CN1766091A - Bacillus subtilis and its uses - Google Patents
Bacillus subtilis and its uses Download PDFInfo
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Abstract
The invention discloses a (Bacillus subtilis) HL259 with CGMCC No.1451 aimed to provide product with broad spectrum sterilization activity and opposite microbe agent that can prevent and cure root rot and damping-off of vegetable, melon crop, peas and alfalfa brought by fusarium, sapromycetin and others. The preparation method needs simple operation, short fermentation period and low invest.
Description
Technical field
The present invention relates to a bacillus subtilis and application thereof, particularly is the microbiobacterial agent of activeconstituents with this bacillus subtilis.
Background technology
Along with the enforcement of vegetable basket project and the adjustment of agricultural planting structure, China beans, melon and cultivated areas such as vegetables, clover present the situation that develops rapidly.Simultaneously because increase international, the domestic market demand amount, nuisanceless, green and Organic farming production base cultivated area and construction of base scale cumulative year after year.Show according to dynamic multiple spot monitoring of anniversary and investigation; because the plantation of continuous cropping year after year of protection ground vegetables; nonstandard crop rotation; soil-borne pathogen accumulates in a large number, multiplies; cause vegetable sprout term samping off (Pythium aphanidermatum), damping-off (Rhizoctoniasolani), blight (Fusarium oxysporum) and mould by corruption, epidemic disease is mould and the compound harm of infecting the soil-borne diseases such as root rot that cause of sickle-like bacteria increases the weight of day by day; cause the serious underproduction or reduce quality product, even total crop failure.Bacterial strains such as some Fusarlum moniliforme and proliferation sickle-like bacteria also can produce polyketone class mycotoxinss such as volt horse mycin, directly influence the quality of agricultural-food and the health of harm humans and animals.Main using of chemical pesticide and chemical fertilizer, the restriction high-yield and high-efficiency Agricultural Development of relying in the production.
Chemical prevention occupies an important position on world's agriculture development history.For centuries, chemical pesticide is being brought into play positive effect in the control of the phytopathy Chinese caterpillar fungus plague of rats, for the stable yields of agricultural, increase production, retrieve a loss and improve quality and made significant contribution.But, influence non-target organism and cause the resistance generation of harmful organism and serious problems such as develop rapidly simultaneously also because of excessive using with pesticide abuse causes soil fertility decline and environmental pollution.Especially in recent years because pesticide residue exceed standard in the agricultural-food, cause person poultry poisoning's incident constantly to take place, the people's quality of life in serious threat, has also limited the foreign export of agricultural products in China.The generation of soil-borne disease potentially contaminated rampant and chemical bactericide has become primary key issue in the agricultural safety in production.
Along with the development of social civilization, the agricultural sustainable development problem makes human understanding to resource and environment that new leap arranged, and agricultural chemicals has been proposed efficient, low toxicity, environmentally friendly requirement.Countries in the world are also in the use of minimizing chemical synthetic pesticide that takes practical steps, just announced to cancel the registration of 91 kinds of chemical pesticides 90 years 20th century as U.S. EPA, Holland, Denmark have just formulated 5 years and the ten year plan that reduces half agricultural chemicals usage quantity in nineteen ninety, and European Union has also formulated similar plan.China has also cancelled the registration of tens kinds of chemical pesticides.For solve agricultural-food improve output and ensure the quality of products between contradiction, reach the great disease and pest of integrated control vegetables, reduce the purpose of chemical pesticide usage quantity, development biological control technology more and more receives national governments, scientific worker and the common people's concern.Bacteria agent (BCA) is good with its low toxicity, environment compatibility, have the ability of building the group at the crop seedling rhizosphere, lasting period is long, harmful organism is difficult for characteristics such as it develops immunity to drugs are paid close attention to widely and paid attention to, advanced in the world at present farming company plays an active part in the research and the marketization of bacteria agent mostly, particularly beneficial bacteria has been obtained certain progress in the control of soil biography, seed-borne disease, and the application that has series product to succeed, obtained remarkable economical and ecological benefits.Biological control is being brought into play more and more important effect as important measures of integrated control in the plant pest comprehensive regulation.
PGPR (promoting the rhizosphere bacteria of plant-growth) is a class biocontrol bacteria that relatively is subjected to extensive concern at present.Also be one of domestic main biological and ecological methods to prevent plant disease, pests, and erosion factor that is applied to the soil-borne disease integrated control, it is by transplanting in root system and preferentially capturing root system or suppress harmful rhizosphere bacteria on the root and growth that harmful fungoid (DRMO) promotes plant.Wherein bacillus (Bacillus spp.) distributes wide with it, easily separated cultivation, can produce heat-resisting siccostabile statospore, storage period is long, characteristics such as easy to use, become a kind of ideal biological and ecological methods to prevent plant disease, pests, and erosion microorganism, after report producing bacillus subtilis such as Johnson in 1945 are given birth to antimicrobial substance, the research worker of various countries is expected to become a kind of biological control factor and expresses very big concern it since over half a century, and on various crop, carried out controlling widely the disease ability test, many successful Application are arranged, in production practice, show tangible disease-preventing and yield-increasing effect, also further confirmed the stability of genus bacillus biocontrol fungicide in product, obviously be better than non-genus bacillus and fungi biocontrol microorganisms with the consistency of chemical pesticide and the consistence aspect of different plant different year preventive effects.
Chinese scholars successively reported respectively have the biological control effect each not the subtilis of homophyletic system biological property, grow correlative study such as condition, antimicrobial substance physicochemical property surely and research that prevention effect, the potential inducing plant of the different soil-borne diseases that cause Different Crop root field planting situation,, sickle-like bacteria mould to corruption etc. produce disease resistance and promote aspects such as growth of seedling.There are some researches show because of genus bacillus can produce endogenous spore, to have extremely strong anti-adversity ability, compare the biological and ecological methods to prevent plant disease, pests, and erosion factor of other types, more help the production of microbial inoculum, formulation processing reaches survives in environment, grows surely and breeding.
Summary of the invention
An object of the present invention is to provide a strain and have the active subtilis of broad-spectrum sterilization.
Subtilis provided by the present invention, be subtilis (Bacillus subtilis) HL259, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 09 01st, 2005, preserving number is CGMCC No.1451.
Subtilis (Bacillus subtilis) HL259 CGMCC № 1451 separates obtaining from the soybean field soil of Keshan County, Qiqihaer City, Heilungkiang.
Second purpose of the present invention provides a kind of active microbiobacterial agent of broad-spectrum sterilization that has.
Microbiobacterial agent provided by the present invention, its activeconstituents are subtilis (Bacillussubtilis) HL259 CGMCC No.1451.
Described microbiobacterial agent can obtain as follows: subtilis (Bacillus subtilis) the HL259 CGMCC № 1451 that ferments in YMA or corn steep liquor substratum, mix the fermented liquid that obtains to obtain microbiobacterial agent with matrix.
Contain sucrose 5g in the described corn steep liquor substratum, Semen Maydis powder 2g, KH
2O
40.05g, (NH
4)
2SO
40.5g, K
2HPO
40.05g, MgSO
40.025g, CaCO
32g, distilled water 1000ml.
Contain N.F,USP MANNITOL 10g in the YMA medium, yeast powder 3g, K
2HPO
40.25g, KH
2PO
40.25g, 0.2g MgSO
4.7H
2O, NaCl 0.1g, CaCO
33g, agar 10g, distilled water 1000ml, pH value 7.2.
In the described fermentation condition, culture temperature can be 25-30 ℃, and air flow can be 1: 0.5-1: 1.6, and stirring velocity can be 200-400rpm, and incubation time can be 24-48h; Described air flow is that per minute feeds the volume of air of fermentor tank and the volume ratio of fermentation cylinder for fermentation liquid.
Wherein, described culture temperature is preferably 28-30 ℃, and air flow is preferably 1: 1-1: 1.2, and stirring velocity is preferably 250-300rpm, and incubation time is preferably 24-30h.
Can add vegetables oil or organic silicone oil during fermentation as foam killer.
The quality percentage composition of described vegetables oil can be 1-2%; The quality percentage composition of described organic silicone oil can be 0.04-0.1%.
Described matrix can be the peat composed of rotten mosses, micro mist lime carbonate, attapulgite or wilkinite.
The viable count of subtilis in the described microbiobacterial agent (Bacillus subtilis) HL259 CGMCC № .1451 can be 5 * 10
9Cfu/g, the pH value can be 6.0-7.8.
