CN105779305A - Special compound microorganism bacterium agent capable of eliminating continuous cropping obstacles of radix pseudostellariae and preparation method of special compound microorganism bacterium agent - Google Patents
Special compound microorganism bacterium agent capable of eliminating continuous cropping obstacles of radix pseudostellariae and preparation method of special compound microorganism bacterium agent Download PDFInfo
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Abstract
The invention discloses a special compound microorganism bacterium agent capable of eliminating continuous cropping obstacles of radix pseudostellariae. The special compound microorganism bacterium agent is prepared from common probiotics and aboriginal probiotics at the volume ratio of 4 to 6, wherein the common probiotics are prepared by mixing lactic acid bacteria, photosynthetic bacteria, saccharomycetes and actinomyces at the volume ratio of 1 to 1 to 1 to 1; the aboriginal probiotics are prepared by mixing burkholderia, pseudomonas and bacillus at the volume ratio of 4 to 1 to 1. The invention further discloses a preparation method of the special compound microorganism bacterium agent capable of eliminating the continuous cropping obstacles of the radix pseudostellariae. The preparation method comprises the following steps: step 1, screening aboriginal antagonistic bacteria; step 2, preparing common compound bacterium liquid; step 3, activating and carrying out expanding culture on the aboriginal probiotics; step 4, preparing the compound microorganism bacterium agent. The special compound microorganism bacterium agent prepared by the method has a good root fixed planting capability and is safe and reliable, has obvious prevention and control effects on the continuous cropping obstacles of the radix pseudostellariae and soil-borne diseases, and has no pathogenic infection capacity on other succession crops.
Description
Technical field
The present invention relates to microorganism and technical field of biological control, particularly relate to a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle and preparation method thereof.
Background technology
Even if continuous cropping obstacle refers under normal field management measure, same plot is planted identical plant for years and is caused the phenomenon that growth and development of plants is bad, pest and disease damage serious, yield and quality declines, also referred to as allelopathic from poison phenomenon or cultivating disease.Medicinal plants generally avoids continuous cropping, according to statistics, there are about the tuber class medical material of 70% and all there is Soil-sickness Problem in various degree, this seriously inhibits the sustainable development of China's modern medicine industry.
Many years of field is it has been observed that continuous cropping Radix Pseudostellariae vine growth and development is bad, and underground part tuber cannot normally expand, and generally after results, same plot must can be planted at interval for 2～4 years again.Early-stage Study is thought, under continuous cropping, rhizosphere microorganism structure of community is unbalance, and namely probiotics reduces, and pathogen amount reproduction grows, and is the principal element causing Radix Pseudostellariae continuous cropping obstacle.Radix Pseudostellariae continuous cropping also results in soil acidification, hardens from poison substance release, soil adhesion, degradation problem under water conservation fertilizer-preserving ability, simultaneously, peasant household is when unclear Radix Pseudostellariae continuous cropping obstacle Forming Mechanism, attempt to maintain yield by lavishment pesticide and fertilizer, often poor effect, the problem such as also result in that production cost strengthens, fertilizer loss is serious and agriculture is residual.So, explore a kind of environmentally friendly continuous cropping obstacle decreasing measure significant.
