CN106148250B - One plant of Costa Rica streptomyces actinomyces and application thereof - Google Patents

Abstract

The present invention provides one plant of Costa Rica streptomyces actinomyces GDMCC 60071, abbreviation LP69, which has the function of producing heteroauxin, produces siderophore and dissolution phosphorus.Simultaneously the present invention also provides the bio-feritlizer and its fertilizing method that are made of actinomyces LP69, which can effectively facilitate the growth of the plants such as tobacco.

Description

One plant of Costa Rica streptomyces actinomyces and application thereof

Technical field

The invention belongs to microbial manure fields, and in particular to one plant of Costa Rica streptomyces actinomyces and its use On the way.

Background technique

Endophytic actinomycetes in plants (Endophytic actinobacteria), which refers to, is present in plant with symbiosis or parasitic method Object organization internal does not cause the microorganism of the obvious illness of plant, during long-term coevolution, Endophytic actinomycetes in plants with The exchange of substance, energy and hereditary information is constantly carried out between host, is formd between many functions special actinomyces and plant Complicated symbiosis, is the important component of plant microecosystem.It colonizes in the intracorporal actinomyces of plant, not vulnerable to outer Boundary's environment influence, with Soluble phosphorus nitrogen fixation, secretion plant growth regulating substance, synthesis siderophore, induction plant generate resistance with And generate the modes such as antimycotic metabolite and promote plant growth, and long-term continuous action, have in agricultural production very big Application potential.It is reported both at home and abroad that there is disease-resistant growth-promoting endogeny rayungus mainly to have: from pteridophyte Pteridium The pteridic acids that isolated Streptomyces hygroscopicus TP-A0451 is generated in aquilinum A and B can promote Kidney bean plumular axis to form adventitious root；Qin etc. is isolated 19 plants from manioca Jatropha curcas L. With generate ACC, heteroauxin, siderophore, Soluble phosphorus and nitrogen fixing capacity endogeny rayungus, wherein 7 plants are capable of mentioning for conspicuousness Plant height, root long and the fresh weight of high Jatropha curcas L. seedling；Li gets one plant from rhizoma alismatis Alisma orientale Streptomyces CNS-42 can generate star p0-357 (staurosporine) with the disease-resistant dual function of growth-promoting.Passari Deng getting 42 plants of endogeny rayungus from medicinal plant, wherein 22 plants have the abilities for producing heteroauxin, 20 plants of heteroauxin Concentration is in 10-32 μ g/mL；All isolated strains, which have, produces ammonia ability, produces ammonia density in 5.2-54mg/mL, 21 plants of bacterium, which have, to be produced Siderophore ability；14 plants of bacterium have phosphate solubilization, and 19 plants of bacterium, which have, produces chitinase activity, and 15 plants of bacterium have the ability for producing HCN, Amplification is to heteroauxin (iaaM) and chitinase (chic) gene from 20 and 19 plants of bacterium, further study show that 2 plants of potentiality Bacterial strain Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) plays the role of capsicum to promote growth.

Melia toosendan (Melia Toosendan Sieb.et Zucc) is Meliaceae Melia deciduous tree, is distributed mainly on China west Southern area, is important the important plant of industrial cut stock trees and water and soil conservation.As traditional Chinese medicine, melia toosendan is in China Medicinal history is long, and bark, root skin, trunk, leaf and fruit can be used as medicine, and is superior to originate from the melia toosendan fruit in Sichuan, therefore Name Fructus meliae toosendan is important river and produces authentic chinese medicinal materials.The toosendanin (Toosendanin) extracted from melia toosendan is to insect, nematode It has inhibiting effect with acarid and microorganism, and not yet find the case where insect develops drug resistance, is made efficiently without residual hazard The important source material of novel pollution-free plant pesticide.During coevolution long-term with host plant, dwell inside melia toosendan Actinomyces may have under host plant and the long-term influence of external environment and generate structure novel, the unique biology of activity The potentiality of active material, and host plant growth is supplied by own metabolism product, host plant is improved to nutrients in environment The absorbability of matter, and inhibit the modes such as phytopathogen and the certain noxious materials of degradation to promote plant by different mechanisms Growth.Therefore, separation obtains the endogeny rayungus with growth-promoting antibacterial action from melia toosendan, and probes into it in agricultural production Application potential meets the demand of modern agriculture sustainable development.