Experimental result shows that subtilis (Bacillus subtilis) HL259 CGMCC № .1451 all has obvious suppression growth effect to the 22 kind of plant pathogenic fungies of 13 genus of four Eumycotinas except that Rhodopseudomonas and the representative pathogens bacterium of 5 important genus, wherein mould to the melon and fruit corruption, Phytophthora capsici, glue born of the same parents sickle-like bacteria, wheat cereal rhizoctonia, clover root rot sickle-like bacteria, verticillium dahliae, cotton-wilt fusarium, the inhibiting rate of Valsa mali and wheat dry thread Pyrenomycetes reaches 72.76%～94.86%.Indoor and greenhouse test shows that this bacterial strain as soil regulation and control, seed treatment and overground part spraying, has good prevention effect to microbial root rot of cause of disease such as soil biography, kind biographies.In meeting next batch of soybean field, Heilongjiang Province and Jilin Province; more serious a plurality of vegetable protecting fields take place the Beijing suburb root rot and the control test has been carried out in the clover field; the result shows that HL259 and meta-bolites thereof can significantly control by Pythium; Phytophthora; the mashed kind that Fusarium and Rhizoctonia etc. cause; damping off; upright withered; diseases such as root-rot; and the double parts of fine fungal disease of controlling; has certain production-increasing function; being particularly useful for having significant diseases prevention seedling protecting effect for the compound root rot that causes that infects of multiple pathogenic bacteria after seed treatment and the soil treatment, is that a strain has the active biocontrol strain of broad-spectrum sterilization.
1451 pairs of crops of subtilis (Bacillus subtilis) HL259 CGMCC № all do not have pathogenic effects, and 1451 pairs of tomato stems of leaf-cutting inoculation method inoculation subtilis (Bacillus subtilis) HL259 CGMCC № are long, stem of Bush Redpepper is long and summer squash stem length has promoter action; 1451 pairs of Cucumis sativus stems of stab inoculation inoculation subtilis (Bacillus subtilis) HL259 CGMCC № are long, fresh weight has promoter action; Dip in root inoculation method inoculation subtilis (Bacillus subtilis) HL259 CGMCC № 1451 pairs of Muskmelon stems length, fresh weight and Herba Medicaginis stem length promoter action is arranged.
Experimental result shows, with subtilis (Bacillus subtilis) HL259 CGMCC № 1451 is that the microbiobacterial agent of activeconstituents can effectively be prevented and treated in vegetables, melon, beans and the ALFALFA PRODUCTION by Plant diseasess such as the microbial root rot of sickle-like bacteria, pythium spp, phytophthora and miliary damping-off, samping off and damping-offs, and the preparation technology of this microbiobacterial agent is simple, fermentation period short, it is low to drop into and be easy to preserve.
Description of drawings
Figure 1A is the optical microscope photograph of subtilis (Bacillus subtilis) HL259 CGMCC № 1451
Figure 1B and Fig. 1 C are the electromicroscopic photograph of subtilis (Bacillus subtilis) HL259 CGMCC № 1451
Fig. 2 analyzes the evolutionary tree result who obtains according to the 16SrRNA sequence with DNAMAN Version 4.0
Fig. 3 is the 16SrRNA gene order source property comparative result of subtilis (Bacillus subtilis) HL259 CGMCC № 1451 and Bacillussubtilis US116
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Separation and the evaluation of embodiment 1, subtilis (Bacillus subtilis) HL259 CGMCC № 1451
1, the separation of subtilis (Bacillus subtilis) HL259 CGMCC № 1451
Gather soil sample 10g from soybean field, Keshan County, Qiqihaer City, Heilungkiang, in the triangular flask of dress 100ml sterilized water and granulated glass sphere, the 20min that vibrates on 100 commentaries on classics/min shaking tables gets 0.5ml and adds in the 4.5ml sterilized water, dilutes 10 successively in adding
-2, 10
-3, 10
-4Doubly, getting above suspension liquid 0.1ml respectively is coated with on YMA, beef extract-peptone (bacteria culture medium), Ma Dingshi (fungi isolation medium), No. 1 (actinomycetes substratum) substratum of improvement Gao Shi, each concentration repeats 3 times, place 28 ℃ of thermostat containers to cultivate 24h, 3d and 5d, carry out bacterium respectively, fungi separates and purifying with actinomycetic.And respectively with Pythium, Phytophthora, Fusarium and Rhizoctonia are the target bacterium, carry out antimicrobial screening short of money.Therefrom filter out subtilis (Bacillus subtilis) the HL259 CGMCC № 1451 that each is had obvious fungistatic effect for the examination bacterium.
2, the evaluation of subtilis (Bacillus subtilis) HL259 CGMCC № 1451
(1) morphological specificity of thalline
Subtilis (Bacillus subtilis) HL259 CGMCC № 1451 soaks on the juice nutrient agar at soil that (soil 1kg adds water 1000ml, high pressure steam (121 ℃) sterilization 30min.Immersion liquid is used double-deck filter paper filtering after adding talcum powder, the pH value is adjusted to neutrality, then according to soil extract 100ml, agar 17g, water 900ml prepares substratum) cultivate 24h after bacterium colony for circular, White-opalescent, along with incubation time increases, the bacterium colony thickening becomes dry, and the edge is irregular, and gauffer shape projection is arranged on the bacterium colony, become creamy, growth cellular material thereon is difficult for disperseing in liquid, observes staff cell (Fig. 2 A) under the opticmicroscope, the great majority motion, flagellum Zhousheng, cell is 0.6～0.8 * 3～5 μ m, and gramstaining is even, and is dyed the bluish voilet gemma in the visible cell.Sem observation is further proved conclusively the shaft-like form of gemma, and cell walls has the clayey layer of thickness homogeneous outward, produces the ellipse gemma, no parasporal crystal (Fig. 2 B and Fig. 2 C).
(2) Physiology and biochemistry of bacterial strain is identified
Subtilis (Bacillus subtilis) HL259 CGMCC № 1451 and subtilis reference culture 1.504 (Chinese culture presevation management committee) are carried out the Physiology and biochemistry proterties detect result such as table 1 under identical experiment condition.
Table 1.HL259 certified variety and result
| Test item | HL259 | Reference culture | Test item | HL259 | Reference culture | ||
| Gram-reaction | + | + | Produce melanochrome | 1% glucose | - | - | |
| 1% tyrosine | - | - | |||||
| The starch hydrolysis | + | + | |||||
| Spore staining | + | + | Utilize | Citrate trianion | + | + | |
| Parasporal crystal | - | - | Propionic salt | - | - | ||
| Mobility | + | + | From NO 3Produce NO 2 | + | + | ||
| Catalase | + | + | Produce indoles | - | - | ||
| Anaerobic growth | - | - | Decompose | Casein | + | + | |
| The V-P reaction | + | + | Tyrosine | - | - | ||
| Egg yolk reaction | - | - | The pH5.7 growth | + | + | ||
| The 7%NaCI growth a | + | + | 50 ℃ of growths | + | + | ||
| Produce acid | Glucose | + | + | Gelatin hydrolysis | + | + | |
| Pectinose | + | + | Aerogenesis | Glucose | - | - | |
| Wood sugar | + | + | |||||
| N.F,USP MANNITOL | + | + | |||||
Annotate: a represents that HL259 also can grow in 10%NaCl."+" expression positive findings; "-" expression negative findings.
(2) HL259 bacterial strain 16SrRNA sequencing and plasmid extract
16SrRNA gene order sequencing result shows that the 16SrRNA gene order of HL259 bacterial strain has the nucleotide sequence of sequence 1 in the sequence table, carry out homology relatively according to genus bacillus 16SrRNA gene order among sequencing result and the Genbank, the result shows that the homology of HL259 and Bacillus subtilis US116 reaches 99.52% (Fig. 3, among the figure, "-" expression during HL259 is capable is identical with the base in the corresponding site of US116, " g, c, a " expression is different with the base in the corresponding site of US116), illustrate that the HL259 bacterial strain belongs to bacillus (Bacillus).Plasmid extracts and electrophoresis result shows that HL259 contains about 4.5kb left and right sides plasmid, can normal growth on the flat board that contains 50 μ g/ml penbritins (Amp).