Being improved the continuous cropping soil of deterioration by microbial bacterial agent or biological organic fertilizer, improve diversity of soil microorganism level, the soil ecosystem of reconstruction health is considered as the main path solving plant continuous cropping obstacle.As shown in Figure 1, the present inventor's early stage from morbidity Radix Pseudostellariae plant and continuous cropping soil separation screening to many pathogen pathogenic by force, such as Fusarium oxysporum, fusarium moniliforme, ankle joint Fusarium spp. etc., and many pathogen strains bacterium shows host specificity, namely only infects Radix Pseudostellariae and does not infect rear stubble other crops such as corn and soybean, Oryza sativa L. etc..So, separation screening the probiotics of antagonism Radix Pseudostellariae specialized pathogen bacterium could can effectively prevent and treat Radix Pseudostellariae cultivating disease.Early-stage Study is it was also found that commercialization microbial inoculum or the bacterial manure of commercial type are extremely limited to the effect of preventing and treating Radix Pseudostellariae continuous cropping obstacle, and this is likely to relevant with the rhizospere competition of probiotics, and these probioticss are not suitable for growing in Radix Pseudostellariae continuous cropping soil.Radix Pseudostellariae continuous cropping causes that soil environment occurs substantially to worsen, and the multiple secondary metabolite of Radix Pseudostellariae sustained release and active ingredient, these compositions can result in the probiotics in commercially available microbial inoculum and surely cannot grow in Radix Pseudostellariae rhizosphere, therefore adopt and need to consider the host specificity of Radix Pseudostellariae soil-borne pathogen and the particularity of Continuous Cropping Soil Environment during microbial bacterial agent preventing and treating Radix Pseudostellariae cultivating disease.
In view of this, the present inventor studies and devises a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle and preparation method thereof, and this case thus produces.
Summary of the invention
It is an object of the invention to provide a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle, for the Soil-sickness Problem of Biological control medicinal plants Radix Pseudostellariae, play the dual purpose promoting tuber growth and controlling disease.
Another object of the present invention is to the preparation method that a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle is provided, mixed by proportion optimizing by general probiotics and original inhabitants' probiotics, purpose is to ensure that probiotics is grown surely in the success of Radix Pseudostellariae rhizosphere, can activating soil nutrient, degraded Radix Pseudostellariae release from poisonous substance matter, Radix Pseudostellariae specialized pathogen bacterium can be prevented and treated again.
To achieve these goals, this invention address that its technical problem is adopted the technical scheme that:
A kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle, is made up of for 4:6 with original inhabitants' probiotics by volume general probiotics；The ratio that described general probiotics is 1:1:1:1 by volume by lactic acid bacteria, photosynthetic bacteria, yeast, actinomycetes is mixed；Described original inhabitants probiotics is mixed by the ratio of burkholderia, pseudomonas, bacillus cereus 4:1:1 by volume.
As the optimal way of embodiment, described lactic acid bacteria is one or more in Lactobacillus plantarum, streptococcus acidi lactici and lactobacillus casei.
As the optimal way of embodiment, described photosynthetic bacteria is one or both in Rhodopseudomonas palustris and hydrogenlike silicon ion.
As the optimal way of embodiment, described yeast is one or both in saccharomyces cerevisiae and Candida utilis.
As the optimal way of embodiment, described actinomycetes are one or both in streptomyces griseus and streptomyces albus.
nullOptimal way as embodiment，Described burkholderia is Burkholderiaambifaria (Burkholderiaambifariasp.nov.,anovelmemberoftheBurkholderiacepaciacomplexincludingbiocontrolandcysticfibrosis-relatedisolates.TomCoenye、EshwarMahenthiralingam、DeborahHenry,et.InternationalJournalofSystematicandEvolutionaryMicrobiology，2001 (51): 1481-1490.) one and in new Burkholderia cepacia or two kinds.
nullOptimal way as embodiment，Described pseudomonas is pseudomonas fluorescens、Pseudomonas putida、Pseudomonasprotegens(Pseudomonasprotegenssp.nov.,widespreadplant-protectingbacteriaproducingthebiocontrolcompounds2,4-diacetylphloroglucinolandpyoluteorin.AlbanRamette,MicheleFrapolli,MarionFischer-LeSaux.et.SystematicandAppliedMicrobiology，2011(34):180-188.)、One or more in Korea S pseudomonas (Pseudomonaskoreensis).
As the optimal way of embodiment, described bacillus cereus is one or more in bacillus amyloliquefaciens, bacillus subtilis, bacillus cereus, Bacillus pumilus, bacillus megaterium, bacillus mycoides.
