CN1554744A - Bacillus subtilis strain - Google Patents

Abstract

The present invention relates to a kind of Bucillus subtilis strain, belongs to the field of microbiological technology. The Bucillus subtilis strain, named as Bucillus subtilis B9601-Y2, is preserved in the preservation number of CGMCC No. 0954. Bucillus subtilis B9601-Y of the present invention may be used in preventing various plant diseases and pollution to edible fungus, may be used in promoting plant growth, controlling soil transmitted plant pathogeny and preserving fresh cut China rose, and has long shelf period, high resistance and no environmental pollution.

Description

A kind of bacillus subtilis strain

Technical field

The invention belongs to microbial technology field, but mainly be a kind of bacterial isolates that wide spectrum control dissimilar diseases of plant and edible mushrooms pollute and can promote plant-growth.

Background technology

Chemical pesticide is the valuable source of controlling plant diseases, but owing to too much, mistake are used, the anxiety of the residual toxicity of causing, environmental pollution and ecosystem destruction is arranged.Therefore, be badly in need of to seek a kind of can alternate, the safer and more effective method of preventing and treating.Have preventive and therapeutic effect although a lot of biological prevention and control agents have been proved, only have a few to be applied to produce, major part still is in research and exploratory stage.The biological prevention and control agent that is applied to produce, every kind of biological prevention and control agent generally can only prevent the disease of a few type, does not have the wide spectrum prevention and control capability, promotes the DeGrain of plant-growth.Edible mushrooms is a big industry of China, but the culture material of edible mushrooms is seriously polluted by filamentous funguss such as Trichodermas in producing, in order to prevent to pollute, mainly rely on the high-temperature sterilization of culture material in the production and when preparing material, add sterilant, so both increased cost, may in edible fungus, contain sterilant again, so be necessary to seek a kind of safer, effective, economic method.Yet, do not see the report of the biological prevention and control agent that is useful on edible mushrooms as yet.

Summary of the invention

Purpose of the present invention provides a kind of just and can be used for preventing and treating dissimilar diseases of plant and edible mushrooms pollution in order to overcome above-mentioned the deficiencies in the prior art, and can promote plant-growth, the bacillus subtilis strain of taking root effectively.

The objective of the invention is to be achieved through the following technical solutions.

A kind of bacillus subtilis strain, called after subtilis (Bacillus subtilis) B9601-Y2, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on June 18th, 2003, preserving number: CGMCC No.0954.

B9601-Y2 bacterium colony circle of the present invention, weak tea brown, bacterium colony central color are slightly dark, protuberance, smooth surface; Thalli morphology is shaft-like, end is round, and Gram-positive produces gemma, peritrichous (electromicroscopic photograph), tool mobility, and sporocyst does not expand, and brood cell's oval, each cell only produce a brood cell, are positioned at somatic cells central authorities, 0.66 * 2.10 (μ m).

B9601-Y2 strain culturing mode of the present invention is:

(1), liquid culture: inoculation in the LB substratum, under 35 ℃ of temperature, was cultivated on the shaking table of rotating speed 120r/m 2～3 days;

(2), solid culture: inoculation in the PSA substratum, was cultivated 3～4 days in 37 ℃ of incubators.

B9601-Y2 of the present invention can the secretory protein antibacterial substance, to rice blast fungus, Rhizoctonia solani Kuhn, fusarium moniliforme, ustilaginoidea virens, the root rotof flax bacterium, gaeumannomyces graminis, the wheat snow mold bacterium, southern corn leaf blight, the broad bean pine root fungus, phytophthora infestans, cotton-wilt fusarium, cotton boll rot pathogens, the cotton anthracnose bacterium, Sclerotinia sclerotiorum, cucumber anthracnose, the pepper anthracnose bacterium, phytophthora capsici, tobacco brown spot pathogen, tobacco black shank bacterium, the ring rot of apple bacterium, the apple scab bacterium, the vanilla pine root fungus, the husky Moindawang of good forage pine root fungus, functions on common pollutant bacteria-trichoderma viride in the edible mushrooms, 24 kinds of important pathogenic fungies on important crop such as trichoderma harzianum and the edible mushrooms have very strong restraining effect, its mechanism of action is to decompose the cell walls of fungi, the cracking plasmalemma exosmoses pathogenic fungi tenuigenin and death.

