CN101575581A - Marine bacillus subtilis 3512A and preparation thereof for separating and curing cucumber fusarium wilt - Google Patents
Marine bacillus subtilis 3512A and preparation thereof for separating and curing cucumber fusarium wilt Download PDFInfo
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Abstract
The invention relates to the prevention of cucumber fusarium wilt and the application of marine microorganisms, in particular to a marine bacillus subtilis 3512A and a preparation thereof for separating and curing the cucumber fusarium wilt. The marine bacillus subtilis 3512A has a preserving number of CCTCC No. M 207051. The preparation of the marine bacillus subtilis 3512A, which is used for separating and curing the cucumber fusarium wilt is prepared by the following steps: (1), directly selecting health living gorgonian organisms from gorgonian growth regions in South China Sea, cutting growth point parts of the gorgonian organisms into small blocks, putting the small blocks into a small bottle, taking the small bottle to a laboratory and grinding a sample and sterile water at a ratio of 1 to 1 to obtain a fresh gorgonian cell extract; (2), using a method combining a plate mutual antagonism screening model and directed screening to screen the marine bacillus subtilis 3512A resisting the cucumber fusarium wilt and co-inhabited with gorgonians; and (3), using the marine bacillus subtilis 3512A as a microorganism seed and fermenting the microorganism seed to prepared the microorganism preparation capable of effectively curing the cucumber fusarium wilt. The invention has simple and convenient operation and can be popularized and applied.
Description
Technical field
The present invention relates to the control of cucumber fusarium axysporum, specifically a marine bacillus subtilis and to the control of cucumber fusarium axysporum.
Background technology
Cucumber fusarium axysporum (Cucumber fusarium Wilt) claim wilt disease, dead arm again, is a kind of global vascular bundle diseases of plant.This disease is caused by Fusarium oxysporum (Fusarium oxysporum), is the vascular bundle diseases of the main soil-borne infection of a class, and melon growing area generally takes place in China.According to statistics, the sickness rate of annual cucumber fusarium axysporum can reach 20%, even up to 80-90%, has caused the serious underproduction of cucumber.At present this type of disease of control mainly is a chemical pesticide, but long-term a large amount of chemical pesticide that uses, not only serious environment pollution, destroy the eubiosis, threaten human health, and cause the resistance of pathogenic bacteria more and more stronger, make disease more and more be difficult to control.Biological control can overcome the drawback that chemical pesticide brings, and the biological control method for cucumber fusarium axysporum mainly contains employing disease-resistant variety, crop rotation and grafting at present.Existing cucumber variety, only the minority kind has certain anti-, anti-sick ability (as middle peasant No. 5, Tianjin spring No. 5 etc.), even cultivate these kinds, disease still takes place every year; The planting type of crop rotation can reduce the occurring degree of blight, but can not fundamentally solve the generation of disease; Though graft technology can be prevented and kill off blight effectively, still, on producing, be difficult to big area and promote.Therefore, by seek novel diseases prevention, the means of curing the disease, reach the harm of controlling cucumber fusarium axysporum effectively, become the problem that presses for solution in the production practice.To this green requirement that can eat vegetables raw of cucumber, the biological control of cucumber fusarium axysporum has been become the focus that people pay close attention to based on advantage such as efficient, the safety of biological pesticide, environmental protection and people.Be used for the bacterium of control of plant disease, the research of withered grass bud pole bacterium (Bacilluss ubtilis) is a lot, and prophylaxis effect is not only arranged, and the significantly short effect of increasing production of giving birth to is arranged.Become the class biocontrol bacteria that suppresses the bacteria-treating disease that has of people's approval.But this bacterioid is used for the biological control of cucumber fusarium axysporum studies the less of report.
Summary of the invention
The present invention relates to cucumber fusarium axysporum control and marine microorganism utilisation technology, a kind of specifically cucumber fusarium axysporum biological control sea bacillus subtilis preparation manufacture method.
Sea bacillus subtilis provided by the present invention, be subtilis Bacillus subtilis3512A, be deposited in Chinese typical culture collection center, address: China on April 23rd, 2007. Wuhan. Wuhan University, preserving number is CCTCC No.M 207051.
