CN105950509A - Biological fungicide and preparation method as well as application thereof - Google Patents

Biological fungicide and preparation method as well as application thereof Download PDF

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CN105950509A
CN105950509A CN201610362310.7A CN201610362310A CN105950509A CN 105950509 A CN105950509 A CN 105950509A CN 201610362310 A CN201610362310 A CN 201610362310A CN 105950509 A CN105950509 A CN 105950509A
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贺字典
高玉峰
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Hebei Normal University of Science and Technology
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Abstract

The invention discloses a biological fungicide. The fungicide comprises a PGPR bacteria solution with the weight percentage of 5 to 10 percent; a PGPR viable count in the fungicide is not less than 1*1010CFU/ml. The fungicide can be prepared into biological fermented bacterial manure and a seed coating agent; the biological fungicide not only has good control effects on fungal diseases such as fusarium wilt of cucumber and plant bacteria diseases such as bacterial stem soft rot of the cucumber, but also can improve a growing environment of vegetables under a continuous cropping condition and enhance the absorption and utilization of the vegetables on hard dissolved phosphorus in soil, thus being good to realizing high-yield, safe and pollution-free production of the vegetables.

Description

A kind of bacteria agent and its preparation method and application
Technical field
The invention belongs to pharmaceutical technology field, relate to microbial bacterial agent, be specifically related to the bacteria agent that main component is PGPR, And also relate to bacteria agent and include the preparation method of biofermentation bacterial manure and seed coat agent, also relate to the application of bacteria agent.
Background technology
Along with the increase of the facility continuous cropping time limit, continuous cropping obstacle is on the rise, and soil-borne pathogen constantly accumulates, and occurs in that soil Give birth to the problems such as salination, nutrient imbalance, heavy metal pollution, soil microenvironment deterioration, have a strong impact on vegetable Yield and quality, continuous cropping obstacle has become restriction vegetable and has produced the key factor of sustainable development, therefore created and be conducive to vegetable raw Long soil environment has become a problem demanding prompt solution.
Ethylene is one of five big endogenous hormones in plant, shows obvious physiological action, i.e. suppress stem under relatively low concentration Elongation growth, promote that the increasing of stem or root is thick and makes stem cross growth triple response.Plant major part in all one's life growth and development stage needs The concentration ratio wanting ethylene is relatively low, and in general plant tissue, ethylene contents is less than 0.11pmol, only on soon ripe and old and feeble rank Section just synthesizes substantial amounts of ethylene.But under the conditions of the soil salinization, heavy metals exceeding standard, arid and pathogen infection, plant The a large amount of synthesizing ethylene of interior ethylene synthase precursor 1-amino-cyclopropane-1-carboxylic acid (ACC), the ethylene continuing a large amount of synthesis can suppress root System's elongation, hinders root growth.
PGPR is a group autotrophic bacteria in rhizosphere surrounding soil, can be by producing direct by ethylene synthase of acc deaminase Precursor ACC is degraded to α-batanone acid and ammonia, to promote the elongation of plant roots, draws more nutrient substance, moisture etc.. Mayak etc. detect the achromobacter growth-promoting functions to Tomato Root System, and the Tomato Root System of result display inoculation achromobacter is in salinity 32.8mm has been extended during for 120mM, and nonvaccinated for 27.4mm, it can be seen that, ACC active bacteria is at hypersaline environment bar The growth of plant can be promoted under part.The acc deaminase activity endogenetic bacteria SS12 that Teng Songshan etc. are separated in halophytes Herba suadeae glaucae Radix Raphani wilt and cucumber fusarium axysporum had antagonism.Liu Weihong etc. use enrichment isolation method, are unique nitrogen with ACC Source gathers soil sample from different vegetable plant strain rhizospheres, filters out 46 strain acc deaminase activated bacterials, wherein XG32 bacterial strain pair Fructus Lycopersici esculenti has obvious growth-promoting functions, and the phytopathogens such as Phytophthora capsici are had certain inhibitory action.
Cucumber fusarium axysporum is the common soil-borne disease of one occurred during green cucumber, the most sick at hinterland supervention of yielding positive results, quilt Evil strain is manifested initially by the side blade of partial blade or plant and wilts sagging noon, like hydropenia shape, sooner or later can recover, after Wilting blade is on the increase, and gradually throughout Herb, sooner or later can not restore, and the most withered；Cucumber anthracnose occurs not in recent years Break the weight that becomes, and is to be caused by the seed-borne fungi introduced, and spring and autumn all has generation, and difficulty of prevention and cure is relatively big, huge to Influence of production Greatly.Fructus Cucumidis sativi bacillary stem soft rot is newfound serious bacterial sexually transmitted disease (STD) evil, preventing and treating in the market on nearly 2 years cucumber productions Chemical bactericide and the agricultural antibiotic of bacteriosis are the most undesirable to the prevention effect of this disease, it would be highly desirable to exploitation this kind of disease of preventing and treating Medicament.
