CN106148250A - One strain Costa Rica streptomyces actinomycetes and application thereof - Google Patents

Abstract

The invention provides strain Costa Rica streptomyces actinomycetes GDMCC 60071, be called for short LP69, these actinomycetes have product heteroauxing, produce siderophore and the effect of dissolved phosphorus.Present invention also offers the bio-feritlizer and fertilizing method thereof being made up of actinomycetes LP69, this bio-feritlizer can effectively facilitate the growth of the plants such as Nicotiana tabacum L. simultaneously.

Description

One strain Costa Rica streptomyces actinomycetes and application thereof

Technical field

The invention belongs to microbial manure field, be specifically related to strain Costa Rica streptomyces actinomycetes and a use thereof On the way.

Background technology

Endophytic actinomycetes in plants (Endophytic actinobacteria) refers to be present in symbiosis or parasitic method plant Inside fabric texture, do not cause the microorganism of the obvious disease of plant, during long-term coevolution, Endophytic actinomycetes in plants with Constantly carry out the exchange of material, energy and hereditary information between host, define between actinomycetes that many functions are special and plant Complicated symbiosis, is the important component part of plant microecosystem.Surely the actinomycetes in plant are grown, outside being susceptible to Boundary's environmental effect, with molten phosphorus nitrogen fixation, secretion plant growth regulating substance, synthesis siderophore, induction plant produce resistance with And produce the mode such as antifungal metabolite and promote plant growing, and long-term continuous action, have the biggest in agricultural production Application potential.The disease-resistant growth-promoting endogeny rayungus that has reported both at home and abroad mainly has: from pteridophyta Pteridium The pteridic acids that in aquilinum, the Streptomyces hygroscopicus TP-A0451 of isolated produces A and B, it is possible to promote that Kidney bean plumular axis forms adventitious root；Isolated 19 strain from Jatropha curcas Jatropha curcas L. such as Qin Having generation ACC, heteroauxing, siderophore, molten phosphorus and an endogeny rayungus of nitrogen fixing capacity, wherein 7 strains can the carrying of significance Plant height, root length and the fresh weight of high Jatropha curcas L. seedling；Li gets a strain from Rhizoma Alismatis Alisma orientale Streptomyces CNS-42 can produce star p0-357 (staurosporine) and have the disease-resistant dual-use function of growth-promoting.Passari Deng getting 42 strain endogeny rayungus from medicinal plants, wherein 22 strains have the ability producing heteroauxing, the heteroauxing of 20 strains Concentration is at 10-32 μ g/mL；All isolated strains have product ammonia ability, and product ammonia density is at 5.2-54mg/mL, and 21 strain bacterium have product Siderophore ability；14 strain bacterium have phosphate solubilization, and 19 strain bacterium have product chitinase activity, and 15 strain bacterium have the ability producing HCN, From 20 and 19 strain bacterium, expand heteroauxing (iaaM) and chitinase (chic) gene, further study show that 2 strain potentiality Bacterial strain Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) have the effect promoting growth to Fructus Capsici.

Fructus Toosendan (Melia Toosendan Sieb.et Zucc) is Meliaceae Melia deciduous tree, is distributed mainly on China west Area, south, is important industrial cut stock trees, is also the important plant of water and soil conservation.As Chinese medicine, Fructus Toosendan is in China Medicinal history is long, and bark, root bark, trunk, leaves and fruit all can be used as medicine, and is superior to originate from the Fructus Toosendan fruit in Sichuan, therefore Name Fructus Toosendan, is important road, the place of production, river Chinese crude drug.The Toosendanin (Toosendanin) extracted from Fructus Toosendan is to insecticide, nematicide There is inhibitory action with acarid and microorganism, and not yet find the situation that insecticide develops immunity to drugs, be to make efficiently without residual hazard The important source material of novel pollution-free plant pesticide.During coevolution long-term with host plant, dwell within Fructus Toosendan Actinomycetes, under the long-term influence of host plant and external environment condition, may possess and produce novel structure, the biology of activity uniqueness The potentiality of active substance, and grown by own metabolism product supply host plant, improve host plant to environment Middle nutrition thing The absorbability of matter, and promote plant by different mechanisms Suppressing phytopathogens and the modes such as some noxious substance of degrading Growth.Therefore, separate from Fructus Toosendan and obtain the endogeny rayungus with growth-promoting antibacterial action, and probe into it in agricultural production Application potential, meets the demand of modern agriculture sustainable development.

