmful apple orchard of pumpkin and muskmelon, peasant can not have to or reduce the dosage of other chemical pesticides, this can be not only that peasant saves
Spending is saved, and is conducive to the export trade of these crops.
Greenhouse test shows that LAS mixture effectively can control droop to infect watermelon, reaches the effect of symptom management generation
Fruit, protection effect have 86.7%, and growth-promoting effect has 33.3%；Field experiment shows that LAS mixture on the one hand can symptom management hair
Raw, preventive effect is maintained at 75.0%, while can promote to appeal the growth of 4 kinds of crops, increasing both production and income, and growth-promoting effect is up to 66.6%；
Comprehensive greenhouse and the experiment of crop field growth-promoting find that LAS mixture can promote to appeal the growth of 4 kinds of crops, improve yield.
Brief description of the drawings
The antagonistic effect figure of Fig. 1 differences biocontrol microorganisms and wilt
The suppression figure that Fig. 2 differences biocontrol microorganisms grow wilt
The growth-promoting design sketch of 30 days after the transplanting of Fig. 3 watermelons
Biomaterial preservation information
JC65, bacillus Bacillus sp., Chinese microorganism strain preservation is preserved on December 15th, 2010
Administration committee's common micro-organisms center, preservation address are the institute of BeiChen West Road, Chaoyang District, BeiJing City No. 13, and the Chinese Academy of Sciences is micro-
Biological study institute, culture presevation number are CGMCC No.4475.
CH01, Bacillus cercus Bacillus cereuse, China Microbiological is preserved on November 17th, 2015
Culture presevation administration committee common micro-organisms center, preservation address are the institute of BeiChen West Road, Chaoyang District, BeiJing City No. 13, China
Institute of microbiology of the academy of sciences, culture presevation number are CGMCC NO.11677.
SM16, Bacillus subtillis Bacillus subtilis, the micro- life of China was preserved on 06 18th, 2009
Thing culture presevation administration committee common micro-organisms center, preservation address is the institute of BeiChen West Road, Chaoyang District, BeiJing City No. 13, in
Institute of microbiology of the academy of sciences of state, culture presevation number are CGMCC No.3127.
Embodiment
Embodiment 1
1. bacterium source：JC65 is by isolated in Cucumber rhizosphere soil；SM16 and CH01 is by tomato healthy tree root soil
In it is isolated.
2. strain separation method：It is (micro- with reference to microbial room of Chinese Academy of Sciences Nanjing soil institute soil using plate dilution method
Biological study method [M] Beijing：Scientific publication is shut out, and 1985.).
3. bacterial strain screening foundation：The antagonism of watermelon blight germ and biocontrol microorganisms measures (opposite culture method, Lin Fu
It is in bacteriolysis [J] the plant pathologies of Li De forts bacillus subtilises (Bacillus subtilis) to plant pathogenic fungi
Journal, 2003,33 (2)：174~177.), biocontrol microorganisms are to watermelon blight prevention and growth-promoting functions greenhouse, field experiment.
4. bacterial strain identification method：Identified using prokaryotes 16S rRNA encoding gene segment sequences method.Identification
It the results are shown in Table 1.
Table 1：Comparison result is sequenced
5. tablet antagonistic experiment method
Method one：The withered germ of water-melon being stored in 4 DEG C is inoculated on PDA plate and is activated, treats that fungi covers with tablet
Uniformly break into the circular fungus block of a diameter of 8mm from bacterium colony outer edge with the card punch of sterilizing afterwards.By inoculated by hypha block new
The center of PDA plate, is inoculated with one plant of biocontrol microorganisms, as shown in Figure 1 respectively in its surrounding away from center equidistant.25 DEG C of cultures, often
Fungi growing state was observed every two days, records the size of inhibition zone.Each processing repeats three times.Record data and processing group information
It is shown in Table 2 and Fig. 1.
Inhibiting rate of the different biocontrol microorganisms of table 2 to wilt
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. inhibiting rate (%)=(R-r)/R*100%
Method two：Take 5.0 × 107The biocontrol microorganisms 1mL of CFU/ml mixes a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices as processing 1 in the PDA of 99mL, takes 5.0
×107The biocontrol microorganisms 5mL of CFU/ml mixes a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices as processing 2 in the PDA of 95mL.Using be not added with the PDA plates of any biocontrol microorganisms as
Control.By the bacterium dish accomplished fluently as tablet center.25 DEG C of cultures, observe fungi growing state and record fungi growth every three days
Situation.Each processing is in triplicate.Processing group information is shown in Table 3 and Fig. 2.