Summary of the invention
For present situation, the invention provides a kind of bacteria agent, this bacteria agent main component is PGPR, it is possible to effectively prevent Harness the Yellow River cucurbit wilt, cucumber anthracnose, cucumber bacterial angular leaf spot and Fructus Cucumidis sativi bacillary stem soft rot.
Present invention also offers the preparation method of this bacteria agent, carry out effectively preventing by making biofermentation bacterial manure and seed coat agent Cucumber fusarium axysporum, cucumber anthracnose, cucumber bacterial angular leaf spot and Fructus Cucumidis sativi bacillary stem soft rot.
In order to realize the purpose of the present invention, the present invention provides following technical scheme:
A kind of bacteria agent, described microbial inoculum includes the PGPR bacterium solution that percentage by weight is 5～10%, containing PGPR in described microbial inoculum Viable bacteria amount is no less than 1 × 1010CFU/ml。
Preferably, described PGPR is germ oligotrophy unit cell bacterium solution.
Wherein, described microbial inoculum can make biofermentation bacterial manure, and described microbial inoculum is composed of the following components in parts by weight: PGPR bacterium Liquid 5～10 parts, edible fungus bran 50～70 parts, tenebrio molitor frass 5～25 parts, fulvic acid 1～5 parts, FeSO4·7H2O 0.01～0.02 Part and ZnSO40.01～0.03 part, wherein, described edible fungus bran can replace with the peat composed of rotten mosses or charcoal；
The preparation method of above-mentioned bacteria agent (biofermentation bacterial manure), comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g, MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) gather the rhizosphere soil of seashore salting plant, weigh 1g Rhizosphere Soil and put in the triangular flask containing 50ml PAF culture fluid, 25 DEG C or 30 DEG C, hunting speed be shaken cultivation 24h under conditions of 200r/min, after enrichment culture 4d, retransfer 1ml bacterium Suspension in 50ml ADF culture fluid, continue 25 DEG C or 30 DEG C, hunting speed be shaken cultivation 48h under the conditions of 200r/min, Isolated and purified for containing acc deaminase activated bacterial, bacteria suspension in gradient dilution ADF culture fluid, coat ADF solid On flat board, cultivating 72h in the calorstat that temperature is 28 DEG C, line separates ,-80 DEG C of preservations after purification；
(3) by the PGPR inoculation after Purification in TSB fluid medium, temperature is 28～30 DEG C, hunting speed For shaken cultivation 24h under conditions of 200r/min, measuring bacteria suspension OD value and reach more than 0.8, bacterium amount is no less than 1010CFU/ml After, adsorb with edible fungus bran, be separately added into tenebrio molitor frass, fulvic acid, FeSO the most again4·7H2O and ZnSO4·7H2O, And by carrying out fermentation with covered rearing with plastic film until tenebrio molitor frass is become thoroughly decomposed completely after water content furnishing 60%, stir with blender Uniformly, pack, obtain biofermentation bacterial manure.
Described bacteria agent can also make seed coat agent, and described microbial inoculum is composed of the following components in parts by weight: PGPR bacterium solution 50～100 Part, nutrition carrier 6～14 parts, kieselguhr 600～800 parts, sodium carboxymethyl cellulose 10～20 parts, sodium lignin sulfonate 60～80 Part, polyvinyl alcohol 10～20 parts, FeSO4·7H2O 1～2 parts and ZnSO4·7H2O 1～3 parts, described nutrition carrier is by humic acid 1～2 part, glutamic acid 2～5 parts and Semen Maydis powder 3～7 parts of compositions, described bacterium is 1～2 × 10 containing PGPR viable bacteria amount in being10CFU/ml。
The preparation method of above-mentioned bacteria agent (seed coat agent), comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g and MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) by PGPR inoculation in TSB fluid medium, temperature be 28～30 DEG C, hunting speed be 200r/min Under conditions of shaken cultivation 24h, measure that bacteria suspension OD value reaches more than 0.8, bacterium amount is 1～2 × 1010During CFU/ml, use Kieselguhr after described nutrition carrier and sterilizing adsorbs, and is then respectively adding described sodium carboxymethyl cellulose sticker, wooden Element sodium sulfonate dispersant, polyvinyl alcohol film former, FeSO4·7H2O and ZnSO4·7H2O, i.e. obtains seed coat agent after mix homogeneously.
Described seed coat agent in use with seed mixing and stirring, make seed coat agent uniformly be wrapped in the surface of the seed, complete seed coat agent Pelleting Cotton seeds to seed, described seed coat agent is 1:10 with the weight ratio of seed.
Described bacteria agent is used for preventing and treating the bacillary stem of cucumber fusarium axysporum, cucumber anthracnose, cucumber bacterial angular leaf spot and/or Fructus Cucumidis sativi Soft rot.Find that this bacterial strain fermentation liquor is equal to cucumber fusarium axysporum, cucumber anthracnose, cucumber bacterial angular leaf spot through overtesting Having preferable prevention effect, the seed coat agent made is in field control effect is tested, and the preventing and treating of the soft corruption of stem bacillary to Fructus Cucumidis sativi is imitated Fruit is 62.67%.