The preparation method of a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle, comprises the following steps:
Step one, original inhabitants Antagonistic Fungi screening:
Be respectively adopted pseudomonas selective medium, Radix Pseudostellariae rhizosphere pseudomonas, bacillus cereus are easily separated by bacillus cereus selective medium, adopt inorganic medium to be easily separated from poisonous substance matter degradation bacteria；
Described pseudomonas selective medium formula is: weigh 20g Soybean-casein digest agar culture medium, add 495.5mL distilled water heated and stirred, after fully dissolving, add 1mL0.1% (wt/vol) crystal violet storing solution, 121 DEG C of autoclaving 15min, when being cooled to about 50 DEG C, adding 3.5mL5% (wt/vol) Nitrofurantoin storing solution in culture medium again, described Nitrofurantoin storing solution DMF makes solvent, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices cools down, standby；
Described bacillus cereus selective medium formula is: every liter containing 5.0g peptone, 1.5g yeast extract, 1.5g Carnis Bovis seu Bubali cream, 5.0gNaCl, 20g agar, pH7.2；
Described minimal media based formulas is: every liter containing NH4NO31.0g, NaCl0.5g, K2HPO4·3H2O1.5g, MgSO47H2O0.5g, (NH4)2SO40.5g, pH7.2；Weigh each component and be dissolved in 1000mL distilled water, 121 DEG C of autoclaving 15min, when being cooled to 45～50 DEG C, add in Radix Pseudostellariae continuous cropping soil identify from poisonous substance matter, described from poisonous substance matter include gallic acid, coumaric acid, 3,4-resorcylic acid, P-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, ferulic acid and benzoic acid, mixing phenolic acid total concentration 1000mg/L, after inoculating for 10% Radix Pseudostellariae rhizosphere soil suspending liquid in enrichment cultivation number generation, screening has the beneficial bacteria from poisonous substance matter degradation capability and antagonism Radix Pseudostellariae pathogen；
Prepared by step 2, general composite bacteria liquid:
Toward the well water of 18L or at least place in the tap water after 2 days and add 300g brown sugar and 50mLMS mother solution, add the 1L EM liquid spawn activated, it is placed in the indoor sealed fermenting 2～3 weeks of 35～37 DEG C, within second day, start every other day to open the venting decompression of hermetic container mouth once from fermentation, sour-sweet taste can be smelt when opening vessel port, and more and more obvious, represent that fermentation spreads cultivation successfully, namely EM composite bacteria liquid is prepared, standby；Successful finished product fermentation liquid has sour-sweet taste, pH3.0～4.0, and viable count is more than 10,000,000,000/mL；
Step 3, original inhabitants' probiotics activation and amplification culture:
Actication of culture: the pseudomonas fluorescens screened, pseudomonas putida, Pseudomonasprotegens, Korea S pseudomonas are inoculated in respectively in pseudomonas selective medium, is placed in 28～32 DEG C of incubators and activates 12～16h；The burkholderia screened and bacillus cereus are inoculated in LB solid culture respectively, are placed in 37 DEG C of incubators and activate 8～16h；
Amplification culture: the various original inhabitants probiotics activated being inoculated in liquid culture and carry out fermentation culture, pseudomonas is placed in amplification culture at 28～32 DEG C, and burkholderia and bacillus cereus are placed in amplification culture at 37 DEG C, when viable count is higher than 1.0 × 109Amplification culture is stopped during CFU/mL, standby；
Prepared by step 4, complex micro organism fungicide:
Original inhabitants' probiotics and burkholderia, pseudomonas, bacillus cereus are mixed and made into original inhabitants' probiotics composite bacteria liquid, i.e. IM composite bacteria liquid for the ratio of 4:1:1 by volume；The ratio that described EM composite bacteria liquid and described IM composite bacteria liquid are 4:6 by volume is mixed and made into Radix Pseudostellariae specific complex microbial bacterial agent.