Summary of the invention

The present invention separates one plant of endogeny rayungus bacterial strain Costa Rica streptomycete from wild melia toosendan skin (Streptomyces costaricanus), abbreviation LP69.The bacterial strain was preserved in the micro- life in Guangdong Province on 08 29th, 2016 Object Culture Collection Center, deposit number are GDMCC No:60071, and preservation address is XianLie Middle Road, GuangZhou City, GuangDong Province 100 5 building, the building of compound the 59th, Guangdong Microbes Inst.

Meanwhile the present invention also provides the nucleotide sequences of the 16s rDNA of the actinomyces, are sequence table SEQ ID NO.1 It is described.

In addition, being prepared by the following steps to obtain the present invention provides a kind of spore bacteria suspension of actinomyces:

Actinomycetes strain GDMCC 60071 is taken, is inoculated on base plate, is placed in 28 DEG C of incubators and cultivates 7 days, to entire After plate covers with mycelia, culture medium is shredded with sterile scalpel, equipped in the seed bottle of sterile barley inoculum, 28 DEG C are cultivated for access 1 week or more, after all wheats cover with the spore of streptomycete LP69, wheat spore under sterile washing is taken out, then by spore Suspension is centrifuged 5min with 10000 revs/min of speed, then the spore concentration of LP69 is adjusted to 10 with sterile water⁸Cfu/mL is obtained Spore bacteria suspension.Cfu is term commonly used in the art, that is, refers to Colony Forming Unit.

The present invention also provides the fertilizing methods of above-mentioned spore bacteria suspension, comprising the following steps:

In plant seedlings transplanting, spore bacteria suspension is used with root water, and laggard at plant shoots transplanting culture 10 days Row pouring root pours into spore suspension 10-50mL, such as 20mL near root.

Meanwhile the present invention also provides the spores of actinomyces GDMCC 60071 and/or actinomyces GDMCC 60071 to hang Liquid prepares the purposes of bio-feritlizer；The bio-feritlizer, which has, to be produced heteroauxin, produces siderophore, dissolution phosphorus, improves Soil Nitrogen member Cellulose content improves soil Determination of Potassium, improves soil iron content and/or improves the purposes of soil phosphorus element content.

In addition, the present invention also provides a kind of bio-feritlizer for tobacco, which contains actinomyces GDMCC 60071；

And a kind of bacteria preparation is provided, contain GDMCC 60071 and acceptable auxiliary material；

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

In addition, the present invention also provides above-mentioned bio-feritlizer, bacteria preparations to produce heteroauxin, produces siderophore, dissolution phosphorus, raising Soil nitrogen element content improves soil Determination of Potassium and/or improves the purposes of soil phosphorus element content.

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

Strain morphology feature: in ISP₄It is grown on culture medium, bacterium colony is closely smaller, edge is irregular, rough surface, bacterium colony Color is in taupe, and aerial hyphae physically well develops with substrate mycelium, and not chromogenic element, aerial hyphae forms close spiral spore Chain.

Physiological and biochemical property: bacterial strain LP69 can be grown in 15 DEG C of -50 DEG C of temperature ranges, cannot be grown in pH < 4.0, It can be grown in 0%-5%NaCl solution, can use D-Fructose, D-Glucose, D-MANNOSE, D- xylose, galactolipin is Sole carbon source cannot utilize L-arabinose, sucrose, gossypose, rhamnose；L-cysteine, Serine, L- figured silk fabrics can be utilized Propylhomoserin cannot be only nitrogen source using L-Histidine and L-Methionine；It cannot make gelatin liquefaction, starch can be hydrolyzed, decompose fiber Element.

Bacterial strain LP69 carries out 16S rDNA sequencing, and column are sequenced and submit the website EzBioCloud (www.ezbiocloud.net) sequence analysis is carried out, and carries out Phylogenetic Analysis using 5.0 software of MEGA.As a result table It is bright, the 16S rDNA nucleotide sequence of bacterial strain LP69 and Costa Rica streptomycete (Streptomyces costaricanus) Homology 99% or more.The experiment such as comprehensive morphological feature, physiological and biochemical property and 16S rDNA sequence homology analysis As a result, identifying that LP69 is Costa Rica streptomycete (Streptomyces costaricanus).

Bacterial strain LP69 can generate heteroauxin, siderophore, insoluble phosphate can be converted to can be utilized by plant can Soluble phosphoric acid salt.