The invention has the beneficial effects as follows: the bacteria agent of the present invention, not only for the fungal diseases such as cucumber fusarium axysporum and Fructus Cucumidis sativi antibacterial Property the bacteriosis such as stem soft rot there is good preventive effect, it is also possible to improve the growing environment of vegetable under Continuous Mono-cropping, strengthen vegetable To the absorption of insoluble phosphorus in soil and utilization.Thus contribute to realizing the high yield of vegetable, safety, No-harmful apple orchard.
Accompanying drawing explanation
Fig. 1 is PGPR Gram’s staining design sketch of the present invention；
Fig. 2 is the bacterial 16 S rDNA collection of illustrative plates of primer 2 7f/1492r amplification；
Fig. 3 is that different bacterial manure is to cucumber fusarium axysporum prevention effect comparison diagram；
Fig. 4 different pharmaceutical stem bacillary to Fructus Cucumidis sativi Disease-Control for Soft-Rotting effect contrast figure.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
By specific embodiment, the present invention will be described in detail below.These embodiments are provided to be able to more thoroughly Understand the present invention, and complete for the scope of the present invention can be conveyed to those skilled in the art.
" comprising " or " including " as mentioned by the middle of description and claim in the whole text is an open language, therefore should be construed to " comprise but be not limited to ".Description subsequent descriptions is to implement the better embodiment of the present invention, and right described description is with description Rule for the purpose of, be not limited to the scope of the present invention.Protection scope of the present invention is when regarding claims institute circle The person of determining is as the criterion.
Embodiment 1
A kind of bacteria agent (biofermentation bacterial manure), described microbial inoculum is composed of the following components in parts by weight: PGPR bacterium solution 5 parts, Edible fungus bran 70 parts, tenebrio molitor frass 22 parts, fulvic acid 2.96 parts, FeSO4·7H2O 0.01 part and ZnSO40.03 part, Described germ oligotrophy unit cell bacterium solution contains viable bacteria amount no less than 1010CFU/ml。
The preparation method of above-mentioned bacteria agent (biofermentation bacterial manure), comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g, MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) gather seashore salting plant reed, Radix Artemisia ordosicae, Herba Xanthii or the rhizosphere soil of Herba suadeae glaucae, weigh 1g Rhizosphere Soil and put into containing 50ml In the triangular flask of PAF culture fluid, 25 DEG C, hunting speed be shaken cultivation 24h under conditions of 200r/min, enrichment culture 4d After, retransfer 1ml bacteria suspension in 50ml ADF culture fluid, continue 25 DEG C, hunting speed be to vibrate under the conditions of 200r/min Cultivate 48h, isolated and purified for containing acc deaminase activated bacterial, bacteria suspension in gradient dilution ADF culture fluid, coating On ADF solid plate, cultivating 72h in the calorstat that temperature is 28 DEG C, line separates ,-80 DEG C of preservations after purification；
(3) by the PGPR inoculation after Purification in TSB fluid medium, temperature is 28～30 DEG C, hunting speed For shaken cultivation 24h under conditions of 200r/min, measuring bacteria suspension OD value and reach more than 0.8, bacterium amount is no less than 1010CFU/ml After, adsorb with edible fungus bran, be separately added into tenebrio molitor frass, fulvic acid, FeSO the most again4·7H2O and ZnSO4·7H2O, And by carrying out fermentation with covered rearing with plastic film until tenebrio molitor frass is become thoroughly decomposed completely after water content furnishing 60%, stir with blender Uniformly, pack, obtain biofermentation bacterial manure.
Embodiment 2
A kind of bacteria agent (biofermentation bacterial manure), described microbial inoculum includes the PGPR bacterium solution that percentage by weight is 8%.Described PGPR bacterium solution is germ oligotrophy unit cell bacterium solution.Described microbial inoculum is composed of the following components in parts by weight: addicted to Fructus Hordei Germinatus oligotrophy unit cell Bacterium bacterium solution 8 parts, the peat composed of rotten mosses 67 parts, tenebrio molitor frass 20 parts, fulvic acid 4.97 parts, FeSO4·7H2O 0.02 part and ZnSO40.01 Part, described germ oligotrophy unit cell bacterium solution contains viable bacteria amount no less than 1010CFU/ml。
The preparation method of above-mentioned bacteria agent (biofermentation bacterial manure), comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g, MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) gather seashore salting plant reed, Radix Artemisia ordosicae, Herba Xanthii or the rhizosphere soil of Herba suadeae glaucae, weigh 1g Rhizosphere Soil and put into containing 50ml In the triangular flask of PAF culture fluid, 30 DEG C, hunting speed be shaken cultivation 24h under conditions of 200r/min, enrichment culture 4d After, retransfer 1ml bacteria suspension in 50ml ADF culture fluid, continue 30 DEG C, hunting speed be to vibrate under the conditions of 200r/min Cultivate 48h, isolated and purified for containing acc deaminase activated bacterial, bacteria suspension in gradient dilution ADF culture fluid, coating On ADF solid plate, cultivating 72h in the calorstat that temperature is 28 DEG C, line separates ,-80 DEG C of preservations after purification；
(3) by the PGPR inoculation after Purification in TSB fluid medium, temperature is 28～30 DEG C, hunting speed For shaken cultivation 24h under conditions of 200r/min, measuring bacteria suspension OD value and reach more than 0.8, bacterium amount is no less than 1010CFU/ml After, adsorb with edible fungus bran, be separately added into tenebrio molitor frass, fulvic acid, FeSO the most again4·7H2O and ZnSO4·7H2O, And by carrying out fermentation with covered rearing with plastic film until tenebrio molitor frass is become thoroughly decomposed completely after water content furnishing 60%, stir with blender Uniformly, pack, obtain biofermentation bacterial manure.