Specific complex microbial bacterial agent of the present invention has several advantages that
1, specific complex microbial bacterial agent of the present invention contains general probiotics and Radix Pseudostellariae rhizosphere original inhabitants' Antagonistic Fungi, wherein original inhabitants' Antagonistic Fungi includes burkholderia, pseudomonas and bacillus cereus, it is all screen to obtain from Radix Pseudostellariae rhizosphere soil, during Field information, its rhizospere competition is good and safe and reliable, and to other crops of rear stubble (such as corn and soybean, Oryza sativa L., vegetable etc.) all without pathogenic infection ability；
2, original inhabitants' Antagonistic Fungi is to be obtained by substantial amounts of screening operation, screens and obtain from 3000 strain soil bacterias, and Radix Pseudostellariae specialized pathogen bacterium (such as Fusarium oxysporum, fusarium moniliforme, ankle joint mycete etc.) is had very strong antagonistic effect.
3, of the present invention original inhabitants Antagonistic Fungi screening time add in culture medium higher concentration from poisonous substance matter, as selection pressure, what Radix Pseudostellariae root system was secreted by the major part original inhabitants' probiotics being sieved to has Degradation and Transformation function from poisonous substance matter and active ingredient (such as Radix pseudostellariae cyclic peptides B).
4, original inhabitants' Antagonistic Fungi of the present invention also has dissolving P capacity except can suppressing pathogen, it is possible to effective activation soil nutrient, improves soil physico-chemical property, promotes that plant nutrient absorption aspect has important function.
5, the present invention is through verifying on the spot for a long time, and formula is reasonable, stable effect, and preventing and treating Radix Pseudostellariae continuous cropping obstacle effect is obvious, improves the yield and quality of continuous cropping Radix Pseudostellariae.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of the pathogen that Radix Pseudostellariae plant diseases root is separated to；Wherein, A: Fusarium oxysporum；B: fusarium moniliforme；C: ankle joint mycete；
Fig. 2 is the opposite culture photo of original inhabitants' Antagonistic Fungi and Radix Pseudostellariae specialized pathogen fungus (fusarium moniliforme)；Wherein, A: only inoculation pathogen fusarium moniliforme；B: original inhabitants' probiotics and fusarium moniliforme opposite culture；
Fig. 3 is that original inhabitants' probiotics dissolving P capacity measures；Wherein, arrow instruction has the indigenous probiotics of dissolving P capacity, and periphery of bacterial colonies forms transparent circle.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments so that the invention will be further described,
New Burkholderia Burkholderiacenocepacia, Korea S pseudomonas Pseudomonaskoreensis, Pseudomonasprotegens and Burkholderiaambifaria on 01 28th, 2016 China Committee for Culture Collection of Microorganisms's common micro-organisms center register preservation, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCCNo.12119, CGMCCNo.12122, CGMCCNo.12121 and CGMCCNo.12120 respectively.
The screening of embodiment 1 original inhabitants' Antagonistic Fungi and qualification thereof
Be respectively adopted pseudomonas selective medium, Radix Pseudostellariae rhizosphere pseudomonas, bacillus cereus are easily separated by bacillus cereus selective medium, adopt inorganic medium (Radix Pseudostellariae containing higher concentration is from poisonous substance matter) to be easily separated from poisonous substance matter degradation bacteria.
The single bacterium colony of random choose on above-mentioned different culture media adopt the method for PDA plate face-off evaluate they antagonistic effects to Radix Pseudostellariae specialized pathogen bacterium (such as Fusarium oxysporum, fusarium moniliforme, ankle joint mycete etc.) respectively.Adopt 16srRNA gene or 16s-23srRNA interval PCR to identify the antibacterial with strong antagonistic ability and carry out Molecular Identification.16srRNA gene PCR amplification upstream and downstream primer is respectively as follows: 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1522r (5'-AAGGAGGTGATCCAGCCGCA-3')；16s-23srRNA gene PCR amplification upstream and downstream primer is respectively as follows: 1407f (5'-TTGTACACACCGCCCGTC-3') and 456r (5'-CCTTTCCCTCACGGTACTG-3').
