CN103571777B - 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 Control Technology field, particularly relate 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 circular or the water stain shape scab of subcircular, do not cave in, after expanding gradually, form light tan irregular shape soft rot spot.The portion's dehydration of later stage disease, 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, later stage cotton hair 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, these wounds can as the passage of pathogenic bacteria.According to Food and Argriculture OrganizationFAO's report of survey display, in recovery process, physical abuse can make fruits and vegetables loss reach 8 ~ 12% improperly.In addition, current China fruit acquisition technique and postpartum fruit condition of storage all very limited, and weaker to the prevention and control of Postharvest diseases.Therefore, the gray mold on shelf lives the operatic circle still can cause serious financial consequences.
In agriculture production, use chemical agent to be the major measure of preventing and treating diversified economy crop gray mold, 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 of Disease management, and current spendable sterilant is very limited.In addition, Botrytis cinerea sporulation quantity is large, and growth cycle is short, is easy to develop immunity to drugs to sterilant, studies have found that, China's ash arrhizus bacteria to various sterilization agent as derosal, iprodione etc. produce serious resistance problems.Therefore, for the prevention and control of gray mold, we are badly in need of finding more efficiently prevention and controls.In addition, because chemical bactericide has broad spectrum, it uses in a large number and not only easily destroys 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 more existing Botrytis cinerea antagonistic microbes at present, 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 tomato caused and lily gray mold (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, have good restraining effect to the pathogen of Botrytis cinerea mycelial growth, spore germination, to plant botrytis especially pears gray mold, there is 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, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 18th, 2013, 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.
Present invention also offers 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.
When spraying biocontrol fungicide, spray to the plant surface globule and flow.
During as prevented and treated pears gray mold, described biocontrol fungicide can be sprayed on pear fruit at shelf time, spraying and flowing to pear fruit surface water drops, can by pear fruit 20 ~ 25 DEG C of moisturizing 20 ~ 24h after spraying 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, is more preferably 1 × 10
8cFU/mL.
Described biocontrol fungicide is prepared by 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 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 select LB liquid nutrient medium.
Wherein, the inoculum size of described seed liquor is preferably 1 ~ 2%(v/v), be preferably 1%(v/v).
In often 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; Adjust ph is 7.0.
As preferred further, in often 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; Adjust ph 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 carried out in fermentor tank, and the temperature of described enlarged culturing is 28 ~ 30 DEG C, and be preferably 30 DEG C, the time of described enlarged culturing is 20 ~ 24h.Under this culture condition, while guarantee 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.After fermented liquid goes out tank, gradient dilution coated plate detects and regulates bacteria suspension concentration to be 1 × 10
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 in dull and stereotyped antagonistic effect has good restraining effect to the pathogen of Botrytis cinerea mycelial growth, spore germination, can be applied to the biological control of plant botrytis.
(2) the pear fruit gray mold controlling experiment effect that illumination box carries out shows, Strain BS1 02 of the present invention can effectively suppress germ at the Spot expansion on pear fruit surface, preventive effect is up to 90%, under holding conditions, the preventive effect of Strain BS1 of the present invention 02 pair of pears gray mold still can reach more than 68%, prevention effect is good, and this Strain BS1 02 therefore can be utilized to prevent and treat pears gray mold.
(3) Strain BS1 02 of the present invention is applied to the control of plant botrytis, the series of problems that the use because of chemical pesticide brings can be overcome completely, thus the No-harmful apple orchard of agricultural-food is conducive to, peasant or can reduce the consumption of other chemical pesticides, this not only can be peasant and reduces expenses, and is conducive to improving the quality of products.
Accompanying drawing explanation
Fig. 1 is the colonial morphology figure of biocontrol microorganisms BS102.