Described cucumber fusarium axysporum biological control with the manufacture method of sea bacillus subtilis preparation is:
1) bacterial strain primary dcreening operation: subtilis Bacillus subtilis 3512A is to separate to obtain from the gorgonian of the South Sea.Concrete grammar is as follows: at South Sea gorgonian growth district, directly choose gorgonian organism healthy, that live, under aseptic condition, clean, get its vegetative point position, be cut into the long fritter of 0.5～1.0cm, in the aseptic sampling bottle of packing into, be placed in the water bottle (or liquid nitrogen container), take back the laboratory, with sample: sterilized water=grind at 1: 1 obtains fresh gorgonian cell extract; To fresh gorgonian cell extract, adopt plate dilution method (scribbling 0.001～0.01% gorgonian in the substratum), the separating marine spore bacteria filling on the plate of above-mentioned special culture media, is coated with strike-off stick, puts in the insulation can then and cultivates; Cultivate after 3～5 days, the pure microorganism of single bacterium colony of growing on the plate behind the purifying of ruling again, is chosen typical single bacterium colony and moves to slant preservation.The present invention is simple to operate, and ideal can obtain needed bacterial strain as a result.
2) bacterial strain sieves again: single bacterium colony slant tube bacterial strain that above-mentioned primary dcreening operation is obtained carries out the plate activation, 28 ℃ 48 hours, adopt plate antagonistic effect method, with the cucumber fusarium axysporum is the target bacterium, the antagonistic effect that on plate, stands facing each other according to a conventional method, heat insulating culture is 48～84 hours in 25～28 ℃ of insulation cans, take out face-off antagonistic effect plate, detect the inhibition zone size of each gorgonian sea bacillus subtilis bacterial strain to cucumber fusarium axysporum target bacterium, bacteriostatic diameter reaches 18mm or 18mm is above for qualified, selects numbering to preserve.
3) sea bacillus subtilis decides to grow test at the cucumber rhizosphere: with sea bacillus subtilis test tube slant bacterial classification, move connect expand numerous, then at 25-30 ℃ of heat insulating culture 36-60 hour, this moment, the sea bacillus subtilis lawn looked very plentiful, can scrape with sterilized water and wash, make bacteria suspension, make the somatic cells number in every milliliter of bacteria suspension reach 1,000,000,000 more than the CFU, promptly reach 10
9Concentration, can with the potted plant soil uniform mixing of cucumber, make the cell count of the subtilis of the test determination in every gram soil reach 10
5More than the CFU, reach more than 100,000 CFU cells in promptly every gram soil; Soil is packed in the basin, and 20 cucumber seeds of every basin sowing by the management of pot experiment ordinary method, are measured once every 2 weeks, inserts in the soil after 6 weeks, and the sea bacillus subtilis cell count is stabilized in 10
6CFU/ restrains native level, illustrates that the sea bacillus subtilis that obtains can grow in the China fir rhizosphere soil surely, and this bacterial strain is an effective strain, can supply with test or is applicable.The subtilis of deciding to grow of separating from soil is not less than the result who inoculates preceding experimental determination, i.e. antibacterial circle diameter 〉=18mm to the antibacterial circle diameter of target bacterium.
The described inhibiting ocean spore bacillus to pathogenic bacteria that obtains is a subtilis, identifies or the 16SrDNA sequential detection proves that it is subtilis (BacillusSubtilis) with traditional bacteriology.
4) sea bacillus subtilis fermentation method for producing:
(1), the Kolle flask seed culture, adopt 500ml volume Kolle flask, dress 60ml substratum, sterilized 30 minutes for 121 ℃, put the inclined-plane after the cooling, the inoculation sea bacillus subtilis, then this Kolle flask was placed in 25～28 ℃ of insulation cans heat insulating culture 48～72 hours, and took out and promptly can be used as seed and use.The substratum that the Kolle flask seed culture is used is the modified starch substratum;
(2), the 10L fermenting organism answers device fermentation, canned substratum is 6L, substratum consists of the modified starch substratum, sterilized 30 minutes, and after the cooling, inoculated two bottles in above-mentioned Kolle flask seed for 121 ℃, under cultivated 36～48 hours, can put jar, this fermented liquid can be used for making microbiobacterial agent.