The suppression that the different biocontrol microorganisms of table 3 grow wilt
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. inhibiting rate (%)=(R-r)/R*100%
Embodiment 2
Bacterial strain JC65, CH01 and SM16 are inoculated into the flat lining outs of LB respectively, 14- is cultivated in 28 DEG C of biochemical cultivation cases
16h, after single bacterium colony is grown, in the test tube of LB nutrient solutions of the picking single bacterium colony access containing 5mL, is placed in 28 DEG C, 200r/min
Shaking table in cultivate, seed liquor is made；Seed liquor is inoculated in the triangle of the LB nutrient solutions equipped with 500mL with 1% inoculum concentration
In bottle, 28 DEG C are placed in, 48h is cultivated in the shaking table of 200r/min, 3 kinds of bacterial concentrations are diluted to 5 × 107According to 1 after CFU/mL:
1:1 ratio is uniformly mixed, and is the complex microbial inoculum.
3 greenhouse Biological control of embodiment
The consistent watermelon transplantation of seedlings of growing way is selected when watermelon seedling grows to the 3-4 leaf phases into the basin alms bowl containing Nutrition Soil, often
10 watermelon seedlings of a processing transplanting.The same day has been transplanted, the 5 × 10 of 20mL are poured with root-pouring method7The corresponding biocontrol microorganisms of CFU/ml, are moved
30 days after cultivation biocontrol microorganisms of teeming again.Using clear water as control.Observation each group growth-promoting situation daily.
It the results are shown in Table 4 and Fig. 3.
Always (average) fresh weight=underground part fresh weight+overground part fresh weight
Growth-promoting effect=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] × 100%
Growth-promoting functions (transplanting 30d) of the 4 composite bacteria agent LAS of table to watermelon
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. biomass increase=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] ×
100%
4 greenhouse diseases prevention experiment of embodiment
When watermelon seedling grows to the 3-4 leaf phases, the consistent watermelon transplantation of seedlings of growing way is selected into the basin alms bowl containing Nutrition Soil, often
10 watermelon seedlings of a processing transplanting.The same day has been transplanted, 5 × 10 are poured to the watermelon seedling for the treatment of group with root-pouring method7The phase of CFU/ml
Biocontrol microorganisms are answered, 5 × 10 are poured to the watermelon seedling for the treatment of group with root-pouring method again immediately6The spore suspension 20mL of a spore/mL.With
Clear water is control.Observation each group diseases prevention situation daily, is observed with control group incidence up to stopping after more than 95%.Diseases prevention information is shown in
Table 5.
Droop disease level standard：
0 grade：Without illness；
1 grade：Yellowing leaf or wilting area ＜ 50%；
2 grades：Yellowing leaf or wilting area ＞ 50%；
3 grades：Blade is wilted or withered, only growing point survival；
4 grades：Whole pearl is withered.
Disease severity (%)=100% × Σ (sick plant number × disease series)/(total plant number × highest disease series)
Protection effect (%)=100% × (control disease severity-processing disease severity)/control disease severity
Prophylaxis effects (transplanting 30d) of the 5 composite bacteria agent LAS of table to watermelon
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. prevention effect %=(control disease severity-processing disease severity)/control disease severity × 100
5 crop field diseases prevention experiment of embodiment
Field experiment is carried out in southern agriculture decorated archway Experimental Base.Variety of watermelon is early good (84-24)
Experiment process is：Biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS, clear water control.Processing method：Transplanting same day processing, 15d, 30d, 75d
Handled respectively in a manner of pouring root.
Each 3 repetitions of processing, each plot area 24m2(6m*4m), line-spacing 100cm, spacing in the rows 40cm.Each
60 watermelons of cell.Specifying information is shown in Table 6.
Prophylaxis effects (transplanting 105d) of the big Tanaka's composite bacteria agent LAS of table 6 to watermelon
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. prevention effect=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] × 100%
The crop field diseases prevention experiment of cucumber, muskmelon and pumpkin is tested with the diseases prevention of watermelon crop field, and specifying information is shown in Table 6,7,8.
Prophylaxis effects (transplanting 105d) of the big Tanaka's composite bacteria agent LAS of table 7 to cucumber
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. prevention effect=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] × 100%
Prophylaxis effects (transplanting 105d) of the big Tanaka's composite bacteria agent LA of table 8 to muskmelon
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. prevention effect=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] × 100%
Prophylaxis effects (transplanting 105d) of the big Tanaka's composite bacteria agent LA of table 9 to pumpkin
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. prevention effect=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] × 100%
7 crop field growth-promoting effect of embodiment
Field experiment is carried out in southern agriculture decorated archway Experimental Base.Variety of watermelon is early good (84-24)
Experiment process is：Biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS, clear water control.Processing method：Transplanting same day processing, 15d, 30d, 75d
Handled respectively in a manner of pouring root.
