CN102174431B - Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon - Google Patents
Bio-control strain for preventing and controlling bacterial fruit blotch of watermelon Download PDFInfo
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- CN102174431B CN102174431B CN 201110004936 CN201110004936A CN102174431B CN 102174431 B CN102174431 B CN 102174431B CN 201110004936 CN201110004936 CN 201110004936 CN 201110004936 A CN201110004936 A CN 201110004936A CN 102174431 B CN102174431 B CN 102174431B
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Abstract
The invention relates to a bio-control strain JC65 for preventing and controlling bacterial fruit blotch of watermelon, and belongs to the technical field of crop disease prevention and control. The bacteria are Bacillus sp.; and the bacteria are subjected to shaking culture for 20 to 24 hours in load balance (LB) culture solution at the temperature of 30 DEG C and at the revolution speed of 180rpm, then the solution is centrifuged for 10 minutes at the revolution speed of 6,000rpm, the wet bacteria and the bacteria preservation solution are prepared into bactericide in a ratio of 1:40 (g/ml), and the total concentration of living bacteria in the finished product is 1*10<9> to 1*10<12>cfu/ml. The bactericide is diluted to the concentration of 1*10<8>cfu/ml in use, the prevention effect on the bacterial fruit blotch of watermelon is 50 to 90 percent, and the yield increasing effect on a sick field is 10 to 200 percent. The growth promoting effect on the disease-free plants can reach 10to 200 percent, and the yield increasing effect on the disease-free field is 5 to 20 percent. The bactericide can be blended with water for spraying or made into compost for use, and can also be diluted for irrigating roots during transplanting.
Description
One, technical field
The present invention relates to a kind of genus bacillus biocontrol strain JC65 that prevents and treats the bacillary fruit blotch disease of watermelon, belong to the farm crop technical field of preventing and curing diseases, be exclusively used in the non-environmental pollution control of the bacillary fruit blotch of watermelon.
Two, background technology
The bacillary fruit blotch of watermelon (Bacterial fruit blotch of watermelon; BFB) be a kind of dangerous quarantine disease on the watermelon; Claim bacterial speck again; This disease was found at Fla. by Crall and Schenck early than 1969, just the fruit blotch symptom was described at that time, did not do the evaluation of pathogenic bacteria; So far, in the many provinces and cities of China, as Xinjiang, Shaanxi, Hebei, Shanxi, Jilin, Fujian, ground such as Gansu all have the report of fruit blotch.This disease main harm fruit causes watermelon fruit rot etc., can cause crushing loss, in case morbidity medication control does not have effect basically.And infected seed will become the important primary source of infection of next year.The yield and quality that has had a strong impact on watermelon of this disease becomes one of difficult problem that needs to be resolved hurrily in the watermelon production.
This disease be by oat wtih watermelon subspecies (
Acidovorax avenaeSubsp.
Citrulli, Aac) cause, the watermelon fruit spot does not have immunological species at present in melon crop.The bacillary fruit blotch of watermelon is a kind of destructive bacterial disease, and field growth period is difficult for coming to light, and morbidity is unexpected, development is rapid, because at present
Also do not find the better method of seed treatment, the chemical agent that is adopted in producing at present has agricultural streptomycin, can kill; New strysolin, bacterium kills, Ye Shuanqing; Jia Ruinong, tiger glue adipic acid coppers etc. are though chemical agent has certain control effect to this disease; But but control effect is not high, and uses chemical bactericide to tend to cause environmental pollution, pesticide residue in a large number, and causes problems such as pathogenic bacteria resistance.Biological control is a kind of means of prevention safely and effectively, meets the direction of agricultural sustainable development.
In the prior art according to this breadboard preservative fluid for thallus patent (publication CN1358838, open day: on July 17th, 2002) method, but thalline room temperature preservation 2～3 years.
Three, summary of the invention
Technical problem:The objective of the invention is the bigger bacillary fruit blotch of watermelon of biological control watermelon industry potential hazard, live through opposing enzyme and test and the warmhouse booth test, this disease effect of microbial inoculum control of the genus bacillus JC65 that screening obtains is better, and preventive effect can reach more than 50%.
Technical scheme
A kind of biocontrol strain JC65 that prevents and treats the bacillary fruit blotch disease of watermelon, for genus bacillus (
Bacillus
Sp.), on December 15th, 2010 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, and Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC NO.4475.