(3), the manufacturing of anti-cucumber fusarium axysporum sea bacillus subtilis microbial preparation, fermented liquid according to claim 3 and the 4 described gorgonian sea bacillus subtilis that obtain, the microscopy thalli growth is good in the laboratory, every milliliter of fermented liquid somatic cells reaches 200,000,000 more than the CFU, to the antagonism antibacterial circle diameter of cucumber fusarium axysporum more than 18mm, do not have assorted bacterium, it is qualified to be, and can make microbial preparation:
1. liquid preparation: the fermented liquid of 10L jar fermentative production directly is potted in the plastic barrel of cleaning and preserves, the mycetocyte number reaches 200,000,000 CFU/ml;
2. solid preparation: fermented liquid mixed with aseptic peat composed of rotten mosses powder mix thoroughly, make water content about 15～20%, the mycetocyte number is at 0.5 hundred million CFU/ml～100,000,000 CFU/ml;
The present invention has following advantage:
1, resulting sea bacillus subtilis 3512A can carry out effectively preventing to cucumber fusarium axysporum, diseases prevention efficient reaches more than 70%, and to the person poultry harmless, free from environmental pollution, avoid the drawback of widely applying chemical pesticide to bring, satisfy people this green requirement of eating vegetables raw of cucumber.Not and can promote the growth of cucumber to increase cucumber yield, can not think a good biocontrol strain or a PGPR bacterial strain, have the potentiality that are developed as high-quality microorganism biological prevention and control agent product or PGPR product.
2, resulting sea bacillus subtilis 3512A, colonization ability is strong in the cucumber rhizosphere soil (carries out the cucumber pot experiment, in 6 weeks behind the inoculation 3512A bacterium, measures its deciding in the cucumber rhizosphere soil and grow density 10
6CFU/ restrains soil, and the 3512A bacterium of deciding to grow of separating from soil is not less than the result of experimental determination to the antibacterial circle diameter of target bacterium, and antibacterial circle diameter reaches 18.00mm), to the cucumber growth unrestraint, and the effect that promotes growth is arranged, help application.
Description of drawings
Related Bacillus subtilis 3512A among the present invention has been deposited in Chinese typical culture collection center, address: China on April 23rd, 2007. Wuhan. and Wuhan University, preserving number is CCTCC No.M 207051.
Fig. 1 is the phylogenetic tree of marine bacteria 3512A (being the Bacillus subtilis 3512A that relates among the present invention) and relevant bacterial strain.
Embodiment
The method that the present invention adopts conventional mutual antagonism screening model of plate and directed screening to combine, its concrete steps: (1) is at ocean coral growth district, directly choose gorgonian organism healthy, that live, under aseptic condition, clean, get its vegetative point position, be cut into the long fritter of 0.5～1.0cm, pack in the aseptic sampling bottle, be placed in the water bottle (or liquid nitrogen container), take back the laboratory, with sample: sterilized water=grind at 1: 1 obtains fresh gorgonian cell extract; (2) to fresh gorgonian cell extract, (substratum is for the wort beef extract-peptone: extractum carnis 0.3%, peptone 0.5%, wort 1%, agar 2%, pH7.0-7.2 to adopt the special culture media plate dilution method.121 ℃, sterilized 20 minutes) in scribble 0.001～0.01% gorgonian), the separating marine spore bacteria filling on the plate of above-mentioned substratum, is coated with strike-off stick, puts in the insulation can then and cultivates; (3) cultivate after 3～5 days, the pure microorganism of single bacterium colony of growing on the plate behind the purifying of ruling again, is chosen typical single bacterium colony and moves to slant preservation; (4) to obtain mainly causing in the cucumber rhizosphere soil harmful fungi be the target bacterial strain to separate, and separation and purification obtains carrying out antagonistic effect between the bacillus marinus 3512A bacterial strain, detects its restraining effect to cucumber fusarium axysporum.Be specially: with the cucumber fusarium axysporum is the target bacterium, and the antagonistic effect that stands facing each other on plate according to a conventional method 25-30 ℃ of insulation 36-60 hour, takes out plate, detects the inhibition zone size of 3512A bacterial strain to the target bacterium.(5) filter out by pot experiment and have efficient antagonistic activity and can in the cucumber rhizosphere soil, grow surely, measure in the soil in position the antagonistic strain that causes harmful fungi (Fusarium oxysporum Schl.f.sp.vasinfectum) effect in the obvious inhibition cucumber rhizosphere soil is arranged, this bacterial strain simultaneously will be to cucumber growth unrestraint effect, or slightly promote the growth effect, select, determine that it is subtilis through the 16SrDNA analysis, resulting bacterial strain is efficient bacterial strain 3512A.