Fig. 2 is biocontrol microorganisms BS102 dull and stereotyped antagonism the pathogen of Botrytis cinerea 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 andpreconcentration
BS102 strains separation is on Haian County of Jiangsu Province wheat wheat head.Get the wheat wheat head to grind, 80 DEG C of water-baths are coated after 10 minutes on LB flat board; Next day, picking colony carried out purifying cultivation.
Bacterial strain BS102 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 comparison result display of sequence in NCBI is the highest with the homology of subtilis (Bacillus subtilis).
This bacterial strain has been kept at the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being 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 fungicide
1, the preparation of biocontrol fungicide
(1) Strain BS1 02 is inoculated in LB nutrient solution, 30 DEG C, 180r/min shaking culture, after 12 ~ 16 hours, in Bechtop, the OD value at 600nm place is surveyed in sampling respectively, returns to zero in OD mensuration process with the nutrient solution not connecing bacterium;
(2) kind of a daughter bacteria liquid is when shaking culture is between 0.5 ~ 0.8 to the OD value of bacterium liquid, be that 1:100 adds and is equipped with in the 200L fermentor tank of fermentation culture with volume ratio by kind of daughter bacteria liquid, 30 DEG C 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 (often 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; Adjust ph is 7.0.
Embodiment 3 BS102 biocontrol strain measures 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 opposite culture method, the pathogen of Botrytis cinerea GZ22 be stored in 4 DEG C (pattern bacterium) is inoculated on PDA flat board and activates, cover with after flat board until fungi and break into from bacterium colony outward flange the circular bacterium block that diameter is 6mm with the punch tool of sterilizing uniformly.By inoculated by hypha block at WA flat board (WA substratum/L: peptone 5g, glucose 10g, gravy medicinal extract 3g, sodium-chlor 5g, agar 20g, pH=7.0) center, is about 25mm place in its surrounding apart from center and inoculates BS102 respectively, test with subtilis type strain PY79, clear water and 0.3 μ g/ml fenhexamid as control group.25 DEG C of cultivations, until mycelia close to the size (counting from bacterial colony center) measuring inhibition zone when covering with flat board.
The antagonism the pathogen of Botrytis cinerea activity of size to BS102 according to inhibition zone 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 on clear water direction; R
2for pathogenic bacteria is at the colony radius 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, suppress circle radius to be 15.0mm, 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 Example 2,10,000r/min, gets supernatant after centrifugal 10min.Supernatant is stand-by after bacterial filter (0.22 μm) filters.
In 50 DEG C of WA substratum, add the spore of the pathogen of Botrytis cinerea GZ22, be 10 to spore final concentration
5individual/ml.Pour in the flat board of 9cm diameter by the WA substratum containing the pathogen of Botrytis cinerea GZ22 spore, 20mL poured into by every flat board.To be cooled solidify after, make a call to 3 holes with the punch tool of 7mm diameter in the symmetric position of flat board, every hole adds aseptic supernatant prepared by 25 μ L respectively, with nutrient solution and fenhexamid for control group.Antagonism circle size (counting from center, hole) is observed after 25 DEG C of static gas wave refrigerator.
(2) interpretation of result
The aseptic supernatant of biocontrol microorganisms BS102 effectively can suppress mycelial growth, suppresses circle radius to be 10.4mm, and the inhibition zone radius of 3 times of concentrated aseptic supernatants is 12.5mm, sees 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 Example 2,10,000r/min, gets supernatant after centrifugal 10min.Supernatant is stand-by after bacterial filter (0.22 μm) 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.Getting the spore suspension 10 μ l being placed in fructose soln is placed on slide glass, often processes 5 times and repeats.Slide is placed in moisture preservation box, 25 DEG C of static gas wave refrigerator, within each 1 hour, observes each treatment group spore germination situation, calculates germination rate.
(2) interpretation of result
The sprouting of biocontrol microorganisms BS102 aseptic supernatant energy strongly inhibited the pathogen of Botrytis cinerea spore, the spore after process can not form germ tube (Fig. 4).Induction adds up the germination rate of each treatment group for 8,16,24 hours after sprouting respectively, and result display is when negative control group germination rate is 100%, and BS102 treatment group spore is still without sprouting.Statistics is in table 1.
