CN105660706A - Application of bacillus cereus BCJB01 in control of root-knot nematodes - Google Patents
Application of bacillus cereus BCJB01 in control of root-knot nematodes Download PDFInfo
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- CN105660706A CN105660706A CN201610054473.9A CN201610054473A CN105660706A CN 105660706 A CN105660706 A CN 105660706A CN 201610054473 A CN201610054473 A CN 201610054473A CN 105660706 A CN105660706 A CN 105660706A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses an application of bacillus cereus BCJB01 in control of root-knot nematodes. A supernatant and a bacterial suspension of the bacillus cereus BCJB01 have a completely lethal effect on second instar larvae of tomato root-knot nematodes and eggs being about to hatch out the second instar larvae. One new strain for controlling the root-knot nematodes is provided, the effect of the bacillus cereus BCJB01 is not lower than that of an existing chemical agent in the market, and great progress in the aspect of control of the root-knot nematodes is achieved.
Description
Technical field
The present invention relates to the Application Areas of bacterial strain, particularly relate to the application of bacillus cereus BCJB01 in control root knot nematode.
Background technology
Root knot nematode disease is a kind of serious Plant nematode parasitics disease, often causes serious financial loss. The control of root knot nematode disease is a difficult problem urgently to be resolved hurrily in current agriculture production. Traditional prophylactico-therapeutic measures mainly comprises chemical prevention, crop rotation and use disease-resistant variety etc., but these prophylactico-therapeutic measuress have its limitation. Utilize microbial control root knot nematode, there is the advantage of Sustainable development, be one of the desirable approach of the sustainable improvement of root knot nematode disease.
Utilize the research report of biocontrol bacteria control root knot nematode, mainly contain rhizosphere growth-promoting bacterium, puncture pasteurella, Pseudomonas alba, bacillus thuringiensis, subtilis, series bacillus, bacillus megaterium, bacillus amyloliquefaciens etc., they have huge potentiality to be exploited in biocontrol of nematodes, and it is worth also cognitive by people gradually. But the effect of existing biocontrol bacteria control root knot nematode still needs to improve to be strengthened, new biocontrol bacteria also needs continual exploitation.
Summary of the invention
The object of the present invention is exactly the defect for above-mentioned existence and provides the application of bacillus cereus BCJB01 in control root knot nematode. The present invention provides the bacterial strain of a kind of control root knot nematode newly, and its effect is not less than existing chemical agent on the market, has great progress in control root knot nematode.
The utilisation technology scheme of the bacillus cereus BCJB01 of the present invention in control root knot nematode is, tomato root-knot eelworm second instar larvae and the ovum that is about to hatch second instar larvae are all had complete lethal effect by the supernatant liquor of bacillus cereus BCJB01 and bacteria suspension.
Bacillus cereus of the present invention (Bacilluscereus) BCJB01, its preserving number is CGMCCNO.10477, depositary institution: CGMCC (is called for short) in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, preservation date is on January 30th, 2015.
Bacillus cereus BCJB01 fermented liquid is prepared into the control that biocontrol fungicide carries out root knot nematode disease.
The preparation of bacillus cereus BacilluscereusBCJB01 fermented liquid is specially: culture temperature 30 DEG C, rotating speed 110RPM, and fermentation time is 36～44h, and fermented liquid pH maintains 7.0, dissolved oxygen amount 20%, air flow 8～12m3/ hour, tank pressure 0.05～0.1KPa, spore production rate is more than 80%, live bacterial count 7 × 109CFU·mL-1;Fermentative medium formula: Semen Maydis powder 2%, bean cake powder 1%, yeast powder 0.3%, CaCl20.1%, MgSO4·7H2O0.035%, Na2HPO4·12H2O0.3%, NaH2PO4·2H2O0.15%, pH7.0～7.2.
Described biocontrol fungicide is suspension agent, is mixed with tensio-active agent, sanitas, suspension aids and thickening material etc. by bacillus cereus BacilluscereusBCJB01 fermented liquid and prepares suspension agent, and wherein suspension aids is Repone K. The suspension aids selective chlorination potassium of the suspension agent of the present invention substitutes now conventional sodium-chlor, can reduce the content of sodium-chlor in edatope in actual applications, prevent salting of soil, and Repone K can be used as potash fertilizer energy utilization absorbed by crops simultaneously. Preferably, suspension aids of the present invention is Repone K, and sanitas is sodium Diacetate, and tensio-active agent is organosilicon BD-3077, and thickening material is xanthan gum.
