CN103571777A - Biocontrol strain BS102 and application thereof in preventing and treating plant gray mold - Google Patents
Biocontrol strain BS102 and application thereof in preventing and treating plant gray mold Download PDFInfo
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Abstract
The invention discloses a biocontrol strain BS102 and application thereof in preventing and treating plant gray mold. The biocontrol strain BS102 is Bacillus subtilis BS102, and the collection number is CGMCC No.7728. The application comprises the following steps: preparing a biocontrol microbial inoculum containing the biocontrol strain BS102; and spraying the biocontrol microbial inoculum on the plant. The biocontrol strain has a strong antagonistic action on botrytis cinerea, and can be used for preventing and treating gray mold. The invention provides a biocontrol method of the gray mold; and the method does not use any chemical pesticide, has the advantages of no toxicity, low tendency to generating drug resistance, high environmental and biological safety and the like, and has wide application prospects.
Description
Technical field
The invention belongs to crop pest prevention and control technical field, relate in particular to a kind of Strain BS1 02 and the application in control plant botrytis thereof.
Background technology
Botrytis cinerea (Botrytis cinerea Pers.:Fr.) can work the mischief to the operatic circle of shelf lives.Infect initial stage fruit surface and produce circle or the water stain shape scab of subcircular, do not cave in, after expanding gradually, form the irregular shape soft rot of light tan spot.Later stage disease portion dehydration, epidermis is shrinkage depression gradually, so that the soft rotten shrinkage of full fruit.Under wet condition, scab surface can produce the cotton hair of loose canescence, on cotton hair of later stage, produces the mould powder of Slate grey, sometimes also can produce irregular shape black particle.The Major Diseases that although the gray mold on shelf lives pears is not pears produces, improper due to harvest maturity and collecting method, can cause physical abuse, and these wounds can be used as the passage of pathogenic bacteria.According to Food and Argriculture OrganizationFAO's report of survey, show, in recovery process, physical abuse can make fruits and vegetables loss reach 8～12% improperly.In addition, at present China's fruit acquisition technique and postpartum fruit condition of storage all very limited, and to postpartum disease prevention and control weaker.Therefore, the gray mold on shelf lives the operatic circle still can cause serious financial loss.
In agriculture production, using chemical agent is the major measure of preventing and treating diversified economy crop gray mold, and common medicament has derosal, iprodione, phonetic mould amine etc.But due to postpartum, the prevention and control of fruit disease are the last line of defenses that disease is controlled, and current spendable sterilant is very limited.In addition, Botrytis cinerea sporulation quantity is large, and growth cycle is short, is easy to sterilant to develop immunity to drugs, and studies have found that, China's ash arrhizus bacteria produces serious resistance problem to various sterilization agent as derosal, iprodione etc.Therefore,, for the prevention and control of gray mold, we are badly in need of finding the more efficiently method of preventing and treating.In addition,, because chemical bactericide has broad spectrum, its a large amount of use is not only easily destroyed micro-ecological environment, but also causes non-target resistance.
In non-chemically property prophylactico-therapeutic measures, biological control is one of the most promising method, and it has the advantages such as environmental friendliness, residual without agriculture, preventive effect is better.Though the report of at present more existing Botrytis cinerea antagonistic microbes, as gamboge ash Streptomycin sulphate (Streptomyces luteogriseus) ECO00001 (patent CN200610048662.1), Parasitism broom bacterium (Bionectria ochroleuca) WY-1(patent CN200910072862.4) etc., but not yet there is the report to genus bacillus.In addition, domestic existing utilize antagonistic bacterium biological control by the gray mold of the tomato causing and lily (as and CN200710014340), but preventive effect is poor, and lacks the research report of other crop gray mold biocontrol strains.
Summary of the invention
The invention provides a kind of Strain BS1 02, the pathogen of Botrytis cinerea mycelial growth, spore germination are had to good restraining effect, plant botrytis especially pears gray mold is had to good prevention effect.
The invention provides a kind of Strain BS1 02, Classification And Nomenclature subtilis (Bacillus subtilis), complete called after subtilis (Bacillus subtilis) BS102, on June 18th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCC No.7728.
This bacterial strain BS102 bacterium colony surface irregularity, opaque on LB substratum, presents shrinkage, white, can form gemma, can move aerobic-type.