Summary of the invention

The present invention separates strain endogeny rayungus bacterial strain Costa Rica streptomycete from wild Fructus Toosendan skin (Streptomyces costaricanus), is called for short LP69.This bacterial strain was preserved in the micro-life in Guangdong Province on 08 29th, 2016 Thing DSMZ, deposit number is GDMCC No:60071, and preservation address is XianLie Middle Road, GuangZhou City, GuangDong Province 100 5th floors, No. 59 building of compound, Guangdong Microbes Inst.

Meanwhile, present invention also offers the nucleotide sequence of this actinomycetic 16s rDNA, for sequence table SEQ ID NO.1 Described.

Additionally, the invention provides a kind of actinomycetic pityrosporion ovale suspension, following steps prepare:

Take actinomycetes strain GDMCC 60071, be inoculated on base flat board, be placed in 28 DEG C of incubators cultivation 7 days, treat whole After flat board covers with mycelia, with sterile scalpel, culture medium is shredded, access in the seed bottle equipped with aseptic barley inoculum, 28 DEG C of cultivations More than 1 week, after the spore that all wheat grain cover with streptomycete LP69, take out wheat grain spore under aseptic washing, then by spore Suspension is with the centrifugation 5min of 10000 revs/min, then with sterilized water, the spore concentration of LP69 is adjusted to 10⁸Cfu/mL, Pityrosporion ovale suspension.Cfu is term commonly used in the art, i.e. refers to colony-forming units.

Present invention also offers the fertilizing method of above-mentioned pityrosporion ovale suspension, comprise the following steps:

When plant seedlings is transplanted, with determine root water use pityrosporion ovale suspension, and plant shoots transplant cultivate 10 days laggard Row pouring root, pours into spore suspension 10-50mL, such as 20mL near root.

Meanwhile, the spore that present invention also offers actinomycetes GDMCC 60071 and/or actinomycetes GDMCC 60071 is the most outstanding Liquid prepares the purposes of bio-feritlizer；Described bio-feritlizer has product heteroauxing, produces siderophore, dissolved phosphorus, raising Soil Nitrogen unit Cellulose content, raising Potassium in Soils constituent content, raising soil iron content and/or the purposes of raising soil phophorus constituent content.

Additionally, present invention also offers a kind of bio-feritlizer for Nicotiana tabacum L., this bio-feritlizer contains actinomycetes GDMCC 60071；

And, it is provided that a kind of bacteria preparation, containing GDMCC 60071 and acceptable adjuvant；

It is solid-state microbial inoculum that described bacteria preparation is preferably, and the viable count of microbial inoculum is (1～5) × 10⁹Cfu/g, preferably 2 × 10⁹cfu/g。

Additionally, present invention also offers above-mentioned bio-feritlizer, bacteria preparation product heteroauxing, produce siderophore, dissolved phosphorus, raising Soil Nitrogen constituent content, raising Potassium in Soils constituent content and/or the purposes of raising soil phophorus constituent content.

The biological property of the LP69 bacterial strain of the present invention is as follows:

Strain morphology feature: at ISP₄Growing in culture medium, bacterium colony is the least, edge is irregular, rough surface, bacterium colony Color is taupe, and aerial hyphae all physically well develops with substrate mycelium, the most chromogenic element, and aerial hyphae forms close spiral spore Chain.

Physiological and biochemical property: bacterial strain LP69 can grow 15 DEG C of-50 DEG C of temperature ranges, pH < can not grow when 4.0, 0%-5%NaCl solution can grow, it is possible to use D-Fructose, D-Glucose, D-MANNOSE, D-xylose, galactose are Sole carbon source, it is impossible to utilize L-arabinose, sucrose, Raffinose, rhamnose；Cys, Serine, L-figured silk fabrics can be utilized Propylhomoserin, it is impossible to utilize L-Histidine and L-Methionine for only nitrogen source；Gelatin liquefaction can not be made, starch can be hydrolyzed, decompose fiber Element.