Document shows that subtilis is movable, Gram-positive, gemma ellipse or column, middle life or middle partially giving birth to, the catalase test positive, V-P tests positive, grows in the 7%-10% sodium-chlor, produce acid from glucose, pectinose, wood sugar and N.F,USP MANNITOL, hydrolyzed starch utilizes Citrate trianion as carbon source, and reduction nitrate becomes nitrite, decompose casein, the egg yolk reaction feminine gender does not form melanochrome on glucose and tyrosine agar, do not utilize propionic salt and do not decompose characteristics such as tyrosine.The part Physiology and biochemistry proterties detected result of above HL259 is compared with the subtilis reference culture, (microbial bacterial institute of Chinese Academy of Sciences division bacteria group is compiled according to " general bacterium authentication method commonly used ", Science Press, 1978), " bacillus " (Cai Miaoying, war Garrick etc. is translated, agricultural press, 1988) and " microbial taxonomy " (Zhang Jizhong, Fudan University in Shanghai press, 1998) key in is retrieved, and determines that the corresponding proterties of HL259 Physiology and biochemistry proterties and subtilis is identical.Simultaneously 16SrRNA sequence and further confirm the biochemical identification result with the evolutionary tree result (Fig. 2) that DNAMAN Version 4.0 analyzes acquisitions according to the 16SrRNA sequence identifies that HL259 is a subtilis.
The bacteriostatic activity (antimicrobial spectrum mensuration) of embodiment 2, subtilis (Bacillus subtilis) HL259 CGMCC № .1451
One, bacteriostatic activity is measured
Adopt mycelial growth rate assay method and spore germination method to detect subtilis (Bacillussubtilis) HL259 CGMCC No.1451 bacterial strain and the 22 kind of plant pathogenic fungies of meta-bolites: miliary damping-off germ Rhizoctonia solani to comprising that four Eumycotinas 13 belong to, Valsa mali Valsa mali, fusarium graminearum Fusarium graminearum, cucumber didymella bryoniae Mycosphaerella melonis, tomato early blight bacterium Alternaria solani, fusarium moniliforme Fusarium moniliforme, cotton-wilt fusarium Fusariun oxysporum f.sp.vasinfectum, botrytis cinerea Botrytis cinerea, pea eggplant shape sickle-like bacteria, Fusarium solani f.sp.pisi, southern corn leaf blight Bipolaris maydis, ring rot of apple bacterium Physalospora piricola, anthrax bacteria Colletotrichumgloeosporioides, verticillium dahliae Verticillium dahliae, paddy rice rice blast fungus Piriculariaoryzae, Phytophthora capsici Phytophthora capsici, melon and fruit pythium spp Pythium aphanidermatum, glue fusarium oxysporum Fusarium subglutinans, rhizoctonia cerealis Rhizoctonia cerealis, clover root rot sickle-like bacteria Fusarium avenaceam, Sporisorium reilianum Sporisoriumholci-sorghi (Rivolta) vanky); Adopt double-layer plate culture method and Oxford cup method to measure the representative pathogens bacterium of 5 important genus: the Agrobacterium during Gracilicutes mainly belongs to (Agrobacterium) k27 germ, cloth gram Bordetella (Ralstonia) solanum solanacearum, Rhodopseudomonas (Pseudomonas) angular leaf spot of cucumber bacterium, xanthomonas (Xanthomonas) rice leaf spot bacteria, cotton angular leaf spot fungus and tomato Streptomyces scabies; The bacteriostatic activity and the antimicrobial spectrum of clavate Bacillaceae (Clavibacter) bacterial canker of tomato during Firmicutes mainly belongs to.
1, subtilis (Bacillus subtilis) HL259 CGMCC № 1451 suppresses the pathogenic fungi effect detection
Adopt plating method, subtilis (Bacillus subtilis) HL259 CGMCC № 1451 activates 48h down for 28 ℃ on 523 inclined-planes, makes bacteria suspension with 0.85% physiological saline, determines that with Mike's turbidimetry final bacteria suspension concentration is 10
9Cfu/ml is standby.For examination fungi 25 ℃ of activation 5d on the PDA flat board, make it form bacterium colony, (diameter 5mm) produces the bacterium cake with punch tool, and mycelia is faced down connects bacterium in the dull and stereotyped central authorities of PDA.The double-deck filter paper (diameter 5mm) of high-temperature sterilization is soaked in subtilis (Bacillus subtilis) HL259 CGMCCNo1451 bacteria suspension, evenly be placed on the PDA flat board, 3 in every ware, distance evenly, the PDA flat board that soaks the sterilized water scraps of paper with placement is contrast, each is handled 4 times and repeats, and measures the fungal colony diameter respectively behind 25 ℃ of following dark culturing 4,5,6, the 7d, and calculates and suppress growth rate.
1451 couples of restraining effect results for the examination pathogenic fungi of subtilis (Bacillus subtilis) HL259 CGMCC № are as shown in table 2.
1451 pairs of restraining effect of table 2. subtilis (Bacillus subtilis) HL259 CGMCC № for the examination pathogenic fungi
| Strains tested | Filter paper method (colony radius cm) | Inhibiting rate (%) | |
| CK | HL259 | ||
| Miliary damping-off germ Rhizoctonia solani | 4.00 | 0.76 | 92.57 |
| Valsa mali Valsa mali | 3.80 | 0.85 | 89.39 |
| Fusarium graminearum Fusarium graminearum | 2.50 | 1.33 | 58.50 |
| Cucumber didymella bryoniae Mycosphaerella melonis | 3.47 | 1.43 | 68.69 |
| Tomato early blight bacterium Alternaria solani | 3.13 | 1.54 | 60.46 |
| Fusarium moniliforme Fusarium moniliforme | 3.23 | 1.33 | 69.60 |
| Cotton-wilt fusarium Fusariun oxysporum f.sp.vasinfectum | 2.77 | 1.05 | 75.77 |
| Botrytis cinerea Botrytis cinerea | 3.97 | 1.45 | 72.62 |
| Pea eggplant shape sickle-like bacteria Fusarium solani f.sp.pisi | 3.62 | 1.09 | 81.09 |
| Southern corn leaf blight Bipolaris maydis | 2.52 | 1.40 | 55.44 |
| Ring rot of apple bacterium Physalospora piricola | 2.27 | 1.18 | 61.58 |
| Anthrax bacteria Colletotrichum gloeosporioides | 2.90 | 1.47 | 59.58 |
| Verticillium dahliae Verticillium dahliae | 3.28 | 1.14 | 76.98 |
| Paddy rice rice blast fungus Piricularia oryzae | 2.85 | 1.64 | 51.49 |
| Phytophthora capsici Phytophthora capsici | 2.77 | 1.05 | 75.77 |
| Melon and fruit pythium spp Pythium aphanidermatum | 3.62 | 1.35 | 72.76 |
| Glue fusarium oxysporum Fusarium subglutinans | 3.25 | 1.18 | 75.27 |
| Rhizoctonia cerealis Rhizoctonia cerealis | 4.00 | 0.68 | 94.86 |
| Clover root rot sickle-like bacteria Fusarium avenaceam | 3.62 | 1.09 | 81.09 |
| Sporisorium reilianum | Adopt the turbidity method to measure 70.88 | ||
Annotate: Sporisorium reilianum adopts the turbidity method to measure, and promptly measures the absorbancy of different treatment under the 650nm wavelength respectively, and compares with the space management that does not add soup and to obtain relative inhibition percentage.
2, subtilis (Bacillus subtilis) HL259 CGMCC № 1451 suppresses the pathogenetic bacteria effect detection
With making bacteria suspension behind subtilis (Bacillus subtilis) HL259 CGMCC № 1451 and the target bacterium activation 48h, bacteria suspension is adjusted to 1.5 * 10 respectively with Mike's turbidimetry
9Cfu/ml, absorption 0.1ml target bacterium bacteria suspension is added on the culture medium flat plate to be paved with the coating shovel.Oxford cup (diameter of Oxford cup is 0.85cm) is placed on the substratum, the bacteria suspension that in each Oxford cup, adds 100 μ l, PDA flat board with the sterilized water of adding 100 μ l in the cup of Tianjin is contrast, 3 Oxford cups of every ware, distance evenly, preserve 16h down for 4 ℃, each is handled 3 times and repeats, and measures antibacterial circle diameter behind 28 ℃ of following dark culturing 24-48h.Subtilis (BACILLUSSUBTILIS) HL259 CGMCC No1451 is to as shown in table 3 for the restraining effect of examination pathogenic fungi, and the antibacterial circle diameter of used contrast is 0cm.
