CN103255077A - Bacillus subtilis YB-04 and microorganism preparation thereof, and application of bacillus subtilis YB-04 in straw degradation - Google Patents

Abstract

The present invention relates to a strain of Bacillus subtilis YB-04 and a preparation method of Bacillus subtilis YB-04 agent, wherein the preservation number of the Bacillus subtilis YB-04 is CGMCC NO.6497. According to the present invention, the Bacillus subtilis YB-04 provides a significant straw degradation effect, provides significant bacterial inhibition effects on wheat root take-all diseases, sheath blight and the like, and presents broad-spectrum bacterial inhibition effects, wherein a degradation rate can be 49% when the Bacillus subtilis YB-04 separately acts on straws, when the Bacillus subtilis YB-04 and fungi mixedly act on straws to achieve moistening and rotten surface of the straw, a degradation rate achieves the maximal value 59%, and a degradation effect of the experiment combination is far superior to a degradation effect of the commercially available bacterial agent; and the Bacillus subtilis YB-04 agent further presents broad-spectrum bacterial inhibition effects, wherein the Bacillus subtilis YB-04 agent provides good bacterial inhibition effects for cotton verticillium dahlia, wheat take-all pathogen, wheat sheath blight and other rhizosphere plant diseases, straws can be degraded, rhizosphere pathogenic bacteria can be inhibited, rhizosphere microorganism community can be regulated, and good development utilization prospects are provided.

Description

Subtilis YB-04, its microbial preparation and the application in straw degradative thereof

Technical field

The present invention relates to a kind of microbial technology field, be specifically related to a kind of subtilis YB-04 ,Its microbial preparation and the application in straw degradative thereof.

Background technology

Straw-returning more and more comes into one's own in recent years, and on the one hand large-scale agriculture production makes stalk need to handle nearby, to save the labour; On the other hand, use the growth that chemical fertilizer is unfavorable for soil fertility for a long time merely.Contain organic nutrients such as enriching De Dan ﹑ Lin ﹑ potassium in the agricultural crop straw, will increase the content of the soil organism behind the straw-returning, improve Tu earth Jie Gou ﹑ culture fertility, have to studies show that in a large number behind the straw-returning that soil nutrient and organic content are had raising.But under field conditions (factors), stalk decomposition speed is slow, if field then stalk decomposition fully also in a large number can influence and broadcast kind of a quality ﹑ and emerge and seedling growth.Stalk also can exert an influence to second stubble crop for disease and pest provides the place of surviving the winter after covering.Along with the development of mechanize, straw-returning has become the integral part of advanced agriculture production, because stalk nature decomposition speed is slow excessively, straw-returning has brought a series of problem, and the further investigation straw degradative will fundamentally solve soil disease, crop straw burning, problems such as pollution of agricultural products.Stalk degraded fast is a kind of realization resource circulation utilization technology, and the potentiality of development and use are arranged in the Sustainable development of agricultural.The microbial straw decomposition agent is the more direction of the quick degradation technique research of stalk, and the stalk decomposition agent mainly is made up of the bacterial strain of strong secretion straw degradative enzyme.Under the comprehensive action of microbial straw decomposition agent cellulosic molecule is thoroughly disintegrated, after the organism mineral was divided solution, detritusization formed organic matter then.Along with continuous researchdevelopment, the composite degradation bacteria strains of people not only can promote the quick degraded of stalk, also develops to the direction that suppresses disease and pest simultaneously.

Subtilis ( Bacillus subtilis), have that growth and breeding is fast, nutrition is simple, can produce heat-resisting degeneration-resistant gemma, good to person poultry harmless, environment compatibility, can suppress plurality of plant diseases, be difficult for making farm crop to develop immunity to drugs.Advantages such as and mass production processes is simple, and cost is also lower, uses conveniently, and the shelf lives is long, some bacterial strain plays good effect in degrading straw, therefore, be a kind of desirable degraded preparation.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of subtilis YB-04 with the effect of antagonism take-all, and has provided concrete cultivation application method.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

Isolated a kind of subtilis YB-04, its deposit number is CGMCC NO.6497.

A kind of microbial preparation is characterized in that, contained activeconstituents is that above-mentioned subtilis YB-04 is or/and its meta-bolites.

Described subtilis YB-04 is applied to straw degradative with liquid formulation, liquid bacterial preparation process: the subtilis YB-04 that preserves is moved on the TSA test tube slant substratum, cultivate down for 28～35 ℃ and obtained thalline in 24～36 hours; Bacterial classification in the test tube is inserted in the LB liquid medium, shake down to cultivate at 28～32 ℃ and made seed liquor in 12～16 hours; Liquid fermentation medium is: get glucose, Semen Maydis powder, soybean cake powder, dipotassium hydrogen phosphate and add water and be mixed with the nutrient solution that contains glucose 1.2%, Semen Maydis powder 1.8%, soybean cake powder 3%, dipotassium hydrogen phosphate 0.2%; Behind fermentation equipment and medium sterilization, seed liquor is inoculated in the above nutrient solution for preparing by 5% inoculum size, at 27～34 ℃, rotating speed 180rpm, PH8.0 condition bottom fermentation cultivate after 36～48 hours at least, collect fermented liquid, namely get liquid bacterial agent.

Described matrix substratum adds water and is mixed with by getting glucose, Semen Maydis powder, soybean cake powder, contain glucose 9～12g/L, Semen Maydis powder 11～16g/L, soybean cake powder 12～16g/L quality make by the high pressure moist heat sterilization than mixing.

