CN101298597B - Sea bacillus subtilis and screening method thereof - Google Patents
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Abstract
The invention relates to an application technology of marine microorganism and a disease control method for fungi in the rhizosphere soil in Chinese fir plantation, in particular to a type of marine bacillus subtilis and a screening method thereof. The microorganism refers to the bacillus subtilis 3728C, the number for preservation of which is CCTCC No. M207052. The method combines a screening model with mutually antagonized plates and directional screening. By the screening method, strains of the marine bacillus subtilis for controlling the harmful fungi in the rhizosphere soil in Chinese fir plantation are used as one of the major components to develop a type of disease-preventing and toxicity-decreasing ecological agent, which solves the problems of continuous planting of Chinese firs.The disease-preventing and toxicity-decreasing ecological agent is a type of microbial preparation with high efficiency, safe and free of toxicity and residues, which can enhance the development of the green ecosystem of the Chinese fir plantation. With satisfactory application and test results in Hunan Huitong Chinese Fir Plantation, the disease-preventing and toxicity-decreasing ecological agent has bright development prospects.
Description
Technical field
The present invention relates to marine microorganism Applied Biotechnology and the disease control of China fir rhizosphere soil fungi, a kind of specifically sea bacillus subtilis and screening method thereof that is used to prevent and treat Chinese Fir Plantation woods rhizosphere soil fungal disease.
Background technology
China fir (Cunninghamia lanceolata) is the commerical tree species of the distinctive fast growing of China, is distributed in southern 17 provinces of China, and 1214.07 ten thousand hectares of existing Chinese Fir Plantation woods areas account for 24% of China's artificial forest total area.Studies show that for many years: along with China fir connects the increase of planting algebraically, the China fir productivity will descend, and China fir occurs and even plants obstacle.The China fir pure forest connects plants, soil fertility descends, China fir seedling damping-off evil increases, toxic substance accumulation in the soil, soil microorganisms quantity reduces year by year, and causing harmful fungi in the soil increases, and environmental degradation is serious day by day, plant on the 3rd generation China fir soil 20 years living Chinese Fir Plantation woodss than plant the same year the 1st generation China fir, its diameter of a cross-section of a tree trunk 1.3 meters above the ground, the height of tree and growing stock reduce by 22%, 30% and 54% respectively.China fir is even planted and is caused that it is a universal phenomenon that productivity descends, it is a lot of to have traced it to its cause, for example pass by soil nutrient transformed and be obstructed, soil nutrient content descends, it is bad that soil physical and chemical property becomes, it is more that the China fir disease and pest increases report, and to causing that China fir connects that to plant one of obstacle be that the soil microorganisms diversity significantly reduces, growth causes harmful pathogenic fungi showed increased to China fir, even plant the Fusarium oxysporum Schl.f.sp.vasinfectum (Fusarium oxysporum f.sp.vasinfectum) that mainly causes in the soil in the harmful bacterium through microbiology evaluation China fir, penicillium bacterium (Eupenicilliumberfeldianum) etc., caused China fir connects plants soil poisoning, makes the withered research report of poisoning particularly outstanding this respect that waits of China fir seedling few.Over nearly 5 years, the China fir pure forest connected fungal pathogens occurs after planting and increase, the toxic substance accumulation, cause that soil poisoning, soil fertility descend, China fir seedling damping-off serious situation has given enough attention, but does not still have ideal, Prevention Technique targetedly, needing badly to have new biological and ecological methods to prevent plant disease, pests, and erosion technology early, solves Chinese Fir Plantation woods soil disease and soil fertility decline problem.
Summary of the invention
The object of the present invention is to provide a kind of sea bacillus subtilis and screening method thereof, the sea bacillus subtilis bacterial classification that the present invention obtains can be used for preventing and treating Chinese Fir Plantation woods rhizosphere soil fungal disease, further can be made into the diseases prevention attenuated microorganisms preparation that is used for Chinese Fir Plantation woods silborne fungal diseases.
Simultaneously, the present invention also provides a kind of and can carry out effective method for screening to the sea bacillus subtilis of China fir rhizosphere soil fungi disease control.
For achieving the above object, the technical solution used in the present invention is:
A kind of sea bacillus subtilis, it was Bacillus subtilis 3728C, had been deposited in Chinese typical culture collection center, address: China on April 23rd, 2007. Wuhan. and Wuhan University, preserving number is CCTCC No.M 207052.