1451 couples of table 3. subtilis (Bacillus subtilis) HL259 CGMCC № supply the examination main diseases
The restraining effect of indigenous bacteria
| Strains tested | Three multiple antibacterial circle diameters (cm) | Average antibacterial circle diameter (cm) | ||
| 1 | 2 | 3 | ||
| Potato rot positive Erwinia carotovora | 2.13 | 2.15 | 2.14 | 2.14 |
| Paddy rice bacterial leaf spot Xanthomonas oryzae | 1.97 | 2.03 | 2.07 | 2.02 |
| K27 Agrobacterium rhizogenes | 1.91 | 1.9 | 1.96 | 1.92 |
| Cucumber angle spot Pseudomonas syringae | 0.85 | 0.85 | 0.85 | 0.85 |
| Cotton angle spot Xanthomonas malvacearum | 1.34 | 1.36 | 1.90 | 1.63 |
| Tomato scab Xanthomonas resicatoria | 1.48 | 1.55 | 1.65 | 1.56 |
| Tomato ulcer Clavibacter michiganensis subsp.michiganensis | 1.50 | 1.42 | 1.58 | 1.50 |
| Eggplant Ralstonia solanacearum strain Ralstonia solanacearum | 1.6 | 1.58 | 1.58 | 1.59 |
Detected result shows, 1451 pairs of potato rot positive of subtilis (Bacillus subtilis) HL259 CGMCC №, paddy rice bacterial leaf spot, K27, tomato ulcer, eggplant green grass or young crops are withered, tomato scab, cotton angle spot produce effect, and are invalid to cucumber angle spot.
Present embodiment is measured the antimicrobial spectrum of subtilis (Bacillus subtilis) HL259 CGMCC № 1451 to comprise that the 22 kind of plant pathogenic fungies that four Eumycotinas 13 belong to and the representative pathogens bacterium of 5 important genus are the target bacterium; The result shows that 1451 pairs of other confession examination target germs except that Rhodopseudomonas of subtilis (Bacillus subtilis) HL259 CGMCC № all have obvious suppression growth effect, show that subtilis (Bacillus subtilis) HL259 CGMCC № 1451 has antimicrobial spectrum widely.Wherein mould to the melon and fruit corruption, Phytophthora capsici, glue born of the same parents sickle-like bacteria, rhizoctonia cerealis, clover root rot sickle-like bacteria, verticillium dahliae, cotton-wilt fusarium, the inhibiting rate of Valsa mali and miliary damping-off germ reaches 72.76%～94.86%.
Two, the security of 1451 pairs of crops of subtilis (Bacillus subtilis) HL259 CGMCC № is measured
At first carry out indoor seed germination security and detect, promptly using concentration is 10
8The seed soaking of the subtilis of cfu/ml (Bacillus subtilis) HL259 CGMCC № 1451 bacteria suspensions is observed the seed germination security after handling several staple crop seeds, with the security of clear and definite subtilis (Bacillus subtilis) HL259 CGMCCNo1451, for the field test application provides theoretical foundation in order to seed treatment.The result shows, germinating energy and the percentage of germination and the contrast no significant difference of the tomato after the bacteria suspension seed soaking is handled, capsicum, muskmelon, cucumber, clover, Kidney bean, summer squash, eight kinds of crop seeds of eggplant, subtilis (Bacillus subtilis) HL259CGMCC No1451 seed soaking is handled also has obvious promoter action to Kidney bean percentage of germination, eggplant germinating energy.Further adopt and dip in the root inoculation method, stab inoculation and leaf-cutting inoculation method detect subtilis subtilis (Bacillus subtilis) HL259 CGMCC No1451 under booth or field test condition have no pathogenicity to above eight kinds of crops, for subtilis (Bacillus subtilis) HL259 CGMCC No1451 further test spread aborning lays the foundation.The result shows, inoculate withered subtilis (Bacillus subtilis) HL259 CGMCC No1451 eight kinds of crops upgrowth situation compared with the control, subtilis (Bacillus subtilis) HL259 CGMCC No1451 does not all have pathogenic effects to eight kinds of crops as can be seen, and leaf-cutting inoculation method inoculation subtilis (Bacillus subtilis) HL259 CGMCC No1451 is long to the tomato stem, stem of Bush Redpepper is long and summer squash stem length has promoter action; Stab inoculation inoculation subtilis (Bacillussubtilis) HL259 CGMCC No1451 is long to Cucumis sativus stem, fresh weight has promoter action; Dip in root inoculation method inoculation subtilis (Bacillus subtilis) HL259 CGMCC No1451 Muskmelon stem length, fresh weight and Herba Medicaginis stem length are had promoter action.
Embodiment 3, be the preparation of the microbiobacterial agent of activeconstituents with subtilis (Bacillus subtilis) HL259 CGMCC No1451
1, plain substratum of the suitableeest product and culture condition determines
In order to determine to be suitable for the solid and the liquid nutrient medium of subtilis (Bacillus subtilis) HL259 CGMCC No1451 growth, compared subtilis (Bacillus subtilis) HL259 CGMCCNo1451 at LB, YMA, YEM, PDA, Jin Shi B, sucrose protein culture medium (sucrose 2.0%, peptone 0.5%K
2HPO
40.05%, MgSO
47H
2O 0.025%, distilled water 1000ml, agar 17.0g), YGM (glucose 2.5%, yeast extract paste 2.0g, K
2HPO
40.25g, KH
2PO
40.25g, MgSO
47H
2O 0.1g, MnSO
40.15g, NaCl 0.05g, FeSO
47H
2O 0.005g, agar 17.0g, distilled water 1000ml), the BPY cultivation proterties and the speed of growth on totally 9 kinds of substratum 523,, subtilis (Bacillus subtilis) HL259CGMCC No1451 is through streak inoculation, cultivate 24-48h down at 28-30 ℃, equal energy grown cultures on 9 kinds of culture medium flat plates, show light tan or light canescence respectively, but colony growth there are differences on the different substratum, on YMA, cultivate bacterial ooze shape in early stage, it is round to form bacterium colony, the bacterium colony thickening is opaque gradually, and gauffer is arranged, thalline bleaches, and the bacterium colony expansion is fast; Next measures the variation of growth curve, OD450 value and the pH value of subtilis (Bacillus subtilis) HL259 CGMCC No1451 in the different liqs substratum, carry out analysis-by-synthesis and determine that YMA medium is suitable for liquid and shakes training, shake in the YMA nutrient solution to train to 12h bacterium amount and reached 10
8Cfu/ml, be 24h the period that its bacterium amount peaks, the bacterium amount is 10
10Cfu/ml; Carry out plain substratum of the suitableeest product and the culture condition screening of subtilis (Bacillus subtilis) HL259 CGMCC No1451 subsequently.Test shows that being suitable for the plain substratum of subtilis (Bacillus subtilis) HL259 CGMCC No1451 product is YMA and corn steep liquor substratum (sucrose 5g, Semen Maydis powder 2g, KH
2PO
40.05g, (NH
4)
2SO
40.5g, K
2HPO
40.05g, MgSO
40.025g, CaCO
32g adds water 1000ml).And then studied and comprised and shake Pei Wendu that initial pH, liquid amount and inoculum size etc. shake the selection test of training condition, determine inclined-plane seed (10
8Cfu/ml) with 1～2% (volume ratio) inoculum size to the shaking table triangular flask, at liquid amount is 20～40% (volume ratios), and culture temperature is 28～32 ℃, and initial pH is 6.0, shaking table speed is to shake training 18～24h under 180～220rpm culture condition, and the bacteriostatic action of its meta-bolites is the most remarkable.Further, studied the fermentation condition of seeding tank: seed (10 in the triangular flask to fermentor tank from the fixed bottle condition of shaking
10Cfu/ml) (30～50L), fermented liquid is inoculated into fermentor tank (800～1000L) with 3～5% (volume ratio) inoculum size in the seeding tank to seeding tank with 1～2% (volume ratio) inoculum size.Cultivate 24～48h down for the culture condition of seeding tank and fermentor tank: 25-30 ℃, air flow is 1: 0.5～1: 1.6 (air flow is defined as per minute and feeds the volume of air of fermentor tank and the volume ratio of fermentation cylinder for fermentation liquid), foam killer is that vegetables oil or the quality percentage composition of 1-2% is the organic silicone oil of 0.04-0.1% with the quality percentage composition, and stirring velocity is 200～400rpm; Then bacterium liquid is made the powder-type microbial inoculum with peat composed of rotten mosses absorption, or with the bacterial sediment of the centrifugal acquisition of fermented liquid by 1: 1 mass ratio and micro mist lime carbonate mixed biocontrol fungicide, wherein subtilis (Bacillus subtilis) HL259CGMCC No1451 bacterium is measured more than or equal to 5 * 10
8Cfu/g.