Description of drawings

Fig. 1 is the form photo of subtilis B9601-Y2 under electron microscope (12000 *).

Embodiment

Be separated to subtilis B9601-Y2 from Shandong Province's Qingzhou City wheatland soil, two kinds of liquid nutrient mediums are adopted in the cultivation of bacterial strain: (1), PS substratum: fresh potato 200g fries in shallow oil juice, and sucrose 15g adds water 1000ml sterilization; (2), the LB substratum: Tryptones 10g, yeast extract 5g, sodium-chlor 10g, water 1000m, it is standby to sterilize.According to the substratum difference, the training method of biocontrol strain can be selected two kinds for use: (1), liquid culture: biocontrol microorganisms is inoculated in LB or the PS substratum, under 35 ℃ of temperature, cultivates 2～3d on the shaking table of rotating speed 120r/m; (2), solid culture: be inoculated in the PSA substratum (fresh potato 200g fries in shallow oil juice, sucrose 15g, agar 17g adds water 1000ml sterilization), in 37 ℃ of incubators, cultivated 3～4 days.The preparation method of bacterium liquid is that the bacterium liquid with liquid culture is diluted to desired concn with clear water, is used for control of plant disease or promotes plant establishment, growth and cutflower preservation (thalline of solid culture is used for the preservation of bacterial classification etc.).B9601-Y2 bacterium colony circle, weak tea brown, bacterium colony central color are slightly dark, protuberance, smooth surface; Thalli morphology is shaft-like, end is round, and Gram-positive produces gemma, peritrichous (electromicroscopic photograph), tool mobility, and sporocyst does not expand, and brood cell's oval, each cell only produce a brood cell, are positioned at somatic cells central authorities, 0.66 * 2.10 (μ m).Fig. 1 is the form photo figure of subtilis B9601-Y2 under electron microscope (12000 *).

The protection effect experiment: biocontrol strain B9601-Y2 is obtained from the laboratory separation by the contriver, is separated from the laboratory with other people by the contriver with saprophytic filamentous fungus (seeing Table 1) for the pathogenic fungi that tries and is so kind as to give.The PSA plate culture medium is adopted in the cultivation of indicator strain.Adopt dull and stereotyped face-off culture method: the indicator bacterium cake that to inoculate 4 diameters on the dull and stereotyped diagonal lines of culture dish be 0.6cm equidistantly, connect biocontrol strain B9601-Y2 as processing in dull and stereotyped central authorities, be contrast not deliver a child fungi-proofing.Cultivated 3～4 days in 28 ℃ of incubators inoculation back, near an end of dull and stereotyped central authorities to the ultimate range of an end of contiguous plate edge diameter as colony growth.Measure the 3rd day of inoculation, what bacterium colony did not grow does not measure, and bacterium colony length is to a time-out, is considered as covering with stopping to measure.Every processing repeats for 3 times, has respectively repeated 4 observed values.Table 1: downtrod filamentous fungus

Bacterial strain formal name used at school bacterial strain formal name used at school

Fusarium moniliforme Gibberella fujikuroi vanilla pine root fungus F.oxysporum

Rhizoctonia solani Kuhn Rhizoctonia solani broad bean pine root fungus F.solani

The husky Moindawang pine root fungus Sda1 F.solani (sda1) of ustilaginoidea virens Ustilaginoidea virens

The husky Moindawang pine root fungus Sda2 F.oxysporum (sda2) of Pyricularia oryzae Magnaporthe grisea

Root rotof flax bacterium bacterium Bipolaris sorokiniana Trichoderma viride

Viride bacterial strain LTR-2

LTR-2

Gaeumannomyces graminis Gaeumannomyces graminis

Var.tritici viride bacterial strain T2 T.viride T2

Wheat snow mold bacterium Microdochium nivale viride bacterial strain F1 T.viride F1

Southern corn leaf blight Bipolaris maydis viride bacterial strain F2 T.viride F2

Phytophthora infestans Phytophthora infestans viride bacterial strain Jb T.viride Jb

Cotton dry thread Pyrenomycetes R.solani trichoderma strain Th-B T.harzianum Th-B

Fusarium?oxysporum f.sp.