The method that biocontrol strain JC65 is used to prepare biocontrol fungicide is:
1) with this inoculation in the LB nutrient solution 30 ℃ cultivate the 180rpm shaking culture, after 12～16 hours in Bechtop the sampling OD value of surveying the 600nm place respectively, return to zero with the LB nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time; Kind of daughter bacteria liquid added with the 1:100 volume ratio be equipped with in the 200L fermentor tank of fermentation culture, 30 ℃ of 250rpm shaking culture 20～24 hours, fermented liquid pH value maintains 7.0; Dissolved oxygen amount 20%, air flow 5-7 m
3/ hour, tank pressure 0.05-0.1KPa.The centrifugal 10min of 6000rpm then, every 1000ml nutrient solution obtain the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline is got wet bacterium and preservative fluid for thallus and is made into microbial inoculum by 1 g:40ml, and the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Cfu/ml.Wherein:
1) seed culture fluid LB prescription: sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L, and regulating the pH value is 7.2;
2) fermentation culture liquid formula: SANMALT-S 2.5g/L, Semen Maydis powder 5 g/L, analysis for soybean powder 5g/L, Tryptones 5 g/L, Calcium dichloride dihydrate 0.5 g/L, manganese sulfate monohydrate 0.5 g/L, potassium hydrogenphosphate 1g/L, regulating the pH value is 7.0;
3) preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
Beneficial effect
Advantage of the present invention and positively effect show: the present invention is the biological prevention and control agent of developing to the bacillary fruit blotch disease of watermelon specially.Owing to be biotechnological formulation; Do not have a series of problems that use brought because of chemical pesticide fully, thereby help the nuisanceless production of watermelon, the peasant can or reduce the consumption of other chemical pesticides; This not only can be the peasant and reduces expenses, and helps the export trade of watermelon.
Biocontrol microorganisms JC65 thalline shows in dull and stereotyped antagonistic effect has good inhibitory effect (Fig. 1 .) to watermelon fruit blotch pathogenic bacteria; This bacterium has proteolytic enzyme (Fig. 2 .) simultaneously, has a liking for plain enzyme (Fig. 3 .) of iron and cellulase activity (Fig. 4 .), and the warmhouse booth test-results shows; Biocontrol fungicide JC65 is remarkable to the growth-promoting functions effect of watermelon; The living weight increase reaches 119.00% (table 1), and biocontrol fungicide JC65 is remarkable to watermelon fruit blotch diseases control, up to 57.63% (table 2); The field experiment result shows that biocontrol fungicide JC65 can significantly reduce the sickness rate of the bacillary fruit blotch of watermelon simultaneously; Its average preventive effect reaches 50.76%, and this preparation has significant growth-promoting functions to watermelon simultaneously, finds in the test; This microbial inoculum reaches 16.96% to the raising the output average energy of anosis field piece, can on average reach 125.30% (table 3) to the raising the output of ill field piece
This microbial inoculum (belongs to this laboratory granted patent with wet bacterium and this breadboard preservative fluid for thallus; Patent No. ZL02112559.7; Publication CN1358838, open day: on July 17th, 2002) be made into microbial inoculum by 1:40 (g/ml), but room temperature preservation 2～3 years.This microbial inoculum can dilute the back in watermelon sowing or move when plucking pouring and use.
Four, description of drawings
Fig. 1.
BacillusSp. the bacillary fruit blotch germ of the dull and stereotyped antagonism watermelon of JC65 effect
;
Fig. 2.
BacillusSp. the JC65 proteolytic enzyme enzyme test effect of living
Fig. 3.
BacillusSp. JC65 cellulose enzyme activity test effect
Fig. 4.
BacillusSp. JC65 has a liking for the plain enzyme enzyme of iron test effect alive
Fig. 5.
BacillusSp. JC65 is to the growth-promoting functions of watermelon
Five, embodiment
A kind of biocontrol strain JC65 that prevents and treats the bacillary fruit blotch disease of watermelon, for genus bacillus (
Bacillus
Sp.), obtain by separating in the pear tree rhizosphere soil of theatre, county, Deqing, Zhejiang Province.