Described culture medium prescription is:
(1) pseudomonas selective medium: weigh 20g Soybean-casein digest agar culture medium, add 495.5mL distilled water heated and stirred, after fully dissolving, adding 1mL0.1% (wt/vol) crystal violet storing solution, 121 DEG C of autoclaving 15min, when being cooled to about 50 DEG C, 3.5mL5% (wt/vol) Nitrofurantoin storing solution is added again (with N in culture medium, dinethylformamide makes solvent), a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices cools down, standby.
(2) bacillus cereus selective medium: every liter containing 5.0g peptone, 1.5g yeast extract, 1.5g Carnis Bovis seu Bubali cream, 5.0gNaCl, 20g agar, pH7.2.
(3) from poisonous substance matter degradation bacteria culture medium: every liter containing NH4NO31.0g, NaCl0.5g, K2HPO4·3H2O1.5g, MgSO47H2O0.5g, (NH4)2SO40.5g, pH7.2.Weigh each component and be dissolved in 1000mL distilled water, 121 DEG C of autoclaving 15min, when being cooled to 45～50 DEG C, add in Radix Pseudostellariae continuous cropping soil identify various from poisonous substance matter (gallic acid, coumaric acid, 3,4-resorcylic acid, P-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, ferulic acid, benzoic acid), mixing phenolic acid total concentration 1000mg/L, after inoculating for 10% Radix Pseudostellariae rhizosphere soil suspending liquid in enrichment cultivation number generation, screening has the beneficial bacteria from poisonous substance matter degradation capability and antagonism Radix Pseudostellariae pathogen.
Obtain 12 strains altogether through Screening and Identification and there is the Radix Pseudostellariae Rhizosphere Soil work probiotics of strong antagonistic ability, including 2 strain burkholderias (Burkholderiaambifaria, new Burkholderia cepacia)；4 pseudomonas (pseudomonas fluorescens, pseudomonas putida, Pseudomonasprotegens, Korea S pseudomonas)；6 bacillus (bacillus amyloliquefaciens, bacillus subtilis, bacillus cereus, Bacillus pumilus, bacillus megaterium, bacillus mycoides).Wherein the strong new Burkholderia cepacia of Antagonistic Fungi, Burkholderiaambifaria, Pseudomonasprotegens, Korea S pseudomonas are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 28th, 2016, and deposit number is respectively as follows: CGMCCNO.12119, CGMCCNO.12120, CGMCCNO.12121, CGMCCNO.12122.
Original inhabitants' Antagonistic Fungi of the present invention is to be obtained by substantial amounts of screening operation, screen from 3000 strain soil bacterias and obtain, Radix Pseudostellariae specialized pathogen bacterium (such as Fusarium oxysporum, fusarium moniliforme, ankle joint mycete etc.) had very strong antagonistic effect, as shown in Figure 2.Original inhabitants' Antagonistic Fungi of the present invention also has dissolving P capacity except can suppressing pathogen, it is possible to effective activation soil nutrient, as it is shown on figure 3, improve soil physico-chemical property, promotes that plant nutrient absorption aspect has important function.
The preparation of embodiment 2 specific complex microbial bacterial agent
A kind of specific complex microbial bacterial agent of the present invention, is mixed by the ratio that general probiotics and original inhabitants' probiotics are 4:6 by volume, and concrete preparation method is as follows:
Prepared by 2.1EM composite bacteria liquid
300g brown sugar and 50mLMS mother solution is added in the well water (tap water uses after need at least placing 2 days) of 18L, add the 1L EM liquid spawn activated, it is placed in the indoor sealed fermenting 2～3 weeks of 35～37 DEG C, within second day, start every other day to open the venting decompression of hermetic container mouth once from fermentation, sour-sweet taste can be smelt when opening vessel port, and more and more obvious, and this is normal phenomenon, namely represent that fermentation spreads cultivation successfully, standby.Successful finished product fermentation liquid has sour-sweet taste, pH3.0～4.0, and viable count is more than 10,000,000,000/mL.