Cotton-wilt fusarium

Vasinfectum Fusarlum moniliforme Fusariumn moniliforme

Cotton boll rot pathogens F.moniliforme Alternariaspp Alternaria sp.

Cotton anthracnose bacterium Collectotrichum gosyipii capsicum sickle-like bacteria Fusarium sp.

Sclerotinia sclerotiorum Sclerotinia sclerotiorum Chinese cabbage sickle-like bacteria Fusarium sp.

Colletotrichum

Cucumber anthracnose

Orbiculare muskmelon sickle-like bacteria Fusarium sp.

Capsicum anthrax-bacilus Colletotrichum capsici Cladosporium Cladosporium sp.

Phytophthora capsici Phytophthora capisci needle mushroom Flammulina velutipes

Tobacco brown spot pathogen Alternaria alternata mushroom Lentinus edodes

Ring rot of apple bacterium Botyosphaeria

berengeriana

Promote plant-growth effect experiment: B9602-Y2 was cultivated 3 days on 37 ℃, 120r/m shaking table in the PS substratum.

Experiment one: to cultivate 10 of bacterium liquid ^-5Concentration is handled paddy rice and wheat, and is contrast with the root-inducing powder of 10mg/mL with clear water.Experiment showed, 10 ^-5Concentration can obviously increase plant height and root is long, and handling than clear water increases plant height 73.12%, increases root long 98.31%.For wheat, 10 ^-5Handling plant height increases by 23.66% than clear water, increases by 40.24% than root-inducing powder, and the root appearance answers increasing amount to reach 43.68% and 22.55% respectively.

Experiment two: adopt 10 ^-5Sizable Ipomoea batatas seedling is got in bacterium liquid, naphthylacetic acid and three processing of clear water, and directly cuttage is in peat that equivalent is housed and mixing with soil matrix, and it is long with total root to calculate radical after 10 days.The result shows that B9601-Y2 can promote that obviously Ipomoea batatas takes root, and this handles radical increases by 22.16% than clear water, and handling than naphthylacetic acid increases by 13.79%, the long corresponding increase by 54.03% and 5.12% respectively of root.

Experiment three: the naphthylacetic acid with B9601-Y2 fermented liquid and 1.5mg/mL is handled, and is blank with the clear water, and field test results shows that this bacterial strain has significantly promoted the Flos Pelargonii rooting rate, and it is long to increase the number of blade, blade wheel number, radical and root.B9601-Y2 promotes the time that plant takes root to be shorter than clear water blank and naphthylacetic acid processing, in cuttage in the time of the 9th day, the cutting of blank is also without any reaction, and all cutting base portions of B9601-Y2 all grow white projection, naphthylacetic acid is handled has only minority cutting base portion adularescent projection, and in the time of the 12nd day, the blank root begins, and 1-2 bar root only, B9601-Y2 opens and grows 3-4 bar root.Compare with blank, the cutting blade wheel number many 1.19 that B9601-Y2 handles is taken turns, and increases by 54.09%, and many 2.05 leaves of the number of blade increase by 33.17%, and many 6.38 of radical increases 65.44%, and the long 2.39cm of root increases 29.70%, the big 0.25cm of maximum blade diameter.