Biocontrol strain
Bacillus sp.The method that JC65 is used to prepare biocontrol fungicide is:
1) with this inoculation in the LB nutrient solution 30 ℃ cultivate the 180rpm shaking culture, after 12～16 hours in Bechtop the sampling OD value of surveying the 600nm place respectively, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time; Kind of daughter bacteria liquid added with the 1:100 volume ratio be equipped with in the 200L fermentor tank of fermentation culture, 30 ℃ of 250rpm shaking culture 20～24 hours, fermented liquid pH value maintains 7.0; Dissolved oxygen amount 20%, air flow 5-7 m
3/ hour, tank pressure 0.05-0.1KPa.The centrifugal 10min of 6000rpm then, every 1000ml nutrient solution obtain the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline is got wet bacterium and preservative fluid for thallus and is made into microbial inoculum by 1 g:40ml, and the viable bacteria total concn is 1 * 109～1 * 1012cfu/ml in the finished product.Wherein:
Seed culture fluid LB prescription: sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L, and regulating the pH value is 7.2;
The fermentation culture liquid formula: SANMALT-S 2.5g/L, Semen Maydis powder 5 g/L, analysis for soybean powder 5g/L, Tryptones 5 g/L, Calcium dichloride dihydrate 0.5 g/L, manganese sulfate monohydrate 0.5 g/L, potassium hydrogenphosphate 1g/L, regulating the pH value is 7.0;
Preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
To combine test correlated results figure below, and carry out embodiment and explain:
(1) the dull and stereotyped antagonistic effect of genus bacillus JC65 thalline
The bacillary fruit blotch pathogenic bacteria of selection watermelon oat wtih (
Acidovorax avenae) FC440 (Jianjun Feng, Erin L. Schuenzel, Jianqiang Li, and Norman W. Schaad. 2009. Multilocus Sequence Typing Reveals Two Evolutionary Lineages of
Acidovorax avenaeSubsp.
Citrulli.Bacteriology.99 (8): 913-920) as supplying the examination pathogenic bacteria.Cultivation 16-24 h to CFU in LB nutrient solution (sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L, regulating pH value is 7.2) is 10 with pathogenic bacteria
8/ ml), add fusing and be cooled in the solid medium about 40 ℃, volume ratio 1:200, every 20mL pours in the flat board of 9 cm diameters with substratum.After to be cooled the solidifying, toothpick inoculation biocontrol microorganisms JC65.Place 28 ℃ of constant incubators to cultivate, record inhibition zone ring footpath behind 24 h.
(2) genus bacillus JC65 bacterial enzyme determination test alive
With biocontrol microorganisms JC65 activation on the LB flat board (sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L; Regulating the pH value is 7.2), cultivate 24 h, be inoculated into proteolytic enzyme respectively for 28 ℃ with toothpick picking list bacterium colony; Have a liking for the plain enzyme of iron, and on the flat board of cellulase activity mensuration, place 28 ℃ of constant incubators to cultivate; Cultivate 2-3 d, measure the size
of transparent circle.
(3) short the giving birth in genus bacillus JC65 greenhouse tested
After the germination of watermelon seeds were sown in pots containing sterile soil in Portland, pot one,? 16/8? H photoperiod.Keep soil comparatively dry before the sowing.Water processing in the time of the watermelon sowing, the bacterium liquid that suspension is good is with 10
8The concentration of CFU/ml slowly pours into watermelon seed and encloses, every strain 20 ml, and after this routine is watered.Place the greenhouse to cultivate, 28 ℃, more than 70% relative humidity, the biocontrol microorganisms inoculation was got treatment group and control group plant respectively after 30 days, carefully rejected root soil, and after cleaning up, natural air drying is added up minister on the ground respectively, underground minister, plant fresh weight.60oC is dried and is measured the plant dry weight behind 72 h.
Living weight increases (%)=(treatment group plant dry weight-adjoining tree dry weight)/adjoining tree dry weight * 100%
(4) genus bacillus JC65 warmhouse booth efficiency test
After the germination of watermelon seeds were sown in pots containing sterile soil in Portland, a pot before planting to keep the soil is dry.Water processing in the time of the watermelon sowing, the bacterium liquid that suspension is good is with 10
8The concentration of CFU/ml slowly pours into watermelon seed and encloses, every strain 20 ml, wherein with clear water as contrast, cultivate with being placed in the greenhouse, 28 ℃, more than 70% relative humidity, 16/8 h photoperiod.Second and third day only waters on a small quantity to keep planting and encloses moistening getting final product, and after this routine is watered.Reuse biocontrol microorganisms in the time of 15 days and spray in plant leaf, treat that the second inoculation antagonism bacterium after 5 days, carries out the inoculation of pathogenic bacteria, inoculum density is 10
7CFU/ml inoculates back 3 days, begins to add up its sickness rate, and calculates preventive effect.