Bacterial strain LP69 carries out 16S rDNA sequencing, and the row that checked order submit EzBioCloud website to (www.ezbiocloud.net) carry out sequence analysis, and utilize MEGA 5.0 software to carry out Phylogenetic Analysis.Result table Bright, bacterial strain LP69 and the 16S rDNA nucleotide sequence of Costa Rica streptomycete (Streptomyces costaricanus) Homology more than 99%.Comprehensive morphological feature, physiological and biochemical property and 16S rDNA sequence homology analysis etc. are tested As a result, identify that LP69 is Costa Rica streptomycete (Streptomyces costaricanus).

Bacterial strain LP69 can produce heteroauxing, siderophore, insoluble phosphate can be converted into can by plant utilize can Soluble phosphoric acid salt.

Prepare barley inoculum, inoculating strain LP69, after wheat grain covers with spore, with to make concentration under aseptic washing be 1 × 10⁸The spore liquid of cfu/mL, as microbial inoculum, when tobacco seedlings is transplanted with determining the use of root water, and is carried out after tobacco seedlings transplants training 10 days Pouring root, every strain 20mL.

By potted plant experiment, demonstrate bacterial strain LP69 and the growth of Nicotiana tabacum L. is had obvious facilitation, Nicotiana tabacum L. plant height, Big stem girth, maximum leaf length, maximum width of blade and fresh weight, dry weight add 23.3% respectively, 18.7%, 17.6%, 11.7%, 28.6%, 28.1%.

Beneficial effect

The Fructus Toosendan endogeny rayungus LP69 that separation screening of the present invention provides has generation heteroauxing, siderophore, molten phosphorus etc. Growth-promoting ability, by the application on tobacco cultivation, it is thus achieved that can promote the effect of the growth of Nicotiana tabacum L., can reduce in planting process The sowing amount of chemical fertilizer, has preferable application potential in agricultural production.

Accompanying drawing explanation

Fig. 1 is the colonial morphology figure of Costa Rica streptomycete LP69.

Fig. 2 is the thalli morphology figure of Costa Rica streptomycete LP69.

Fig. 3 is Costa Rica's streptomycete LP69 phylogenetic tree built based on 16S rDNA gene order.

Fig. 4 is the pot test effect that Costa Rica streptomycete LP69 promotes tobacco growing by pouring root mode.The wherein left side For LP69 root irrigation, the right is clear water comparison.

Detailed description of the invention

The present invention is further described below in conjunction with accompanying drawing and concrete example.Relate in the present invention is main former auxiliary Material, reagent and instrument and equipment are the most known in the art.But the present invention is not limited to following embodiment.

Embodiment one: the separation of Fructus Toosendan endogeny rayungus LP69, screen and identify

1. the separation of Fructus Toosendan endogeny rayungus

Pick up from the root of the wild Fructus Toosendan in Ya'an Sichuan province Yucheng District, stem, leaf, really, the tissue such as skin take back laboratory, first use tap water Plant sample is rinsed well, then cleans 10min with ultrasonic (15 kilo hertzs), remove plant tissue surface impurity, make suitable repairing After cutting, first clean 30s, aseptic water washing three times with 0.1%Tween 20；75% soak with ethanol 5min, aseptic water washing three times； 2% sodium hypochlorite soaks 5min, aseptic water washing three times；10% sodium bicarbonate rinsing 10min.The sample handled well is placed in paving Have in the sterile petri dish of aseptic filter paper, after blotting the moisture of plant tissue surface, after pulverizing sample with sterile crushing machine, take suitable The sample tissue that amount pretreatment is good uniformly dispenses in Gause I culture medium, positive horizontalization plate, cultivates more than 7 weeks for 28 DEG C.Wait to put After line bacterium grows, with aseptic bamboo let picking at ISP₄Purification in culture medium, bacterial strain LP69 conservation after purification is in ISP₄Test tube is oblique Face, 4 DEG C of preservations.Last, all over cleaning the sterilized water 200 μ L of sample, after coating LB solid medium, is placed in 28 DEG C of cultivations, and 7 Generate if any bacterium colony on it rear plate, it was demonstrated that surface sterilization is the most thorough；If growing without bacterium colony, then can be depending on the bacterial strain separated For endophyte.

