CN101063093A - Bacterial strain PX35 preventing and curing glasshouse vegetable epidemic disease - Google Patents
Bacterial strain PX35 preventing and curing glasshouse vegetable epidemic disease Download PDFInfo
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- CN101063093A CN101063093A CN 200710021284 CN200710021284A CN101063093A CN 101063093 A CN101063093 A CN 101063093A CN 200710021284 CN200710021284 CN 200710021284 CN 200710021284 A CN200710021284 A CN 200710021284A CN 101063093 A CN101063093 A CN 101063093A
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Abstract
The invention discloses a fungi-proofing strain PX35 to prevent and cure warm house vegetable plague (Phytophthora capsici) in preventing and curing disease domain of farm crop, which is characterized by the following: attesting PX35 as Bacillus sp. ; culturing on LB medium for 10 h; generating single bacterial colony; culturing for 24 h; turning the bacterial colony to lacte; possessing uniform texture and smooth surface; displaying short and straight rod-like; generating germ; secreting cellulose, proteinase enzymatic activity and iron element; possessing important meaning for preventing disease; making the average prevention and cure effect at 75-90%; increasing output of diseased filed by 30-96%; allocating bacterial agent with the wet bacterium and thallus preserved liquid at 1 : 40 (g/ml); keeping at room temperature for 2-3 years. This PX35 strain possesses good effect.
Description
One, technical field
The present invention relates to prevent and treat the single bacterial strain of curing glasshouse vegetable epidemic disease, belong to the farm crop technical field of preventing and curing diseases, be exclusively used in the non-environmental pollution control of vegetable epidemic disease.
Two, technical background
The cultivated area of the ground of China's protection at present vegetables constantly enlarges, owing to protect the suitable mould growth of epidemic disease of temperature and humidity condition in the ground, adds the continuous cropping of same kind, causes eqpidemic disease to increase the weight of year by year.The chemical control to vegetable epidemic disease at present mainly also is to use chemical pesticide, as: phosethyl Al, metaxanin, Pu Like, Amici reach etc.But, use chemical pesticide not only can produce pesticide residue, and along with the resistance of using phytophthora year by year of chemical pesticide is also increasing gradually, the Phytophthora capsici bacterial strain of resistance occurred Ridmil gold is had as Michigan, USA, and discovered when a bacterial strain develops immunity to drugs to Ridmil gold and can not lose original other characteristics simultaneously.(M.HausbeckR.Fulbright and R.Hammerschmidt.An integrated approach to manage phytophthora blighton Michigans vine crops.)。The vegetable grower can only strengthen the usage quantity of agricultural chemicals for this reason, and thus, agricultural chemicals uses and degree of dependence presents the vicious cycle state.A large amount of uses of agricultural chemicals make in the vegetables that the pesticide residue problem that exceeds standard is outstanding.In May, 2000, The Institute for the Control of Agrochemicals of the Ministry of Agriculture,PRC organized the agriculture chemical examination institution of 5 provinces and cities such as Beijing, Shanghai, Chongqing, Shandong and Zhejiang, to 50 vegetable varieties, the pesticide residue of 1293 samples are carried out sampling Detection, the pesticide residue exceeding standard rate reaches 30%, and the high person of residual concentration is several times even tens times of the permission residual quantity.The severe overweight of pesticide residue in the vegetables causes intoxication accident to happen occasionally.According to Ministry of Health's statistical figure, China in 1999 is because food supply retail shop's dish food poisoning dish that pesticide residue cause has 37, and the example of acute poisoning can also cause our attention, and the situation of chronic poisoning and retention toxicosis we are just unknown, its result can be more fearful in fact.China exports to the vegetables of developed countries such as Japan, Korea S and is not often return because pesticide residue do not reach their standard, and this causes very big loss for many vegetable growers.Should widely popularize the plantation pollution-free vegetable, the standard production standard reduces the consumption of chemical pesticide as far as possible for this reason.