The present invention has actively useful effect:

Isolated subtilis YB-04 CGMCC NO. 6497 has tangible degradation effect to stalk, and obvious to fungistatic effects such as wheat root gaeumannomyces graminis disease, banded sclerotial blights, finds that it shows the comparatively fungistatic effect of wide spectrum.When acting on stalk separately, degradation rate can reach 49%, and when during in stalk, making the more moistening corruption in stalk surface with the fungi mixing effect, degradation rate has reached maximum value 59%, and the Degradation of experiment combination is far superior to the Degradation of commercially available microbial inoculum.Just the stalk after the solution carries out content of cellulose mensuration, the content of cellulose of finding each test group all descends to some extent, become 44.7% with content of cellulose in the stalk after 3 kinds of fungies combination degraded, as seen the Mierocrystalline cellulose in the stalk is degraded in a large number, the one-piece construction of stalk is upset, thereby stalk obtains good degraded.

Subtilis YB-04 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 3rd, 2012, it abbreviates CGMCC as, the address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the deposit number of this subtilis YB-04 is CGMCC NO.6497.

Description of drawings

Fig. 1 is the colony characteristics figure of bacterial strain YB-04;

Fig. 2 is the molecular evolution tree graph of bacterial strain YB-04;

Fig. 3 is the design sketch of P3, P4, the independent degrading straw of P5;

Fig. 4 is that P3, P4, P5 are respectively at the design sketch of YB-04, L8 combined degradation stalk;

Fig. 5 is the design sketch of P3, P4, P5 mixing degrading straw;

Fig. 6 is P3, P4, P5 and the design sketch of YB-04, L8 mixing degrading straw;

Fig. 7 is the design sketch of commercially available microbial inoculum 1 degrading straw;

Fig. 8 is the design sketch of commercially available microbial inoculum 2 degrading straws.

Embodiment

In order to make the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used for the present invention being described and being not used in the scope of the present invention of placing restrictions on, NM concrete experimental technique in the following example, experimental technique carries out routinely usually.

Separation, the screening of embodiment 1 subtilis YB-04

Sample collecting: the corn that sample collecting is rotting from a plurality of fields, base, Yuanyang, academy of agricultural sciences, Henan Province and wheat stalk (acquisition time: 6-2010 October in 2010; Concrete collecting location: base, Yuanyang, academy of agricultural sciences, Henan Province, gather the people: Xie Maofang), take back with freshness protection package.

The isolation and purification of subtilis: get the rotten stalk of adopting, weigh 1g and put into the triangular flask that contains the 100mL sterilized water, 2h fully vibrates on 28 ℃ of constant temperature oscillators.To dilute the spread plate method with 10 ^-1-10 ^-7Sample liquid coat in the LB flat board, cultivated one day, and carried out purifying at the LB flat board respectively according to colonial morphology for 37 ℃.And substratum is inverted in 37 ℃ of constant temperature culture spends the night, last picking form, color and luster, size and the similar single bacterium colony of subtilis are transferred on the LB plate culture medium, behind 37 ℃ of cultivation 2d, record its numbering, select single colony inoculation again and to the LB slant medium, preserve.

Table 1 has big transparent circle bacterial strain

Separate through the pedotheque microorganism, isolate the similar bacterial isolates of 20 strains altogether, through repeated screening relatively altogether effect thin 3 strains preferably, called after YB-04, L4 and L8 respectively.This 3 strain bacterium all can vigorous growth on its corresponding screening culture medium, as shown in table 1, produce hyaluronic circle greater than 20mm in starch degradation screening culture medium and proteolytic degradation screening culture medium, show that YB-04 is rich in the enzyme of degraded starch and protein, has the potentiality of good degrading straw.

The evaluation of embodiment 2 subtilis YB-04

(1)Form and Physiology and biochemistry classification are identified Adopt colonial morphology to observe and microscopic observation method evaluation YB-04 bacterial strain, and searching document is determined its kind.Individual morphological feature comprises gramstaining anti-Ying ﹑ gemma Guan Cha ﹑ thalline size etc.; Colony's morphologic observation, observe Jun fall Xing Zhuan ﹑ bacterium colony Biao face Te Zheng ﹑ Jun fall Bian Yuan ﹑ bacterium colony color and whether secrete soluble pigment etc.; Giving birth to the biochemical examination of reason tests ﹑ and crosses the oxide compound enzyme and survey and decide ﹑ V.P examination and test ﹑ sugar fermentation examination and test the examination of ﹑ starch water solution and test that ﹑ gelatin liquefaction ﹑ Citrate trianion is sharp to try to test ﹑ salt tolerance test etc. with the ﹑ indoles.