Embodiment 3
A kind of bacteria agent (seed coat agent), described microbial inoculum is composed of the following components in parts by weight: PGPR bacterium solution 61 parts, nutrition 14 parts of carrier, 800 parts of kieselguhr, sodium carboxymethyl cellulose 20 parts, sodium lignin sulfonate 80 parts, polyvinyl alcohol 20 parts, FeSO4·7H2O 2 parts and ZnSO4·7H2O 3 parts, described nutrition carrier is by humic acid 2 parts, 5 parts of glutamic acid and Semen Maydis powder 7 Part composition.
The preparation method of above-mentioned bacteria agent (seed coat agent), comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g and MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) by PGPR inoculation in TSB fluid medium, temperature be 28～30 DEG C, hunting speed be 200r/min Under conditions of shaken cultivation 24h, measure that bacteria suspension OD value reaches more than 0.8, bacterium amount is 1～2 × 1010During CFU/ml, use Kieselguhr after described nutrition carrier and sterilizing adsorbs, and is then respectively adding described sodium carboxymethyl cellulose sticker, wooden Element sodium sulfonate dispersant, polyvinyl alcohol film former, FeSO4·7H2O and ZnSO4·7H2O, i.e. obtains seed coat agent after mix homogeneously.
Described seed coat agent in use with seed mixing and stirring, make seed coat agent uniformly be wrapped in the surface of the seed, complete seed coat agent Pelleting Cotton seeds to seed, described seed coat agent is 1:10 with the weight ratio of seed.
Embodiment 4
A kind of bacteria agent (seed coat agent), described microbial inoculum is composed of the following components in parts by weight: germ oligotrophy unit cell bacterium solution 100 parts, nutrition carrier 10 parts, 787 parts of kieselguhr, sodium carboxymethyl cellulose 15 parts, sodium lignin sulfonate 70 parts, poly-second Enol 15 parts, FeSO4·7H2O 1 part and ZnSO4·7H2O 2 parts, described nutrition carrier is by humic acid 2 parts, 3 parts of glutamic acid With Semen Maydis powder 5 parts composition.
The preparation method of above-mentioned bacteria agent (seed coat agent), comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g and MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) by germ oligotrophy unit cell inoculation in TSB fluid medium, it is 28～30 DEG C, hunting speed in temperature For shaken cultivation 24h under conditions of 200r/min, measure that bacteria suspension OD value reaches more than 0.8, bacterium amount is 1～2 × 1010CFU/ml Time, adsorb with the kieselguhr after described nutrition carrier and sterilizing, be then respectively adding described sodium carboxymethyl cellulose sticker, Sodium lignin sulfonate dispersant, polyvinyl alcohol film former, FeSO4·7H2O and ZnSO4·7H2O, i.e. obtains kind of a clothing after mix homogeneously Agent.Described seed coat agent in use with seed mixing and stirring, make seed coat agent uniformly be wrapped in the surface of the seed, complete seed coat agent Pelleting Cotton seeds to seed, described seed coat agent is 1:10 with the weight ratio of seed.
In order to prove effectiveness of the invention, applicant carried out and test as follows:
One, the separation of PGPR bacterium, purification and qualification
Enrichment culture containing acc deaminase activity PGPR: gather seashore salting plant reed, Radix Artemisia ordosicae, Herba Xanthii or the root of Herba suadeae glaucae Border soil, weighs 1g Rhizosphere Soil in the 250ml triangular flask containing 50ml PAF culture fluid, 25 DEG C or 30 DEG C, 200r/min shakes Swing cultivation 24h.2d, in transfer l ml bacteria suspension to another 50mlPAF culture fluid, cultivates 24h under equal conditions.3d, From PAF culture fluid, shift 1ml bacteria suspension in 50ml DF culture fluid, under the same terms, cultivate 24h.4d, retransfers 1ml bacteria suspension, in 50mlADF culture fluid, cultivates 48h under the same terms, for containing acc deaminase activated bacterial point From purification.Gradient dilution (10-3—10-7Power dilutes) bacteria suspension in ADF culture fluid, coat ADF solid plate, 28 DEG C Cultivating 72h in calorstat, line separates ,-80 DEG C of preservations after purification.