2.2 original inhabitants' probiotics activation and amplification culture
1) actication of culture
Pseudomonas fluorescens, pseudomonas putida, Pseudomonasprotegens, Korea S pseudomonas are inoculated in pseudomonas Selective solid culture medium respectively, are placed in 28～32 DEG C of incubators and activate 12～16h.
Other indigenous probiotics (including burkholderia, bacillus cereus) is inoculated in LB solid culture respectively, is placed in 37 DEG C of incubators and activates 8～16h.
2) amplification culture
The various original inhabitants probiotics activated being inoculated in liquid culture and carry out fermentation culture, pseudomonas is placed in amplification culture at 28～32 DEG C, and other original inhabitants' probioticss are placed in amplification culture at 37 DEG C, when viable count is higher than 1.0 × 109Amplification culture is stopped during CFU/mL, standby.
Prepared by 2.3 complex micro organism fungicides
Original inhabitants' probiotics and burkholderia, pseudomonas, bacillus cereus are mixed and made into original inhabitants' probiotics composite bacteria liquid (IM composite bacteria liquid) for the ratio of 4:1:1 by volume.
Further, the ratio that EM composite bacteria liquid and IM composite bacteria liquid are 4:6 by volume is mixed and made into Radix Pseudostellariae specific complex microbial bacterial agent.
Embodiment 3 specific complex microbial bacterial agent preventing and treating Radix Pseudostellariae continuous cropping obstacle field efficacy is evaluated
First by specific complex microbial bacterial agent fermenting and preparing biological fertilizer together with organic materials of preparation, it can be used as base fertilizer that Radix Pseudostellariae continuous cropping soil is improved in advance, play furrow afterwards wholely, plantation Radix Pseudostellariae, and start timely and appropriate discovery pouring root seedling stage from Radix Pseudostellariae and impose special complex microorganism liquid bacterial agent, carry out volume variance harvest time and compare.Concrete prophylactico-therapeutic measures is as follows:
Prepared by the 3.1 organic base fertilizer of microorganism
Organic materials bean cypress, Testa oryzae, drying pig manure, fish flour, brown sugar by weight 2:1:1:1:0.03, are carried out mixing and mixes thoroughly, add appropriate specific complex microbial liquid microbial inoculum, degree of drying adjustment according to material adds the amount of complex microorganism liquid bacterial agent, water content control (is pinched easily agglomerating but not water outlet with hands to be advisable) 30%, after fully mixing thoroughly, pack or barrelling carry out closing anaerobic fermentation, 37 DEG C, 48h, ferment complete, and room temperature places 2～4 weeks with after-ripening.Accessing appropriate special microorganism liquid bacterial agent in above-mentioned anaerobic fermentation product again, in equipment that is airtight and that ventilate, 37 ° of fermentation 48h, prepare special microorganism fertilizer, total viable count 1.0～5.0 hundred million/g.
3.2 soil improvements
Process group: after preceding crop results, carry out first time wholely, every mu applies the microbial organic fertilizer 1200kg fermenting (starting fermentation at least in advance for 1 month) in advance, deep ploughing is ploughed under in soil, harrowing even furrow, cover black mulch film, furrow irrigation is spread and sunk in, and namely water is micro-wet (but not flooding to furrow face) to furrow face cuts off the water, overlay film ferments at least 1 month, repairs, to improve, the Continuous Cropping Soil Environment worsened.
Matched group: applying the equivalent bed material for fermentative microorganism fertilizer, the later stage does not impose special microorganism liquid bacterial agent, other field management measures keep consistent with process group.