2, the preparation of subtilis (Bacillus subtilis) HL259 CGMCC No1451 microbiobacterial agent (HL259 microbial inoculum)
Subtilis (Bacillus subtilis) HL259 CGMCC No1451, cultivates 24h down in 30 ℃ and carries out actication of culture on YMA medium through streak inoculation; Then with inclined-plane seed (10
8Cfu/ml) inoculum size with 1～2% (volume ratio) is seeded in the YMA nutrient solution of shaking table triangular flask (volume is 1000ml), and liquid amount is 30% (volume ratio), and culture temperature is 28 ℃, and initial pH is 6.0, and shaking table speed is 200rpm, and shaking the training time is 24h; Afterwards with seed in the triangular flask (10
10Cfu/ml) be seeded in the interior corn steep liquor substratum of seeding tank (volume 50L) with 2% (volume ratio) inoculum size, cultivate 24h down for 28 ± 1 ℃, air flow is 1: 1-1: 1.2, and foam killer organic silicone oil 0.04%, stirring velocity is 260rpm; Be inoculated into fermented liquid in the seeding tank in the corn steep liquor nutrient solution of fermentor tank (volume is 1000L) with 5% (volume ratio) inoculum size, culture condition is 28 ± 1 ℃, and air flow is 1: 1-1: 1.2, and foam killer organic silicone oil 0.04%, stirring velocity is 260rpm, cultivates 24h.Bacterium liquid is made the powder-type microbial inoculum with peat composed of rotten mosses absorption, or with the bacterial sediment of the centrifugal acquisition of fermented liquid by 1: 1 mass ratio and micro mist lime carbonate are mixed must biocontrol fungicide, wherein, the subtilis of powder-type microbial inoculum (Bacillussubtilis) HL259 CGMCC No1451 bacterium amount is 5 * 10
9Cfu/g.Further by microscopic examination, means such as plate count detect the living bacteria count in the microbial inoculum, and pH value and assorted bacterium rate are determined the microbial inoculum quality product.The microbial inoculum product quality analysis is to cooperate than turbid, microscopy and three kinds of methods of plate count.Turbidimetry and microscopy are easy, quick, use in the detection of the bacteria containing amount of fermented liquid and purity in fermenting processs such as triangular flask, seeding tank and fermentor tank.Get fermented liquid 500ml during than turbid check, compare with blank liquid nutrient medium, measure the OD650 of fermented liquid, OD650 is higher than at 1.0 o'clock, fermented liquid should dilute, each sample is established 3 repetitions, finds corresponding bacteria containing amount by the OD650 that measures on " bacteria containing amount (cfu/ml)-OD650 " typical curve.Get fermented liquid 500ml during the microscopy thalli morphology, compare with pure strain, smear with sarranine dyeing, is examined under a microscope thalli morphology, compares with pure strain.When final final product quality check, adopt colony counting method.Detected result shows that the living bacteria count of powder-type biocontrol fungicide is 8 * 10
9Cfu/g, pH value 7.2, assorted bacterium rate 0%.
The effect of embodiment 4, subtilis (Bacillus subtilis) HL259 CGMCC No1451 microbial inoculum control watermelon damping-off, samping off and blight
Damping-off, samping off, blight are main diseases in the watermelon growing process, and present embodiment is measured subtilis (Bacillus subtilis) HL259 CGMCC No1451 microbial inoculum to the influence of watermelon (Xing Nongyi number) seedling rate, growth indexes and to the prevention effect of seedling blight, samping off and middle and later periods blight by field test.Concrete grammar is as follows:
For reagent product: subtilis (Bacillus subtilis) the HL259CGMCC No1451 powder-type microbial inoculum of embodiment 3 steps 2 preparation, contrast No. 1, biocontrol fungicide (available from Soil Fertilizer Working Station, Beijing, activeconstituents is a waxy Bacillus, and living bacteria count is 10
8Cfu/g), No. 2 (available from Soil Fertilizer Working Station, Beijing, activeconstituents is a subtilis to the contrast biocontrol fungicide, and living bacteria count is 10
8Cfu/g).
7 processing of this test, 4 repetitions amount to 28 sub-districts.Random alignment.
Handle 1: 40 concentration of 1:HL259 microbial inoculum and be used for the watermelon seed seed dressing.
Handle 1: 50 concentration of 2:HL259 microbial inoculum and be used for each son seed dressing of watermelon.
Handle 1: 60 concentration of 3:HL259 microbial inoculum and be used for each son seed dressing of watermelon.
Handle 4: the seed dressing in No. 11: 50 of contrast biocontrol fungicide.
Handle 5: the seed dressing in No. 21: 50 of contrast biocontrol fungicide.
Handle the seed dressing in 1: 200 of 6:50% thiram wettable powder.
Handle 7: without fungicide-treated seed (contrast)
Every sub-district area 46.8M
2, spacing in the rows * line-spacing=36cm * 65cm.Experimental field soil is silty loam.Organic (%): 1.2828 alkali-hydrolyzable nitrogens: 93.56mg/kg rapid available phosphorus: 55.95mg/kg available potassium: 69.2mg/kgpH value: 8.09.Preceding crop is a watermelon.
A few days ago according to seed dressing or seed dressing pharmacopoeia ratio, respectively that each sub-district is required medicament and seed are mixed in the plastics bag, adopt manual shake mode in sowing, with medicament evenly wrap by or stick to seed-coat.Adopt direct-seeding to sow.After planting 7 days investigation seedling rates, random sampling 4 points in every sub-district are fixed 5 strains at every, and each is handled and investigates 80 strains altogether.Sow random sampling 20 strain watermelon seedling in every sub-district after 20 days, investigation and record stem are thick, climing length, and blade is long, leaf is wide, main growth indexes such as root length.Each handle that the sub-district medicament is emerged to watermelon and the watermelon growing index influence the result shown in table 4 and table 5, show that the HL259 microbial inoculum handles watermelon seed with 1: 40 and the seed dressing of 1: 50 concentration, the promoter action of watermelon seedling rate and growth indexes all is better than other processing.
Table 4. different treatment sub-district watermelon seedling seedling rate (%) investigation result
| 3 | 100.0 | 96.3 | 100.0 | 86.7 | 83.9 | 89.7 | 83.9 |
| 4 | 100.0 | 96.3 | 92.6 | 100.0 | 92.9 | 96.3 | 96.3 |
| On average | 100.0 | 97.2 | 95.3 | 94.8 | 93.2 | 94.6 | 89.3 |
Table 5. different treatment sub-district watermelon growing index investigation result
| 7 | 1 | 0.70 | 74.40 | 14.83 | 16.49 | 21.50 |
| 2 | 0.71 | 72.80 | 14.53 | 16.38 | 20.68 | |
| 3 | 0.68 | 70.50 | 14.30 | 17.05 | 25.45 | |
| 4 | 0.66 | 66.30 | 14.50 | 16.55 | 24.15 | |
| On average | 0.69 | 70.90 | 14.45 | 16.60 | 22.95 |
Sow after 21 days, random searching 40 strains of every sub-district, the dead strain number of record morbidity calculates the result that prevents and treats that watermelon founds withered and samping off compared with the control.Sow after 60 days, every sub-district random sampling 20 strains, the record diseased plant rate, and, calculate the prevention effect of blight with disease index by following grade scale investigation state of an illness radix.
Grade scale is as follows:
0 grade-plant strain growth is normal.
1 grade-diseased plant is shorter slightly than normal melon seedling, cuts open cauline bundle flavescence brown, and melon seedling produces and is suppressed.
2 grades-diseased plant is shorter slightly than normal melon seedling, cuts open cauline bundle flavescence brown, sooner or later acts normally, and noon, melon seedling was wilted, and influenced watermelon production.
3 grades-diseased plant obviously shortens than normal melon seedling, and it is withered to wilt, and extracts the disease seedling and observes root browning look, and watermelon loses commodity value.