Prepare barley inoculum, inoculating strain LP69, after wheat covers with spore, with concentration is made under sterile washing is 1 × 10⁸The spore liquid of cfu/mL is used in tobacco seedlings transplanting with root water as microbial inoculum, and is carried out after tobacco seedlings transplanting training 10 days Pouring root, every plant of 20mL.

By potted plant experiment, demonstrating bacterial strain LP69 has an apparent facilitation to the growth of tobacco, tobacco plant height, most Big stem girth, maximum leaf be long, maximum width of blade and fresh weight, dry weight have increased separately 23.3%, 18.7%, 17.6%, 11.7%, 28.6%, 28.1%.

Beneficial effect

The melia toosendan endogeny rayungus LP69 that separation screening of the present invention provides, which has, generates heteroauxin, siderophore, Soluble phosphorus etc. Growth-promoting ability is obtained the effect that can promote the growth of tobacco, can be reduced in planting process by the application on tobacco cultivation The sowing amount of chemical fertilizer has preferable application potential in agricultural production.

Detailed description of the invention

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 constructed 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 control.

Specific embodiment

The present invention is further described below in conjunction with attached drawing and specific example.The main original being related in the present invention is auxiliary Material, reagent and instrument and equipment are known in the art.But the present invention is not limited to following embodiments.

Embodiment one: separation, screening and the identification of melia toosendan endogeny rayungus LP69

1. the separation of melia toosendan endogeny rayungus

It picks up from the tissues such as root, stem, leaf, fruit, the skin of the wild melia toosendan in Ya'an Sichuan province Yucheng District and takes back laboratory, first use tap water Plant sample is rinsed well, then cleans 10min with ultrasonic (15 kilo hertzs), plant tissue surface impurity is removed, makees appropriate repair After cutting, 30s first is cleaned with 0.1%Tween 20, aseptic water washing is three times；75% ethyl alcohol impregnates 5min, and aseptic water washing is three times； 2% sodium hypochlorite impregnates 5min, and aseptic water washing is three times；10% sodium bicarbonate rinses 10min.The sample handled well is placed in paving Have in the sterile petri dish of aseptic filter paper, after the moisture for blotting plant tissue surface, after crushing sample with sterile crushing machine, takes suitable It measures the sample tissue that pre-process uniformly to dispense on Gause I culture medium, positive horizontalization plate, 28 DEG C are cultivated 7 weeks or more.Wait put After line bacterium grows, with sterile bamboo stick picking in ISP₄It is purified on culture medium, bacterial strain LP69 conservation after purification is in ISP₄Test tube is oblique Face, 4 DEG C of preservations.Last after being coated on LB solid medium, is placed in 28 DEG C of cultures all over the 200 μ L of sterile water of cleaning sample, and 7 It is generated on plate if any bacterium colony after it, it was demonstrated that surface sterilization is not thorough；It, can be depending on the bacterial strain separated if no bacterium colony is grown For endophyte.

2. the growth-promoting functional screening of melia toosendan endogeny rayungus LP69

2.1 produce the measurement of heteroauxin ability

Bacterial strain produces the measurement of heteroauxin ability, the method is as follows:

A) nitrogenous culture solution is sub-packed in test tube, every pipe 4mL, and 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 into above-mentioned culture medium, 140rmp/min, is cultivated 7 days by 28 DEG C；

D) fermentation liquid 8000rmp/min is centrifuged 5min；

E) supernatant 2mL is taken, the developing solution of 4mL is added, mixes well, 25 DEG C of dark treatments, develop the color 30min, in 530nm wave Long lower measurement absorbance value A；

F) not connect the fluid nutrient medium of bacterium for control zeroing, with concentration 0, the heteroauxin mark of 5,20,40,60mg/L Quasi- liquid ibid does mark song, calculates the concentration of heteroauxin in fermentation liquid；

2.2 produce the measurement of siderophore ability

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

B) it takes 0.015g cetyl trimethylammonium bromide to be dissolved in 8mL distilled water, obtains solution b；

C) a liquid is added slowly in b liquid, is uniformly mixed, obtains dye liquor c；

D) two ethanesulfonic acid of 10 × MM9 salting liquid 20mL and 6.04g piperazine is added in the triangular flask for filling 150mL distilled water PH to 6.8 is adjusted with 50%NaOH after mixing, 3.2g agar powder is added, obtains culture medium d；

E) by dye liquor c, the CaCl of culture medium d and 1mmoL/L₂, the MgSO of 1mmoL/L₄, 20% glucose, 10% junket Argine Monohydrochloride when each solution temperature is down to 50-60 DEG C, takes 200 μ L CaCl respectively in 115 DEG C of sterilizing 20min₂, 4mL MgSO₄, 6mL casamino acid, 2mL glucose be added culture medium d, further along bottle wall be added dye liquor c, mix well and be sure not to produce Anger is steeped up to blue detection culture medium, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices.