The concrete preparation method of described suspension agent is: in bacillus cereus BacilluscereusBCJB01 fermented liquid, add Repone K 5%～8% by mass percentage, sodium Diacetate 0.1%～0.5%, organosilicon BD-30770.1%～0.3%, the obtained suspension agent of xanthan gum 0.2%～0.3%, BCJB01 live bacterial count 5 × 10 in gained suspension agent9cfu·mL-1. The consumption of above each component is all taking the quality of the fermented liquid of BCJB01 bacterium as radix.
Described biocontrol fungicide is pulvis, bacillus cereus BacilluscereusBCJB01 is mixed with protective material, then the female powder of spraying dry preparation, then it is carrier taking turfy soil, chitosan is synergistic agent, fatty alcohol-polyoxyethylene ether is wetting agent, 2-naphthalene sulfonic acid formaldehyde polymer sodium salt is dispersion agent, fluorescent brightener CBS is uv-protector, female for BCJB01 powder is made pulvis, protective material of wherein spraying is white carbon black, the mixture of glycerine and tween 80, protective material concentration is 14～20%, in female powder, viable bacteria yield is on average more than 60%, viable count is at 15,000,000,000/more than g.
Described pulvis is grouped into by following one-tenth by weight:
Female powder 5～10 weight part
Turfy soil 55～77 weight part
Chitosan 1～2.5 weight part
2-naphthalene sulfonic acid formaldehyde polymer sodium salt 3～5 weight part
Fatty alcohol-polyoxyethylene ether 3～5 weight part
Fluorescent brightener CBS 0.2～0.5 weight part.
The application useful effect of the bacillus cereus BCJB01 of the present invention in control root knot nematode is: tomato root-knot eelworm second instar larvae is all had lethal effect by BCJB01 fermented supernatant fluid and bacteria suspension thereof, lethal effect reduces along with the reduction of diluent concentration, strengthens along with the prolongation in treatment time. The prevention effect of BCJB01 suspension agent in the present invention when every mu of consumption 4L is best, and anti-effect is 91.59%. The prevention effect of BCJB01 pulvis when every mu of consumption 8kg is 90.40%. The prevention effect of tomato root-knot eelworm is all better than the B418 bacterial strain preparation of commercialization by BCJB01 suspension agent in the present invention and pulvis. Wherein, under identical using dosage, BCJB01 suspension agent prevention effect relatively B418 suspension agent improve 1.37 times, BCJB01 pulvis prevention effect relatively B418 pulvis improve 1.11 times. Anti-effect during BCJB01 suspension agent mu consumption 2L in the present invention is anti-effect during 76.64%, BCJB01 pulvis mu consumption 4kg is 78.12%, and all best with prevention effect on Vehicles Collected from Market chemical agent 10% fosthiazate granule is close. Show that the BCJB01 suspension agent in the present invention and pulvis are the good microorganism nematode killing agents of effect.
Accompanying drawing explanation
Fig. 1 show bacillus cereus (Bacilluscereus) BCJB01 to the lethal processes of root knot nematode second instar larvae ovum.
Fig. 2 show the lethal second instar larvae of BCJB01.
Embodiment:
In order to understand the present invention better, the technical scheme of the present invention is described in detail, but the present invention is not limited thereto below with specific examples.
Bacillus cereus of the present invention (Bacilluscereus) BCJB01, its preserving number is CGMCCNO.10477, depositary institution: CGMCC (is called for short) in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Embodiment 1
BCJB01 strains separation and qualification
1, the ovum that the present invention relates to tomato root-knot eelworm second instar larvae and be about to hatch second instar larvae all has the BCJB01 bacterial strain of lethal effect, is located away from the white horticultural vegetable field soil in Shandong province Liaocheng City Gaotang County. Culture presevation number is CGMCCNO.10477.