Pollution-free vegetable becomes the main flow that world vegetables produce already.As far back as the twenties in 20th century, just begin to develop pollution-free vegetable abroad, its main mode of production is soilless culture.According to incompletely statistics, single in the world country with NFT (NFT) cultivation pollution-free vegetable just reaches 76.In New Zealand, fruit vegetableses such as tomato more than half, cucumber are soilless cultures.Developed countries such as Japan, Holland, the U.S. adopt modern water planting greenhouse, produce pollution-free vegetable throughout the year.Research and the production of China pollution-free vegetable start from nineteen eighty-two, hold national biological control meeting this year, and Jiangsu Province takes the lead in proposing to replace chemical pesticide control with biological control.Nineteen eighty-three, under the support energetically of national plant protection master station, the whole nation 23 provinces, cities have carried out research, demonstration and the popularization of pollution-free vegetable.Logical research practice is in a few years explored a pollution-free vegetable production art of overlapping integrated control disease and pest, minimizing pesticidal contamination.The whole nation in 1985 is promoted pollution-free vegetable and is produced area 40,000 hm2 (600,000 mu), and almost all there is the pollution-free vegetable production base all parts of the country in each city so far.During this time, developed some successful biological bactericides, as agricultural streptomycin, jinggangmeisu, the former can be made into the 200mg/L liquid irrigating root and prevent and treat vegetable bacterial soft rot, bacterial wilt, and the latter is made into 1000-1500 times of liquid control vegetables anthrax, oidium.But the biocontrol strain of higher preventive effect does not appear eqpidemic disease is had as yet.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 does not have the biocontrol strain preparation present situation of higher preventive effect at the greenhouse eqpidemic disease, provide exploitation a kind of single bacterial strain of preventing and treating vegetable epidemic disease, be exclusively used in control capsicum, tomato, cucumber isothermal chamber vegetable epidemic disease, the preventive effect height of bacterial strain, and the volume increase function is arranged.
Technical scheme
The bacterial strain PX 35 of control curing glasshouse vegetable epidemic disease was bacillus (Bacillus sp.), was deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center on 01 26th, 2007, and culture presevation number is CGMCCNO.1930.PX35 10h on the LB substratum produces single bacterium colony, cultivates 24h bacterium colony oyster white, the quality homogeneous, and circle, there is serration at the edge, median rise, smooth surface, thalline is the short straight rod shape, G
+, produce gemma.Bacterial strain PX 35 can be secreted cellulase-producing, proteolytic enzyme enzymic activity and product and have a liking for the iron element, and is significant to the eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion.
With the bacterial strain PX 35 of above-mentioned control curing glasshouse vegetable epidemic disease at 37 ℃ of 180rpm shaking culture of PDA nutrient solution, 20～24h, the centrifugal 10min of 6000rpm then, get the wet bacterium of PX35 and preservative fluid for thallus (this breadboard preservative fluid for thallus patent, publication CN1358838, open day: be to be made into microbial inoculum at 1: 40 by mass volume ratio on July 17th, 2002), the viable bacteria total concn was 1 * 10 in the microbial inoculum finished product
9～1 * 10
12Cfu/ml.Used PDA nutrient solution compound method is: be cut into small pieces to be put in the water after peeling with the 200g potato and boil, boil 30min after the boiling, then filter with four layers of hospital gauze, filtrate adds the 20g common sucrose, be settled to 1000ml, the pH value transfers to 7.2-7.4,121 ℃ of sterilization 20min.
Beneficial effect
The present invention compares with existing vegetable epidemic disease Prevention Technique, its advantage and positively effect show: the present invention is specially at the biocontrol strain of vegetable epidemic disease exploitation, thereby significantly improves comparing prevention effect with other broad spectrum biological disinfection agent at present aspect the control vegetable epidemic disease.Owing to be biological bacterial strain preparation, do not have a series of problems that use brought because of chemical pesticide fully, thereby help the nuisanceless production of vegetables, the peasant can or reduce other consumptions of preventing and treating the eqpidemic disease chemical pesticide, this not only can be the peasant and reduces expenses, and helps the outlet of vegetables.Simultaneously, PX35 bacterial strain preparation increases production function in addition, can be the peasant and increases income.
The PX35 bacterial strain (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.