The screening method of described sea bacillus subtilis is:
1) bacterial strain primary dcreening operation: Bruguiera conjugata (Brugiera gymnorrhiza) the young root root table soil with mangrove forest (Mangrove forest) is the sample that separates bacterial classification, the Bruguiera conjugata root childhood sample that will have root table soil is cut into the long segment of 0.5-4cm, then be put in the triangular flask that fills seawater, triangular flask is placed in the 70-90 ℃ of water-bath, thermal treatment 15-20 minute (to kill the non-sporeformer in the root table pedotheque, reach effective separation high reactivity purpose bacterial strain), take out cooling, be coated with plate with dilution plate separation method, carry out primary dcreening operation, the primary dcreening operation plate is placed in the 25-30 ℃ of insulation can heat insulating culture 36-60 hour, with morphologic observation picking list bacterium colony, and numbering;
Described its Bruguiera conjugata young root is prop root childhood.Root childhood of this moment is flexible, screen casing fiber hollow, spongy, there is inflation to hold the effect of oxygen, the effect (old root is lignifying, does not have desirable bacterial strain and can screen) of united symbiosis is arranged, with the sample of this pedotheque between bacillus marinus in the China fir Gen Genbiao childhood soil of this moment and Bruguiera conjugata young root as the screening and separating bacterial classification, with after the directed screening method of combining is carried out bacterial screening, can obtain the purpose bacterial strain through mutual antagonistic effect screening.
The substratum that described primary dcreening operation is used adopts beef extract-peptone wort agar substratum, be exactly specifically with etc. the beef extract-peptone nutrient agar of weight, with etc. the pol of weight be the wort agar substratum of Baume 7 degree, bottling respectively, the sterilization cooling, face time spent difference heating and melting, mix the use of falling the plate in the sterilisable chamber.
2) bacterial strain sieves again: it is the typical bacterium colony of subtilis that above-mentioned primary dcreening operation is obtained morphological specificity, move and receive in the slant tube that fills beef extract-peptone agar, picking list bacterium colony is 5-10 respectively, is positioned over 25-30 ℃ of heat insulating culture 36-60 hour, uses for multiple sieve;
Multiple sieve adopts plate antagonistic effect method, the method that the mutual antagonistic effect screening model of described plate combines with directed screening, be that to obtain mainly causing harmful fungi in the Chinese Fir Plantation woods rhizosphere soil be the target bacterial strain to separate, with obtain carrying out the method that the antagonistic effect screening combines with directed screening between the bacillus marinus bacterial strain in the Bruguiera conjugata young root root table soil of separation and purification, filter out highly active bacillus marinus.
Be specially: with China fir rhizosphere soil disease fungi is the target bacterium, the antagonistic effect that on plate, stands facing each other according to a conventional method, 25-30 ℃ of insulation 36-60 hour, take out plate, detect the inhibition zone size of primary dcreening operation bacterial strain to the target bacterium, antibacterial circle diameter reach 16mm or 16mm above for qualified, preserve according to qualifications;
Described China fir rhizosphere soil disease fungi is Fusarium oxysporum Schl.f.sp.vasinfectum (Fusariumoxysporum f.sp.vasinfectum).
3) screening fixed sea bacillus subtilis again can carry out deciding to grow test in the potted plant soil of the annual seedling of China fir;
Surely grow test: 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 China fir seedling, 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 the annual seedling of every pot transplanting China fir by the management of pot experiment ordinary method, inserts that 2-16 is after week in the soil, and the sea bacillus subtilis cell count is stabilized in 10
5CFU/ 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 of experimental determination, antibacterial circle diameter 〉=16.6mm 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.
The present invention has following advantage:
1, with clearly defined objective for the sample of screening high-efficiency prevention and control China fir rhizosphere soil sickle-like bacteria cause of disease bacterium, adopt mangrove forest Bruguiera conjugata prop root childhood (having root table soil), carry out separation screening with this type of sample by the inventive method, can obtain to have the good sea bacillus subtilis of pathogenic fungi disease such as Fusarium oxysporum in the high-efficiency prevention and control China fir rhizosphere soil.