The sea bacillus subtilis 3512A that is used to prevent and treat cucumber fusarium axysporum is in China's typical culture collection center preservation, and its preserving number is CCTCC No.M207051.Sea bacillus subtilis 3512A has typical subtilis feature, gemma column, middle life, does not expand.Thalline is shaft-like or rod-short, Gram-positive, and bacterium colony is faint yellow, opaque, dry, surface irregularity, the edge is irregular.Hydrolyzed starch, the catalase positive is utilized Citrate trianion, can be in 7%NaCl and 45 ℃ of growth, the 16S rDNA sequence of molecular biology identification 3512A bacterial strain and the sequence similarity of subtilis are up to 99.4%.The 3512A bacterial strain can grow (＞10 surely at cucumber rhizosphere soil middle-high density
6CFU/g soil), cucumber fusarium axysporum there is good prevention effect (bacteriostasis rate＞60%, preventive effect reaches more than 70%);
By the sea bacillus subtilis preparation of the produced control cucumber fusarium axysporum of the present invention, be a kind of efficient, nontoxic, safety, noresidue, can promote the microbial preparation of cucumber green ecological system development, rising.
With sea bacillus subtilis (Bacillus subtilis) 3512A that screening method of the present invention obtains, have the effect of high-efficiency prevention and control cucumber fusarium axysporum, and to cucumber growth unrestraint effect and have good promotes growth effect, environmentally friendly.
The manufacture method step of the sea bacillus subtilis preparation of control cucumber fusarium axysporum is as follows:
1, sample collecting: subtilis Bacillus subtilis 3512A is to separate to obtain from the gorgonian of the South Sea.Concrete grammar is as follows: at South Sea gorgonian growth district, directly choose gorgonian organism healthy, that live, under aseptic condition, clean, get its vegetative point position, be cut into the long fritter of 0.5～1.0cm, in the aseptic sampling bottle of packing into, be placed in the water bottle (or liquid nitrogen container), take back the laboratory, screening purpose bacterium to be separated is used.
2, bacterial strain primary dcreening operation: sample: sterilized water=grind at 1: 1 obtains fresh gorgonian cell extract; To fresh gorgonian cell extract, adopt plate dilution method (scribbling 0.001～0.01% gorgonian in the substratum), the separating marine spore bacteria filling on the plate of above-mentioned special culture media, is coated with strike-off stick, puts in the insulation can then and cultivates; Cultivate after 3～5 days, the pure microorganism of single bacterium colony of growing on the plate behind the purifying of ruling again, is chosen typical single bacterium colony and moves to slant preservation
3, bacterial strain sieves again: single bacterium colony slant tube bacterial strain that above-mentioned primary dcreening operation is obtained carries out the plate activation, cultivated 48 hours for 28 ℃, adopt plate antagonistic effect method, with the cucumber fusarium axysporum is the target bacterium, the antagonistic effect that on plate, stands facing each other according to a conventional method, heat insulating culture is 48～84 hours in 25～28 ℃ of insulation cans, take out face-off antagonistic effect plate, detect the inhibition zone size of each gorgonian sea bacillus subtilis bacterial strain to cucumber fusarium axysporum target bacterium, bacteriostatic diameter reaches 18mm or 18mm is above for qualified, selects numbering to preserve.
