CN102399712B - Bacterial strain CHS55 for controlling cucumber powdery mildew and downy mildew - Google Patents
Bacterial strain CHS55 for controlling cucumber powdery mildew and downy mildew Download PDFInfo
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Abstract
The invention which relates to a bacterial strain CHS55 for controlling cucumber powdery mildew and downy mildew belongs to the technical field of plant protection. The bacterial strain CHS55 is subjected to oscillation culturing for 18-20h in an LB nutrient solution under conditions that the temperature is 32DEG C and the oscillation speed is 250rpm, and is centrifuged for 15min according to a rotation speed of 4000rpm, a bacterial inoculum is prepared by mixing above wet bacterium with a thalline preservation liquid in the lab of the inventors according to a ratio of 1:40 (g/ml), and the total concentration of live bacteria in the bacterial inoculum is 1*10<9>-1*10<12>CFU/mL. The bacterial inoculum which is diluted 100-1000 times is sprayed or sprinkled on transplanted cucumber of the four-leaf stage, and is once used according to an interval period of 10d/7d when weather factors are suitable for the early epidemic cucumber downy mildew, and control efficiencies of the bacterial inoculum to the cucumber downy mildew reach 62.31% and 65.79% respectively. In middle and late stages of greenhouse tests, the bacterial inoculum has a strong control effect on the powdery mildew, the control efficiency reaches more than 70%, and the control efficiency of the crude protein extract of the bacterial inoculum to the cucumber downy mildew reaches 70.33%.
Description
One, technical field
The present invention relates to a kind of biocontrol strain CHS55 that prevents and treats warmhouse booth powdery mildew of cucumber and oidium, belong to the plant protection technology field, be exclusively used in oidium and the Powdery Mildew non-environmental pollution control of Folium Cucumidis sativi section.
Two, background technology
Cucumber downy mildew and Powdery Mildew be warmhouse booth Folium Cucumidis sativi section two kinds of their pathogenic bacterias of fungal disease of normal generation all be obligate parasite, and the specialization selectivity to host plant is very strong, its sporocyst can be propagated with wind and rain, cucumber beetle, agricultural utensil etc., all is a kind of main air infection diseases.
Along with China's warmhouse booth area sharply increases, the plantation of anti-season cucumber greenhouse cultivation is in enormous quantities to be occurred, so that its germ can be infected cucumber throughout the year; Fall ill when serious, it is withered that full leaf is tawny, causes at last plant withered early dead, shortens and receive the melon phase, has a strong impact on cucumber yield, and loss can reach 20% to 60%, and this disease is that warmhouse booth affects one of main restricting factor of cucumber yield.
This disease peasant sprays chemical agent according to experience, spray chemical agent one week one time, but the speed that resistance produces is very fast, at present many businessmans transfer to sight on green, the ecological product biological pesticide, but mostly be in the laboratory study development phase, registration very little.And because two kinds of pathogenic bacterias are live body bacterial parasites, mostly finish the test of simple indoor measurement, carry out heliogreenhouse in China main cucumber cultivation district or test the biological and ecological methods to prevent plant disease, pests, and erosion research of drug effect broad spectrum test very little in the open.In sum, what develop new, more efficient and safe microbial bactericide control cucumber downy mildew and Powdery Mildew is the needs that improve output, and the while is the needs of agricultural safety Sustainable development especially, symbol social development demand.
Three, summary of the invention
1 technical problem:
For the present situation that cucumber downy mildew is not had the biological prevention and control agent of higher preventive effect in the prior art, a kind of single Biocontrol Strain and microbial inoculum thereof of preventing and treating cucumber downy mildew is provided, this microbial inoculum can reach more than 60% its preventive effect.
2 technical schemes
The biocontrol strain CHS55 of control warmhouse booth cucumber downy mildew, be Bacillus licheniformis (Bacillus licheniformis), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 1st, 2011, culture presevation number is CGMCC NO.4738, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences.