2, the simple and clear easily row of screening method of the present invention, the screened yield height.More than select mangrove forest Bruguiera conjugata (Bruguiera gymnorrhiza) prop root childhood (having root table soil) to be the sample of separation screening purpose bacterial strain, adopt the laboratory aseptic technique, with Bruguiera conjugata prop root childhood (having root table soil) through 85 ℃, after the thermal treatment in 15 minutes, separate the purpose bacterial strain with dish dilution method, strain number, and between the target bacterial strain China fir rhizosphere caused by soil evil fungi Fusarium oxysporum Schl.f.sp.vasinfectum (Fusarium oxysorum f.sp.vasinfectum), carry out the plate antagonistic effect, filter out the numbering bacterial strain of high antagonistic activity, identify or 16S rDNA sequential detection proves that it is subtilis (Bacillussubtilis) through traditional bacteriology.
3. The selection result is with a high credibility.The described sea bacillus subtilis that obtains carries out the annual seedling pot experiment of China fir, can high-density in the China fir rhizosphere soil grow surely (insert in the soil after 12 weeks, sea bacillus subtilis rhizosphere decide grow density and reach 10
5More than the CFU/ gram soil), high reactivity suppresses pathogenic bacteria (separate the bacterial strain inhibition pathogenic bacteria activity that reaches 95% or more of growing surely from rhizosphere and be not less than experimental determination antibacterial circle diameter 16.6mm).And the sea bacillus subtilis that the requirement inoculation is grown surely is to China fir growth unrestraint effect, or has a bacterial strain that promotes China fir growth effect, these are had in the bacterial strain of antagonism target bacterial strain select excellent preservation, resulting bacterial strain is effective strain, thereby has finished the screening of the sea bacillus subtilis of preventing and treating Chinese Fir Plantation woods rhizosphere soil fungal disease.
4.Bacillus subtilis 3728C effect is good.Resulting sea bacillus subtilis 3728C, active strong (the antibacterial circle diameter 16.66mm) that suppresses sickle-like bacteria in the China fir soil, colonization ability (carries out the pot experiment of the annual seedling of China fir by force in the China fir rhizosphere soil, in 12 weeks behind the inoculation subtilis, measure its deciding in China fir seedling rhizosphere soil and grow density 10
5CFU/ restrains soil, the efficient bacterial strain that it obtains for screening, the subtilis 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, antibacterial circle diameter 16.66mm) to China fir growth unrestraint, and the effect that promotes growth is arranged, and environmentally friendly, make ecological agent, effective at Chinese Fir Plantation woods application test, help application.
Description of drawings
Related Bacillus subtilis 3728C 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 207052.
Fig. 1 is the phylogenetic tree of marine bacteria 3728 (being the Bacillus subtilis 3728C 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: Bruguiera conjugata (Burguiera gymmorrhiza) prop root childhood of 1. gathering the coastal mangrove forest in the torrid zone or subtropical zone (Mangrove forest) forest zone is (flexible, screen casing fiber hollow, spongy, there is inflation to hold the effect of oxygen.Do not get old root, because of old root lignifying gradually, hardening.) have a sample of soil; 2. after handling in 85 ℃, 15 minutes, separate required bacterial strain with dish dilution method; 3. be target mainly to cause harmful fungi Fusarium oxysporum Schl.f.sp.vasinfectum in the Chinese Fir Plantation woods rhizosphere soil, filter out the isolating sea bacillus subtilis of Bruguiera conjugata root table pedotheque by the plate antagonistic effect with efficient antagonistic activity; 4. filter out by pot experiment and grow surely in the China fir rhizosphere soil that has efficient antagonistic activity and can on Chinese Fir Plantation woods soil, plant the China fir seedling, measure in the soil in position the antagonistic strain that causes harmful fungi (Fusarium oxysporum Schl.f.sp.vasinfectum) effect in the obvious inhibition China fir rhizosphere soil is arranged, this bacterial strain simultaneously will be to the China fir unrestraint effect of growing, or slightly promote the growth effect, select, determine that it is subtilis through the 16SrDNA analysis, resulting bacterial strain is efficient bacterial strain 3728C.