4, grow test surely: with sea bacillus subtilis test tube slant bacterial classification, move connect expand numerous, then at 25-30 ℃ of heat insulating culture 36-60 hour, this moment, the sea bacillus subtilis lawn looked very plentiful, can scrape with sterilized water and wash, make bacteria suspension, make the somatic cells number in every milliliter of bacteria suspension reach 1,000,000,000 more than the CFU, promptly reach 10
9Concentration, can with the potted plant soil uniform mixing of cucumber, the cell count of the subtilis of the test determination in every gram soil is reached more than the 105CFU, reach in promptly every gram soil more than 100,000 CFU cells; Soil is packed in the basin, and 20 cucumber seeds of every basin sowing by the management of pot experiment ordinary method, insert in the soil after 6 weeks, and the sea bacillus subtilis cell count is stabilized in 10
6CFU/ restrains native level, illustrate that the sea bacillus subtilis that obtains can grow surely in the cucumber rhizosphere soil, this bacterial strain is an effective strain, through measuring growth unrestraint effect to cucumber, (plant height, chlorophyll content, the weight contrast that more do not connect bacterium as a result increase to some extent and the effect of certain promotion growth is arranged, test and potted plant each 4 basin of contrast, the pot experiment time is not shorter than three months).Measuring chlorophyll content adopts the acetone extraction method, respectively surveys once in the seedling phase and the phase of yielding positive results respectively, gets all the 2nd leaf of unfolded (functional leaf) mensuration chlorophyll contents of cucumber plant.After measured, the chlorophyll content of cucumber leaves contrasts highly by 5% behind the inoculation sea bacillus subtilis, and difference reaches conspicuous level.Surely, the subtilis that grows of separating from soil is not less than the result who inoculates preceding experimental determination, antibacterial circle diameter 〉=18mm to the antibacterial circle diameter of target bacterium.After testing, this sea bacillus subtilis can be grown on the inorganic phosphorus substratum and be produced the obvious transparent circle, and the effect that it has phosphorus decomposing is described, promptly can impel the phosphorus of insoluble in the soil to become the phosphorus of solubility, and then promotes the absorption of cucumber to phosphoric.Can significantly promote the growth of water planting cucumber seedling root system after its aseptic ferment filtrate dilutes 1000 times in addition, certain growth stimulation is described.Sea bacillus subtilis 3512A shows to the promotion of cucumber growth and the result of volume increase that in the test of cucumber rhizosphere colonization and it this bacterial strain can be for carrying out expanding test for further applying.
5, sea bacillus subtilis fermentation: (1), Kolle flask seed culture, adopt 500ml volume Kolle flask, dress 60ml substratum, sterilized 30 minutes for 121 ℃, put the inclined-plane after the cooling, the inoculation sea bacillus subtilis then was placed on this Kolle flask in 25～28 ℃ of insulation cans heat insulating culture 48～72 hours, took out promptly to can be used as seed and use.The substratum that the Kolle flask seed culture is used is the modified starch substratum, and it consists of: Zulkovsky starch 2%, extractum carnis 0.5%, yeast extract paste 0.5%, NaCl0.5%, pH nature.
(2), 10L fermenting organism reactor fermentation, canned substratum is 6L, substratum consists of the modified starch substratum, promptly Zulkovsky starch 2%, extractum carnis 0.5%, yeast extract paste 0.5%, NaCl0.5%, all the other be water, pH naturally.Sterilized 30 minutes for 121 ℃, after the cooling, inoculate two bottles in above-mentioned Kolle flask seed, after seed washes with the 200ml sterilized water, use Aseptic technique and be seeded in the 10L volume bio-reactor, the culture condition of fermentative production is: tank pressure 0.05Mpa, 28 ℃ ± 1 ℃ of jar temperature, ventilation is 6L/min, and stirring velocity is 200rpm.Cultivated with this understanding 36～48 hours, and can put jar, this fermented liquid can be used for making microbiobacterial agent.
(3), the manufacturing of anti-cucumber fusarium axysporum sea bacillus subtilis microbial preparation, the fermented liquid of the above-mentioned gorgonian sea bacillus subtilis that obtains, the microscopy thalli growth is good in the laboratory, every ml fermented liquid somatic cells reaches 200,000,000 more than the CFU, to the antagonism antibacterial circle diameter of cucumber fusarium axysporum more than 18mm, do not have assorted bacterium, it is qualified to be, and can make microbial preparation:
1. liquid preparation: the fermented liquid of 10L jar fermentative production directly is potted in the plastic barrel of cleaning and preserves, the mycetocyte number reaches 200,000,000 CFU/ml;
2. solid preparation: with 121 ℃ of sterilizations of peat composed of rotten mosses powder 30min, mix with fermented liquid behind the airing and mix thoroughly, about 15～20%, the mycetocyte number is at 0.5 hundred million CFU/ml～100,000,000 CFU/ml;
Obtain to be used to prevent and treat the sea bacillus subtilis 3512A effective strain of cucumber fusarium axysporum with the present invention, have following feature after measured:
(1) morphological specificity of thalline
Marine bacteria 3512A has typical sporeformer feature, the thalline rod-short, and Gram-positive, peritrichous has gemma, gemma ellipse, middle life, does not expand.The light khaki color of bacterium colony, opaque, surface irregularity, edge are irregular.