The HL259 microbial inoculum is prevented and treated result such as table 6 to the upright withered and samping off of watermelon, and the HL259 microbial inoculum is handled watermelon seed with 1: 40,1: 50 and concentration seed dressing in 1: 60, and the watermelon samping off is had the better prevention effect, is respectively 81.66%, 77.08% and 72.49%; The damping-off preventive effect is respectively 88.89%, 88.89% and 72.22%.The HL259 microbial inoculum is to the prevention effect such as the table 6 of blight, and protection effect is respectively 91.24%, 83.00% and 75.95%, is better than other processing.
Adopt Duncan ' s multiple range test method that samping off, damping-off and blight prevention effect have been carried out analyzing relatively, to estimate the significance of difference of preventing and treating three kinds of main diseases of watermelon in this test between the chemicals treatment.ANOVA showed significant, three kinds of different seed dressing concentration of HL259 microbial inoculum are not remarkable to the prevention effect difference of samping off and damping-off; Prevention effect to blight all shows significant difference on 5% and 1% level, and also exists bigger difference with other each processing on 5% and 1% level.
Watermelon growing is normal during the whole test, does not find poisoning.Illustrate that the HL259 microbial inoculum handles watermelon seed to watermelon growing safety with 1: 40 and the seed dressing of 1: 50 concentration.Do not find the detrimentally affect of reagent agent in the investigation, illustrate that this medicament is environmentally safe non-target organism.The pesticide-seeds ratio of seed seed dressing is 1: the 40-50 prevention effect is better.
The control of table 6.HL259 microbial inoculum watermelon blight, samping off and damping-off test-results
| Handle | Repeat | Investigation strain number | Blight | Investigation strain number | Samping off | Damping-off | |||||||||
| 0 grade | 1 grade | 2 grades | 3 grades | Disease index | Prevention effect (%) | Morbidity strain number (strain) | Sickness rate (%) | Prevention effect (%) | Morbidity strain number (strain) | Sickness rate (%) | Prevention effect (%) | ||||
| 1 | 1 | 20 | 19 | 1 | 0 | 0 | 0.56 | 94.40 | 40 | 2 | 5.00 | 63.32 | 0 | 0.00 | 100.00 |
| 2 | 20 | 19 | 1 | 0 | 0 | 0.56 | 94.4 | 40 | 1 | 2.50 | 81.66 | 0 | 0.00 | 100.00 | |
| 3 | 20 | 18 | 2 | 0 | 0 | 1.11 | 89.49 | 40 | 1 | 2.50 | 81.66 | 2 | 5.00 | 55.56 | |
| 4 | 20 | 19 | 0 | 1 | 0 | 1.11 | 86.67 | 40 | 0 | 0.00 | 100.0 0 | 0 | 0.00 | 100.00 | |
| On average | 20 | 0.83 | 91.24 | 40 | - | 2.50 | 81.66 | - | 1.25 | 88.89 | |||||
| 2 | 1 | 20 | 18 | 2 | 0 | 0 | 1.11 | 88.9 | 40 | 1 | 2.50 | 81.66 | 1 | 2.50 | 77.78 |
| 2 | 20 | 18 | 1 | 1 | 0 | 1.67 | 84.19 | 40 | 1 | 2.50 | 81.66 | 1 | 2.50 | 77.78 | |
| 3 | 20 | 17 | 3 | 0 | 0 | 2.22 | 78.98 | 40 | 1 | 2.50 | 81.66 | 0 | 0.00 | 100.00 | |
| 4 | 20 | 18 | 1 | 1 | 0 | 1.67 | 79.95 | 40 | 2 | 5.00 | 63.32 | 0 | 0.00 | 100.00 | |
| On average | 20 | 1.67 | 83.00 | 40 | - | 3.13 | 77.08 | - | 1.25 | 88.89 | |||||
| 3 | 1 | 20 | 17 | 2 | 1 | 0 | 2.22 | 77.80 | 40 | 1 | 2.50 | 81.66 | 3 | 7.50 | 33.33 |
| 2 | 20 | 16 | 3 | 1 | 0 | 2.78 | 73.67 | 40 | 3 | 7.50 | 63.32 | 1 | 2.50 | 77.78 | |
| 3 | 20 | 17 | 2 | 1 | 0 | 2.2 | 78.98 | 40 | 2 | 5.00 | 63.32 | 1 | 2.50 | 77.78 | |
| 4 | 20 | 17 | 2 | 0 | 1 | 2.22 | 73.35 | 40 | 1 | 2.50 | 81.66 | 0 | 0.00 | 100.00 | |
| On average | 20 | 2.34 | 75.95 | 40 | - | 4.38 | 72.49 | - | 3.13 | 72.22 | |||||
| 4 | 1 | 20 | 16 | 2 | 2 | 0 | 333 | 66.7 | 40 | 2 | 5.00 | 63.32 | 2 | 5.00 | 55.56 |
| 2 | 20 | 16 | 2 | 1 | 1 | 3.89 | 63.16 | 40 | 2 | 5.00 | 63.32 | 4 | 10.00 | 11.11 | |
| 3 | 20 | 15 | 3 | 2 | 0 | 5.00 | 52.65 | 40 | 3 | 7.50 | 44.97 | 1 | 2.50 | 77.78 | |
| 4 | 20 | 15 | 3 | 2 | 0 | 3.89 | 53.30 | 40 | 1 | 2.50 | 81.66 | 2 | 5.00 | 55.56 | |
| On average | 20 | 4.03 | 58.95 | 40 | - | 5.00 | 63.32 | - | 5.63 | 50.00 | |||||
| 5 | 1 | 20 | 15 | 2 | 2 | 1 | 5.0 | 50.00 | 40 | 2 | 5.00 | 63.32 | 0 | 0.00 | 100.00 |
| 2 | 20 | 15 | 3 | 1 | 1 | 4.44 | 57.95 | 40 | 4 | 10.00 | 26.63 | 1 | 2.50 | 77.78 | |
| 3 | 20 | 11 | 5 | 4 | 0 | 7.22 | 31.63 | 40 | 3 | 7.50 | 44.97 | 4 | 10.00 | 11.11 | |
| 4 | 20 | 14 | 3 | 2 | 1 | 5.56 | 33.25 | 40 | 0 | 0.00 | 100.0 0 | 1 | 2.50 | 77.78 | |
| On average | 20 | 5.56 | 43.21 | 40 | - | 5.63 | 58.73 | - | 3.75 | 66.67 | |||||
| 6 | 1 | 20 | 14 | 3 | 1 | 2 | 6.11 | 38.90 | 40 | 0 | 0.00 | 100.0 0 | 2 | 5.00 | 55.56 |
| 2 | 20 | 12 | 3 | 3 | 2 | 8.33 | 21.12 | 40 | 3 | 7.50 | 44.97 | 2 | 5.00 | 55.56 | |
| 3 | 20 | 12 | 4 | 3 | 1 | 7.22 | 31.63 | 40 | 4 | 10.00 | 44.97 | 1 | 2.50 | 77.78 | |
| 4 | 20 | 13 | 4 | 2 | 1 | 6.11 | 26.65 | 40 | 3 | 7.50 | 63.32 | 2 | 5.00 | 55.56 | |
| On average | 20 | 6.94 | 29.58 | 40 | - | 6.25 | 63.32 | - | 4.38 | 61.12 | |||||
| 7 | 1 | 20 | 11 | 3 | 3 | 3 | 10.00 | - | 40 | 5 | 12.50 | - | 4 | 10.00 | - |
| 2 | 20 | 11 | 2 | 4 | 3 | 10.56 | - | 40 | 6 | 15.00 | - | 3 | 7.50 | - | |
| 3 | 20 | 10 | 3 | 5 | 2 | 10.56 | - | 40 | 4 | 10.00 | - | 6 | 15.00 | - | |
| 4 | 20 | 12 | 3 | 3 | 2 | 8.33 | - | 40 | 7 | 17.50 | - | 5 | 12.50 | - | |
| On average | 20 | - | - | 40 | - | 13.63 | - | - | 11.25 | - | |||||
The effect of embodiment 5, the climing rot of subtilis (Bacillus subtilis) HL259 CGMCC No.1451 microbial inoculum control cucumber
The climing rot of cucumber is the important disease during cucumber produces, the main harm basal part of stem, nascent oil stain shape scab, the surface bleaches, and slight crack occurs, overflows jelly sometimes, this disease from the autumn to the vernal aspect to low temperature damage to crops in period, rapid spread under especially many wet conditions.Present embodiment is measured the prevention effect of HL259 microbial inoculum to Tianjin spring No. 2 (Tianjin cucumber institute) climing rots of cucumber by field test.Concrete grammar is as follows:
For reagent product: subtilis (Baciillus subtilis) the HL259CGMCC No.1451 biocontrol fungicide of embodiment 3 steps 2 preparation, contrast No. 1, biocontrol fungicide (available from Soil Fertilizer Working Station, Beijing, activeconstituents is a waxy Bacillus, and living bacteria count is 10
8Cfu/g).