The sea bacillus subtilis 3728C that is used to prevent and treat Chinese Fir Plantation woods rhizosphere soil fungal disease is in China's typical culture collection center preservation, and its preserving number is CCTCC No.M207052.Sea bacillus subtilis 3728C 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 3728C bacterial strain and the sequence similarity of subtilis are up to 99.5%.The 3728C bacterial strain can grow (＞10 surely at China fir rhizosphere soil middle-high density
5CFU/g soil), Chinese Fir Plantation woods rhizosphere soil fungal disease there is tangible prevention effect (bacteriostasis rate＞60%, preventive effect reaches more than 60%);
Prevent and treat the sea bacillus subtilis that Chinese Fir Plantation woods rhizosphere soil causes the Bruguiera conjugata rhizosphere of harmful fungi by what the present invention screened, with this bacterial strain is that one of main ingredient is developed into and overcomes China fir and connect the diseases prevention attenuation ecological agent plant obstacle, be a kind of efficient, nontoxic, safety, noresidue, can promote the microbial preparation of Chinese Fir Plantation woods green ecological system development, through good, rising together with the geographic application test result of Chinese Fir Plantation woods in the Hunan.
Sea bacillus subtilis (Bacillus subtilis) 3728C that obtains with screening method of the present invention, effect with high-efficiency prevention and control Chinese Fir Plantation woods rhizosphere soil fungal disease, the activity intensity big (antibacterial circle diameter reaches 16.6mm) that suppresses Fusarium oxysporum in the China fir soil, and to China fir growth unrestraint effect and have certain promotes growth effect, environmentally friendly.
The screening method step of sea bacillus subtilis of preventing and treating Chinese Fir Plantation rhizosphere fungal disease (sickle-like bacteria) is as follows:
1, sample collecting: prepare aseptic sampling bottle in the laboratory, sterilized scissors, cotton ball soaked in alcohol, bring to the torrid zone, mangrove forest area, subtropics, choose the Bruguiera conjugata tree, get Bruguiera conjugata prop root childhood (having root table soil), use sterilized scissors, the long root segment of the about 3cm of clip tip of a root leading portion prop root childhood, put in the aseptic sampling bottle, take back to the laboratory, the usefulness of screening purpose bacterium to be separated with the ice chest preservation, the Bruguiera conjugata prop root of being adopted childhood is wanted flexible sense, its screen casing fiber hollow, spongy, childhood root the thickness diameter like people's pinkie shape, Bruguiera conjugata prop root childhood surface will have the soil (promptly sticking in the ooze on the root, not the soil that can fall down through shake) of state of nature.
2, bacterial strain primary dcreening operation: with Bruguiera conjugata root childhood (having root table soil) sample, with sterile scissors Bruguiera conjugata prop root childhood is cut into the long segment of 2cm, then be put in the triangular flask that fills seawater and (use the 500mL triangular flask, dress seawater 100mL, sterilized 20 minutes for 121 ℃), and this triangular flask is placed in 85 ℃ of water-baths, thermal treatment 15-20 minute, kill non-spore bacteria, take out cooling, be coated with plate with dilution plate separation method, carry out primary dcreening operation, the primary dcreening operation plate was placed in 28 ℃ of insulation cans heat insulating culture 48 hours, with morphologic observation picking list bacterium colony, and numbering.The substratum that primary dcreening operation is used adopts beef extract-peptone wort agar substratum, specifically just with etc. the beef extract-peptone nutrient agar of weight, with etc. the pol of weight be the wort agar substratum of Baume 7 degree, bottling respectively, the sterilization cooling, face time spent difference heating and melting, mix the use of falling the plate in the sterilisable chamber.Bruguiera conjugata root childhood (having root table soil) sample suspension, be coated with this plate with dilution method, 28 ℃ of heat insulating culture, the single bacterium colony that grows on the ware, colonial morphology is very typical, sea bacillus subtilis on this plate, single colonial morphology of formation (48 hours): bacterium colony limited growth, the periphery irregular, colony diameter 2-4mm, shallow khaki color, surface irregularity, drying, obviously and typically fold is arranged, sea bacillus subtilis is grown unique on this plate, and the single colony morphology characteristic of other that grow out on the plate has tangible difference therewith, is easy to distinguish.
Above-described two kinds of substratum are:
(1) the beef extract-peptone nutrient agar consists of: extractum carnis 3g, peptone 5g, water 1000mL, agar 20g, pH7.0-7.2.121 ℃, sterilized 20 minutes, standby;
(2) the wort agar substratum consists of: pol is the wort 1000mL of Baume 7 degree, agar 20g, pH7.0-7.2.121 ℃, sterilized 20 minutes, standby;
The preparation of wort:
(i) first preparation malt meal: get vulgare (preferably results) then seed and soaked 24 hours, place in the water basket, the upper cover cloth that wets, insulation about 20 ℃, wash by water every day 1-2 time (drip washing), during to long for wheat length 1-1.5 times of bud, can shine (baking) and dry grind into powder.