2. the growth-promoting functional screening of Fructus Toosendan endogeny rayungus LP69

2.1 produce heteroauxing ability measures

Bacterial strain produces heteroauxing ability and measures, and method is as follows:

A) nitrogenous culture fluid is sub-packed in test tube, often pipe 4mL, 121 DEG C, 20min sterilizing；

B) the L-Trp solution 1mL of the 2.5mg/mL of filtration sterilization is added；

C) strains tested is accessed above-mentioned culture medium, 140rmp/min, cultivates 7 days by 28 DEG C；

D) fermentation liquid 8000rmp/min, centrifugal 5min；

E) taking supernatant 2mL, add the nitrite ion of 4mL, fully mix, 25 DEG C of dark treatment, develop the color 30min, at 530nm ripple Long lower mensuration absorbance A；

F) with the fluid medium that do not connects bacterium for comparison zeroing, with concentration 0,5,20,40, the heteroauxing mark of 60mg/L Quasi-liquid ibid does mark song, calculates the concentration of heteroauxing in fermentation liquid；

2.2 produce siderophore ability measures

A) take 0.012g chromazurine to be dissolved in 10mL distilled water, and with the FeCl of 2mL 1mmoL/L₃·6H₂O solution mixes, Obtain solution a；

B) take 0.015g cetyl trimethylammonium bromide to be dissolved in 8mL distilled water, obtain solution b；

C) a liquid is added slowly in b liquid, mix homogeneously, obtains dye liquor c；

D) 10 × MM9 saline solution 20mL and 6.04g piperazine two ethyl sulfonic acid are added in the triangular flask filling 150mL distilled water Regulate pH to 6.8 with 50%NaOH after mix homogeneously, add 3.2g agar powder, obtain culture medium d；

E) by dye liquor c, culture medium d and the CaCl of 1mmoL/L₂, the MgSO of 1mmoL/L₄, the glucose of 20%, the cheese of 10% Argine Monohydrochloride is respectively at 115 DEG C of sterilizing 20min, when each solution temperature is down to 50-60 DEG C, takes 200 μ L CaCl₂, 4mL MgSO₄, 6mL casamino acid, 2mL glucose adds culture medium d, adds dye liquor c further along bottle wall, and fully mixing is sure not to produce Angry bubble i.e. obtains blue detection culture medium, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices.

F) inoculating strains tested with aseptic bamboo let, 5 bacterium cakes equidistantly inoculated by every plate, and every strain bacterium is repeated 3 times, 28 DEG C, cultivate 3 My god, observed and recorded crocus transparent circle size.

2.3 phosphate solubilization measure

Strains tested is inoculated in Meng Jinna culture medium, be placed in 28 DEG C and cultivate 10 days, observe with or without molten phosphorus circle and molten phosphorus circle Size.Determining its solvability to Phos with the size of molten phosphorus circle, be worth the biggest, phosphate solubilization is strong, otherwise, molten phosphorus energy Power is weak, does not produce the bacterial strain of molten phosphorus circle without phosphate solubilization；

4 identification of strains

4.1 strain morphology

(1) colony morphology characteristic: by inoculation at ISP₄In culture medium, it is placed in 28 DEG C and cultivates 5 days, observe the bacterium of bacterial strain Fall form.

(2) morphological features: by inoculation at ISP₄In culture medium, then enter nothing at the position oblique cutting being connected to strain The coverslip (inserted sheet method) of bacterium, is placed in 28 DEG C and cultivates 5 days, take out and be coated with the coverslip of mycelia and simply dye by azaleine Microscopy, by viewed form result microphotograph, as shown in Figure 2.4.2 bacterial strain LP69 physiological and biochemical properties

(1) utilization of carbon source test: by inoculation on the basal medium containing different carbon source, be placed in 28 DEG C of incubators Middle cultivation 5 days, continuous passage 3 times, can grow bacterium colony for the positive.