The bacterial strain of purification is compiled " common bacteria system identification handbook " micro-life in (1999) and the Chinese Academy of Sciences according to the elegant pearl in east and Cai Miaoying Thing is write the method for introductions such as " general antibacterial commonly uses authentication method " (1978) and is carried out morphological feature and physiological and biochemical property Mensuration.Bacterial strain thalline is shaft-like, (0.3～0.5) μ m (1.3～1.5) μm, and Gram’s staining is negative, without pod membrane, without spore, There are motor capacity, several flagellums；Bacterium colony is rounded, light green, smooth surface, moistening, protuberance (as shown in Figure 1).
Use antibacterial to extract test kit (Tian Gen bio tech ltd) and extract bacterial genomes DNA, with DNA as template, With universal primer 27f and 1492r, 16Sr DNA sequence is expanded.PCR reaction system is Premix12.5 μ L, 10 μm oL L-1 The each 1 μ L of primer, template DNA 1 μ L, with ddH2O complements to 25 μ L.PCR reaction condition: 94 DEG C of denaturations 5min； 94 DEG C of degeneration 30s；55 DEG C of annealing 1min；72 DEG C extend 2min；35 circulations, extend 10min after 72 DEG C.Amplified production Through 1% agarose gel electrophoresis isolation identification, PCR primer directly carries out two-way order-checking (as shown in Figure 2).By sequencing result After splicing, take a complete sequence on GenBank, carry out sequence alignment, reference similarity, the bacterial strain pair that homology is the highest Aimed strain carries out taxonomic identification.Through comparison, the bacterial strain of the present invention and germ oligotrophy unit cell (Stenotrophomonas Maltophilia) similarity is 100%, it may be determined that for germ oligotrophy unit cell.
Two, PGPR bacterial strain produces acc deaminase determination of activity
By each inoculation after purification in TSB fluid medium, 28 DEG C, 200rpm shaken cultivation 24h, 4 DEG C, 5000rpm Centrifugal 10min collects thalline, abandons supernatant, with centrifugal twice of DF fluid medium washing.Thalline is resuspended to ADF liquid Culture medium, 28 DEG C, 200r/min cultivates 24h, produces acc deaminase with induction.Measure with reference to Honma and Saleh method Acc deaminase activity.The acc deaminase of germ oligotrophy unit cell (Stenotrophomonas maltophilia) after measured Activity is between 56～324nmol a-batanone acids/mg/h, and the strains A CC deaminase active that wherein present invention prepares is the highest, For 324nmol a-batanone acid/mg/h.
Three, PGPR Salt resistant test
In LB culture medium, it is separately added into the NaCl of 2%, 5%, 7% variable concentrations, takes the young strain liquid inoculation in age cultivating 24h, Cultivate 3d and 7d for 28 DEG C, compared with nonvaccinated control tube, estimate growing state, if growth is then positive.Result shows this Bright bacterial strain all can grow in the LB culture medium that salinity is 2%, 5%, 7%.
Four, the PGPR biological activity determination to pathogen
Different PGPR fermentation liquid is measured to cucumber bacterial angular leaf spot bacterium, Fructus Cucumidis sativi bacillary stem Bacteria erwinia 2 by Odontothrips loti Plant pathogenetic bacteria and cucumber fusarium axysporum, the bacteriostatic activity of 2 kinds of pathogenic fungi of cucumber anthracnose.By the pathogenic bacteria and not after activation With PGPR be inoculated in respectively in NB fluid medium, in 28 DEG C, shaken cultivation 24～36h under the conditions of 126rpm, then After utilizing ultraviolet-uisible spectrophotometer (wavelength 650nm) to measure absorbance, adjust the OD of bacterial suspension650It is 0.8, concentration About 109cfu/ml.Draw 100 μ L pathogenetic bacteria suspensions in NA planar surface, smoothen with spreader, each flat board is put Putting 3 Oxford cups, be charged with 100 μ L supplies examination PGPR fermentation liquid, and each process is repeated 3 times, to add in 3% Raw 600 times of liquid of rhzomorph wettable powder and blank cultures are comparison, cultivate in 28 DEG C of constant incubators, measure and press down after 48～72h Bacterium loop diameter.
Fungus card punch (d=0.6mm) beats pure culture biscuits involvng inoculation in PDA plate central authorities on the bacterium colony cultivating 3d.There is mycelia One faces down, after cultivating 24h, with 4 cattle of decussation Rotating with Uniform at the 30mm of distance center up and down of culture dish Tianjin cup, injects bud PGPR fermentation liquid 50 μ L, cultivates in 28 DEG C of constant incubators.Bacterium colony mycelia to be compareed will be covered with whole flat During plate, measure the diameter of bacterium colony, calculate bacteriostasis rate (Liu Yongfeng etc., 2007) by formula below.