Blank group: be not applied to the bed material of fermentative microorganism fertilizer, the later stage does not impose special microorganism liquid bacterial agent yet, and other field management measures keep consistent with process group.
3.3 Radix Pseudostellariae plantations
Plant the last week, carry out second time wholely, whole good furrow face is opened by line-spacing 15 centimetres the craspedodrome bar ditch of deep 7～10 centimetres.Before plantation, using compound fertilizer 20kg, phosphate fertilizer 25kg, potash fertilizer 10kg to apply after mixing in plantation ditch for every mu and make seed manure, a little blindings, then tiltedly arrange in furrow bank by spacing in the rows 5～7 centimetres by seed ginseng a little, and head-up, bud is from Horizon about 5cm, light earthing for bud.Planting in late November to December, every mu with seed ginseng 25～35 kilograms.
3.4 impose bacterium solution
In mid or late Febuary, after coming up, pouring root applies the specific complex microbial inoculums 1 time of 100 times of dilutions, mid-March and mid-April, then pouring root applies each 1 time of the specific complex microbial inoculums of 50 times of dilutions respectively, occurs resisting soil-borne disease.According to Radix Pseudostellariae growing way and incidence, adjusting pouring root number of times, if there being disease symptom, can suitably increase pouring root number of times.Matched group and blank group pouring root apply the water of equivalent.
3.5 results and volume variance compare
Wither the time of falling Seedling in late June Radix Pseudostellariae plant, select fine day to excavate Radix Pseudostellariae.Deep-cut more than 15 centimetres with spud, Radix Pseudostellariae tuber is completely dug out, it is to avoid damage tuber, sort out, clean, dry, claim fresh weight.
Result of the test shows for many years, compared with continuous cropping Radix Pseudostellariae (matched group and blank group), uses the special microorganism microbial inoculum invented and can significantly inhibit incidence, improve Radix Pseudostellariae per mu yield, increase production about 30～65%.Visible, special microorganism microbial inoculum of the present invention is notable to preventing and treating Radix Pseudostellariae continuous cropping obstacle field efficacy, and stable effect, market application foreground is huge.
All deformation that those of ordinary skill in the art can directly derive from the disclosure of invention or associate, are all considered as protection scope of the present invention.
Claims (9)
1. the specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle, it is characterised in that: it is made up of for 4:6 by volume with original inhabitants' probiotics general probiotics；The ratio that described general probiotics is 1:1:1:1 by volume by lactic acid bacteria, photosynthetic bacteria, yeast, actinomycetes is mixed；Described original inhabitants probiotics is mixed by the ratio of burkholderia, pseudomonas, bacillus cereus 4:1:1 by volume.
2. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described lactic acid bacteria is one or more in Lactobacillus plantarum, streptococcus acidi lactici and lactobacillus casei.
3. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described photosynthetic bacteria is one or both in Rhodopseudomonas palustris and hydrogenlike silicon ion.
4. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described yeast is one or both in saccharomyces cerevisiae and Candida utilis.
5. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described actinomycetes are one or both in streptomyces griseus and streptomyces albus.
6. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described burkholderia is the one in Burkholderiaambifaria and new Burkholderia cepacia or two kinds.
7. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described pseudomonas is one or more in pseudomonas fluorescens, pseudomonas putida, Pseudomonasprotegens, Korea S pseudomonas.
8. a kind of specific complex microbial bacterial agent eliminating Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: described bacillus cereus is one or more in bacillus amyloliquefaciens, bacillus subtilis, bacillus cereus, Bacillus pumilus, bacillus megaterium, bacillus mycoides.