(2) physiological and biochemical property
Starch hydrolysis, salt tolerance and utilization of carbon source, measurement result sees Table 2.The marine bacteria 3512A catalase positive can be grown for 45 ℃, and 7%NaCl growth can hydrolyzed starch; Utilize glucose, raffinose, wood sugar, sucrose, N.F,USP MANNITOL, pectinose, fructose, xylan, dextrin, starch, malonate, cellobiose, gelatin; Citrate trianion can be utilized, semi-lactosi, inositol, sorbose, rhamnosyl, synanthrin, formate, acetate, propionic salt can not be utilized.
(3) molecular biology identification
The sequence similarity of 16S rDNA sequence of marine bacteria 3728 and subtilis (Bacillus Subtilis) is up to 99.5%, in phylogenetic tree (evolutionary tree), also be on the same branch, see accompanying drawing with subtilis (Bacillus Subtilis).
(4) marine bacteria 3512A is to the restraining effect of cucumber fusarium axysporum
Marine bacteria 3512A has very strong bacteriostatic activity to Fusarium oxysporum, and antibacterial circle diameter reaches 18.3mm, 10 times of no fermented liquid dilutions, and its bacteriostasis rate reaches 70.83%.
Application examples
The sea bacillus subtilis 3512A test tube slant bacterial classification that above-mentioned screening is obtained, move connect expand numerous, then at 28 ℃ of heat insulating culture 48h, this moment, lawn looked very plentiful, can scrape with sterilized water and wash, make bacteria suspension, make the somatic cells number in every milliliter of bacteria suspension reach 1,000,000,000 more than the CFU, promptly reach 10
9Concentration, can with the potted plant soil of cucumber (damp brown earth) uniform mixing, make the cell count of the subtilis of the test determination in every gram soil reach 10
5More than the CFU, reach more than 100,000 CFU cells in promptly every gram soil; Soil is packed in the basin, 20 cucumber seeds of every basin sowing, and by the management of pot experiment ordinary method, the marine bacteria number in soil, grown surely of results of regular determination weekly.Surely grow and measure to adopt dilution-plate method, get the cucumber rhizosphere soil a little, dilute with sterilized water, add on the wort agar flat board at beef extract-peptone and be coated with 28 ℃ of heat insulating culture 48h.Select the colonies typical consistent with connecing 3512A form and count, spot-check the anti-microbial activity to cucumber fusarium axysporum simultaneously, the bacterium colony that should reach more than 90% has anti-microbial activity.The result proves that this separates from gorgonian sea bacillus subtilis bacterial strain 3512A, can grow surely in the cucumber rhizosphere soil, inoculated for 6 weeks after, measure cell count and can be stabilized in 10
6The level of CFU/ gram soil, cell count is stabilized in 10 after 10 weeks
5～10
6The level of CFU/ gram soil is illustrated as effective strain.
Sea bacillus subtilis 3512A fermentation: (1), Kolle flask seed culture, adopt 500ml volume Kolle flask, dress 60ml substratum, sterilized 30 minutes for 121 ℃, put the inclined-plane after the cooling, the inoculation sea bacillus subtilis then was placed on this Kolle flask in 25～28 ℃ of insulation cans heat insulating culture 48～72 hours, took out promptly to can be used as seed and use.The substratum that the Kolle flask seed culture is used is the modified starch substratum, and it consists of: Zulkovsky starch 2%, extractum carnis 0.5%, yeast extract paste 0.5%, NaCl0.5%, pH nature.