(2) nitrogen source utilizes test: by inoculation on the basal medium containing different nitrogen sources, be placed in 28 DEG C of incubators Middle cultivation 5 days, continuous passage 3 times, can grow bacterium colony for the positive.

(3) growth temperature: by inoculation in ISP₄In culture medium, be respectively placed in 5 DEG C, 15 DEG C, 25 DEG C, 35 DEG C, 45 DEG C, The incubator of 55 DEG C is cultivated 5 days, has seen whether that bacterium colony is formed.

(4) Salt tolerance: by inoculation in containing 0%, 3%, 5%, 7%, 10%, 15%, 20% ISP₄Culture medium On, after being placed in 28 DEG C of incubators cultivation 5 days, see whether that bacterium colony is formed.

(5) pH resistance test: inoculation is respectively 3.0 in pH value, 4.0,5.0,6.0,7.0,8.0,9.0,10.0, 11.0 ISP₄In culture medium, after being placed in 28 DEG C of incubators cultivation 5 days, see whether that bacterium colony is formed.

(6) Starch Hydrolysis reaction: by bacterial strain dibbling in the culture medium containing soluble starch, be repeated 3 times, in 28 DEG C of trainings Supporting in case and cultivate 3 days, after forming bacterium colony, drip Lu Geershi iodine liquid, covered by whole plate on flat board, flat board is blueness, As periphery of bacterial colonies occurs with or without transparent circle, illustrating that starch is hydrolyzed, transparent circle size shows the size of its hydrolysis starch ability.As Occur without transparent circle, then say that this bacterium is without amylatic ability.

(7) cellulose decomposition: by inoculation to Cellulose and congo red differential medium, be placed in 28 DEG C of incubators cultivation 5 My god, if producing Clear & Transparent circle around bacterial strain, then prove the positive strain with degraded cellulose ability, vice versa.

(8) gelatin liquefaction: by bacterial strain percutaneous puncture-inoculation in gelatin culture medium, is placed in 28 DEG C of incubators cultivation 5 days, then is placed in Observed result after 30min in 4 DEG C of refrigerators, if culture medium is liquefaction is that gelatin liquefaction is positive, if culture medium is still solid, shaped State is then negative for gelatin liquefaction.

The Molecular Identification of 4.3 bacterial strains

4.3.1 reagent: lysozyme, E.C. 3.4.21.64,10 × TAE, 1 × TE, 3mol/L sodium acetate (pH4.8-5.2), 70% second Alcohol, (saturated phenol: chloroform: isoamyl alcohol=25:24:1), Mix.

4.3.2 primer

Primer A:5 '-AGAGTTTGATCCTGGCTCAG-3 ' (identical with 16S rRNA 5 ' end 8-27 site base)； Primer B:5 '-TTAAGGTGATCCAGCCGCA-3 ' (identical with 16S rRNA 3 ' end 1523-1504 site base)；

4.3.3 endogeny rayungus DNA extraction

Take a little thalline in aseptic 1.5mL Eppendorf pipe, add 20 μ l (50mg/mL) final concentrations and reach 2mg/ The lysozyme of mL, puts into 37 DEG C of shaking tables, 200rmp/min, 1-2h；Add 20%SDS 50 μ L and E.C. 3.4.21.64 5 μ L (20mg/ ML), 55 DEG C of shaking tables are put in mixing, and 200rmp/min processes 1-2h；Add 550 μ L (phenol: chloroform: isoamyl alcohol=25:24:1) Extracting, 12000rmp/min is centrifuged 10min, draws supernatant (in triplicate)；Add 800 μ L dehydrated alcohol, 80 μ L 3mol/L second Acid sodium (pH4.8-5.2), mixing, precipitates DNA, 1h, 12000rmp/min, 10min in 4 DEG C, abandons supernatant；Add 200 μ L 70% Ethanol, cleans tube wall 1-2 time, 12000rmp/min, centrifugal 5min, abandons supernatant；After ethanol volatilization is dry, add 50 μ L 1 × TE Dissolving DNA, and in-20 DEG C of preservations；1% agarose gel electrophoresis detection.