9. the preparation method of the specific complex microbial bacterial agent of an elimination Radix Pseudostellariae continuous cropping obstacle as claimed in claim 1, it is characterised in that: comprise the following steps:
Step one, original inhabitants Antagonistic Fungi screening:
Be respectively adopted pseudomonas selective medium, Radix Pseudostellariae rhizosphere pseudomonas, bacillus cereus are easily separated by bacillus cereus selective medium, adopt inorganic medium to be easily separated from poisonous substance matter degradation bacteria；
Described pseudomonas selective medium formula is: weigh 20g Soybean-casein digest agar culture medium, add 495.5mL distilled water heated and stirred, after fully dissolving, add 1mL0.1% (wt/vol) crystal violet storing solution, 121 DEG C of autoclaving 15min, when being cooled to about 50 DEG C, adding 3.5mL5% (wt/vol) Nitrofurantoin storing solution in culture medium again, described Nitrofurantoin storing solution DMF makes solvent, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices cools down, standby；Pseudomonas fluorescens, pseudomonas putida, Pseudomonasprotegens and Korea S pseudomonas is screened by described pseudomonas selective medium；
Described bacillus cereus selective medium formula is: every liter containing 5.0g peptone, 1.5g yeast extract, 1.5g Carnis Bovis seu Bubali cream, 5.0gNaCl, 20g agar, pH7.2；Bacillus cereus is screened by described bacillus cereus selective medium；
Described minimal media based formulas is: every liter containing NH4NO31.0g, NaCl0.5g, K2HPO4·3H2O1.5g, MgSO47H2O0.5g, (NH4)2SO40.5g, pH7.2；Weigh each component and be dissolved in 1000mL distilled water, 121 DEG C of autoclaving 15min, when being cooled to 45～50 DEG C, add in Radix Pseudostellariae continuous cropping soil identify from poisonous substance matter, described from poisonous substance matter include gallic acid, coumaric acid, 3,4-resorcylic acid, P-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, ferulic acid and benzoic acid, mixing phenolic acid total concentration 1000mg/L, after inoculating for 10% Radix Pseudostellariae rhizosphere soil suspending liquid in enrichment cultivation number generation, screening has the beneficial bacteria from poisonous substance matter degradation capability and antagonism Radix Pseudostellariae pathogen；
Prepared by step 2, general composite bacteria liquid:
Toward the well water of 18L or at least place in the tap water after 2 days and add 300g brown sugar and 50mLMS mother solution, add the 1L EM liquid spawn activated, it is placed in the indoor sealed fermenting 2～3 weeks of 35～37 DEG C, within second day, start every other day to open the venting decompression of hermetic container mouth once from fermentation, sour-sweet taste can be smelt when opening vessel port, and more and more obvious, represent that fermentation spreads cultivation successfully, namely EM composite bacteria liquid is prepared, standby；Successful finished product fermentation liquid has sour-sweet taste, pH3.0～4.0, and viable count is more than 10,000,000,000/mL；
Step 3, original inhabitants' probiotics activation and amplification culture:
Actication of culture: the pseudomonas fluorescens screened, pseudomonas putida, Pseudomonasprotegens, Korea S pseudomonas are inoculated in respectively in pseudomonas selective medium, is placed in 28～32 DEG C of incubators and activates 12～16h；The burkholderia screened and bacillus cereus are inoculated in LB solid culture respectively, are placed in 37 DEG C of incubators and activate 8～16h；
Amplification culture: the various original inhabitants probiotics activated being inoculated in liquid culture and carry out fermentation culture, pseudomonas is placed in amplification culture at 28～32 DEG C, and burkholderia and bacillus cereus are placed in amplification culture at 37 DEG C, when viable count is higher than 1.0 × 109Amplification culture is stopped during CFU/mL, standby；
Prepared by step 4, complex micro organism fungicide:
Original inhabitants' probiotics and burkholderia, pseudomonas, bacillus cereus are mixed and made into original inhabitants' probiotics composite bacteria liquid, i.e. IM composite bacteria liquid for the ratio of 4:1:1 by volume；The ratio that described EM composite bacteria liquid and described IM composite bacteria liquid are 4:6 by volume is mixed and made into Radix Pseudostellariae specific complex microbial bacterial agent.
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