Experiment showed, that this bacterial strain of B9602-Y2 can reduce the onset speed of the late blight of potato, the preventive effect of capsicum epidemic disease is reached 82.3%; Result of treatment reaches 65.8%, and 1: 100 ratio of B9601-Y2 and peat applies the muskmelon base portion, prevents and treats the blight effect and reaches 80.03%.And the muskmelon plant there is the growth of promotion effect, promotes early flowering 5-7 days.Aforementioned germ disease is had wide practical use.It also has very strong restraining effect to contaminated bacteria-Trichoderma common in the edible mushrooms.B9601-Y2 has the plant-growth of promotion and takes root, and improves the phytomass effect.With 10 very much one to 1,000 ten thousand/ the root of fermented liquid seed dressing can increasing paddy rice and wheat long, plant increases, improve Ipomoea batatas, Chinese China pink, and Caulis Hederae Sinensis take root, improve the Chinese rose rooting of cuttings and sprout, and effect is better than producing at present and goes up 0.2% root-inducing powder commonly used and handle.10 very much during bacterium liquid pouring culture medium, paddy rice plant height and root are long to improve 49.75% and 73.12% and 69.81% and 98.31% than root-inducing powder and clear water respectively, during 100,000/ferment filtrate pouring culture medium, paddy rice plant height and root length are handled raising 61.50% and 86.71% and 95.65% and 128.47% than root-inducing powder and clear water.When the bacterium liquid of same concentration was handled wheat, plant height and root are long handled raising 40.24% and 23.66% and 22.55% and 47.68% than root-inducing powder and clear water respectively; When handling the Ipomoea batatas cutting, the long processing with clear water than 1.5mg/mL naphthylacetic acid respectively of radical and root increases by 13.79% and 22.16% and 5.12% and 54.03%.When getting the cuttage of bacterium liquid to dip in, nandina certain herbaceous plants with big flowers blade wheel number, the number of sheets, radical and root are long to increase by 10.06% and 54.09%, 5.92% and 33.17%, 10.33% and 65.44% and 5.38% and 29.70% than 1.5mg/mL naphthylacetic acid and clear water respectively.During with 100,000/fermented liquid and filtrate cuttage Caulis Hederae Sinensis, promote root length to increase by 321.09% and 681.25%, 285.71% and 631.25% than naphthylacetic acid and clear water blank.Dip in Chinese rose with bacterium liquid and transplant a cutting, it can promote axillalry bud length than clear water contrast increase 137.45%-200.49%, and radical and total root are long to reach 41.67% and 112.20% and 187.29% and 230.21% respectively than 0.2% root-inducing powder and clear water processing.In addition, this bacterial strain 10 very much one to 1,000 ten thousand/ fermented liquid cuttage Chinese rose fresh cutting flower, can obviously prolong cut-flower in bottle freshness date 5-7 days.To rice blast fungus, sheath blight fungus, little total eclipse germ, the wheat snow mold bacterium, cotton-wilt fusarium, the red rot bacterium, anthrax bacteria, Sclerotinia sclerotiorum, tobacco brown spot pathogen, tobacco black shank bacterium, the ring rot of apple bacterium, the broad bean pine root fungus, 24 kinds of important plant pathogenic fungis such as the husky Moindawang of good forage pine root fungus all have obvious restraining effect, particularly to rice blast fungus, sheath blight fungus, the root rotof flax bacterium, gaeumannomyces graminis, tobacco black shank bacterium, Sclerotinia sclerotiorums etc. have splendid inhibition effect, and these germs are close on substratum and can not grow.B9601-Y2 still has the obvious suppression effect to viride and the trichoderma harzianum that causes the edible mushrooms green mould, in addition, this bacterial strain also all has remarkable restraining effect to being common in secondary bacterial strain Alternariaspp of plant surface and sickle-like bacteria, alleviates the effect that the Plant Secondary Materials cause of disease is assisted morbidity thereby have.

B9601-Y2 is a genus bacillus from soil, it and plant root are in close relations, and a lot of soil is passed the phytopathy originals to be had preferably control action kou, takes into account the long and cut rose flower freshening effect of plant growth-promoting, energy storage tolerance, good stress resistance, again apply in the environment and also can not cause environmental pollution, have good DEVELOPMENT PROSPECT.

Claims (3)

1, a kind of bacillus subtilis strain, it is characterized in that, called after subtilis (Bacillus subtilis) B9601-Y2, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation day: on June 18th, 2003, preserving number: CGMCC No.0954.

2, a kind of bacillus subtilis strain according to claim 1 is characterized in that, B9601-Y2 bacterium colony circle, weak tea brown, bacterium colony central color are slightly dark, protuberance, smooth surface; Thalli morphology is shaft-like, end is round, and Gram-positive produces gemma, peritrichous (electromicroscopic photograph), tool mobility, and sporocyst does not expand, and brood cell's oval, each cell only produce a brood cell, are positioned at somatic cells central authorities, 0.66 * 2.10 (μ m).

3, a kind of bacillus subtilis strain according to claim 1 is characterized in that, B9601-Y2 strain culturing mode is:

(1), liquid culture: inoculation in LB or PS substratum, under 35 ℃ of temperature, was cultivated on the shaking table of rotating speed 120r/m 2～3 days;

(2), solid culture: inoculation in the PSA substratum, was cultivated 3～4 days in 37 ℃ of incubators.

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