(2)Molecular Identification: extracting genome DNA is with reference to following steps: 1. cultivate the bacterial cultures of 5mL to state of saturation, get the centrifugal 1min of culture 12000rpm of 1.2mL.2. throw out adds the sterilized water of 500 μ L, blows and beats repeatedly with suction pipe and makes it resuspended.The Proteinase K that adds the 20mg/ μ L of the 10%SDS of 30 μ L and 10 μ L, mixing is in 37 ℃ of insulation 1h.3. the NaCL that adds 100 μ L5mol/L, fully mixing adds 80 μ LCTAB/NaCL solution again, and mixing is bathed 10min in 60 ℃ of temperature.4. the chloroform that adds 750 μ L: primary isoamyl alcohol (24:1), mixing leaves standstill 5min, and the centrifugal 5min of 12000rpm gets supernatant.5. add isopyknic chloroform in supernatant: primary isoamyl alcohol (24:1), extracting once again.6. draw the freezing Virahol that supernatant adds 0.6 times of volume.Mix up to DNA precipitating gently, 4 ℃ leave standstill 2-4h.7. the centrifugal supernatant of abandoning precipitates with the washing of 1mL75% alcohol, and centrifugal 5min abandons supernatant, makes the alcohol volatilization fully, is resuspended in the 30 μ L deionized waters.The upstream and downstream primer that is used for amplification 16Sr dna sequence dna is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, is respectively 16SF:5 ‵-AGAGTTTGATCATGGCICAG-3 ‵, 16SR: ‵-TAGGGTTACCTTGTTACGACTT-3 ‵.Be template with the genomic dna, utilize primer 16SF and 16SR to carry out pcr amplification.The PCR reaction system is 25 μ l:10 * PCR Buffer 2.5 μ l, template 1 μ l, dNTP (10 mM each) 0.5 μ l, primer 16SF (10 mM) 1 μ l, 16SR (10 mM) 1 μ l, archaeal dna polymerase (5 U/ μ l) 0.25 μ l adds deionized water to 25 μ l.Pcr amplification condition: 94 ℃ of 5 min; 94 ℃ of 1 min; 55 ℃ of 30 sec; 72 ℃ of 1 min; 33 circulations; 72 ℃ of 10 min.The PCR reaction product adopts 1% agarose gel electrophoresis to detect.After 1400bp size purpose band adopts DNA to reclaim test kit (AXYGEN company) recovery purifying, (Takara PMD-19T) is connected with the T-carrier, the transformed competence colibacillus cell, carry out the screening of blue hickie at the flat board that contains Amp, IPTG, X-Gal then, and carry out bacterium colony PCR and filter out recombinant plasmid.Empirical tests is that the positive colony of purpose fragment is delivered to Shanghai and given birth to the biological company limited of worker and check order.Sequencing result adopts BLAST to carry out sequence identity relatively by retrieval Genbank (http://www.nebi.nlm.nih.gov/Blast/).Simultaneously, download Bacillus strain 16SrDNA sequence, and use CLUSTAL X (Version 1.8) software to carry out contraposition and arrange, be aided with artificial check and correction, the sequence that obtains is used MEGA3.1 molecular evolution genetic analysis software analysis.Calculate on the genetic distance basis in Kimura 2-parameter method, adopt adjacent method to make up the NJ phylogenetic tree, the degree of confidence of each branch of genealogical tree is carried out 1000 circulations altogether with bootstrapping method of inspection (bootstrap) check.

(3) bacterial strain YB-04 morphological specificity: the bacterial strain that will sieve the better YB-04 of effect is again cultivated 1d under 28 ℃ in the LB culture plate, observe its colonial morphology and color.YB-04 is well-grown on the LB substratum, and bacterium colony is creamy white, and not seeing has pigment to produce, and colonial morphology is irregular, there is protuberance on the surface, and forms irregular fold, (see figure 2).Produce slight fishy odor after cultivating 24d, bacterium colony becomes one and mucus is arranged when provoking.Gramstaining (the G that is positive ⁺).

(4) Physiology and biochemistry of bacterial strain YB-04 is identified: with reference to the method for " the outstanding Bacteria Identification handbook of uncle (the 8th edition " and " common bacteria identification handbook ", measure and observe its part physiological and biochemical index.

By table 2 as seen, bacterial strain YB-04 can make gelatine liquefication, starch hydrolysis, nitrate reduction; Milk is peptonized, can produce catalase.Can not make cellulose hydrolysis, the glucose aerogenesis.Can utilize glucose, fructose, semi-lactosi, wood sugar, maltose, raffinose and N.F,USP MANNITOL.Bacterial strain YB-04 tolerance NaCl concentration is 7%, as pH＜5 or pH〉bacterial strain can not be grown 9 the time, and Citrate trianion utilizes positive, the gelatine liquefication positive.According to " uncle outstanding Bacteria Identification handbook (the 8th edition) and " common bacteria identification handbook ", the morphological specificity of YB-04 and physiological and biochemical property all meet subtilis ( Bacillus subtilis) criteria for classification.

The physiological and biochemical property of table 2 bacterial strain YB-04

Annotate: "+": positive findings Positive results; “ – ": negative findings Negative results

(5) the Molecular Identification result of bacterial strain YB-04: correlated series in YB-04 sequence and the GenBank database is carried out BLAST analyze, the result shows 130 homologous sequences with this sequences match, choose the 16S rDNA sequence construct phylogenetic tree of the higher and representative bacterial strain of homology, shown in Fig. 3, bacterial strain YB-04 with Bacillus subtilis(OYKN9W5701S) be in same branch in the evolutionary tree, homology is 100%.Comprehensive above-mentioned morphology, physiological and biochemical property and YB-04 and relevant bacterial strain 16SrDNA sequence homology analysis result are subtilis with the YB-04 identification of strains (Bacillus subtilis).