As can be seen from Table 1 and Table 2, the bacteriostasis rate of cucumber fusarium axysporum is by the fermentation liquid of part bacterial strain such as CRG-2 bacterial strain 85.96%, the bacteriostasis rate to cucumber anthracnose is 60.24%；JPG-4 is 60.24% to the bacteriostasis rate of cucumber anthracnose； AHG-6, LWG-2 are 85.96% to the bacteriostasis rate of cucumber fusarium axysporum；CRG-8 is to cucumber bacterial angular leaf spot bacterium and Fructus Cucumidis sativi The antibacterial circle diameter of bacillary stem Bacteria erwinia is respectively 47.00mm and 46.67mm；LWG-5 is to cucumber bacterial angular leaf spot bacterium Antibacterial circle diameter be 20mm；CRG-2 is 15.33mm to the antibacterial circle diameter of cucumber bacterial angular leaf spot bacterium；AHG-5 pair The antibacterial circle diameter of Fructus Cucumidis sativi bacillary stem Bacteria erwinia is 48.33mm；The inhibition zone of CRG-2 stem bacillary to Fructus Cucumidis sativi Bacteria erwinia A diameter of 48.33mm.Visible separated bacterial strain has certain inhibitory action to pathogenic fungi.
Table 1 PGPR fungistatic effect to pathogenic fungi
Note: in table, AHG-1～AHG-6 is the PGPR bacterial strain separated from Radix Artemisia ordosicae rhizosphere soil, LWG-1～LWG-6 is from phragmites communis rhizosphere The PGPR bacterial strain separated in soil, CRG-1～CRG-5 and CRG-8 is the PGPR bacterial strain separated from Herba Xanthii rhizosphere soil, JPG-4, JPG-10, JPG-11 are the PGPR bacterial strains separated from Herba suadeae glaucae rhizosphere soil.Lower same.
Table 2 PGPR fungistatic effect to pathogenetic bacteria
Five, PGPR produces the ability of heteroauxing (IAA)
With reference to Bric (1991) method, LB culture medium is sub-packed in test tube after sterilizing, adds aseptic tryptophan solution 1ml, Access 24h and cultivate bacterium solution 0.1ml, after cultivating 12h, draw the nitrite ion that 2ml bacterium solution adds 4ml, observe color change, Determine that PGPR produces IAA ability.Final qualification result determines that this bacterial strain produces heteroauxing (IAA).
Six, the use of biofermentation bacterial manure
After the biofermentation bacterial manure of the present invention and seedling medium are used by different proportion, measure cucumber seedling growth character and (be shown in Table 3).Artificial vaccination Fusarium oxysporum Fructus Cucumidis sativi specialized form, Investigate incidence, calculate disease index and measure bacterial manure to cucumber fusarium axysporum Prevention effect.
Cucumber fusarium axysporum grade scale:
0 grade of ground, underground part are the most asymptomatic；
1 grade of cotyledon wilting or true leaf are slightly wilted, fibrous root less than 10% browning；
2 grades of heavier wiltings of cotyledon, main root browning or fibrous root 10%～50% browning；
3 grades of cotyledons and true leaf are all wilted, main root 1/2 browning；
4 grades of main root major part brownings and overground part are wilted or withered.
The impact on cucumber seedling growth character of table 3 bacterial manure
From table 3 it can be seen that after bacterial manure is mixed by different proportion with seedling medium, cucumber seedling growth all can be promoted, improve Seedling vigorous index.Fructus Cucumidis sativi plant height is the highest when bacterial manure is 7:3 with the ratio of substrate, for 61.6cm；Cucumis sativus stem is slightly when with 9:1 The thickest, for 0.65cm, compare difference with comparison notable, notable with stem rough error heteropole in 1:9, for 0.48cm, with 3: 7,4:6 and 2:8 significant difference；Fructus Cucumidis sativi root length is the longest when ratio is 9:1, for 32.25cm, the root processed with other Long difference reaches pole significant level, with 5:5 and 6:4 root length significant difference；Cucumber leaves number is most when ratio is 6:4, Be 6, with compare 2,9:1,3:7,2:8 and 1:9 Leaf number difference extremely notable, and compare 1 and 8:2 blade Number significant difference；Fructus Cucumidis sativi fibrous root number at most, is 28 when 1:9, with compare 2,5:5,6:4,8:2,3:7,4: In 6 and 7:3, fibrous root number difference is extremely notable, with 2:8 fibrous root number significant difference；Second leaf leaf area of Fructus Cucumidis sativi is at composite interstitial substance Maximum when being 9:1 with the ratio of soil, for 180.28cm2, all reach with the seedling medium phase specific leaf area diversity of other ratios Pole significant level；The maximum when ratio of the 3rd leaf leaf area of Fructus Cucumidis sativi is 6:4, for 179.37cm2, with being combined of other ratios Substrate leaf area diversity all arrives pole significant level.From the point of view of comprehensive seedling vigorous index, when the ratio of bacterial manure and seedling medium is 9:1 Seedling vigorous index is maximum, is best suitable for the growth of cucumber seedling.