(ii) malt amylase: every 1000g Fructus Hordei Germinatus dry powder is added 3 kilograms in water, and 55 ℃ of saccharification add iodine liquid and detect, till when extremely not being blueness (about 6-7 hour), boiled again 30 minutes, make protein precipitation, transfer pH to neutral, after filtered through gauze, filtrate is poured in the graduated cylinder, standing over night, siphonage sucking-off supernatant liquor is measured it with saccharometer then, is diluted with water to needed Baume 7 degree packing triangular flasks, sterilized 20 minutes, and cooled off standby for 121 ℃.
3, bacterial strain sieves again: it is the typical bacterium colony of subtilis that above-mentioned primary dcreening operation is obtained morphological specificity, move and receive in the slant tube that fills beef extract-peptone agar, single bacterium colony 5-10 of the typical plentiful stalwartness of picking respectively, move and number after connecing the inclined-plane, be positioned over 28 ℃ of heat insulating culture 48 hours, and used for multiple sieve.Multiple sieve adopts plate antagonistic effect method, with China fir rhizosphere soil disease fungi Fusarium oxysporum Schl.f.sp.vasinfectum (Fusarium oxysporum f.sp.vasinfectum) is the target bacterium, tentatively think subtilis (numbered) what primary dcreening operation had obtained, the antagonistic effect that on plate, stands facing each other according to a conventional method, 28 ℃ of insulations 72 hours, take out plate, detect the inhibition zone size of primary dcreening operation bacterial strain to Fusarium oxysporum Schl.f.sp.vasinfectum, antibacterial circle diameter reach 16mm above for qualified, preserve according to qualifications.Bacterial strain that this multiple sieve obtains, that can carry out the China fir rhizosphere soil decides to grow test.
4, grow test surely: screen fixed sea bacillus subtilis again and can carry out deciding to grow test in the potted plant soil of the annual seedling of China fir.With sea bacillus subtilis test tube slant bacterial classification, moving on the big inclined-plane of the Kolle flask of receiving 250mL (slant medium is all used the beef extract-peptone nutrient agar) expands numerous, then 28 ℃ of heat insulating culture 48 hours, this moment, sea bacillus subtilis 3728C 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 China fir seedling, make the cell count of the subtilis of the test determination in every gram soil reach 10
5CFU above (being to reach more than 100,000 CFU cells in every gram soil).Soil is packed in the basin, two annual seedlings of China fir of every pot transplanting, by the management of pot experiment ordinary method, the sea bacillus subtilis cell count that insert this moment in the soil of potted plant China fir shoot root border, fall after rising, the quantity increase of subtilis is very fast in first three all China fir rhizosphere soil, reaches 10
6-10
7CFU/ restrains soil, and three weeks descended later to some extent, progressively tended towards stability, and the sea bacillus subtilis cell count is stabilized in 10
5CFU/ restrains native level, illustrate that the sea bacillus subtilis that obtains can grow surely in the China fir rhizosphere soil, through measuring growth unrestraint effect to the China fir seedling, (contrast that plant height, leading thread more do not connect bacterium increases to some extent and the effect of certain promotion growth is arranged, test and potted plant each three to five basin of contrast, the pot experiment time is not shorter than three months).Surely grow the bacterial strain of success of the test, can further make classical bacteriology and identify and 16S rDNA sequential analysis, final trial integrated data as a result determines that screening obtains the name of bacterial strain, and 98% result is consistent with primary dcreening operation identification of morphology result generally speaking.So far the step of experiment screening method is all finished, and this bacterial strain is an effective strain, can supply with testing or being applicable.
With the effective strain of screening technique acquisition of the present invention for the marine bacteria 3728 of control Cunninghamia Lanceolata Plantations rhizosphere soil fungal disease, have after measured following feature:
1, the morphological feature of marine bacteria 3728: the feature with typical Bacillus, can produce gemma, the gemma form is column, middle life, does not expand, the somatic cells form is shaft-like or rod-short, Gram-positive, that peritrichous, bacterium colony are is faint yellow, dry, opaque, rough surface, edge are irregular.