Biocontrol strain CHS55 for the preparation of the method for biocontrol fungicide is:
Described biocontrol strain CHS55 is inoculated into 32 ℃ of cultivation 250rpm shaking culture in the LB nutrient solution, takes a sample respectively in Bechtop after 12～16 hours and surveys the OD value at 600nm place, returns 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, to plant daughter bacteria liquid and add with 1: 500 volume ratio and be equipped with in the 200L fermentor tank of fermentation culture, 32 ℃ of 250rpm shaking culture 20～24 hours, fermented liquid pH value maintains 7.0, dissolved oxygen amount 20%, air flow 5-7m
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, get wet bacterium and preservative fluid for thallus by 1g: 40ml is made into microbial inoculum, 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: maltose 1.2g/L, Semen Maydis powder 3.2g/L, analysis for soybean powder 3.2g/L, Tryptones 2.0g/L, Calcium dichloride dihydrate 0.5g/L, manganese sulfate monohydrate 0.5g/L, dipotassium hydrogen phosphate 1.0g/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 nonionic surface active agent of 0.001mol/L.
Microbial inoculum dilution 100-1000 doubly after after cucumber is transplanted 4 leaf phases begin to spray and process or sprinkling irrigation is processed, be fit to cucumber downy mildew popular early stage at weather element, gap periods of 10d/7d is used once, can be reached for respectively 62.31% and 65.79% to the preventive effect of cucumber downy mildew.Find that in the greenhouse test middle and later periods control has stronger effect to Powdery Mildew for it, preventive effect can reach more than 70%, and its crude protein extracting solution can reach 70.33% to the cucumber downy mildew preventive effect.
3 beneficial effects
Advantage of the present invention and positively effect show: the present invention is specially for two kinds of disease oidium of Folium Cucumidis sativi section and the sick biological prevention and control agent of developing of Powdery Mildew.Owing to be biotechnological formulation, the series of problems that brings less than the use because of chemical pesticide fully, thereby be conducive to the nuisanceless production of vegetables in greenhouse booth, the peasant can or reduce the consumption of other chemical pesticides, this not only can be the peasant and reduces expenses, and reduce the residual of chemical agent, be conducive to the outlet of vegetables.
The greenhouse experiment shows: this bacteria preparation can significantly reduce the generation of cucumber downy mildew.In the cucumber seeds post-emergence spraying, perhaps when transplanting, fill with root/spraying and process after this microbial inoculum can dilute 100 times.Under greenhouse experiment, can reach 60% for cucumber downy mildew, and its crude protein extracting solution can reach 70.33% to the cucumber downy mildew preventive effect; Find that in the greenhouse test middle and later periods control has stronger effect to Powdery Mildew for it, preventive effect can reach more than 70%.
Four embodiments
The preparation of embodiment 1CHS55 biocontrol fungicide
The present invention relates to a kind of biocontrol strain CHS55 that prevents and treats the warmhouse booth cucumber downy mildew, this bacterial strain is that the cucumber health soil separation at Yangzhou cucumber booth in 2009 obtains.
1) described biocontrol strain CHS55 is inoculated into 32 ℃ of cultivation 250rpm 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, to plant daughter bacteria liquid and add with 1: 500 volume ratio and be equipped with in the 200L fermentor tank of fermentation culture, 32 ℃ of 250rpm shaking culture 20～24 hours, fermented liquid pH value maintains 7.0, dissolved oxygen amount 20%, air flow 5-7m
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, get wet bacterium and preservative fluid for thallus by 1g: 40ml is made into microbial inoculum, 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: maltose 1.2g/L, Semen Maydis powder 3.2g/L, analysis for soybean powder 3.2g/L, Tryptones 2.0g/L, Calcium dichloride dihydrate 0.5g/L, manganese sulfate monohydrate 0.5g/L, dipotassium hydrogen phosphate 1.0g/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 nonionic surface active agent of 0.001mol/L.
Embodiment 2 cucumber downy mildew greenhouse efficiency tests
The four leaf phases after cucumber is transplanted are with biocontrol microorganisms CHS55 (10
7CFU/mL) cucumber leaves is processed in spraying, inoculates bacterium of downy mildew of cucumber (Psedoperonspora cubensis) sporangia suspension behind the 5d, and inoculum density is 10
5Individual sporocyst/ml, the control treatment group is the clear water contrast.Each treatment group 24 strain cucumber seedling, four repetitions.Disease progression is investigated in the morbidity of 10d control group, calculates disease severity and preventive effect.(reference: NY/T 1156.7-2006 farm-chemical indoor determination test rule sterilant the 7th part: control cucumber downy mildew test pot-culture method)
Oidium pathogenic bacteria Pseudoperonospora cubensis (Berkely et Curtis) Rosto wzew, public, see reference literature " the soup coin. research [J] microbiology of downy mildew of garpe in vitro inoculation method circular, 1994, (06). ".