The present invention also provides the application of described Strain BS1 02 in control plant botrytis.
Described plant is specifically as follows pears.
The kind of pears can be imperial crown.
Described application specifically comprises:
(1) preparation is containing the biocontrol fungicide of described Strain BS1 02;
(2) described biocontrol fungicide is sprayed on described plant.
While spraying biocontrol fungicide, spray to the plant surface globule mobile.
When pears gray mold is prevented and treated, can described biocontrol fungicide be sprayed on pear fruit at shelf time, spray to pear fruit surface water drops and flow, spraying can be by 20～25 ℃ of moisturizing 20～24h of pear fruit after end.
For reaching good prevention effect, in described biocontrol fungicide, the concentration of described Strain BS1 02 is preferably 1 * 10
8～10
10cFU/mL, more preferably 1 * 10
8cFU/mL.
Described biocontrol fungicide can be prepared by the following method:
(1) described Strain BS1 02 is cultured to OD in substratum
600be 0.5～0.8, obtain seed liquor;
(2) seed liquor is inoculated in to enlarged culturing in fermentation culture, regulates concentration to 1 * 10 of Strain BS1 02 in fermented liquid
8～10
10cFU/mL, obtains described biocontrol fungicide.
Described substratum can be selected LB liquid nutrient medium.
Wherein, the inoculum size of described seed liquor is preferably 1～2%(v/v), be preferably 1%(v/v).
In every liter, the formula of described fermentation culture is: glucose, 18～22g; Pidolidone, 2.4～2.6g; L-asparagine, 2.4～2.6g; KH
2pO
4, 0.8～1.2g; MgSO
47H
2o, 0.4～0.6g; KCl, 0.4～0.6g; Yeast powder, 0.8～1.2g; L-Phe, 2～3 * 10
-3g; MnSO
4h
2o, 5～6 * 10
-3g; CuSO
45H
2o, 0.16～0.18 * 10
-3g; FeSO
47H
2o, 0.15～0.17 * 10
-3g; Regulating pH value is 7.0.
As further preferred, in every liter, the formula of described fermentation culture base fluid is: glucose, 20g; Pidolidone, 2.5g; L-asparagine, 2.5g; KH
2pO
4, 1g; MgSO
47H
2o, 0.5g; KCl, 0.5g; Yeast powder, 1g; L-Phe, 2 * 10
-3g; MnSO
4h
2o, 5 * 10
-3g; CuSO
45H
2o, 0.16 * 10
-3g; FeSO
47H
2o, 0.15 * 10
-3g; Regulating pH value is 7.0.This fermentation culture is improved by Landy substratum, is more conducive to the propagation of subtilis of the present invention.
In the present invention, described enlarged culturing is to carry out in fermentor tank, and the temperature of described enlarged culturing is 28～30 ℃, is preferably 30 ℃, and the time of described enlarged culturing is 20～24h.Under this culture condition, when guaranteeing bacterium vigor, make it have rate of propagation faster.
In enlarged culturing process, fermented liquid pH value maintains 7.0, dissolved oxygen amount 20%, air flow 5-7m
3/ hour, rotating speed 240～260r/min, tank pressure 0.05-0.1KPa.It is 1 * 10 that fermented liquid goes out after tank that gradient dilution coated plate detects and regulate bacteria suspension concentration
8～10
10cFU/mL.
Compared with prior art, beneficial effect of the present invention is:
(1) Strain BS1 02 of the present invention is subtilis, biocontrol microorganisms BS102 thalline and supernatant show the pathogen of Botrytis cinerea mycelial growth, spore germination are had to good restraining effect, can be applied to the biological control of plant botrytis in dull and stereotyped antagonistic effect.
(2) the pear fruit gray mold control test effect that illumination box carries out shows, Strain BS1 02 of the present invention can effectively suppress germ in the scab expansion on pear fruit surface, preventive effect is up to 90%, under holding conditions, the preventive effect of 02 pair of pears gray mold of Strain BS1 of the present invention still can reach more than 68%, prevention effect is good, therefore can utilize this Strain BS1 02 control pears gray mold.
(3) Strain BS1 02 of the present invention is applied to the control of plant botrytis, can overcome the series of problems that the use because of chemical pesticide brings completely, thereby be conducive to the nuisanceless production of agricultural-food, peasant can or reduce the consumption of other chemical pesticides, this not only can be peasant and reduces expenses, and is conducive to improve the quality of products.