Embodiment 3 YB-04 microbiobacterial agent preparation methods

(1) strain culturing is inoculated into subtilis YB-04 on the TSA test tube slant substratum with preparation, cultivates down for 28～35 ℃ and obtains thalline in 24～36 hours;

(2) the seed liquor preparation inserts the test tube strains of step (1) preparation in the TSB nutrient solution, shakes down to cultivate at 28～32 ℃ and makes seed liquor in 12～16 hours;

(3) fermention medium preparation is got glucose, Semen Maydis powder, soybean cake powder, dipotassium hydrogen phosphate respectively and is added water and be mixed with and contain glucose 1.2%, Semen Maydis powder 1.8%, soybean cake powder 3%, dipotassium hydrogen phosphate 0.2% nutrient solution;

(4) liquid fermenting production to fermentation equipment and medium sterilization after, seed liquor is inoculated in the nutrient solution that step (3) prepares by 5% inoculum size, at 27～34 ℃, rotating speed 180rpm, PH8.0 condition bottom fermentation is cultivated after 36～48 hours at least, collects fermented liquid, namely gets liquid bacterial agent.

The enzyme activity determination of embodiment 4:YB-04 bacterial strain

The preparation of seed liquor: fungi is inoculated in respectively on the PDA test tube slant, and 28 ℃ of constant temperature culture to test tube slants cover with spore.Add the 10mL sterilized water and fully shake, the gained bacteria suspension is seed liquor.Microbionation is in the test tube that 8mL LB nutrient solution is housed, and 37 ℃ of constant temperature culture 24h gained bacteria suspensions are the bacterium seed liquor.

Seed liquor is inoculated in liquid produces in the enzyme substratum, 37 ℃, 180r/min ferments.With the centrifugal 10min of fermented liquid 5000r/min, the gained supernatant was crude enzyme liquid after fermentation was finished.

Diastatic activity measuring method: according to document (Peng Huayuan, Lu Hongmei, Ceng Xiangqin. the mutagenesis screening of high yield zytase bacterial strain and product enzymatic property [J] thereof. brewing science and technology, 2006,10:34-36.) in test tube, add the 1mL crude enzyme liquid, 70 ℃ of water-bath 15min add the citrate buffer of 1mL pH5.06 again, add the NaOH of 4mL 0.4moL/L in the contrast.Test tube is incubated 10min in 40 ℃ of water-baths, adds the 2mL starch solution and shake up, put into 40 ℃ of water-baths immediately and react 5min, take out the back and add the NaOH of 4mL 0.4moL/L rapidly with termination reaction.Extract reaction solution 2 mL and add 2 mL3,40 ℃ of water-bath 5min of 5-dinitrosalicylic acid are settled to 25mL after the cooling.Measure its light absorption value at the 520nm place, reference standard curve calculation enzyme is lived.

The enzymic activity of each bacterial strain of table 3

Protease activity measuring method: according to document (Xu Ying, what on National Day, Chen Qi and etc. buffer system is to producing the influence [J] of elastoser genus bacillus EL31410 fermentation. Journal of Agricultural Biotechnology, 2004,1(6): 709-713.) add the crude enzyme liquid of 1mL in the test tube, blank places 40 ℃ of water-bath preheating 5min in contrast, add 2% casein solution of 1mL then, accurately be incubated 10min.Draw supernatant 1mL after adding the trichoroacetic acid(TCA) solution 15min of 2mL 0.4M immediately, add sodium carbonate solution and the 1mL fehling reagent of 5mL0.4M, 20min develops the color in 40 ℃ of water-baths.Measure light absorption value under the 680nm, reference standard curve calculation enzyme is lived.

The bacterial strain that will have big transparent circle carries out enzymatic production, and enzyme activity determination the results are shown in Table 3.As seen to reach best result in second day in fermentation be not 113.47 u/mL and 134.95 u/mL for YB-04 amylase and protease activity, is higher than L4 and L8 bacterial strain with batch fermentation, can predict that YB-04 is rich in the straw degradative enzyme, has the prospect of exploitation degraded preparation.

Embodiment 5:YB-04 degradation bacteria liquid fermenting stalk condition of enzyme production is optimized

The straw degradative process mainly be the enzyme that produces of various bacterium to the digestibility and utilization process of stalk, enzymatic productivity therefore commonly used is weighed degradation bacteria strains to the ability of utilizing of stalk, the size of enzymatic productivity is general to be weighed with the size alive of the unit enzyme in the fermented liquid.The present invention has studied the fermented stalk enzymatic productivity of bacterial strain YB-04 under the liquid condition, has indicated the factor that influences these strain enzyme-producings.

(1) fermentation time is to the influence of degradation bacteria strains enzymatic productivity: fermentation time is one of factor that influences strain enzyme-producing, and the fermentation initial stage is along with the enzyme of the prolongation fermented liquid of time is lived generally in rising trend; Along with exhausting and the Degradation of zymoprotein of nutritive substance, descending will appear in the enzyme work of fermented liquid.Inoculum size by 1%, the pH nature, shaking speed 180r/min, 37 ℃ are carried out fermentation culture.Took a sample once in every 24h hour, METHOD FOR CONTINUOUS DETERMINATION 5d, research enzyme that degradation bacteria strains produces is lived and is concerned over time.

(2) influence of the degradation bacteria strains enzymatic productivity of pH: different microorganisms has different tolerances to pH, so its product enzyme process is subjected to the influence of pH bigger.Bacterium generally adapts to growth and product enzyme under alkaline environment.The pH value of YB-04 strain enzyme-producing substratum is set 5 levels (7 ﹑, 7.5 ﹑, 8 ﹑, 8.5 ﹑ 9).Inoculum size according to 1%, 37 ℃ of 180r/min of shaking table carry out fermentation culture.The fermented liquid of getting fermentation 48h carries out enzyme activity determination, the enzyme work of research fermented liquid and the variation relation of pH.