5 processing are established in this test, and 4 repetitions amount to 20 sub-districts.Adopt district's group arrangement at random.Each booth sub-district area 15.5m
2, spacing in the rows * line-spacing=30cm * 90cm.Experimental field soil is silty loam.Preceding crop is a cucumber.
1: 20 dilution proportion of processing 1:HL259 microbial inoculum is used for cucumber and sprays root.
1: 10 dilution proportion of processing 2:HL259 microbial inoculum is used for cucumber and sprays root.
1: 5 dilution proportion of processing 3:HL259 microbial inoculum is used for cucumber and sprays root.
Handle 4: No. 11: 5 dilution proportion of contrast biocontrol fungicide sprays root.
Handle 5: the clear water contrast
Investigation state of an illness radix before the dispenser, 20d investigation dispenser last time result after the dispenser, calculated population preventive effect.The preventive effect investigation of climing rot is with reference to the method for relevant in the medicine inspecting institute of the Ministry of Agriculture " pesticide field efficacy medicine test criterion " (two) " control cucurbits fusarium wilt test of pesticide effectiveness criterion ".Adopt the sub-district generaI investigation in the test, the record diseased plant rate is calculated prevention effect with diseased plant rate.
No. 1,3 different weaker concns of HL259 microbial inoculum and contrast biocontrol fungicide are as shown in table 7 to the climing rot prevention effect of cucumber, the HL259 microbial inoculum sprays Radix Cucumidis sativi stem according to 1: 20,1: 10 and 1: 5 dilution proportion dosage respectively, and the preventive effect of the climing rot of cucumber is respectively 53.31%, 60.01% and 86.60%; The contrast biocontrol fungicide is 53.24% with 1: 5 dilution proportion dosage spraying back preventive effect No. 1.The HL259 microbial inoculum is handled (handle 4) significantly better than No. 1, contrast biocontrol fungicide with 1: 5 dilution proportion according to the preventive effect of 1: 10 and 1: 5 respectively as can be seen from the results.The HL259 microbial inoculum preventive effect according to 1: 20 dilution dosage respectively is suitable with processing 4.
Adopt Duncan ' s multiple range test method that climing rot prevention effect has been carried out analyzing relatively, prevent and treat the significance of difference of the climing rot of cucumber for No. 1 to estimate HL259 microbial inoculum various dose in this test and contrast contrast biocontrol fungicide.ANOVA showed significant, handle 3 (1: 5) on 5% level and other processing have significant difference, on 1% level, handle 3 (1: 5) and handle 2 (1: 10) difference not remarkable, but and contrast between No. 1, the biocontrol fungicide and have significant difference.Cucumber growth is normal during the whole test, does not find poisoning.Illustrate that the HL259 microbial inoculum sprays processing cucumber plant root to plant growth safety for examination concentration.Do not find the detrimentally affect of reagent agent in the investigation, illustrate that this medicament is environmentally safe non-target organism.The HL259 microbial inoculum carries out rhizome portion soil at the climing rot their early stage of cucumber and sprays development and the propagation that this disease can be effectively controlled in processing, it is the beneficial agents of the climing rot of control cucumber, the crop normal growth is not had bad harm, and prevention effect significantly is better than routine administration kind and application dose.The HL259 microbial inoculum can spray Radix Cucumidis sativi stem soil according to 1: 10 and 1: 5 Dilution ratio dosage.
That shows the 7.HL259 microbial inoculum sprays the climing rot result of Radix Cucumidis sativi stem soil control cucumber with different Dilution ratio dosage
| Handle | Repeat | Total strain number (strain) | Diseased plant number (strain) before the dispenser | Diseased plant rate % | Diseased plant number (strain) after the dispenser | Diseased plant rate % | Protection effect % |
| ck | 1 | 72 | 1 | 1.39 | 5 | 6.94 | - |
| 2 | 72 | 5 | 6.94 | 7 | 9.72 | - | |
| 3 | 72 | 4 | 5.56 | 6 | 8.33 | - | |
| 4 | 72 | 1 | 1.39 | 3 | 4.17 | - | |
| On average | 72 | 2.75 | 3.82 | 5.25 | 7.29 | - | |
| 1 | 1 | 108 | 1 | 0.93 | 3 | 2.78 | 46.68 |
| 2 | 108 | 4 | 3.70 | 6 | 5.56 | 46.40 | |
| 3 | 108 | 3 | 2.78 | 5 | 4.63 | 46.68 | |
| 4 | 108 | 3 | 2.78 | 4 | 3.70 | 73.48 | |
| On average | 99 | 2 | 2.55 | 4.50 | 4.17 | 53.31 | |
| 2 | 1 | 108 | 3 | 2.78 | 5 | 4.63 | 46.68 |
| 2 | 108 | 1 | 0.93 | 3 | 2.78 | 46.68 | |
| 3 | 108 | 3 | 2.78 | 4 | 3.70 | 73.48 | |
| 4 | 108 | 0 | 0.00 | 1 | 0.93 | 73.20 | |
| On average | 108 | 1.75 | 1.62 | 3.50 | 3.01 | 60.01 | |
| 3 | 1 | 108 | 2 | 1.85 | 3 | 2.78 | 73.20 |
| 2 | 108 | 1 | 0.93 | 1 | 0.93 | 100.00 | |
| 3 | 108 | 0 | 0.00 | 1 | 0.93 | 73.20 | |
| 4 | 108 | 0 | 0.00 | 0.00 | 0.00 | 100.00 | |
| On average | 108 | 0.75 | 0.70 | 1.25 | 1.16 | 86.60 | |
| 4 | 1 | 108 | 2 | 1.85 | 4 | 3.70 | 46.68 |
| 2 | 108 | 2 | 1.85 | 3 | 2.78 | 73.20 | |
| 3 | 108 | 4 | 3.70 | 6 | 5.56 | 46.40 | |
| 4 | 108 | 1 | 0.93 | 3 | 2.78 | 46.68 | |
| On average | 108 | 2.25 | 2.08 | 4.00 | 3.71 | 53.24 |
The effect of embodiment 6, HL259 microbial inoculum control Phytophthora capsici disease
Capsicum epidemic disease is a kind of destructive disease during capsicum produces, in China south and the north distribution is arranged all, at greenhouse and booth and open country generation is arranged all, especially the pepper seedling of booth transplanting is injured the most serious, often cause the seedling phase dead in flakes, become the strain phase to be injured and to cause fallen leaves and whole strain wilting to wither, need to provide effective prophylactico-therapeutic measures in the production.Present embodiment is measured the HL259 microbial inoculum to capital grind capsicum No. 3, the prevention effect of eggplant door pimento capsicum epidemic disease by field test.Concrete grammar is as follows:
For reagent product: the HL259 powder-type microbial inoculum of embodiment 3 steps 2 preparation, No. 1 (available from Soil Fertilizer Working Station, Beijing, activeconstituents is a waxy Bacillus, and living bacteria count is 10 to contrast biocontrol fungicide
8Cfu/g).
5 processing are established in this test, and 4 repetitions amount to 20 sub-districts.Adopt district's group arrangement at random.Each booth sub-district area 15.5m
2, spacing in the rows * line-spacing=30cm * 90cm.Experimental field soil is silty loam.Preceding crop is a capsicum.
Handle 1: 50 dilution proportion of 1:HL259 microbial inoculum and be used for capsicum blade spraying and root irrigation;
Handle 1: 20 dilution proportion of 2:HL259 microbial inoculum and be used for capsicum blade spraying and root irrigation;
Handle 1: 10 dilution proportion of 3:HL259 microbial inoculum and be used for capsicum blade spraying and root irrigation;
Handle 4: No. 11: 50 dilution proportion of contrast biocontrol fungicide is used for capsicum blade spraying and root irrigation;
Handle 5: No. 11: 20 dilution proportion of contrast biocontrol fungicide is used for capsicum blade spraying and root irrigation;
Handle 6: No. 11: 10 dilution proportion of contrast biocontrol fungicide is used for capsicum blade spraying and root irrigation.