PX35 bacterial strain preparation can be sprayed on the field piece of fertilizer before transplant in the greenhouse, bury in the soil in whole ground process with fertilizer then, also can irritate root and use when planting seedling.After planting seedling, bacterial strain works by multiple Biocontrol Mechanism, thereby reaches the effect of preventing and treating eqpidemic disease.
Test shows, using PX35 bacterial strain preparation is 75～90% to the average prevention effect preventive effect of vegetable epidemic disease, and sick field volume increase 30～96% obtains effect of increasing production about 10% at anosis Tian Yineng.
Four description of drawings
The PX35 microbial inoculum is handled after 10 days control and the short fruit of coming into force to capsicum epidemic disease in Fig. 1 greenhouse test
Fig. 2 PX35 microbial inoculum is handled after 60 days the prevention effect (Huaian, Jiangsu, 2006) to the field capsicum epidemic disease
Five embodiments
The separation of 1 bacterial strain and evaluation
A kind of control curing glasshouse vegetable epidemic disease (Phytophthora capsici) biocontrol strain PX35 is exclusively used in control capsicum, tomato, cucumber isothermal chamber vegetable epidemic disease.The contriver is from the morbidity Tanaka of capsicum epidemic disease, gathered the sick healthy tree of capsicum, and in rhizosphere, root, leaf encloses, be separated to 1484 bacterial strains in the leaf, therefrom filters out the 1 strain bacterial strain good to the capsicum epidemic disease biocontrol effect, it is numbered PX35.Cellulase-producing, proteolytic enzyme, chitinase activity and the product of PX35 bacterial strain are had a liking for plain mensuration of iron to be studied, and it greenhouse and land for growing field crops preventive effect research have been carried out, find that PX35 bacterial strain preparation has good preventing disease production-increasing effect, has bigger potential development to become business-like viable bacteria biological prevention and control agent.
The PX35 bacterial strain is Bacillus sp.; PX35 10h on the LB substratum produces single bacterium colony, cultivates 24h bacterium colony oyster white, the quality homogeneous, and circle, there is serration at the edge, median rise, smooth surface, thalline is the short straight rod shape, G
+, produce gemma.The PX35 bacterial strain can be secreted cellulase-producing, protease activity and product and have a liking for the iron element, and is significant to the eqpidemic disease biological and ecological methods to prevent plant disease, pests, and erosion.
2 greenhouse preventive effect experiments
The PX35 fungicide preparation: dull and stereotyped activation PX35 bacterial strain, choose LB nutrient solution (1% peptone, 0.5% yeast extract, 1% NaCl of single colony inoculation to 5ml, pH 7.2), 37 ℃, the 180rmp 20h that vibrates, be transferred in the LB nutrient solution of 500ml, 37 ℃, the 180rmp 24h that vibrates.With the centrifugal 10min of thallus suspension liquid 6000g that shakes, collect thalline, with sterilized water preparation 10
8Cfu/ml antagonism bacterium suspension.
The pathogenic bacteria preparation: pathogenic strains Phytophthora capsici CA1 (Wang Yuanchao, Ding Guoyun, Ma Zhichao, etc.The clone and the sequential analysis in cotton phytophthora and phytophthora capsici ribosomal gene ITS zone, Agricultural University Of Nanjing's journal, 2000,23 (3): 33-36) the V8 substratum (Zheng Xiaobo. phytophthora and investigative technique thereof [M]. Beijing: Chinese agriculture press, 1997) on, cultivate 3d for 25 ℃, the mycelia piece of cutting 4mm*4mm is transferred in the V8 nutrient solution, 10～12 of every 100ml, cultivate 3d for 25 ℃, outwell nutrient solution, change aseptic tap water, every 12h changes once, change 3～4 times, when examining under a microscope a large amount of zoosporangiums, 4 ℃ of cold shock 1～2h stimulate zoospore to produce.Collect spore liquid,, spore liquid is adjusted to 1*10 with the blood counting chamber counting
5Individual zoospore/ml is standby.
Prepare pepper seedling: substrate seedling, 25 ℃ of 16/8h photoperiods, seedling length to 3～4 true leaf (about 30d) are transplanted.