(3) temperature is to the influence of degradation bacteria strains enzymatic productivity: temperature also is one of index that influences occurring in nature production by biological enzyme, and generally about 30 ℃, the product enzyme temperature of bacterium is generally about 37 ℃ for the product enzyme temperature of the fungi of speaking more greatly.Fungi produces the enzyme substratum and is set at (24 ﹑, 26 ﹑, 28 ﹑, 32 ﹑ 34) 5 temperature levels respectively, and Production by Bacteria enzyme substratum is set at (30 ﹑, 35 ﹑, 37 ﹑, 42 ﹑ 47) 5 temperature levels respectively.Inoculum size according to 1%, the pH nature, 180r/min carries out fermentation culture.Measure enzyme behind the fermentation 48h and live, the enzyme of the research fermented liquid relation with leavening temperature of living.

(4) inoculum size is to the influence of degradation bacteria strains enzymatic productivity: what of bacterial classification colony are the size of inoculum size influence, thereby influence production of enzyme, but are not that inoculum size is the bigger the better.Be subjected to the influence of dissolved oxygen and nutritive substance, study suitable inoculum size and just can make fermented liquid have the highest enzyme work.On pH nature, 37 ℃ on 28 ℃ of bacteriums of fungi, under the 180r/min condition, inoculum size is set (0.5% ﹑, 1% ﹑, 1.5% ﹑, 2% ﹑ 2.5%) 5 levels, the enzyme of measuring behind the fermentation 48h is lived, the research fermentation broth enzyme relation with inoculum size of living.

(5) nitrogenous source is to the influence of degradation bacteria strains enzymatic productivity: nitrogenous source is that microorganism growth is necessary, the C/N of stalk is the suitableeest C/N that 50:1 is far longer than microorganism growth, therefore in the fermentation culture certain nitrogenous source will be arranged, different nitrogen sources also has different influences to enzymatic productivity.Nitrogenous source in this research ferment product is that yeast soaks powder, uses yeast extract paste respectively, peptone, and Tryptones substitutes.By 1% inoculum size, at the pH nature, 37 ℃ on 28 ℃ of bacteriums of fungi are fermented under the 180r/min condition, and the enzyme of measuring behind the fermentation 48h is lived, the research fermentation broth enzyme relation with the carbon source addition of living.

(6) interpretation of result: single factor optimization experiment of the fermented liquid of YB-04 bacterial strain being carried out aspects such as enzyme work, pH, temperature, inoculum size, nitrogenous source utilization, as can be seen: 1. YB-04, the fermentation of L1 ﹑ L8 bacterium namely reached product enzyme maximum value after 2 days, one-way analysis of variance finds that the enzyme behind bacterial strain YB-04, the L1 ﹑ L8 fermentation 2d is lived and there is notable difference in other sky, and therefore fermenting was the best fermentation time of YB-04 two strain bacterium in two days; 2. along with the variation of pH, the unit enzyme of fermented liquid is lived and is constantly changed, and visible pH value can have influence on the product enzyme process of bacterial strain.The result shows YB-04, when pH 7.5, produce the proteolytic enzyme enzyme live best result Wei 135.01U/mL, through one-way analysis of variance find YB-04 when pH 7.5 institute's amylase that produces and other level there were significant differences, the optimal pH that proteolytic enzyme is produced in fermentation is 7.5, and it is 8 that diastatic optimal pH is produced in fermentation; 3. under the condition of different temperatures, the enzyme work of fermented liquid has obvious variation.Therefore optimum temperuture determines it also is the important factor of fermentation condition optimization.YB-04 Dian Fen Mei ﹑ proteolytic enzyme in the time of 37 ℃ all has the highest enzyme to live, and diastatic activity is respectively: 142.59U/mL, protease activity is respectively: 133.14 U/mL draw the YB-0437 ℃ of difference during with 40 ℃ not remarkable through variance analysis.Consider that based on economic condition fermentation condition is defined as 37 ℃; 4. along with the enzyme of the increase fermented liquid of inoculum size live in rising trend, but enzyme is lived and is begun again to descend when inoculum size reaches a certain amount of.The inoculum size of YB-04 is that 1% o'clock enzyme is alive the highest, and diastatic activity is respectively 137.81 U/mL, and protease activity is respectively 154.09 U/mL.Draw YB-04 and under 1% inoculum size condition, with other level notable difference arranged through variance analysis, thus the suitableeest inoculum size be 1%; 5. the YB-04 diastatic activity is up to 151.36U/mL when soaking powder with yeast and be nitrogenous source, and protease activity is 121.16U/mL when being nitrogenous source with trypsinase.The enzyme of YB-04 fermented liquid is lived when soaking powder and be nitrogenous source with yeast as can be known through variance analysis, and there were significant differences with other level, so the suitableeest nitrogenous source of YB-04 is that yeast soaks powder.

The antibacterial general survey of embodiment 6:YB-04 is fixed

Be the target bacterium with 10 kinds of pathogenic bacterias, adopt the method for dull and stereotyped face-off to measure the YB-04 bacterial strain to the antagonistic action of these 10 kinds of pathogenic bacterias, measure its antimicrobial spectrum.