4.3.4 raw unwrapping wire 16S rRNA gene amplification in

16S rRNA gene amplification condition: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 1min, 56 DEG C of annealing 1min, 72 DEG C are prolonged 2min, 30 circulations, 72 DEG C of overall elongation 10min.PCR primer is through the raw work EZ Spin Column PCR Product in Shanghai Purification Kit UNlQ-1 pillar PCR primer purification kit (SK1142-N) purification, is carried out by operating guidance, pure Changing product send Jin Weizhi bio tech ltd, Suzhou to check order.

4.3.5 16S rRNA gene sequencing and phylogenetic tree build

After checking order, gained sequence submits to EzBioCloud website (www.ezbiocloud.net) to carry out sequence analysis, Choose the highest 16S rRNA gene order having delivered bacterial strain of similarity to carry out as reference sequence, employing Clustal X software Multiple Sequence Alignment is analyzed, and by MEGA5.0 software with neighbouring method (Neighbor-Joining) phylogenetic tree construction, determines The classification position of this endogeny rayungus.

5. experimental result

By detection, screen a strain and produce heteroauxing ability, produce siderophore, phosphate solubilization all preferably endogeny rayungus Bacterial strain, named LP69.This bacterial strain is at ISP₄Growing in culture medium, bacterium colony is the least, edge is irregular, rough surface, bacterium The color that falls is taupe, and aerial hyphae all physically well develops with substrate mycelium, the most chromogenic element, and aerial hyphae forms flexible, spiral, top The spore chain of end curling, presents typical streptomycete feature (Fig. 1, Fig. 2).Bacterial strain LP69 can 15 DEG C of-50 DEG C of temperature ranges Growth, pH < can not grow when 4.0, can grow in 0%-5%NaCl solution, it is possible to use D-Fructose, D-Glucose, D-MANNOSE, D-xylose, galactose are sole carbon source, it is impossible to utilize L-arabinose, sucrose, Raffinose, rhamnose；Can utilize Cys, Serine, Valine, it is impossible to utilize L-Histidine and L-Methionine for only nitrogen source；Gelatin can not be made Liquefaction, can hydrolyze starch, decomposition of cellulose.

Bacterial strain LP69 carries out 16S rDNA sequencing, and the row that checked order submit EzBioCloud website to (www.ezbiocloud.net) sequence analysis is carried out, comparative result display bacterial strain LP69 and Costa Rica streptomycete The homology of the 16S rDNA nucleotide sequence of (Streptomyces costaricanus) is more than 99%, and passes through MEGA5.0 software is with neighbouring method (Neighbor-Joining) phylogenetic tree construction (Fig. 3).Comprehensive morphological feature, physiology are raw Change the experimental result such as feature and 16S rDNA sequence homology analysis, identify that LP69 is Costa Rica streptomycete (Streptomyces costaricanus)。

Bacterial strain LP69 has stronger molten phosphorus and produces heteroauxing, the ability (table 1) of siderophore.

Table 1 endogeny rayungus LP69 growth-promoting capability result

Example 2 Costa Rica streptomycete LP69 promotes tobacco growing effect experimental

1. prepared by inoculating strain pityrosporion ovale suspension

Bacterial strain LP69 is inoculated on Gause I culture medium flat plate, is placed in 28 DEG C of incubators cultivation 7 days, treat whole flat After plate covers with mycelia, with sterile scalpel, culture medium is shredded, access in the seed bottle equipped with aseptic barley inoculum, cultivate 1 for 28 DEG C More than week, after the spore that all wheat grain cover with Costa Rica streptomycete LP69, take out wheat grain spore under aseptic washing, so After spore suspension 10000rmp/min is centrifuged 5min, then with sterilized water, the spore concentration of LP69 is adjusted to 10⁸cfu/mL.High Number culture medium of family name is by soluble starch 20g；KNO₃1g, K₂HPO₄·3H₂O 0.5g, MgSO₄·7H₂O 0.5g, NaCl 0.5g, FeSO₄·7H₂O 0.01g, tap water 1000mL prepare.