(2), 10L fermenting organism reactor fermentation, canned substratum is 6L, substratum consists of the modified starch substratum, promptly Zulkovsky starch 2%, extractum carnis 0.5%, yeast extract paste 0.5%, NaCl0.5%, all the other be water, pH naturally.Sterilized 30 minutes for 121 ℃, after the cooling, inoculate two bottles in above-mentioned Kolle flask seed, after seed washes with the 200ml sterilized water, use Aseptic technique and be seeded in the 10L volume bio-reactor, the culture condition of fermentative production is: tank pressure 0.05Mpa, 28 ℃ ± 1 ℃ of jar temperature, ventilation is 6L/min, and stirring velocity is 200rpm.Cultivated with this understanding 36～48 hours, and can put jar, this fermented liquid can be used for making microbiobacterial agent.
(3), the manufacturing of anti-cucumber fusarium axysporum sea bacillus subtilis microbial preparation, the fermented liquid of the above-mentioned gorgonian sea bacillus subtilis that obtains, the microscopy thalli growth is good in the laboratory, every ml fermented liquid somatic cells reaches 200,000,000 more than the CFU, to the antagonism antibacterial circle diameter of cucumber fusarium axysporum more than 18mm, do not have assorted bacterium, it is qualified to be, and can make microbial preparation:
1. liquid preparation: the fermented liquid of 10L jar fermentative production directly is potted in the plastic barrel of cleaning and preserves, the mycetocyte number reaches 200,000,000 CFU/ml;
2. solid preparation: with 121 ℃ of sterilizations of peat composed of rotten mosses powder 30min, mix with fermented liquid behind the airing and mix thoroughly, about 15～20%, the mycetocyte number is at 0.5 hundred million CFU/ml～100,000,000 CFU/ml.
The effect (plant height, chlorophyll content, the weight contrast that more do not connect bacterium as a result increase to some extent, test and potted plant each 4 basin of contrast, the pot experiment time is not shorter than three months) that after measured cucumber is had certain promotion growth.Measuring chlorophyll content adopts the acetone extraction method, respectively surveys once in the seedling phase and the phase of yielding positive results respectively, gets all the 2nd leaf of unfolded (functional leaf) mensuration chlorophyll contents of cucumber plant.The chlorophyll content of cucumber leaves contrasts highly by 5% behind the inoculation sea bacillus subtilis 3512A, and difference reaches conspicuous level.
After testing, this sea bacillus subtilis can be grown on the inorganic phosphorus substratum and be produced the obvious transparent circle, and the effect that it has phosphorus decomposing is described, promptly can impel the phosphorus of insoluble in the soil to become the phosphorus of solubility, and then promotes the absorption of cucumber to phosphoric.Can significantly promote the growth of water planting cucumber seedling root system after its aseptic ferment filtrate dilutes 1000 times in addition, certain growth stimulation is described.
Table 13512A is to the prevention effect of the phase cucumber fusarium axysporum of yielding positive results
Table 23512A is to the influence (g) of cucumber yield
Data are 8 multiple mean+SD that 4 * 4 Latin squares are handled in the table.
*The expression significant difference,
*Expression difference is extremely remarkable.
Claims (5)
1. a marine bacillus subtilis, it is characterized in that: its for the sea bacillus subtilis Bacillus subtilis 3512A of gorgonian commensalism, be deposited in Chinese typical culture collection center on April 23rd, 2007, address: China. Wuhan. Wuhan University, preserving number is CCTCC No.M207051.