Table 4 YB-04 bacterial strain antimicrobial spectrum

Pathogenic bacteria	Fungistatic effect	Pathogenic bacteria	Fungistatic effect
				The solanum cinerea bacterium	＋＋	Cotton boll rot pathogens	＋＋
The cucumber parasitica	＋＋＋	Fusarium graminearum	＋＋
				Marssonina coronaria	＋＋	The corn bacterial wilt	＋＋
Gaeumannomyces graminis	＋＋＋	Valsa mali	＋＋
				Rhizoctonia cerealis	＋＋＋	Verticillium dahliae	＋＋＋
Intestinal bacteria	＋	The bacterial wilt of tomato bacterium	＋＋

Annotate: " +++": antibacterial band is more than the 10mm; " ++ ": the antibacterial 5～10mm that is with;

"+": antibacterial band＜5mm; "-": antibacterial band＜3mm

Determine YB-04 to the antibacterial band of 10 kinds of pathogenic bacterias, the result is as shown in table 4, finds that it shows the comparatively fungistatic effect of wide spectrum, and is wherein better to rhizosphere disease fungistatic effects such as verticillium dahliae, gaeumannomyces graminis, wheat hypochnuss.Show that YB-04 not only can degrading straw, can also suppress the rhizosphere pathogenic bacteria, regulate rhizospheric microorganism group, have fine exploitation prospect.

Embodiment 7: the test of bacterial classification combination degraded wheat stalk

(1) co-culture experiments of degradation bacteria: relation is very complicated between the microorganism of the ecosystem, and interdependence is vied each other again.By with two or more inoculation on same flat board, observe whether there is antagonistic action between them, could determine whether a plurality of bacterial strains can co-cultivation.The bacterial strain that this experiment obtains screening is inoculated on the PDA flat board in twos, by the observation of colony diameter, analyzes between two kinds of bacterium whether can produce antagonistic action.

(2) bacterial strain uses therefor: YB-04 for protection bacterial strain subtilis in the patent of the present invention ( BaciLLus stbtiLis), P4 be black-koji mould ( Aspergillus niger), P3 be aspergillus oryzae ( AspergiLLus oryzae) bacterial strain, P5 be Penicillium chrysogenum ( Penicillium chrysogenum) bacterial strain, L8 be bacillus amyloliquefaciens ( B. amyLaLiquefaiens) bacterial strain, separate in the stalk that rots by this laboratory.Microbial inoculum 1 and microbial inoculum 2 are respectively available from Hebi hundred favour bio tech ltds.

(3) degrading straw test: accurately take by weighing 2g and put into culture dish at 60 ℃ of fresh wheat stalks that are dried to constant weight, carry out the effect research of strain degradation wheat stalk, the making of bacteria suspension is with reference to aforementioned seed liquor manufacture method.Single culture is degraded to: add 1mL bacteria suspension and moisturizing in the culture dish that is placed with the 2g stalk to 10mL; Combined degradation is: be placed with and add bacterium liquid in the plate of stalk, every kind of bacterium liquid is 1mL, and moisturizing is to 10mL.

(4) degradation effect analysis: after degraded had been tested, by appearance features, and the changes in weight situation of stalk was estimated the degradation effect of stalk.The stalk of Fu Laning, can be black yellow according to color (Huang ﹑ Wei Huang ﹑ He Huang ﹑) be divided into (1 ﹑, 2 ﹑, 3 ﹑ 4) level Four, smell (Mei Wei ﹑ An Wei ﹑ Jiu Wei ﹑ putrid taste according to stalk) is divided into (1 ﹑, 2 ﹑, 3 ﹑ 4) level Four, rot according to feel softening degree (Ying ﹑ Wei Ruan ﹑ Ruan ﹑) be divided into (1 ﹑, 2 ﹑, 3 ﹑ 4) level Four, the progression of three kinds of features is summed up the rotten degree that is stalk, level is said more big, and rotten degree is more big ^[80]Observed a straw degradative situation in per 5 days, the progression of record straw degradative.

The mycelium on stalk surface is clean with distilled water flushing, and 60 ℃ of weight that are dried to constant weight weighing residue stalk are calculated the straw degradative rate, and will remains stalk and carry out content of cellulose mensuration.Content of cellulose measuring method: take by weighing the 0.1g stalk in test tube, add acetic acid and nitric acid mixed solution (volume ratio is 1/1) 5mL, cover lid heats 30min in boiling water bath, is transferred to that adding distil water is diluted to 45 mL in the big centrifuge tube, the centrifugal supernatant that goes in cooling back, 60 ℃ of oven dry.Get sample after the oven dry and add that (massfraction is 10% sulfuric acid in the mixed solution, concentration is the potassium bichromate of 0.1mol/L) 10 mL, shake up back boiling water bath 15min, all be transferred to titration in the clean triangular flask, the adding massfraction is 20% KI solution 5 mL, with the Sulfothiorine titration of 0.2mol/L to solution just shown blue 2 and half a minute in nondiscoloration, make a blank that does not add stalk in addition.

Content of cellulose calculation formula: X=k (a-b)/(n * 24)

In the formula: k is the concentration of Sulfothiorine, and a is the volume of the used sodium sulfate of blank titration, and b is the volume of solution Sulfothiorine that titration consumes, and n is the quality of rice husk.

(5) be total to the culture experiment result

Table 5 is culture experiment strain growth speed altogether

On flat board, be contrast with the single culture cultivation with two kinds of different inoculation, the bacterium colony size of bacterial strain on flat board sees Table 5.Speed when the growth velocity during with the bacterial strain co-cultivation and single culture is carried out difference analysis, analytical results shows that the P value is all greater than 0.05, therefore the growth velocity of co-cultivation and the single culture property of there are differences not, so show and do not have antagonistic action between the bacterial strain, can co-cultivation.