Germ sporangia suspension preparation method (reference: Du Xinglan. research [D] Agricultural University Of Hebei of downy mildew of garpe and Powdery Mildew biological control, 2008): gather at dusk the sick leaf (not executing within the next few days agricultural chemicals) of the serious fresh cucumber downy mildew of morbidity, take back the laboratory with curling stone, wash away foreign material and original armful of ascus on the blade face, absorbent cotton with immersion encases petiole, place 18 ℃ of dark moisturizings of incubator to cultivate, wait to grow fresh sporocyst behind the 24h.Get the sporocyst that newly grows with the sterilized water brush, double gauze filters, and 12000rpm 15min is centrifugal, and abandoning supernatant is diluted to 10 with aqua sterilisa
5Individual sporocyst/ml, dark culturing 2-3h in 18 ℃ of incubators has treated to use when a large amount of zoosporangiums occur.
Data presentation, CHS55 is 57.27% to the cucumber downy mildew preventive effect.
Table 1: biocontrol fungicide CHS55 is to cucumber downy mildew biocontrol effect (10d is processed in spraying) in the greenhouse
The greenhouse test preventive effect of 3 pairs of powderys mildew of cucumber of embodiment
The four leaf phases after cucumber is transplanted are with biocontrol microorganisms CHS55 (10
7CFU/mL) cucumber leaves is processed in spraying, 28 ℃ of control room temperatures and indoor relative humidity 85%, and visible Powdery Mildew occurs behind the 10d, and the control treatment group is the clear water contrast.Each treatment group 24 strain cucumber seedling, four repetitions.Disease progression is investigated in the morbidity of 10d control group, calculates disease severity and preventive effect.(reference: NY/T 1156.4-2006 farm-chemical indoor determination test rule sterilant the 4th part: control wheat powdery mildew test pot-culture method)
The powdery mildew of cucumber pathogenic bacteria is the monofilament shell Erysiphe [Sphaerothecafuliginea (Schl.) Poll=S.cucurbitae (Jacz.) Zhao] of Ascomycotina, its imperfect stage returns the powder spore of Deuteromycotina to belong to [Oidium sp.], see reference literature " Zhang Xuehui. indoor powdery mildew of cucumber Prevention Method [J]. Hebei Normal University Journal; 2005, (02). ".
The preparation of conidial suspension (Du Xinglan. research [D] Agricultural University Of Hebei of downy mildew of garpe and Powdery Mildew biological control, 2008)): gather fresh conidium on the cucumber plant blade. use aseptic water washing, make conidial suspension (8*10
4Individual/mL).Should be limited in the 2h to finishing inoculation from the conidial suspension preparation.
Grade scale with reference to the State Standard of the People's Republic of China (pesticide field efficacy medicine test criterion () bactericidal agent for preventing and treating cucumber is sick from powder, GBFF17980.30-2000):
0 grade: without scab; 1 grade: lesion area accounts for below 5% of one-piece blade area; 3 grades: lesion area accounts for the 6%-10% of one-piece blade area; 5 grades: lesion area accounts for the 11%-20%:7 level of one-piece blade area: sick area accounts for 21%～40%:9 level of one-piece blade area: the scab face autumn accounts for more than 40% of one-piece blade area:
Table 2: biocontrol fungicide CHS55 is to powdery mildew of cucumber biocontrol effect (10d is processed in spraying) in the greenhouse
Embodiment 4 booth field tests detect the CHS55 biocontrol fungicide to the preventive effect of cucumber downy mildew
The four leaf phases after cucumber is transplanted are with biocontrol microorganisms CHS55 (10
7CFU/mL) cucumber leaves is processed in spraying; The oidium symptom appears in booth behind the 3-5d, and control group arranges clear water and processes.Each treatment group 60 strain, four repetitions.3d to 15d investigates respectively disease progression, calculates disease severity and preventive effect.
Cucumber downy mildew grade scale according to GB/T17980.26-2000 " bactericidal agent for preventing and treating cucumber downy mildew field control effectiveness test criterion () ": 0 grade: without scab, 1 grade, lesion area accounts for below 5% of whole leaf area; 3 grades, lesion area accounts for 6%～10% of whole leaf area; 5 grades, lesion area accounts for 11%～25%, 7 grades of whole leaf area, and lesion area accounts for 26%～50% of whole leaf area; 9 grades, lesion area accounts for more than 50% of whole leaf area.
Investigation method: every leaf of every strain is all investigated, and young leaves is also investigated.