2, identification of strains
Adopt the method that physiological and biochemical property measures and 16SrDNA sequence alignment analysis combines that bacterial strain BCJB01 is accredited as bacillus cereus (Bacilluscereus). Simultaneously, single bacterium colony of separation and purification is carried out the qualification of seven important features such as V.P test, mycoderm formation test, lecithinase test, starch hydrolysis experiment, casein hydrolysis test, polychrom, cellobiose simultaneously, measures according to " the common bacteria system identification handbook " all write with reference to east elegant pearl, Cai Miaoying etc.
Embodiment 2
BCJB01 is to the lethal effect of tomato root-knot eelworm second instar larvae
The preparation of 1.BCJB01 fermented liquid, carries out according to the following steps:
(1) slant strains: adopt solid LB substratum, be seeded on Tube propagation base by bacillus cereus BCJB01, cultivates 24 hours for 30 DEG C.
(2) shaking flask bacterial classification: adopt LB liquid medium, be seeded in LB liquid medium by test tube strains, puts on shaking table 30 DEG C, shaking culture 15 hours.
(3) liquid fermenting: adopt substratum, Semen Maydis powder 2%, bean cake powder 1%, yeast powder 0.3%, CaCl20.1%, MgSO4·7H2O0.035%, Na2HPO4·12H2O0.3%, NaH2PO4·2H2O0.15%, pH7.0～7.2. 121 DEG C of sterilizings 20 minutes, are inoculated in the fermentor tank of 10 liters (inoculum size is 5%wt), 30 DEG C of cultivations by the seed of (2), dissolved oxygen amount 20%, air flow 8～12m3/ hour, tank pressure 0.05～0.1KPa, stirring velocity is 110RPM, and incubation time is 36 ~ 44 hours; Fermentation is terminated when bacillus exfoliation 80%.
2. the preparation of root knot nematode second instar larvae
Root knot nematode picks up from the experiment field of morbidity throughout the year, gets tomato old complaint, and flowing water is cleaned, choose under putting anatomical lens and get fresh egg capsule, put into the sterilizing culture dish of diameter 9cm, with 0.5% clorox surface sterilization 3 minutes, after aseptic washing 3 times, put into 24 well culture plates. It is put in 25 DEG C of incubator dark culturing, treats 2 instar larvae hatchings, changed water every 24 hours 1 time, it is ensured that the 2 instar larvae vigor every time selected are consistent. Being diluted by nematode suspension is 300 mL-1, 4 DEG C of preservations, for subsequent use.
3.BCJB01 the lethality rate of tomato root-knot eelworm second instar larvae is measured
The fermented liquid of the BCJB01 respectively LB liquid nutrient medium cultivated, centrifugal, obtained supernatant liquor stoste and thalline. Thalline aqua sterilisa is made into containing 109CFU·mL-1Bacteria suspension, bacteria suspension and supernatant liquor stoste are diluted to 5 and 10 times of liquid, each draw 50 micro-liters, add 24 hole dishes (micro-rising nematode suspension containing 100), compare with clear water, repeat 9 times. Nematode vigor is observed respectively in 12,24 and 36h. Test-results is in table 1.
The lethality rate of tomato root-knot eelworm second instar larvae is measured by table 1BCJB01
。
As shown in Table 1, tomato root-knot eelworm second instar larvae is all had lethal effect by BCJB01 fermented supernatant fluid and bacteria suspension thereof, and lethal effect reduces along with the reduction of diluent concentration, strengthens along with the prolongation in treatment time.
The lethal processes of root knot nematode second instar larvae ovum is asked for an interview shown in Figure of description Fig. 1 by BCJB01. The second instar larvae that BCJB01 is lethal is asked for an interview shown in Figure of description Fig. 2.
Embodiment 3
BCJB01 preparation research and application
The present invention is taking Repone K or ammonium sulfate as suspension aids, and sodium Diacetate is sanitas, and organosilicon BD-3077 is tensio-active agent, and xanthan gum is thickening material, and BCJB01 is made suspension agent, and effective thalline quantity is 5 × 109cfu·mL-1。
The present invention is taking the mixture of white carbon black, glycerine and fatty alcohol-polyoxyethylene ether as spraying protective material; BCJB01 is made female powder; then it is carrier taking turfy soil; chitosan is synergistic agent; fatty alcohol-polyoxyethylene ether is wetting agent, and 2-naphthalene sulfonic acid formaldehyde polymer sodium salt is dispersion agent, and fluorescent brightener CBS is uv-protector; female for BCJB01 powder is made pulvis, and effective thalline quantity is 7.0 × 108cfu·g-1。
In the present invention, BCJB01 suspension agent and pulvis are to the control of tomato root-knot eelworm
Examination nematode is supplied to be Meloidogyne incognita (Meloidogyneincongnita)
Examination crop is supplied to be tomato (improvement hair powder 802F1)
Reagent agent is BCJB01 suspension agent in the present invention, pulvis, B418 suspension agent, pulvis and 10% fosthiazate granule.