Table 1 each treatment group the pathogen of Botrytis cinerea spore germination rate
Embodiment 4 biocontrol microorganisms illumination box control pears gray mold effect assessment
(1) test method
Pears surface spray inoculation 10
8cFU/ml(is containing 0.05%Tween20) BS102 to globule flowing (the every fruit of about 5 ~ 7ml), be placed in incubator, 25 DEG C of moisturizing 24h.Next day, prick the dark hole of 1mm with sterile toothpick, then inoculate 6mm botrytis cinerea block (10
5/ ml, containing 0.05%Tween20) to wound location.After inoculation, pears are placed in 25 DEG C, 90% humidity, within 4 days, observe percent incidence afterwards and measure lesion diameter.Test is established and is not inoculated pathogenic bacteria and only inoculate pathogenic bacteria control treatment group, and each treatment group inoculates 15 pears.Test is imperial crown for examination Pear varieties.
(2) interpretation of result
Table 2 each treatment group illumination box control pears gray mold effect
In illumination box, carry out BS102 prevent and treat pears gray molds test-results and show, biocontrol microorganisms BS102 significantly can suppress the growth of the pathogen of Botrytis cinerea GZ22 on pears and cause a disease (Fig. 5).Prevention effect on imperial crown is 90.24%(table 2).
Embodiment 5 biocontrol microorganisms is used for the control of pears gray mold
(1) test method
The biocontrol microorganisms BS102 bacterial strain fermentation liquor of Example 2, with dense fog type atomizer in pears surface spray inoculation 10
8cFU/ml(is containing 0.05%Tween20) BS102 to globule flowing (the every fruit of about 5 ~ 7ml), be placed in incubator, 25 DEG C of moisturizing 24h.Next day, sterile toothpick pricks 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 location.After inoculation, pears are placed in fruit tray, then pallet is put into carton, carton is placed in 4 DEG C, under ventilation condition, observe percent incidence after 4 weeks and measure lesion diameter.Test is established and is not inoculated pathogenic bacteria spore and fenhexamid control treatment group, and each treatment group inoculates 12 pears.Whole experiment repetition 3 times.Test is imperial crown for examination Pear varieties.
(2) interpretation of result
Imperial crown gray mold effect is prevented and treated under table 3 BS102 holding conditions
As shown in table 3, under holding conditions, by spraying biocontrol microorganisms BS102, significantly can suppress the growth of the pathogen of Botrytis cinerea GZ22 on pears and causing a disease, can about 68% be reached, close to the prevention effect of the sterilant fenhexamid of control grey mold to the average preventive effect of imperial crown pears gray mold.
Claims (9)
1. a Strain BS1 02, is characterized in that, called after subtilis (Bacillus subtilis) BS102, deposit number is CGMCC No.7728.
2. the application of Strain BS1 02 as claimed in claim 1 in control plant botrytis.
3. apply as claimed in claim 2, it is characterized in that, described plant is pears.
4. apply as claimed in claim 3, it is characterized in that, the kind of described pears is imperial crown.
5. the application as described in any one of claim 2 ~ 4, 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. apply as claimed in claim 5, it is characterized in that, in described biocontrol fungicide, the concentration of described Strain BS1 02 is 1 × 10
8~ 10
10cFU/mL.
7. apply as claimed in claim 6, it is characterized in that, described biocontrol fungicide is 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 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. apply as claimed in claim 7, it is characterized in that, the inoculum size of described seed liquor is 1 ~ 2%.
9. apply as claimed in claim 7, it is characterized in that, in often 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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| CN114410528B (en) * | 2022-01-25 | 2023-07-21 | 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 storage period of Yulu fragrant pears and preparation method and application thereof |
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