Investigation state of an illness radix before the dispenser; 15 days investigation dispenser last time results after the dispenser, the calculated population preventive effect.The preventive effect investigation of eqpidemic disease is with reference to the method for relevant " preventing and treating melon eqpidemic disease test of pesticide effectiveness criterion " in the medicine inspecting institute of the Ministry of Agriculture " pesticide field efficacy medicine test criterion " (two).Adopt 5 sampling methods in the test, the record disease index calculates prevention effect.Wherein, the disease index stage division is: 0 grade, and healthy no disease; 1 grade, overground part only leaf, fruit has scab; 3 grades, terrestrial stem, branch have the brown rot spot; 5 grades, basal part of stem has the brown rot spot; 7 grades, terrestrial stem, branch and basal part of stem all have the brown rot spot; 9 grades, complete stool is withered.
The prevention effect that 3 different weaker concns of HL259 microbial inoculum and No. 1 different weaker concns of contrast biocontrol fungicide are ground No. 3 capsicum epidemic diseases of capsicum to the capital is as shown in table 8, the HL259 microbial inoculum is irritated the root dispenser according to 1: 50,1: 20 and 1: 10 dilution proportion dosage respectively, and the preventive effect of capsicum epidemic disease is respectively 45.74%, 73.46% and 83.46%; No. 1, biocontrol fungicide of contrast irritates with 1: 50,1: 20 and 1: 10 dilution proportion dosage that preventive effect is 37.60%, 56.96% and 58.70% behind the root; Illustrate that the HL259 microbial inoculum handles for No. 1 significantly better than the contrast biocontrol fungicide according to the filling root dispenser preventive effect of 1: 20 and 1: 10 respectively.The HL259 microbial inoculum is respectively according to 1: 50,1: 20 and 1: 10 dilution proportion dosage spraying blade, and the preventive effect of capsicum epidemic disease is respectively 56.33%, 66.53% and 74.48%; No. 1, biocontrol fungicide of contrast is 42.64%, 52.25% and 59.97% with preventive effect behind 1: 50,1: 20 and 1: the 10 dilution proportion dosage spraying blade; Illustrate that the HL259 microbial inoculum handles for No. 1 significantly better than the contrast biocontrol fungicide according to the spray pesticide preventive effect of 1: 20 and 1: 10 respectively.
3 different weaker concns of HL259 microbial inoculum and No. 1 different weaker concns of contrast biocontrol fungicide are as shown in table 9 to the prevention effect of eggplant door pimento capsicum epidemic disease, the HL259 microbial inoculum is irritated the root dispenser according to 1: 50,1: 20 and 1: 10 dilution proportion dosage respectively, and the preventive effect of capsicum epidemic disease is respectively 77.32%, 81.58% and 86.35%; No. 1, biocontrol fungicide of contrast irritates with 1: 50,1: 20 and 1: 10 dilution proportion dosage that preventive effect is 49.19%, 54.17%, 57.47% behind the root.Illustrate that the HL259 microbial inoculum handles for No. 1 significantly better than the contrast biocontrol fungicide according to the filling root dispenser preventive effect of 1: 50,1: 20 and 1: 10 respectively.The HL259 microbial inoculum is respectively according to 1: 50,1: 20 and 1: 10 dilution proportion dosage spraying blade, and the preventive effect of capsicum epidemic disease is respectively 53.85%, 59.52% and 70.87%; No. 1, biocontrol fungicide of contrast is 39.54%, 51.44% and 47.52% with preventive effect behind 1: 50,1: 20 and 1: the 10 dilution proportion dosage spraying blade.Illustrate that the HL259 microbial inoculum handles for No. 1 significantly better than the contrast biocontrol fungicide according to the preventive effect of 1: 50,1: 20 and 1: 10 respectively.
Chili growth is normal during the whole test, does not find poisoning.Illustrate that the HL259 microbial inoculum sprays processing pepper plant blade or irritates root to plant growth safety for examination concentration.Do not find the detrimentally affect of reagent agent in the investigation, illustrate that this medicament is environmentally safe non-target organism.The HL259 microbial inoculum carries out the spraying of root root irrigation and leaf portion at the capsicum epidemic disease their early stage and handles development and the propagation that can effectively control this disease, it is the beneficial agents of control capsicum epidemic disease, the crop normal growth is not had bad harm, and prevention effect significantly is better than No. 1, contrast microbial inoculum.To be the HL259 microbial inoculum spray capsicum, root irrigation rhizome portion soil according to 1: 20 and 1: 10 Dilution ratio dosage to use-pattern preferably.
Table 8.HL259 microbial inoculum grinds the prevention effect of capsicum No. 3 to the capital
| Handle | Spray pesticide | Irritate the root dispenser | |||
| Disease index (%) | Prevention effect (%) | Disease index (%) | Prevention effect (%) | ||
| CK | 35.50 | ||||
| HL259 microbial inoculum biocontrol microorganisms 1 (cfu/mL) | 1∶50 1∶20 1∶10 1∶50 1∶20 1∶10 | 14.50 11.88 9.06 20.36 16.95 14.21 | 56.33 66.53 74.48 42.64 52.25 59.97 | 19.26 9.42 5.87 22.15 15.28 14.66 | 45.74 73.46 83.46 37.60 56.96 58.70 |
Table 9.HL259 microbial inoculum is to the prevention effect of eggplant door pimento
| Handle | Spray pesticide | Irritate the root dispenser | |||
| Disease index (%) | Prevention effect (%) | Disease index (%) | Prevention effect (%) | ||
| CK | 43.60 | ||||
| The HL259 microbial inoculum | 1∶50 1∶20 | 20.12 17.65 | 53.85 59.52 | 9.89 8.03 | 77.32 81.58 |
| Biocontrol microorganisms 1 (cfu/mL) | 1∶10 1∶50 1∶20 1∶10 | 12.70 26.36 21.17 22.88 | 70.87 39.54 51.44 47.52 | 5.95 22.15 19.98 18.54 | 86.35 49.19 54.17 57.47 |
Sequence table
<160>1
<210>1
<211>1448
<212>DNA
＜213〉subtilis (Bacillus subtilis)
<400>1
tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg 60
acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg 120
gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg cttcggctac 180
cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360
gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg 420
ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc taactacgtg 480
ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 540
ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600
ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720
gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780
gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaaa cgcattaagc 840
actcccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc 900
acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960
catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt 1260
ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320
gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380
agagtttgta acacccgaag tcggtgaggt aaccttttag gagccagccg ccgaaggtgg 1440
gacagatg 1448
Claims (10)
1, subtilis (Bacillus subtilis) HL259 CGMCC No.1451.
2, a kind of microbiobacterial agent, its activeconstituents are subtilis (Bacillus subtilis) HL259 CGMCC No.1451.
3, a kind of method for preparing the described microbiobacterial agent of claim 2, be subtilis (Bacillus subtilis) the HL259 CGMCC No.1451 that in YMA or corn steep liquor substratum, ferments, the fermented liquid that obtains mixed obtaining microbiobacterial agent with matrix.
4, method according to claim 3 is characterized in that: contain sucrose 5g in the described corn steep liquor substratum, Semen Maydis powder 2g, KH
2PO
40.05g, (NH
4)
2SO
40.5g, K
2HPO
40.05g, MgSO
40.025g, CaCO
32g adds water 1000ml.
5, according to claim 3 or 4 described methods, it is characterized in that: in the described fermentation condition, culture temperature is 25-30 ℃, and air flow is 1: 0.5-1: 1.6, and stirring velocity is 200-400rpm, incubation time is 24-48h; Described air flow is that per minute feeds the volume of air of fermentor tank and the volume ratio of fermentation cylinder for fermentation liquid.
6, method according to claim 5 is characterized in that: described culture temperature is 28-30 ℃, and air flow is 1: 1-1: 1.2, and stirring velocity is 250-300rpm, incubation time is 24-30h.
7, method according to claim 5 is characterized in that: in the described method, add vegetables oil or organic silicone oil during fermentation as foam killer.
8, method according to claim 7 is characterized in that: the quality percentage composition of described vegetables oil is 1-2%; The quality percentage composition of described organic silicone oil is 0.04-01%.
9, method according to claim 3 is characterized in that: described matrix is the peat composed of rotten mosses, micro mist lime carbonate or attapulgite or wilkinite.
10, method according to claim 3 is characterized in that: the viable count of subtilis in the described microbiobacterial agent (Bacillus subtilis) HL259 CGMCC No.1451 is 5 * 10
9Cfu/g, the pH value is 6.0-7.8.
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