Disease severity %=[∑ (sick grade blade number * represent the progression)/total leaf number number * the highest level value that represents] * 100;
Biological control effect %=(contrast sickness rate-processing sickness rate)/contrast sickness rate * 100
Investigation result shows, biocontrol fungicide CHS55, and the preventive effect that spraying is processed cucumber downy mildew reaches 62.31%, and spraying/filling root is processed simultaneously preventive effect and is reached 65.79%.
Table 3: biocontrol fungicide CHS55 is to cucumber downy mildew biocontrol effect (8d is processed in spraying) in the greenhouse
Table 4: biocontrol fungicide CHS55 is to cucumber downy mildew biocontrol effect (fill with root/spraying and process 8d) in the greenhouse
Embodiment 5CHS55 crude protein liquid is to the preventive effect of cucumber downy mildew
According to the literature, Bacillus licheniformis can produce corresponding albumen mostly, the author attempts being separated to a certain amount of albumen (reference: Sun Qili with 80% ammonium sulfate precipitation method, Xu Jingyou. Bacillus licheniformis (BL) antibacterial protein is to restraining effect and the protection effect of Sclerotinia sclerotiorum. the journal .Vol.28 No.3 Sep.2007 of Yangzhou University), and adopting the Xylene Brilliant Cyanine G method to detect its concentration, concentration is approximately 2mg/ml.40 times of liquid dilutions with this albumen dilution spray, and find that result for the treatment of is 60.08%.
Table 5: the CHS55 crude protein is to cucumber downy mildew preventive effect (8d is processed in spraying) in the booth field test
Annotate: be the blade before oidium does not have to occur the period of spraying.
Table 6: booth field CHS55 crude protein is to cucumber downy mildew result for the treatment of (8d is processed in spraying)
Annotate: be that 3d began to spray blade after oidium occured the period of spraying.
Claims (4)
1. biocontrol strain CHS55 who prevents and treats the warmhouse booth cucumber downy mildew, for Bacillus licheniformis (
Bacillus licheniformis), being preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 1st, 2011, culture presevation number is CGMCC NO. 4738, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences.
2. use the biocontrol fungicide of the biocontrol strain CHS55 preparation of the described control warmhouse booth of claim 1 cucumber downy mildew.
3. the described biocontrol strain CHS55 of claim 1 is for the preparation of the method for biocontrol fungicide:
1) the described biocontrol strain CHS55 of claim 1 is inoculated into 32 ℃ of cultivation 250 rpm shaking culture in the LB nutrient solution, in Bechtop, takes a sample respectively after 12～16 hours and survey the OD value at 600 nm places, 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 1:500 volume ratio be equipped with in the 200L fermentor tank of fermentation culture, 32 ℃ of 250 rpm 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 0.05-0.1 KPa, centrifugal 10 min of 6000 rpm then, per 1000 ml nutrient solutions obtain the wet bacterium of 13～16 g, 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.
4. method according to claim 3, wherein:
1) seed culture fluid LB prescription: sodium-chlor 10 g/L, Tryptones 10 g/L, yeast soak powder 5 g/L, and regulating the pH value is 7.2;
2) fermentation culture liquid formula: maltose 1.2 g/L, Semen Maydis powder 3.2 g/L, analysis for soybean powder 3.2 g/L, Tryptones 2.0 g/L, Calcium dichloride dihydrate 0.5 g/L, manganese sulfate monohydrate 0.5 g/L, dipotassium hydrogen phosphate 1.0 g/L, regulating the pH value is 7.0;
3) preservative fluid for thallus is 0.01 mol/L phosphate buffered saline buffer, and also containing concentration is the Tween20 nonionic surface active agent of 0.001 mol/L.
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| CN112400870A (en) * | 2020-11-27 | 2021-02-26 | 云南省微生物发酵工程研究中心有限公司 | Bacillus licheniformis water dispersible granule and application thereof |
| CN114080874B (en) * | 2021-11-17 | 2023-04-14 | 山东省蚕业研究所 | Method for relieving soil salinization by crop rotation of iced vegetables and suaeda salsa |
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Address after: 111 Baizhu Road, Baima Town, Lishui District, Nanjing City, Jiangsu Province 211225 Patentee after: Nanjing Nongda Bio-source Pesticide Creation Co.,Ltd. Address before: College of plant protection, Nanjing Agricultural University, 6 Tongwei Road, Zhongshan Gate, Nanjing, Jiangsu, 210095 Patentee before: Nanjing Nongda Bio-source Pesticide Creation Co.,Ltd. |