Accompanying drawing explanation
Fig. 1 is the colonial morphology figure of biocontrol microorganisms BS102.
Fig. 2 is the dull and stereotyped antagonism the pathogen of Botrytis cinerea of biocontrol microorganisms BS102 mycelial growth.
Fig. 3 is that the aseptic supernatant of biocontrol microorganisms BS102 suppresses the pathogen of Botrytis cinerea mycelial growth.
Fig. 4 is that the aseptic supernatant of biocontrol microorganisms BS102 suppresses the pathogen of Botrytis cinerea spore germination.
Fig. 5 is BS102 biocontrol fungicide illumination box control imperial crown gray mold effect.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
The collection of embodiment 1 BS102 biocontrol strain, separation and evaluation
BS102 strains separation is on Haian County, Jiangsu Province wheat wheat head.Get the wheat wheat head and grind, 80 ℃ of water-baths are coated on LB flat board after 10 minutes; Next day, picking colony carried out purifying cultivation.
Bacterial strain BS102 is bacterium colony surface irregularity, opaque on LB substratum, presents shrinkage, white (see figure 1), can form gemma, can move aerobic-type.
Extract the DNA of this bacterial strain, amplification 16S rDNA sequence, checks order, and the 16S rDNA of this bacterial strain is as shown in SEQ ID No.1, and the homology of the comparison result shows of sequence in NCBI and subtilis (Bacillus subtilis) is the highest.
This bacterial strain has been kept at the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preservation date is on June 18th, 2013, and deposit number is: CGMCC No.7728.
The preparation of embodiment 2 biocontrol fungicides
1, the preparation of biocontrol fungicide
(1) Strain BS1 02 is inoculated in LB nutrient solution, 30 ℃, 180r/min shaking culture, the OD value that after 12～16 hours, 600nm place is surveyed in sampling respectively in Bechtop, does not return to zero to connect the nutrient solution of bacterium in OD mensuration process;
(2) when shaking culture to the OD value of bacterium liquid is to be kind of a daughter bacteria liquid between 0.5～0.8 time, kind of daughter bacteria liquid be take to volume ratio to be equipped with in the 200L fermentor tank of fermentation culture as 1:100 adds, 30 ℃ of 250r/min shaking culture 20～24 hours, fermented liquid pH value maintains 7.0, dissolved oxygen amount is 20%, air flow 5-7m
3/ hour, tank pressure is 0.05-0.1KPa.After fermented liquid goes out tank, gradient dilution coated plate detects and regulates bacteria suspension concentration to be about 1 * 10
9cfu/ml.
The formula (every liter) of fermentation culture is: glucose, 20g; Pidolidone, 2.5g; L-asparagine, 2.5g; KH
2pO
4, 1g; MgSO
47H
2o, 0.5g; KCl, 0.5g; Yeast powder, 1g; L-Phe, 2 * 10
-3g; MnSO
4h
2o, 5 * 10
-3g; CuSO
45H
2o, 0.16 * 10
-3g; FeSO
47H
2o, 0.15 * 10
-3g; Regulating pH value is 7.0.
Embodiment 3 BS102 biocontrol strains are measured the antagonistic activity of the pathogen of Botrytis cinerea
1, BS102 measures the dull and stereotyped antagonistic activity of the pathogen of Botrytis cinerea
(1) test method
Adopt face-off culture method, by being stored in the pathogen of Botrytis cinerea GZ22 (pattern bacterium) in 4 ℃, being inoculated on PDA flat board and activating, the punch tool with sterilizing after fungi is covered with flat board breaks into from bacterium colony outward flange the circular bacterium piece that diameter is 6mm uniformly.By inoculated by hypha block at the dull and stereotyped (WA substratum/L: peptone 5g of WA, glucose 10g, gravy medicinal extract 3g, sodium-chlor 5g, agar 20g, pH=7.0) center, in its surrounding, apart from center, about 25mm inoculates respectively BS102 in place, and it is control group that subtilis type strain PY79, clear water and 0.3 μ g/ml fenhexamid are take in test.25 ℃ of cultivations, approach until mycelia the size (counting from bacterial colony center) of measuring inhibition zone while covering with flat board.