Root irrigation: test front 25 ~ 30d, cultivate tomato seedling in soil sterile-processed in seedling pan, to 2 ~ 3 leaf phases, select the tomato seedling that growing way is consistent, transplant to, in the soil (throughout the year the field soil of morbidity) of nematode, the strain of every basin 5, waters reagent agent simultaneously. If B418 bacterium agent and 10% fosthiazate granule are comparison medicament, clear water is blank, and often 10 repetitions are established in process, and dispenser every other week 1 time in time transplanting, even executes 3 times, 60d after last medicine, cleans tomato root, investigation records root knot quantity, statistics prevention effect.
State of an illness classification is carried out by following standard; By formulae discovery disease index and relative control effect.
0 grade is that root system is complete, without root knot.
1 grade for there being a small amount of root knot, root is downright bad less than 25%.
2 grades account for the 26% ~ 50% of root system number for root knot number.
3 grades account for the 51% ~ 75% of root system number for root knot number.
4 grades is that root knot spy is many and relatively big, accounts for the 76% ~ 100% of root system number.
Sick level index=∑ (plant number at different levels × this grade of numerical value)/(total strain number × 4) × 100.
Prevention effect (%)=(comparison disease index-process disease index)/comparison disease index × 100
Data statistic analysis: statistical study EXCEL2003 and SPSS19.0 software carry out, multiple comparisons adopts the new multipole difference method of inspection of Deng Ken Shi. Test-results refers to table 2
In table 2 the present invention, BCJB01 suspension agent and pulvis are to the prevention effect of tomato root-knot eelworm
。
As shown in Table 2, the prevention effect of the BCJB01 suspension agent in the present invention when every mu of consumption 4L is best, and anti-effect is 91.59%. The prevention effect of BCJB01 pulvis when every mu of consumption 8kg is 90.40%. The prevention effect of tomato root-knot eelworm is all better than the B418 bacterial strain preparation of commercialization by BCJB01 suspension agent in the present invention and pulvis. Wherein, under identical using dosage, BCJB01 suspension agent prevention effect relatively B418 suspension agent improve 1.37 times, BCJB01 pulvis prevention effect relatively B418 pulvis improve 1.11 times. Anti-effect during BCJB01 suspension agent mu consumption 2L in the present invention is anti-effect during 76.64%, BCJB01 pulvis mu consumption 4kg is 78.12%, and all best with prevention effect on Vehicles Collected from Market chemical agent 10% fosthiazate granule is close. Show that the BCJB01 suspension agent in the present invention and pulvis are the good microorganism nematode killing agents of effect.
Claims (9)
1. bacillus cereus BCJB01 is preventing and treating the application in root knot nematode.
2. application according to claim 1, it is characterised in that, tomato root-knot eelworm second instar larvae and the ovum that is about to hatch second instar larvae are all had complete lethal effect by the supernatant liquor of bacillus cereus BCJB01 and bacteria suspension.
3. application according to claim 1, it is characterised in that, bacillus cereus (Bacilluscereus) BCJB01, its preserving number is CGMCCNO.10477.
4. application according to claim 1, it is characterised in that, bacillus cereus BCJB01 fermented liquid is prepared into the control that biocontrol fungicide carries out root knot nematode disease.
5. application according to claim 4, it is characterised in that, the preparation of bacillus cereus BacilluscereusBCJB01 fermented liquid is specially: culture temperature 30 DEG C, rotating speed 110RPM, fermentation time is 36～44h, and fermented liquid pH maintains 7.0, dissolved oxygen amount 20%, air flow 8～12m3/ hour, tank pressure 0.05～0.1KPa, spore production rate is more than 80%, live bacterial count 7 × 109CFU·mL-1; Fermentative medium formula: Semen Maydis powder 2%, bean cake powder 1%, yeast powder 0.3%, CaCl20.1%, MgSO4·7H2O0.035%, Na2HPO4·12H2O0.3%, NaH2PO4·2H2O0.15%, pH7.0～7.2.