Each 3 repetitions of processing, each plot area 24m2(6m*4m), line-spacing 100cm, spacing in the rows 40cm.Each
60 watermelons of cell.Specifying information is shown in Table 10.
The crop field Biological control of cucumber, muskmelon and pumpkin is shown in Table 11,12,13 with watermelon crop field Biological control, specifying information.
Growth-promoting functions (transplanting 35d) of the big Tanaka's composite bacteria agent LAS of table 10 to watermelon
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. biomass increase=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] ×
100%
Growth-promoting functions (transplanting 35d) of the big Tanaka's composite bacteria agent LAS of table 11 to cucumber
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. biomass increase=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] ×
100%
Growth-promoting functions (transplanting 35d) of the big Tanaka's composite bacteria agent LAS of table 12 to muskmelon
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. biomass increase=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] ×
100%
Growth-promoting functions (transplanting 35d) of the big Tanaka's composite bacteria agent LAS of table 13 to pumpkin
Note：1. numerical value is average value, between different letter expression processing under the level of signifiance of P=0.05, otherness is notable.
2. biomass increase=[(processing mean fresh-clear water control mean fresh)/clear water control mean fresh] ×
100%.
Claims (3)
1. it is a kind of prevent watermelon, cucumber, pumpkin or Muskmelon Fusarium wilt microbial composite prevention watermelon, cucumber, pumpkin or
Muskmelon Fusarium wilt and promote its plant growth in terms of application；The microbial composite is made of following 3 kinds of bacteriums, respectively
It is：Bacillus JC65（Bacillus sp.）, Chinese microorganism strain preservation pipe is preserved on December 15th, 2010
Reason committee common micro-organisms center, culture presevation number are CGMCC No. 4475；Bacillus cercus CH01（Bacillus
cereuse）, China Committee for Culture Collection of Microorganisms's common micro-organisms is preserved on November 17th, 2015
Center, culture presevation number are CGMCC NO. 11677；Bacillus subtillis SM16 is（Bacillus subtilis）, in
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation within 06 18th, 2009
Number it is CGMCC No. 3127.
2. application according to claim 1, it is characterised in that being diluted pouring root when crop is transplanted and sows makes
With.
3. a kind of microbial composite for preventing watermelon, cucumber, pumpkin or Muskmelon Fusarium wilt is preparing prevention watermelon, cucumber, south
Application in the complex microbial inoculum of its plant growth of melon or Muskmelon Fusarium wilt and promotion；The microbial composite by with
Lower 3 kinds of bacteriums composition, is respectively：Bacillus JC65（Bacillus sp.）, on December 15th, 2010 is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, culture presevation number are CGMCC No. 4475；Wax-like gemma
Bacillus CH01（Bacillus cereuse）, Chinese microorganism strain preservation pipe is preserved on November 17th, 2015
Reason committee common micro-organisms center, culture presevation number are CGMCC NO. 11677；Bacillus subtillis SM16 is
（Bacillus subtilis）, China Committee for Culture Collection of Microorganisms was preserved on 06 18th, 2009
Common micro-organisms center, culture presevation number are CGMCC No. 3127.
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| CN107586735B (en) * | 2017-08-01 | 2020-04-28 | 环境保护部南京环境科学研究所 | Biocontrol strain CH01 for efficiently preventing and treating citrus canker and application thereof |
| 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 |
| CN108396002B (en) * | 2018-03-07 | 2019-12-06 | 山东新禾丰作物营养有限公司 | Bacillus licheniformis and application thereof in preventing and treating sweet melon fusarium wilt |
| US11555946B2 (en) * | 2019-12-19 | 2023-01-17 | Basf Corporation | Systems and methods for modeling disease severity |
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| CN102174431A (en) * | 2011-01-12 | 2011-09-07 | 南京农业大学 | Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon |
| CN102321554A (en) * | 2011-08-30 | 2012-01-18 | 新疆农业科学院微生物应用研究所 | Bacillus cereus and its application of being as plant growth-promoting rhizobacteria |
| CN102524303A (en) * | 2011-08-31 | 2012-07-04 | 南京农业大学 | Mixture for preventing blight and verticillium wilt of cotton |
| CN103667158A (en) * | 2013-12-27 | 2014-03-26 | 中国热带农业科学院香料饮料研究所 | Bacillus cereus and application of bacillus cereus |
| CN106305033A (en) * | 2015-07-11 | 2017-01-11 | 镇江贝思特有机活性肥料有限公司 | Method for controlling watermelon fusarium wilt |
| CN104988101A (en) * | 2015-08-03 | 2015-10-21 | 黑龙江省科学院微生物研究所 | Bacillus cereus and application of bacillus cereus as agricultural biological agent |
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