According to the size of inhibition zone, the antagonism the pathogen of Botrytis cinerea activity of BS102 is assessed: 0mm unrestraint effect; < 2mm has slight restraining effect; The medium restraining effect of 2-5mm; The restraining effect that > 5mm is stronger.
Inhibiting rate calculates according to following formula:
Inhibiting rate=100 (R
1-R
2)/R
1, R
1for the colony radius of contrast pathogenic bacteria in clear water direction; R
2for the colony radius of pathogenic bacteria in bacterium direction.
(2) interpretation of result
As shown in Figure 2, dull and stereotyped antagonistic effect shows: biocontrol microorganisms BS102 has stronger inhibition to the pathogen of Botrytis cinerea mycelial growth, suppressing circle radius is 15.0mm, and percentage mycelial inhibition is 71.4%, and negative control PY79, clear water and fenhexamid treatment group are without antagonistic effect.
2, the aseptic supernatant of biocontrol microorganisms BS102 suppresses the determination of activity of the pathogen of Botrytis cinerea mycelia
(1) test method
The biocontrol microorganisms BS102 bacterial strain fermentation liquor of getting embodiment 2,10,000r/min, gets supernatant after centrifugal 10min.Supernatant is stand-by after bacterial filter (0.22 μ m) filters.
To the spore that adds the pathogen of Botrytis cinerea GZ22 in 50 ℃ of WA substratum, to spore final concentration be 10
5individual/ml.WA substratum containing the pathogen of Botrytis cinerea GZ22 spore is poured in the flat board of 9cm diameter, and every flat board is poured 20mL into.After to be cooled solidifying, with the punch tool of 7mm diameter, in dull and stereotyped symmetric position, make a call to the aseptic supernatant that ，Mei hole, 3 holes adds respectively 25 μ L to prepare, take nutrient solution and fenhexamid as control group.After 25 ℃ of static cultivations, observe antagonism circle size (Cong Kong is counted at center).
(2) interpretation of result
The aseptic supernatant of biocontrol microorganisms BS102 can effective inhibition mycelial growth, and suppressing circle radius is 10.4mm, and the inhibition zone radiuses of 3 times of concentrated aseptic supernatants are 12.5mm, see Fig. 3.
3, the aseptic supernatant of biocontrol microorganisms BS102 suppresses the pathogen of Botrytis cinerea spore germination determination of activity
(1) test method
The biocontrol microorganisms BS102 bacterial strain fermentation liquor of getting embodiment 2,10,000r/min, gets supernatant after centrifugal 10min.Supernatant is stand-by after bacterial filter (0.22 μ m) filters.
In above-mentioned aseptic supernatant, add fructose, make fructose final concentration to 10mM, add the spore of the pathogen of Botrytis cinerea GZ22.Control group is the nutrient solution containing 10mM fructose.Get the spore suspension 10 μ l that are placed in fructose soln and be placed on slide glass, every processing repeats for 5 times.Slide is placed in moisture preservation box, 25 ℃ of static cultivations, and each observes each treatment group spore germination situation for 1 hour, calculates germination rate.
(2) interpretation of result
The sprouting of the aseptic supernatant energy of biocontrol microorganisms BS102 strongly inhibited the pathogen of Botrytis cinerea spore, the spore after processing can not form germ tube (Fig. 4).Induction is added up respectively the germination rate of each treatment group for 8,16,24 hours after sprouting, and result shows when negative control group germination rate is 100%, and BS102 treatment group spore is still without sprouting.Statistics is in Table 1.
Each treatment group the pathogen of Botrytis cinerea spore germination rate of table 1
Embodiment 4 biocontrol microorganisms illumination box control pears gray mold effect assessments
(1) test method
Pears surface spray inoculation 10
8cFU/ml(is containing 0.05%Tween20) BS102 to the globule, flow till (the approximately every fruit of 5～7ml), be placed in incubator 25 ℃ of moisturizing 24h.Next day, with aseptic toothpick, prick the dark hole of 1mm, then inoculate 6mm botrytis cinerea piece (10
5/ ml, containing 0.05%Tween20) to wound site.After inoculation, pears are placed in to 25 ℃, 90% humidity, observes afterwards morbidity per-cent for 4 days and measures scab diameter.Test is established and is not inoculated pathogenic bacteria and only inoculate pathogenic bacteria control treatment group, 15 pears of each treatment group inoculation.Test is imperial crown for examination pears kind.