The disease severity%=[Σ (sick level strain number * represent progression)/plant sum * the highest grade value of representing] * 100%;
Preventive effect%=(contrast sickness rate-processing diseases prevention rate)/contrast sickness rate * 100%;
Living weight increases%=(control group living weight-treatment group living weight) control group living weight * 100%;
(5) genus bacillus JC65 field experiment
This test is divided into two Da Qu; Be respectively ill field piece and anosis field piece, treat watermelon move pluck when, with amount and the normal root water mixing of this preparation with every mu of consumption 6-10 L; Carry out pouring at plant rhizosphere; Subsequently respectively move pluck the back 30 d, 60 d carry out tracking processing with above-mentioned identical method and consumption.When results, the incidence of statistics plant and the per mu yield of each field piece.Thereby calculate separately preventive effect and the short situation of giving birth to.
The disease severity%=[Σ (sick level strain number * represent progression)/plant sum * the highest grade value of representing] * 100%;
Preventive effect%=(contrast sickness rate-processing diseases prevention rate)/contrast sickness rate * 100%;
Living weight increases%=(control group living weight-treatment group living weight) control group living weight * 100%;
Test-results:
(1) biocontrol microorganisms JC65 antagonism pathogenic bacteria test-results
Biological and ecological methods to prevent plant disease, pests, and erosion genus bacillus JC65 thalline shows the dull and stereotyped preferably effect (Fig. 1 .) that suppresses.
(2) the dull and stereotyped enzyme activity determination test of genus bacillus JC65 thalline
Biological and ecological methods to prevent plant disease, pests, and erosion genus bacillus JC65 thalline has protease activity, cellulase activity, and it is plain active to have a liking for iron.(Fig. 2, Fig. 3, Fig. 4)
(3) short the giving birth in genus bacillus JC65 greenhouse tested
The short test-results of giving birth in greenhouse shows that genus bacillus JC65 can significantly promote the growth of watermelon, shows the fresh weight of watermelon; Dry weight, root is long, and stem is long; Compare with contrast, significant increase is all arranged, the result calculates genus bacillus JC65 can reach 119% to the living weight increase of watermelon.
(4) genus bacillus JC65 warmhouse booth efficiency test
Test-results shows that genus bacillus JC65 can significantly reduce the sickness rate of the bacillary fruit blotch of watermelon, and its average preventive effect reaches 57.63%.
(5) genus bacillus JC65 field experiment
Field experiment result shows; Genus bacillus JC65 can significantly reduce the sickness rate of the bacillary fruit blotch of watermelon, and its average preventive effect reaches 50.76%, and this preparation has significant growth-promoting functions to watermelon simultaneously; Find in the test; This microbial inoculum reaches 16.96% to the raising the output average energy of anosis field piece, can on average reach 125.30% to the raising the output of ill field piece, and therefore above result can explain that this biotechnological formulation has significant biological and ecological methods to prevent plant disease, pests, and erosion and short life ability.
Claims (2)
1. biocontrol strain JC65 who prevents and treats the bacillary fruit blotch of watermelon, for genus bacillus (
BacillusSp.), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 15th, 2010, culture presevation number is CGMCC NO.4475.
2. the biocontrol fungicide for preparing with the said biocontrol strain JC65 of claim 1.
3. the application of the said biocontrol fungicide of claim 2 in the bacillary fruit blotch of control watermelon.
4. the said biocontrol strain JC65 of claim 1 is used to prepare the method for biocontrol fungicide,
1) the said biocontrol strain JC65 of claim 1 is inoculated into 30 ℃ of cultivation 180rpm shaking culture in the LB nutrient solution, in Bechtop, takes a sample respectively after 12～16 hours and survey the OD value at 600nm place, return to zero with the nutrient solution that does not connect bacterium in the mensuration process;
2) when being cultured to the OD value between 0.5～0.8 the time; Kind of daughter bacteria liquid added with the 1:100 volume ratio be equipped with in the 200L fermentor tank of fermentation culture, 30 ℃ of 250rpm shaking culture 20～24 hours, fermented liquid pH value maintains 7.0; Dissolved oxygen amount 20%, air flow 5-7 m
3/ hour, tank pressure 0.05-0.1KPa, the centrifugal 10min of 6000rpm then, every 1000ml nutrient solution obtain the wet bacterium of 13～16g, and the wet bacterium of every gram contains 4 * 10
10～10
15Individual thalline is got wet bacterium and preservative fluid for thallus and is made into microbial inoculum by 1 g:40ml, and the viable bacteria total concn is 1 * 10 in the finished product
9～1 * 10
12Cfu/ml.