6. application according to claim 4, it is characterized in that, described biocontrol fungicide is suspension agent, is mixed with tensio-active agent, sanitas, suspension aids and thickening material etc. by bacillus cereus BacilluscereusBCJB01 fermented liquid and prepares suspension agent, and wherein suspension aids is Repone K.
7. application according to claim 6, it is characterized in that, the concrete preparation method of described suspension agent is: in bacillus cereus BacilluscereusBCJB01 fermented liquid, add Repone K 5%～8% by mass percentage, sodium Diacetate 0.1%～0.5%, organosilicon BD-30770.1%～0.3%, the obtained suspension agent of xanthan gum 0.2%～0.3%, BCJB01 live bacterial count 5 × 10 in gained suspension agent9cfu·mL-1。
8. application according to claim 4, it is characterized in that, described biocontrol fungicide is pulvis, bacillus cereus BacilluscereusBCJB01 is mixed with protective material, then the female powder of spraying dry preparation, then it is carrier taking turfy soil, chitosan is synergistic agent, fatty alcohol-polyoxyethylene ether is wetting agent, 2-naphthalene sulfonic acid formaldehyde polymer sodium salt is dispersion agent, fluorescent brightener CBS is uv-protector, female for BCJB01 powder is made pulvis, protective material of wherein spraying is white carbon black, the mixture of glycerine and tween 80, protective material concentration is 14～20%, in female powder, viable bacteria yield is on average more than 60%, viable count is at 15,000,000,000/more than g.
9. application according to claim 8, it is characterised in that, described pulvis is grouped into by following one-tenth by weight:
Female powder 5～10 weight part
Turfy soil 55～77 weight part
Chitosan 1～2.5 weight part
2-naphthalene sulfonic acid formaldehyde polymer sodium salt 3～5 weight part
Fatty alcohol-polyoxyethylene ether 3～5 weight part
Fluorescent brightener CBS 0.2～0.5 weight part.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106386882A (en) * | 2016-09-07 | 2017-02-15 | 马鞍山纽泽科技服务有限公司 | Environment-friendly type biological pesticide |
| CN106490046A (en) * | 2016-10-31 | 2017-03-15 | 郑州思辩科技有限公司 | A kind of prevent and treat agricultural chemicals of root knot nematode disease and preparation method thereof |
| CN107164290A (en) * | 2017-07-20 | 2017-09-15 | 青岛中达农业科技有限公司 | Aerosporus and its killing root-knot nematode application |
| CN108157390A (en) * | 2018-01-04 | 2018-06-15 | 南京师范大学 | Application and its method of the compound 3,3- dimethyl octane in prevention plant root-knot nematode |
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| CN106490046A (en) * | 2016-10-31 | 2017-03-15 | 郑州思辩科技有限公司 | A kind of prevent and treat agricultural chemicals of root knot nematode disease and preparation method thereof |
| CN107164290A (en) * | 2017-07-20 | 2017-09-15 | 青岛中达农业科技有限公司 | Aerosporus and its killing root-knot nematode application |
| CN108251328A (en) * | 2017-12-16 | 2018-07-06 | 山东省科学院生态研究所 | A kind of microbial bacterial agent for preventing vegetables Meloidogyne incognita disease and its application |
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| CN109136123A (en) * | 2018-07-23 | 2019-01-04 | 云南大学 | One plant of nematode biocontrol microorganisms and its application |
| CN109136123B (en) * | 2018-07-23 | 2021-05-25 | 云南大学 | Nematode biocontrol bacterium and application thereof |
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| CN110551659B (en) * | 2019-09-16 | 2021-11-30 | 中国农业科学院蔬菜花卉研究所 | Bacillus cereus strain with anti-nematode activity and application thereof |
| CN112868672A (en) * | 2021-01-28 | 2021-06-01 | 湖北省生物农药工程研究中心 | Bacillus thuringiensis suspending agent for killing meloidogyne incognita and application thereof |
| CN114831117A (en) * | 2022-04-29 | 2022-08-02 | 华南农业大学 | Application of sodium diacetate in preventing and treating plant root-knot nematode diseases |
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