The prevention effect of cucumber fusarium axysporum is contrasted by different bacterial manure, the results are shown in Table 4 and Fig. 3, from table 4, it can be seen that this Invention bacterial manure, bacillus cereus bacterial manure, gliocladium germ be fertile and Trichoderma spp. bacterial manure is respectively 70.24% to the prevention effect of cucumber fusarium axysporum, 53.57%, 64.29% and 50.0%, it can also be seen that use the bacterial manure of the present invention prevention effect to cucumber fusarium axysporum from Fig. 3 Most preferably.
Table 4 bacterial manure prevention effect to cucumber fusarium axysporum
Seven, the use of seed coat agent
The seed coat agent of the present invention is stirred with seed, makes the uniform coating of spore powder at the surface of the seed, i.e. complete biocontrol microorganisms pair The Pelleting Cotton seeds of cucumber seeds, seed coat agent is 1:10 (w/w) with the weight ratio of seed.
The field efficacy of bacillary to Fructus Cucumidis sativi for different chemical agents stem soft rot is contrasted, by 9 kinds of chemical agents and 2 The field efficacy planting biocontrol agent stem bacillary to Fructus Cucumidis sativi soft rot is found out: the biocontrol agent used by this experiment and commercially available antibacterial pair The prevention effect of Fructus Cucumidis sativi bacillary stem soft rot is all below 65%.Wherein bronopol, Thiodiazole-copper, hydrogen bromine isocyanuric acid, second Bulbus Allii The seed coat agent preventive effect of element and bacillus cereus and the present invention is higher, and respectively 63.43%, 62.67%, 63.43%, 61.60%, 60.00% With 62.67% (being shown in Table 5 and Fig. 4).
The field efficacy of table 5 different pharmaceutical stem bacillary to Fructus Cucumidis sativi soft rot
Described be only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize description of the invention in The equivalent structure of Rong Suozuo or equivalence flow process conversion, or directly or indirectly it is used in other relevant technical fields, the most in like manner include In the scope of patent protection of the present invention.

Claims (9)

1. a bacteria agent, it is characterised in that described microbial inoculum includes the PGPR bacterium solution that percentage by weight is 5～10%, described Containing PGPR viable bacteria amount no less than 1 × 10 in microbial inoculum10CFU/ml。
Bacteria agent the most according to claim 1, it is characterised in that described PGPR bacterium solution is germ oligotrophy unit cell Bacterium solution.
Bacteria agent the most according to claim 1, it is characterised in that described microbial inoculum is composed of the following components in parts by weight: PGPR bacterium solution 5～10 parts, edible fungus bran 50～70 parts, tenebrio molitor frass 5～25 parts, fulvic acid 1～5 parts, FeSO4·7H2O 0.01～0.02 part and ZnSO4·7H2O 0.01～0.03 part.
Bacteria agent the most according to claim 3, it is characterised in that described edible fungus bran can be with the peat composed of rotten mosses or charcoal Replace.
Bacteria agent the most according to claim 1, it is characterised in that described microbial inoculum is composed of the following components in parts by weight: PGPR bacterium solution 50～100 parts, nutrition carrier 6～14 parts, kieselguhr 600～800 parts, sodium carboxymethyl cellulose 10～20 parts, Sodium lignin sulfonate 60～80 parts, polyvinyl alcohol 10～20 parts, FeSO4·7H2O 1～2 parts and ZnSO4·7H2O 1～3 parts, institute State nutrition carrier to be made up of humic acid 1～2 parts, glutamic acid 2～5 parts and Semen Maydis powder 3～7 parts, described bacterium solution is lived containing PGPR Bacterium amount is 1～2 × 1010CFU/ml。
6. the preparation method of the bacteria agent as described in claim 3 or 4, it is characterised in that described method includes as follows Step:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g, MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) gather the rhizosphere soil of seashore salting plant, weigh 1g Rhizosphere Soil and put in the triangular flask containing 50ml PAF culture fluid, 25 DEG C or 30 DEG C, hunting speed be shaken cultivation 24h under conditions of 200r/min, be then enriched with cultivating after 4d, retransfer 1ml Bacteria suspension in 50ml ADF culture fluid, continue 25 DEG C or 30 DEG C, hunting speed be shaken cultivation 48h under the conditions of 200r/min, Isolated and purified for containing acc deaminase activated bacterial, bacteria suspension in gradient dilution ADF culture fluid, coat ADF solid On flat board, cultivating 72h in the calorstat that temperature is 28 DEG C, line separates ,-80 DEG C of preservations after purification；
(3) by the PGPR inoculation after Purification in TSB fluid medium, temperature is 28～30 DEG C, hunting speed For shaken cultivation 24h under conditions of 200r/min, measuring bacteria suspension OD value and reach more than 0.8, bacterium amount is no less than 1010CFU/ml After, adsorb with edible fungus bran, be separately added into tenebrio molitor frass, fulvic acid, FeSO the most again4·7H2O and ZnSO4·7H2O, And by carrying out fermentation with covered rearing with plastic film until tenebrio molitor frass is become thoroughly decomposed completely after water content furnishing 60%, stir with blender Uniformly, pack, obtain biofermentation bacterial manure.