2. the separating screening method of the described sea bacillus subtilis of claim 1 is characterized in that:
(1) bacterial strain primary dcreening operation: from South China Sea gorgonian growth district, directly choose the gorgonian organism growth point position of healthy work, be cut into the long fritter of 0.5～1.0cm, pack in the aseptic sampling bottle, take back the laboratory, with volume ratio sample: sterilized water=0.5-1: 1 grinds, and obtains fresh gorgonian cell extract; Adopt the screening method of conventional genus bacillus, with the method that mutual antagonism screening model of plate and directed screening combine, be about to gorgonian cell extract bottle and place 80 ℃ of waters bath with thermostatic control, thermal treatment 10～15 minutes, take out cooling, be coated with plate with dilution-plate method, carry out primary dcreening operation, the primary dcreening operation plate was placed in 25～28 ℃ of insulation cans heat insulating culture 48～72 hours, single bacterium colony with morphological specificity choosing colony fold tool typical case bacillus subtilis form, move and connect the inclined-plane preservation, number respectively, so that multiple sieve;
(2) bacterial strain sieves again: single bacterium colony slant tube bacterial strain that above-mentioned primary dcreening operation is obtained carries out the plate activation, 28 ℃, 24-60 hour, adopt plate antagonistic effect method, with the cucumber fusarium axysporum is the target bacterium, the antagonistic effect that on plate, stands facing each other according to a conventional method, heat insulating culture is 48～84 hours in 25～28 ℃ of insulation cans, take out face-off antagonistic effect plate, detect the inhibition zone size of each gorgonian sea bacillus subtilis bacterial strain to cucumber fusarium axysporum target bacterium, bacteriostatic diameter reaches 18mm or 18mm is above for qualified, selects numbering to preserve.
3, a kind of control cucumber fusarium axysporum preparation is characterized in that: the fermented liquid with the described sea bacillus subtilis Bacillus of claim 1 subtilis 3512A is an active ingredient.
4, according to the described control cucumber fusarium axysporum of claim 3 preparation, it is characterized in that: the thalline of described fermented liquid is good at laboratory microscopy thalli growth, every ml fermented liquid somatic cells reaches 200,000,000 more than the CFU, to the antagonism antibacterial circle diameter of cucumber fusarium axysporum at 18mm or more than the 18mm, there is not assorted bacterium, it is qualified to be, and can make microbial preparation;
1. liquid preparation: the fermented liquid of fermentative production directly is potted in the Plastic Bottle of cleaning or the bucket preserves, the mycetocyte number reaches 200,000,000 CFU/ml, can be directly as liquid preparation;
2. solid preparation: fermented liquid mixed with aseptic peat composed of rotten mosses powder mix thoroughly, make water content 15～20%, the mycetocyte number is solid preparation at 0.5～100,000,000 CFU/ml.
5, according to the described control cucumber fusarium axysporum of claim 3 preparation, it is characterized in that: the fermentation flow process of described sea bacillus subtilis Bacillus subtilis 3512A is,
1), the Kolle flask seed culture, adopt 500ml volume Kolle flask, dress 60ml modified starch substratum, substratum is formed: Zulkovsky starch 1-2%, extractum carnis 0.3-0.8%, yeast extract paste 0.3-0.8%, NaCl 0.3-0.8%, natural pH; The inclined-plane is put in 121 ℃ of sterilizations 15-30 minute after the cooling, the inoculation sea bacillus subtilis then was placed on this Kolle flask in 25～28 ℃ of insulation cans heat insulating culture 48～72 hours, takes out promptly to can be used as seed and use;
(2), adopt the 10L fermenting organism to answer the device fermentation, canned modified starch substratum is 6L, 121 ℃ of sterilizations 15-30 minute after the cooling, are inoculated two bottles in above-mentioned Kolle flask seed; The culture condition of fermentative production is: tank pressure 0.05Mpa, and 28 ℃ ± 1 ℃ of jar temperature, ventilation is 4-7L/min, stirring velocity is 180-300rp m; Cultivated with this understanding 36～48 hours, and can put jar, this fermented liquid can be used for making microbiobacterial agent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102465096A (en) * | 2010-11-15 | 2012-05-23 | 中国科学院沈阳应用生态研究所 | Microbial agent as well as preparation method and application thereof |
| CN103371041A (en) * | 2012-04-20 | 2013-10-30 | 中国农业大学 | Method for screening bio-control bacteria of cucumber fusarium wilt |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102465096A (en) * | 2010-11-15 | 2012-05-23 | 中国科学院沈阳应用生态研究所 | Microbial agent as well as preparation method and application thereof |
| CN102465096B (en) * | 2010-11-15 | 2015-04-15 | 中国科学院沈阳应用生态研究所 | Microbial agent as well as preparation method and application thereof |
| CN103371041A (en) * | 2012-04-20 | 2013-10-30 | 中国农业大学 | Method for screening bio-control bacteria of cucumber fusarium wilt |
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