2, the physiological and biochemical property of marine bacteria 3728: hydrolyzed starch, gelatin liquefaction, the catalase positive is utilized citrate, can be 3%, 5%, 7%NaCl and 45 ℃ of growths. Glucose, gossypose, wood sugar, sucrose, sweet mellow wine, arabinose, fructose can be utilized, galactolipin, sorbose, rhamnose, synanthrin, formates, acetate, propionate can not be utilized.
3, molecular biology identification, the sequence similarity of the 16S rDNA sequence of marine bacteria 3728 and bacillus subtilis (Bacillus Subtilis) is up to 99.5%, in phylogenetic tree (chadogram), also be on the same branch with bacillus subtilis (Bacillus Subtilis), see accompanying drawing.
4, in the China fir rhizosphere soil, can high density surely grow (＞105CFU/g soil), to Cunninghamia Lanceolata Plantations rhizosphere soil fungal pathogens: Fusarium oxysporum Schl.f.sp.vasinfectum and penicillium etc. has very strong inhibitory action, and Young And Middle-aged Stands of Chinese Fir is established rot fine preventive effect.
Claims (5)
1. sea bacillus subtilis, it is characterized in that: it is subtilis 3728C (Bacillus subtilis 3728C), be deposited in Chinese typical culture collection center on April 23rd, 2007, address: China. Wuhan. Wuhan University, preserving number is CCTCC No.M 207052.
2. the screening method of a sea bacillus subtilis is characterized in that:
1) bacterial strain primary dcreening operation: the Bruguiera conjugata young root root table soil with mangrove forest is the sample that separates bacterial classification, the Bruguiera conjugata root childhood sample that will have root table soil is cut into the long segment of 0.5-4cm, then put into the triangular flask that fills seawater, triangular flask places 70-90 ℃ of water-bath, thermal treatment 10-20 minute, take out cooling, be coated with plate with dilution plate separation method, carry out primary dcreening operation, the primary dcreening operation plate is placed in the 25-30 ℃ of insulation can heat insulating culture 36-60 hour, with morphologic observation picking list bacterium colony, numbering;
The substratum that described primary dcreening operation is used adopts beef extract-peptone wort agar substratum, specifically just with etc. the beef extract-peptone nutrient agar of weight, with etc. the pol of weight be Baume, the wort agar substratum of 7 degree, bottling respectively, time spent difference heating and melting is faced in the sterilization cooling, mixes the use of falling the plate in the sterilisable chamber;
2) bacterial strain sieves again: it is the typical bacterium colony of subtilis that above-mentioned primary dcreening operation is obtained morphological specificity, move and receive in the slant tube that fills beef extract-peptone agar, picking list bacterium colony is 5-10 respectively, is positioned over 25-30 ℃ of heat insulating culture 36-60 hour, uses for multiple sieve;
Multiple sieve adopts plate antagonistic effect method, with China fir rhizosphere soil disease fungi is the target bacterium, the antagonistic effect that on plate, stands facing each other according to a conventional method, 25-30 ℃ of insulation 60-84 hour, take out plate, detect the inhibition zone size of primary dcreening operation bacterial strain to the target bacterium, antibacterial circle diameter reach 16mm above for qualified, preserve according to qualifications.
3. according to the described screening method of claim 2, it is characterized in that: described its Bruguiera conjugata young root is prop root childhood.
4. according to the described screening method of claim 2, it is characterized in that: screen fixed sea bacillus subtilis again and carry out deciding to grow test in the potted plant soil of the annual seedling of China fir;
Surely grow test: 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, scrape to wash with sterilized water, 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 with the potted plant soil uniform mixing of China fir seedling, makes 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 the annual seedling of every pot transplanting China fir by the management of pot experiment ordinary method, inserts in the soil after 12 weeks, and the sea bacillus subtilis cell count is stabilized in 10
5CFU/ 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, and supply is with testing or applying.
5. according to the described screening method of claim 2, it is characterized in that: described China fir rhizosphere soil disease fungi is a Fusarium oxysporum Schl.f.sp.vasinfectum.
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| Title |
|---|
| .杉木人工林不同发育阶段根际与非根际土壤微生物变化杉木人工林不同发育阶段根际与非根际土壤微生物变化趋.林业科学35 1.1999,35(1),53-59. |
| 杨承栋 |
| 焦如珍 |
| 焦如珍;杨承栋;.杉木人工林不同发育阶段根际与非根际土壤微生物变化杉木人工林不同发育阶段根际与非根际土壤微生物变化趋.林业科学35 1.1999,35(1),53-59. * |
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