7. the preparation method of a bacteria agent as claimed in claim 5, it is characterised in that described method comprises the steps:
(1) first preparing PAF culture medium, ADF culture fluid and TSB fluid medium, described PAF culture medium prescription is: Peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g and glycerol 10ml, pH value 7.5；ADF Culture fluid formula is: KH2PO44.0g, Na2HPO46.0g, MgSO4.7H2O 0.2g, glucose 2.0g, gluconic acid 2.0g, Citric acid 2.0g, ammonium sulfate 2.0g and trace element 0.1ml, comprise FeSO in the every 100ml of described trace element4·7H2O 1mg, H3BO310 μ g, MnSO4·H2O 11.19 μ g, ZnSO4·7H2O 124.6 μ g, CuSO4·5H2O 78.22 μ g and MoO310 μ g, PH value is 7.2；The formula of described TSB fluid medium is: tryptone 17g, soya peptone 3g, NaCl 5g, glucose 2.5g and K2HPO42.5g, its pH value is 7.5；
(2) by PGPR inoculation in TSB fluid medium, temperature be 28～30 DEG C, hunting speed be 200r/min Under conditions of shaken cultivation 24h, measure that bacteria suspension OD value reaches more than 0.8, bacterium amount is 1～2 × 1010During CFU/ml, use institute State the kieselguhr after nutrition carrier and sterilizing to adsorb, be then respectively adding described sodium carboxymethyl cellulose sticker, lignin Sodium sulfonate dispersant, polyvinyl alcohol film former, FeSO4·7H2O and ZnSO4·7H2O, i.e. obtains seed coat agent after mix homogeneously.
The preparation method of bacteria agent the most according to claim 7, it is characterised in that described seed coat agent in use with seed Mixing and stirring, makes seed coat agent uniformly be wrapped in the surface of the seed, completes the seed coat agent Pelleting Cotton seeds to seed, described kind Clothing agent is 1:10 with the weight ratio of seed.
9. the purposes of the bacteria agent described in any one of Claims 1 to 5, it is characterised in that described bacteria agent is harnessed the Yellow River for anti- Cucurbit wilt, cucumber anthracnose, cucumber bacterial angular leaf spot and/or Fructus Cucumidis sativi bacillary stem soft rot.
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CN107201229B (en) * 2017-01-24 2019-11-15 河北省科学院生物研究所 A kind of bioremediation agents and its preparation and application with improvement soil and regulating and controlling microbial population structure function
CN107201229A (en) * 2017-01-24 2017-09-26 河北省科学院生物研究所 A kind of bioremediation agents and its preparation and application with improved soil and regulating and controlling microbial population structure function
CN108179013A (en) * 2017-08-09 2018-06-19 上海师范大学 A kind of alkaline land soil biological modification agent and preparation method thereof
CN108179013B (en) * 2017-08-09 2020-08-07 上海师范大学 Saline-alkali soil biological improver and preparation method thereof
CN108863618A (en) * 2018-09-19 2018-11-23 贵州大学 A kind of tea tree selenium-rich liquid bacterial manure and the preparation method and application thereof
CN110819557A (en) * 2019-10-10 2020-02-21 中国农业大学 Cellulose degradation composite microbial inoculum and preparation method and application thereof
CN110819557B (en) * 2019-10-10 2021-07-09 中国农业大学 Cellulose degradation composite microbial inoculum and preparation method and application thereof
CN110604033B (en) * 2019-11-05 2021-08-24 隆尧怡东农牧有限公司 Preparation method and application of matrix for preventing and treating soft rot of vegetables
CN110604033A (en) * 2019-11-05 2019-12-24 隆尧怡东农牧有限公司 Preparation method and application of matrix for preventing and treating soft rot of vegetables
CN110845271A (en) * 2019-11-25 2020-02-28 滨州学院 Soil remediation microbial agent and preparation method thereof
CN113151118A (en) * 2021-06-02 2021-07-23 湖北省农业科学院中药材研究所 Stenotrophomonas maltophilia for preventing and treating gray mold of paris polyphylla and application of stenotrophomonas maltophilia
CN113307686A (en) * 2021-06-21 2021-08-27 广东石油化工学院 Soil antifouling compound fertilizer and preparation method thereof
CN114455999A (en) * 2022-01-27 2022-05-10 宁夏农林科学院荒漠化治理研究所（宁夏防沙治沙与水土保持重点实验室） Special microbial agent for newly-repaired terrace soil in loess hilly area and preparation method thereof

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Application publication date: 20160921