(6) straw degradative interpretation of result

Table 6 strain combinations degrading straw scheme

Test number	Bacterium liquid	Moisturizing (mL)	Total liquid volume added (mL)	Stalk amount (g)
					1	Blank	10	10	2
2	YB-04+L8	8	10	2
					3	YB-04 +P3+ L8	7	10	2
4	YB-04 +P4+ L8	7	10	2
					5	YB-04+P5+ L8	7	10	2
6	YB-04+P3+ P4+ L8	6	10	2
					7	YB-04+P3+ P5 + L8	6	10	2
8	YB-04+P4+ P5+ L8	6	10	2
					9	P3+ P4+P5	7	10	2
10	YB-04 +P3+P4+P5+ L8	5	10	2
					11	Microbial inoculum 1	1g	10	2
12	Microbial inoculum 2	1g	10	2

Annotate: microbial inoculum 1/ microbial inoculum 2 is available from Hebi hundred favour bio tech ltds; Bacterium liquid all adds 1ml. in every kind of combination

Introducing in the previous methods is the Study on degradation that stalk is carried out in the combination of 5 strain bacterium, and assembled scheme sees Table 6, and the degraded progression in the straw degradative process is as shown in table 7.As seen from the above table, during off-test, the stalk that bacterial classification makes up degraded in twos and the stalk of single degraded have reached identical rotten progression, but the speed that the degradation speed of stalk greater than single culture is during the bacterial classification combination.And add fashionablely as genus bacillus, the degradation speed of stalk is faster than the experimental group degradation speed that does not add genus bacillus, and it is deep to rot.The combination of 3 fungal strains and 2 strain gemma makes the rotten progression of stalk reach 12 grades in the test, and the rotten progression of two kinds of commercially available microbial inoculums reaches 9 grades of 8 ﹑ respectively, visible P3+P4+P5+(YB-04+L8) respond well to straw degradative.By Fig. 3-8 as can be known, when having only the fungi effect with stalk, though a large amount of mycelium is adhered on the stalk surface, the stalk surface is dry, and the more moistening corruption in stalk surface is more obvious after adding bacterium.The utilization of fungi (P3+P4+P5) mixing effect stalk behind stalk obviously acts on stalk greater than single fungi, and corruptions such as tangible blackening have also appearred in the test batch stalk that adds bacterium.It is composite effective not as mixing in the research to the degradation effect of stalk to observe these two kinds of microbial inoculums.

Table 7 straw degradative progression

Annotate: the rotten degree of the more big explanation of progression is more obvious

Table 8 straw degradative rate

Test number	Heavy behind the straw degradative	The straw degradative rate
			P3	0.90	55%
P3+（YB-04+L8）	0.89?	55.5%
			P4	1.01	49.5%
P4+（YB-04+L8）	0.93	53.5%
			P5	1.28	36%
P5+（YB-04+L8）	1.20	40%
			P3+ P4+（YB-04+L8）	0.87	56.5%
P3+ P5+（YB-04+L8）	0.89	55.5%
			P4+ P5+（YB-04+L8）	0.92	54%
P3+P4+P5	0.87	56.5%
			P3+P4+P5+（YB-04+L8）	0.82	59%
Microbial inoculum 1	1.35	32.5%
			Microbial inoculum 2	1.10	45%

The variation of table 9 content of cellulose

Test number	Content of cellulose before the degraded	Degraded back content of cellulose
			P3	51.16%	47.2%
P3+（YB-04+L8）	51.16%	47.3%
			P4	51.16%	50.2%
P4+（YB-04+L8）	51.16%	50.5%
			P5	51.16%	50.3%
P5+（YB-04+L8）	51.16%	50.8%
			P3+ P4+（YB-04+L8）	51.16%	46.4%
P3+ P5+（YB-04+L8）	51.16%	48.4%
			P4+ P5+（YB-04+L8）	51.16%	50.9%
P3+P4+P5	51.16%	44.7%
			P3+P4+P5+（YB-04+L8）	51.16%	42.9%
Microbial inoculum 1	51.16%	50.5%
			Microbial inoculum 2	51.16%	48.6%

Content of cellulose sees Table 8 and table 9 in straw degradative rate and the degraded back stalk.As can be known, fungi and bacterium mixing effect are behind stalk, the degradation rate of stalk acts on the degradation rate of stalk separately apparently higher than fungi, when 5 kinds of bacterium mixing effects during in stalk, the degradation rate of stalk has reached maximum value, but being not the compound action that is far longer than single fungi and bacterium, is not to be simple addition during visible bacterial strain mixing degrading straw.More as can be known, the Degradation of experiment combination is far superior to the Degradation of commercially available microbial inoculum by the degradation rate of testing combination and commercially available microbial inoculum.

Embodiment 8 straw degradatives simulation field experiment

(1) test method: in flowerpot (add the thick vegetable garden soil of 8cm among the 15cm * 12cm), above even spreading wheat stalk 100g.Bacterium liquid with different treatment sprays stalk, and bacterium liquid and treatment process are seen embodiment 3, and consumption is 10mL.The back is till overlying soil on the stalk is just in time filled flowerpot then, put into 25 ℃ of greenhouses degraded 30d after, measure the degradation rate of stalk, and respectively handle nitrogen phosphorus potassium and organic content in the soil.Be contrast not add the stalk that bacterium liquid handles.