(2) interpretation of result
Each treatment group illumination box control pears gray mold effect of table 2
In illumination box, carry out BS102 control pears gray mold test-results and show, biocontrol microorganisms BS102 can significantly suppress the pathogen of Botrytis cinerea GZ22 growth and cause a disease (Fig. 5) on pears.Prevention effect on imperial crown is 90.24%(table 2).
Embodiment 5 biocontrol microorganisms are for the control of pears gray mold
(1) test method
Get the biocontrol microorganisms BS102 bacterial strain fermentation liquor of embodiment 2, with dense fog type atomizer, at pears surface spray, inoculate 10
8cFU/ml(is containing 0.05%Tween20) BS102 to the globule, flow till (the approximately every fruit of 5～7ml), be placed in incubator 25 ℃ of moisturizing 24h.Next day, aseptic toothpick is pricked the dark hole of 1mm, with then inoculating 20 μ l (1 * 10
5conidium/ml) spore suspension of the pathogen of Botrytis cinerea GZ22 is to wound site.After inoculation, pears are placed in fruit tray, then pallet is put into carton, carton is placed in to 4 ℃, under ventilation condition, after 4 weeks, observe morbidity per-cent and measure scab diameter.Test is established and is not inoculated pathogenic bacteria spore and fenhexamid control treatment group, 12 pears of each treatment group inoculation.Whole experiment repeats 3 times.Test is imperial crown for examination pears kind.
(2) interpretation of result
Under table 3 BS102 holding conditions, prevent and treat imperial crown gray mold effect
As shown in table 3, under holding conditions, by spraying biocontrol microorganisms BS102, can significantly suppress the growth of the pathogen of Botrytis cinerea GZ22 on pears and cause a disease, average preventive effect to imperial crown pears gray mold can reach 68% left and right, approaches the prevention effect of the sterilant fenhexamid of control grey mold.
Claims (9)
1. a Strain BS1 02, is characterized in that, called after subtilis (Bacillus subtilis) BS102, and deposit number is CGMCC No.7728.
2. the application of Strain BS1 02 as claimed in claim 1 in control plant botrytis.
3. application as claimed in claim 2, is characterized in that, described plant is pears.
4. application as claimed in claim 3, is characterized in that, the kind of described pears is imperial crown.
5. the application as described in claim 2～4 any one, is characterized in that, described application comprises:
(1) preparation is containing the biocontrol fungicide of described Strain BS1 02;
(2) described biocontrol fungicide is sprayed on described plant.
6. application as claimed in claim 5, is characterized in that, in described biocontrol fungicide, the concentration of described Strain BS1 02 is 1 * 10
8～10
10cFU/mL.
7. application as claimed in claim 6, is characterized in that, described biocontrol fungicide can be prepared by the following method:
(1) described Strain BS1 02 is cultured to OD in substratum
600be 0.5～0.8, obtain seed liquor;
(2) seed liquor is inoculated in to enlarged culturing in fermentation culture, regulates concentration to 1 * 10 of Strain BS1 02 in fermented liquid
8～10
10cFU/mL, obtains described biocontrol fungicide.
8. application as claimed in claim 7, is characterized in that, the inoculum size of described seed liquor is 1～2%.
9. application as claimed in claim 7, is characterized in that, in every liter, the formula of described fermentation culture is: glucose, 18～22g; Pidolidone, 2.4～2.6g; L-asparagine, 2.4～2.6g; KH
2pO
4, 0.8～1.2g; MgSO
47H
2o, 0.4～0.6g; KCl, 0.4～0.6g; Yeast powder, 0.8～1.2g; L-Phe, 2～3 * 10
-3g; MnSO
4h
2o, 5～6 * 10
-3g; CuSO
45H
2o, 0.16～0.18 * 10
-3g; FeSO
47H
2o, 0.15～0.17 * 10
-3g.
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| CN114410528A (en) * | 2022-01-25 | 2022-04-29 | 山西农业大学食品科学与工程学院(山西省农业科学院农产品贮藏保鲜研究所) | Biocontrol bacterial liquid for Yulu bergamot pears during storage period and preparation method and application thereof |
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| CN103571777B (en) | 2015-06-17 |
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