Pepper seedling is transplanted in the basin alms bowl (diameter 70mm, high 90mm) of filling matrix every alms bowl three strains, place 16/8h photoperiod greenhouse, the PX35 microbial inoculum is irritated root, every alms bowl 50ml two days later, irritate root and connect pathogenic bacteria two days later, at the mould zoospore suspension (10 of seedling basal part of stem inoculation 2ml epidemic disease
5Zoospore/ml).The 2ml sterilized water is watered in contrast.Add up the state of an illness after meeting PX35 microbial inoculum 30d, state of an illness classification 0-5 level: 0 grade asymptomatic; 1 grade blade is slightly wilted, and begins to occur the brown spot on the stem; The 30-50% of 2 grades of whole plant has symptom; The 50-70% of 3 grades of whole plant has symptom; The 70-90% of 4 grades of whole plant has symptom; 5 grades of whole strain death.Each handles 18 young plants, repeats 3 times.Meanwhile, 6 young plants of each processing are uprooped, cleaned and dry, add up fresh weight respectively, dry weight increases biomass, and root is long, and stem is long.
Seedling stage results from pot experiment test show (table 1, Fig. 1), the sickness rate of only inoculating the Phytophthora capsici control group reaches 72.2%, the sickness rate that the PX35 microbial inoculum is handled is 11.8%, the prevention effect of PX35 reaches 83.5%.The PX35 microbial inoculum can promote chili growth, and biomass has increased by 42.63% compared with the control, and root, stem length also have certain growth.
Table 1 biocontrol microorganisms PX35 preparation is used the back 30 days potted plant preventive effects to capsicum epidemic disease
| Handle | Sickness rate (%) | Preventive effect (%) | Fresh weight (g) | Increase biomass (%) | Root long (cm) | Stem long (cm) |
| The PX35 contrast | 11.8％ 72.2％ | 83.5％ | 6.29±0.66 4.41±0.55 | 42.63％ | 18.91±1.44 16.74±0.45 | 14.71±1.31 14.60±0.89 |
Annotate: 1. fresh weight, root length, stem long data are Mean ± SE in the table
2. preventive effect (%)=(contrast disease index-processings disease index)/contrast disease index * 100%
3. increase biomass (%)=(treatment group fresh weight-control group fresh weight)/control group fresh weight * 100%
4. experiment is total to triplicate.
3 field practical application experiments
The PX35 fungicide preparation: with the PX35 bacterial strain at 37 ℃ of 180rpm shaking culture of PDA nutrient solution, 20～24h, the centrifugal 10min of 6000rpm then, get wet bacterium and this breadboard preservative fluid for thallus (publication CN1358838, open day: on July 17th, 2002) be made into microbial inoculum by 1: 40 (g/ml), the viable bacteria total concn was 1 * 10 in the microbial inoculum finished product
9～1 * 10
12Cfu/ml.
PDA nutrient solution compound method: be cut into small pieces to be put in the water after peeling with the 200g potato and boil, boil 30min after the boiling, then filter with four layers of hospital gauze, filtrate adds the 20g common sucrose, is settled to 1000ml, and the pH value transfers to 7.2-7.4, then 121 ℃ of 20min that sterilize.
Carried out field eqpidemic disease efficiency test demonstration in Huaian, Jiangsu in 2005,2006 with the microbial inoculum of aforesaid method gained.Spraying is used before crop is transplanted, and also can make the compost use, or the root use is irritated in the dilution back when transplanting.Field experiment show (table 2,3, Fig. 2), spraying was used before crop was transplanted, 60d after the capsicum transplantation of seedlings, the sickness rate of PX35 microbial inoculum treatment group is respectively 3.08%, 6.2%, preventive effect is 75.81%, 89.8%., stimulation ratio is respectively: 40.00% and 95.90%, and there were significant differences for the stimulation ratio of PX35 microbial inoculum and zinc manganese ethylenebisdithiocarbamate treatment group.
Table 2 biocontrol microorganisms PX35 microbial inoculum is to the field biocontrol effect (Huaian, Jiangsu, 2005) of capsicum epidemic disease
| Handle | Dispenser dosage | Sickness rate (%) | Preventive effect (%) | Mean yield (Kg/ sub-district) | Stimulation ratio (%) |
| Contrast zinc manganese ethylenebisdithiocarbamate PX35 | ---500g/ mu 3L/ mu | 15.62a 6.57b 3.08c | --- 57.13b 75.81ab | 31.62b 33.21b 44.30a | --- 5.03b 40.00a |
Annotate: be illustrated in significant difference on 5% level with the different lowercase persons of mark behind the column data.