2. take Nicotiana tabacum L. cloud and mist 87 planting seed in seedlings nursing plate, cultivate under natural conditions, water every day once, as Nicotiana tabacum L. children When the plant height of Seedling is 10cm, chooses health and grow fine and consistent tobacco seedling, carry out transfer.Spore suspension is in tobacco seedlings With determining the use of root water during transplanting, and carry out pouring root after tobacco seedlings transplants training 10 days, near root, pour into spore suspension 20mL, make For the Nicotiana tabacum L. of blank then adds the sterilized water of equivalent.

3. pouring root is after 90 days, measures Major Nutrient in the plant height of Nicotiana tabacum L., Root morphology, fresh weight, dry weight, the number of blade and soil Constituent content.

4. Nicotiana tabacum L. surface is that tobacco growing situation embodies the most intuitively.Nicotiana tabacum L. results from pot experiment test shows, bacterial strain The tobacco growing situation of LP69 root irrigation is substantially better than matched group, the plant height of bacterial strain LP69 root irrigation Nicotiana tabacum L., maximum stem girth, Maximum leaf length and maximum width of blade add 23.3%, 18.7%, 17.6% and 11.7% than CK respectively；Nicotiana tabacum L. fresh weight and dry weight are also 28.6%, 28.1% (table 2, Fig. 4) is added respectively than CK.

The table 2 inoculating strain LP69 impact on tobacco growing

5. the development degree of root system is in close relations with the ability that plant absorbs nutrition.From table 3, through bacterial strain LP69 pouring root After process, total root length of tobacco root, root total surface area, average root diameter, total root volume, total root hair number and CK result, exist Significant difference (P < 0.05).

The table 3 inoculating strain LP69 impact on tobacco root

Note:^*Indicate significant difference (LSD, P < 0.05), lower with.

The table 4 inoculating strain LP69 impact on soil main nutrient elements

After bacterial strain LP69 root irrigation, significantly improve the content of alkali slag in soil, active potassic and available P, respectively than CK improves 17.1%, 7.1%, 28.2% (table 4).

In sum, the present invention is isolatable from Fructus Toosendan endogeny rayungus Costa Rica streptomycete (Streptomyces Costaricanus) LP69 can effectively facilitate tobacco growing.

The preparation of embodiment 3 bio-feritlizer

1, microbial inoculum makes

1. flat board strain (one-level kind): Gause I culture medium is (by soluble starch 20g；KNO₃1g, K₂HPO₄·3H₂O 0.5g, MgSO₄·7H₂O 0.5g, NaCl 0.5g, FeSO₄·7H₂O 0.01g, tap water 1000mL), inoculate Costa Rica Cultivate at 28 DEG C after streptomycete (Streptomyces costaricanus) P69 and make first order seed in 7 days.

2. kernel culture (two grades of kinds): with sterile scalpel, the culture medium covering with mycelia is shredded, access equipped with aseptic wheat In the seed bottle that grain is planted, cultivate more than 1 week, after the spore that all wheat grain cover with Costa Rica streptomycete LP69, take for 28 DEG C Go out wheat grain spore under aseptic washing, then spore suspension 10000rmp/min is centrifuged 5min, then with sterilized water by LP69's Spore concentration is adjusted to 10⁸cfu/mL。

3. solid fermentation: with Testa Tritici as substrate (carrier), by weight volume ratio add, Semen Maydis powder 1%, soybean cake powder 1%, KH₂PO₄0.1%, CaCO₃0.1%, pH 7.0, add water regulation Testa Tritici water content to 60%, 121 DEG C, 0.1Mpa steam sterilization 30min；P69 spore suspension is inoculated in solid medium by the inoculum concentration of 20% (volume ratio), 28 DEG C of quiescent culture 9 days After, take out natural air drying, send out for Costa Rica streptomycete (Streptomyces costaricanus) P69 solid-state after pulverizing Yeast-like fungi agent, microbial inoculum viable count is 2 × 10⁹cfu/g。

4. preparation preserves: under microbial inoculum aseptic condition, normal temperature drying preserves does not affects its growth-promoting activity half a year.