5. method according to claim 4, wherein:
1) LB nutrient solution prescription: sodium-chlor 10g/L, Tryptones 10g/L, yeast soak powder 5g/L, and regulating the pH value is 7.2;
2) fermentation culture liquid formula: SANMALT-S 2.5g/L, Semen Maydis powder 5 g/L, analysis for soybean powder 5g/L, Tryptones 5 g/L, Calcium dichloride dihydrate 0.5 g/L, manganese sulfate monohydrate 0.5 g/L, potassium hydrogenphosphate 1g/L, regulating the pH value is 7.0;
3) preservative fluid for thallus is the 0.01mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 non-ionics of 0.001mol/L.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604869B (en) * | 2012-03-23 | 2013-05-29 | 南京农业大学 | Bio-control bacteria strain 1JN2 for preventing and treating bacterial fruit blotches of watermelons and application thereof |
| CN103614306B (en) * | 2013-08-27 | 2016-06-15 | 石河子大学 | One saccharomycete strain and application thereof |
| CN104894008B (en) * | 2015-05-13 | 2018-04-13 | 中国农业科学院植物保护研究所 | One plant of melon bacterial fruit blotch biological and ecological methods to prevent plant disease, pests, and erosion Bacillus amyloliquefaciens strain and its application |
| CN105961441B (en) * | 2016-06-18 | 2019-05-14 | 南京农业大学 | The application of bacillus JC65 and its volatile materials in plant growth-promoting |
| CN106967632A (en) * | 2017-02-23 | 2017-07-21 | 南京农业大学 | It is a kind of efficiently to prevent and treat various crop gray mold and the microorganism mixture of capsicum resistance be lifted |
| CN106754567B (en) * | 2017-02-23 | 2018-05-01 | 南京农业大学 | A kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS of efficiently prevention various crop droop |
| CN113061559A (en) * | 2021-05-10 | 2021-07-02 | 南京农业大学 | Composite biological agent and application thereof in preventing and treating rice blast and promoting rice growth |
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|---|---|---|---|---|
| CN1358838A (en) * | 2002-01-18 | 2002-07-17 | 南京农业大学 | Preservation method for live bacterial preparation |
| CN101215543A (en) * | 2008-01-18 | 2008-07-09 | 新疆农业科学院微生物应用研究所 | Strain for preventing and curing muskmelon bacteroidal spot disease and application thereof in field disease prevention |
| CN101864377A (en) * | 2010-03-16 | 2010-10-20 | 南京农业大学 | Biocontrol bacterial strain SM16 for preventing pear die-back |
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2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1358838A (en) * | 2002-01-18 | 2002-07-17 | 南京农业大学 | Preservation method for live bacterial preparation |
| CN101215543A (en) * | 2008-01-18 | 2008-07-09 | 新疆农业科学院微生物应用研究所 | Strain for preventing and curing muskmelon bacteroidal spot disease and application thereof in field disease prevention |
| CN101864377A (en) * | 2010-03-16 | 2010-10-20 | 南京农业大学 | Biocontrol bacterial strain SM16 for preventing pear die-back |
Non-Patent Citations (3)
| Title |
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| Flávio H. V. Medeiros et al..Management of melon bacterial blotch by plant beneficial bacteria.《Phytoparasitica》.2009,第37卷(第5期),453-460. * |
| Xiaodong Wang et al..Screening of plant epiphytic yeasts for biocontrol of bacterial fruit blotch (Acidovorax avenae subsp. citrulli) of hami melon.《Biological Control》.2009,第50卷(第2期),164-171. * |
| 贺字典，高玉峰.生防菌在植物病害防治中的研究进展(综述).《河北职业技术师范学院学报》.2003,第17卷(第2期),56-59. * |
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