Table 3 biocontrol microorganisms PX35 microbial inoculum is to the diseases prevention and the effect of increasing production (Huaian, Jiangsu, 2006) of field capsicum epidemic disease
| Handle | Dispenser dosage | Sickness rate (%) | Preventive effect (%) | Mean yield (Kg/ sub-district) | Stimulation ratio (%) |
| Contrast zinc manganese ethylenebisdithiocarbamate PX35 | --- 500g/667m2 3L/667m2 | 60.7a 23.1b 6.2c | --- 61.9b 89.8a | 30.88b 32.12b 60.50a | --- 4.01b 95.90a |
Annotate: be illustrated in significant difference on 5% level (P＜0.05) with the different lowercase persons of mark behind the column data.
Claims (6)
1, a kind of bacterial strain PX 35 of preventing and treating curing glasshouse vegetable epidemic disease, bacterial strain PX 35 is bacillus (Bacillus sp.), be deposited at China Committee for Culture Collection of Microorganisms common micro-organisms center on 01 26th, 2007, culture presevation number is CGMCC NO.1930.
2, the application of the bacterial strain PX 35 of the described control curing glasshouse vegetable epidemic disease of claim 1 aspect the control curing glasshouse vegetable epidemic disease.
3, use the biocontrol fungicide of the bacterial strain PX 35 preparation of the described control curing glasshouse vegetable epidemic disease of claim 1.
4, prepare the method for biocontrol fungicide with the bacterial strain PX 35 of the described control curing glasshouse vegetable epidemic disease of claim 1, it is characterized in that:
With the bacterial strain PX 35 of the described control curing glasshouse vegetable epidemic disease of claim 1 at 37 ℃ of 180rpm shaking culture of PDA nutrient solution, 20～24h, the centrifugal 10min of 6000rpm then, getting wet bacterium of PX35 and preservative fluid for thallus is to be made into microbial inoculum at 1: 40 by mass volume ratio, and the viable bacteria total concn is 1 * 10 in the microbial inoculum finished product
9～1 * 10
12Cfu/ml.
5, according to claim 4ly prepare the method for biocontrol fungicide, it is characterized in that used PDA nutrient solution compound method is with bacterial strain PX 35:
Be cut into small pieces to be put in the water after peeling with the 200g potato and boil, boil 30min after the boiling, then filter with four layers of hospital gauze, filtrate adds the 20g common sucrose, is settled to 1000ml, and the pH value transfers to 7.2-7.4,121 ℃ of 20min that sterilize.
6, the application of the described biocontrol fungicide of claim 3 aspect the control curing glasshouse vegetable epidemic disease.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2255660A1 (en) * | 2009-05-27 | 2010-12-01 | Nau Biogenic Pesticides R&D Co., Ltd | Biocontrol agent against soil-borne diseases |
| CN102154193A (en) * | 2011-01-20 | 2011-08-17 | 天津市植物保护研究所 | Method for inducing Phytophthora capsici to produce large amount of sporangia |
| 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 |
-
2007
- 2007-04-26 CN CNB2007100212842A patent/CN100557013C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2255660A1 (en) * | 2009-05-27 | 2010-12-01 | Nau Biogenic Pesticides R&D Co., Ltd | Biocontrol agent against soil-borne diseases |
| WO2010135893A1 (en) * | 2009-05-27 | 2010-12-02 | 南京农大生物源农药创制有限公司 | Microbial agent ps and psx for preventing and curing soil-borne disease of plants and preparation method and use thereof |
| CN102154193A (en) * | 2011-01-20 | 2011-08-17 | 天津市植物保护研究所 | Method for inducing Phytophthora capsici to produce large amount of sporangia |
| CN102154193B (en) * | 2011-01-20 | 2012-07-25 | 天津市植物保护研究所 | Method for inducing Phytophthora capsici to produce large amount of sporangia |
| 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 |
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