2, microbial inoculum P69 Field information

Test plant: flue-cured tobacco cloud 87.

When cured tobacco seedling is transplanted, by following 4 process, cured tobacco seedling is processed: more solito is transplanted by CK；Process 1 Nutrition Soil Dip in root；Process 2 microbial inoculum P69 Nutrition Soils and dilute 10 times；Process 3 microbial inoculum P69 Nutrition Soils dilution 50 times and dip in root；Process 4 microbial inoculums P69 Nutrition Soil dilutes 100 times and dips in root, carries out the mensuration (table 5 of flue-cured tobacco economical character in group phase of tobacco growth and Topping Stage With table 6).

The table 5 microbial inoculum LP69 impact on a flue-cured tobacco group phase economical character

In group the phase of cloud and mist 87, the microbial inoculum LP69 each diluent within 100 times all can be effectively improved the strain of flue-cured tobacco The economical character indexs such as height and the number of blade.

The table 6 microbial inoculum LP69 impact on flue-cured tobacco Topping Stage economical character

At the Topping Stage of cloud and mist 87, the microbial inoculum LP69 each diluent within 100 times is equally effectively improved flue-cured tobacco The economical character indexs such as plant height, the number of blade, pitch and stem girth.

Claims (10)

1. strain Costa Rica streptomycete (Streptomyces costaricanus), deposit number is GDMCC60071.

2. a nucleotide sequence of actinomycetic 16s rDNA described in claim 1, described in sequence table SEQ ID NO.1.

3. an actinomycetic pityrosporion ovale suspension, is prepared by following steps:

Take the actinomycetes strain described in claim 1, be inoculated on base flat board, be placed in 28 DEG C of incubators cultivation 7 days, treat whole After flat board covers with mycelia, with sterile scalpel, culture medium is shredded, access in the seed bottle equipped with aseptic barley inoculum, 28 DEG C of cultivations More than 1 week, after the spore that all wheat grain cover with streptomycete LP69, take out wheat grain spore under aseptic washing, then by spore Suspension is with the centrifugation 5 minutes of 10000 turns per minute, then with sterilized water, the spore concentration of LP69 is adjusted to 10⁸Cfu/ milli Rise, obtain pityrosporion ovale suspension.

4. a fertilizing method for pityrosporion ovale suspension described in claim 3, comprises the following steps:

When plant seedlings is transplanted, use pityrosporion ovale suspension with determining root water, and fill after plant shoots is transplanted and cultivated 10 days Root, pours into spore suspension 10-50 milliliter near root, such as 20 milliliters.

5. the equal suspension of actinomycetic spore described in actinomycetes described in claim 1 and/or claim 3 prepares the use of bio-feritlizer On the way.

Purposes the most according to claim 5, it is characterised in that described bio-feritlizer have product heteroauxing, produce siderophore, Dissolved phosphorus, raising Soil Nitrogen constituent content, raising Potassium in Soils constituent content and/or the purposes of raising soil phophorus constituent content.

7. the bio-feritlizer for Nicotiana tabacum L., it is characterised in that containing the actinomycetes described in claim 1.

8. a bacteria preparation, containing GDMCC 60071 and acceptable adjuvant.

Bacteria preparation the most according to claim 8, it is characterised in that described bacteria preparation is solid-state microbial inoculum, the viable count of microbial inoculum For (1～5) × 10⁹Cfu/ gram, preferably 2 × 10⁹Cfu/ gram.

The most according to claim 8 or claim 9, bacteria preparation have product heteroauxing, produce siderophore, dissolved phosphorus, raising Soil Nitrogen Constituent content, raising Potassium in Soils constituent content and/or the purposes of raising soil phophorus constituent content.