(2) interpretation of result

In the pot experiment, the degradation rate of stalk sees Table 10, and nitrogen phosphorus potassium and organic content behind the straw degradative in the soil see Table 11.By table 10 as seen, degradation rate under the natural condition in the stalk 30d is 55.55%, and the degradation rate of the various processing of adding bacterium liquid all is higher than the degradation rate under the natural condition, and wherein the straw degradative rate of 5 kinds of bacterium combined treatment is 79.61%, reach maximum value, be significantly higher than the degradation rate under the natural condition.

Table 10 simulation field experiment straw degradative rate

Test number	Behind the straw degradative heavy (g)	Straw degradative rate (%)
			Contrast	44.45	55.55m
P3	35.08	64.92jk
			P4	35.08	64.92jk
P5	36.44	63.56l
			YB-04+L8	37.44	62.56kl
P3+（YB-04+L8）	31.42	68.58ghi
			P4+（YB-04+L8）	32.04	67.96hij
P5+（YB-04+L8）	34.26	65.74ij
			P3+ P4+（YB-04+L8）	23.17	76.83ab
P3+ P5+（YB-04+L8）	25.90	74.1bcd
			P4+ P5+（YB-04+L8）	28.32	71.68def
P3+P4+P5	24.94	75.06bc
			P3+P4+P5+（YB-04+L8）	20.39	79.61a
Microbial inoculum 1	30.09	69.91fgh
			Microbial inoculum 2	26.18	73.19

Annotate: lowercase is significance analysis on 0.05 level

By table 11 as seen, stalk can make nitrogen phosphorus potassium and the organic content in the soil increase after degraded in soil, nitrogen phosphorus potassium and organism increased value in the more big soil of straw degradative rate are more big, do not add that nitrogen phosphorus potassium and organic content are respectively 31mg/mL ﹑ 42mg/mL ﹑ 240 mg/mL ﹑ 0.1% in the soil of stalk, N-P-K content has reached 58 mg/mL ﹑, 52 mg/mL ﹑, 674 mg/mL in the soil behind 5 kinds of bacterium combination degrading straw, 1.3%, the content of nitrogen phosphorus potassium is significantly higher than contrast in the soil, illustrate that also 5 kinds of potted plant stalk effects of bacterium combination degraded are best, provide scientific basis for further developing the combination degradation bacteria.

  

Nitrogen phosphorus potassium and organic content in table 11 soil

Test number	Ammonia-state nitrogen mg/kg	Speed phosphorus (mg/kg)	Speed potassium (mg/kg)	Organism (%)
					Do not add the stalk contrast	31i	42i	240q	0.1h
Add the stalk contrast	33i	45gh	290o	0.3g
					P3	36h	47ef	320l	0.6de
P4	34i	46fg	315m	0.5ef
					P5	34i	45gh	300n	0.5ef
YB-04+L8	33i	45gh	280p	0.4fg
					P3+（YB-04﹑L8）	45e	47ef	365h	0.7cd
P4+（YB-04+L8）	43f	46fg	350j	0.5ef
					P5+（YB-04+L8）	40g	46fg	320l	0.5ef
P3+ P4+（YB-04+L8）	52b	50bc	386f	1.0b
					P3+ P5+（YB-04+L8）	45e	49cd	455d	0.8c
P4+ P5+（YB-04+L8）	50c	49cd	370g	0.6de
					P3+P4+P5	52b	51ab	545b	1.0b
P3+P4+P5+（YB-04+L8）	58a	52a	674a	1.3a
					Microbial inoculum 1	36h	46fg	370g	0.5ef
Microbial inoculum 2	47d	48de	505c	0.9b

Claims (6)

1. subtilis YB-04, its deposit number is CGMCC NO.6497.

2. a microbial preparation is characterized in that, contained activeconstituents is that the described subtilis YB-04 of claim 1 is or/and its meta-bolites.

3. according to the described microbiobacterial agent of claim 2, it is characterized in that described microbial inoculum is for liquid.

4. the described microbiobacterial agent preparation method of claim 3 comprises the steps:

(1) strain culturing and preparation: subtilis YB-04 is inoculated on the TSA test tube slant substratum, cultivates down for 28～35 ℃ and obtained thalline in 24～36 hours;

(2) seed liquor preparation: the test tube strains of step (1) preparation is inserted in the TSB nutrient solution, shake down to cultivate at 28～32 ℃ and made seed liquor in 12～16 hours;

(3) fermention medium preparation: get glucose, Semen Maydis powder, soybean cake powder, dipotassium hydrogen phosphate respectively and add water and be mixed with the nutrient solution that contains glucose 1.2%, Semen Maydis powder 1.8%, soybean cake powder 3%, dipotassium hydrogen phosphate 0.2%;

(4) liquid fermenting production: behind fermentation equipment and medium sterilization, seed liquor is inoculated in the nutrient solution that step prepares by 5% inoculum size, after 27～34 ℃ of bottom fermentations are cultivated at least 36～48 hours, collects fermented liquid and namely get microbial inoculum.

5. the application of the described bacillus subtilis strain YB-04 of claim 1 in the crop material degraded.

6. the described bacillus subtilis strain YB-04 of claim 1 is in control wheat rhizosphere banded sclerotial blight and the application in gaeumannomyces graminis disease.

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