F) it being inoculated with strains tested with sterile bamboo stick, every plate is equidistantly inoculated with 5 bacteria cakes, and every plant of bacterium is repeated 3 times, and 28 DEG C, culture 3 It, observes and records crocus transparent circle size.

The measurement of 2.3 phosphate solubilizations

Strains tested is inoculated on Meng Jinna culture medium, 28 DEG C is placed in and cultivates 10 days, whether there is or not Soluble phosphorus circle and Soluble phosphorus circles for observation Size.Its solvability to Phos is determined with the size of Soluble phosphorus circle, is worth bigger, and phosphate solubilization is strong, conversely, Soluble phosphorus energy Power is weak, does not generate the bacterial strain of Soluble phosphorus circle without phosphate solubilization；

The identification of 4 bacterial strains

4.1 strain morphology

(1) colony morphology characteristic: by strain inoculated in ISP₄On 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 strain inoculated in ISP₄On culture medium, then enter nothing in the position oblique cutting for being connected to strain The coverslip (inserted sheet method) of bacterium is placed in 28 DEG C and cultivates 5 days, takes out the coverslip for being covered with mycelia and is simply dyed with azaleine Microscopy, by observed form result microphotograph, as shown in Figure 2.4.2 bacterial strain LP69 physiological and biochemical properties

(1) utilization of carbon source is tested: by strain inoculated on the basal medium containing different carbon source, being placed in 28 DEG C of incubators Middle culture 5 days, continuous passage 3 times, can grow bacterium colony is the positive.

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

(3) growth temperature: by strain inoculated in ISP₄On culture medium, be respectively placed in 5 DEG C, 15 DEG C, 25 DEG C, 35 DEG C, 45 DEG C, It is cultivated 5 days in 55 DEG C of incubator, has seen whether that bacterium colony is formed.

(4) Salt tolerance: by strain inoculated in the ISP for containing 0%, 3%, 5%, 7%, 10%, 15%, 20%₄Culture medium On, it is placed in 28 DEG C of incubators after cultivating 5 days, has seen whether that bacterium colony is formed.

(5) pH resistance test: by strain inoculated in pH value be respectively 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0, 11.0 ISP₄On culture medium, it is placed in 28 DEG C of incubators after cultivating 5 days, has seen whether that bacterium colony is formed.

(6) Starch Hydrolysis reacts: by bacterial strain dibbling on the culture medium containing soluble starch, being repeated 3 times, trains in 28 DEG C It supports and is cultivated 3 days in case, after forming bacterium colony, Lu Geershi iodine solution is added dropwise on plate, entire plate is covered, plate is blue, If periphery of bacterial colonies is whether there is or not transparent circle appearance, illustrate that starch is hydrolyzed, transparent circle size shows that it hydrolyzes the size of starch ability.Such as No transparent circle occurs, then says ability of the bacterium without hydrolysis starch.

(7) cellulose decomposition: it in strain inoculated to Cellulose and congo red differential medium, will be placed in 28 DEG C of incubators and cultivate 5 It is proved to be the positive strain with degraded cellulose ability, vice versa if generating Clear & Transparent circle around bacterial strain.

(8) it gelatin liquefaction: by bacterial strain percutaneous puncture-inoculation in gelatin culture medium, is placed in 28 DEG C of incubators and cultivates 5 days, then be placed in It is observed after 30min in 4 DEG C of refrigerators as a result, if culture medium is positive for gelatin liquefaction in liquefaction, if culture medium is still solid-like State is then gelatin liquefaction feminine gender.

The Molecular Identification of 4.3 bacterial strains

4.3.1 reagent: lysozyme, Proteinase K, 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 ' (holds the site 8-27 base identical) with 16S rRNA 5 '； Primer B:5 '-TTAAGGTGATCCAGCCGCA-3 ' (holds the site 1523-1504 base identical) with 16S rRNA 3 '；

4.3.3 endogeny rayungus DNA is extracted

It takes a little thallus in sterile 1.5mL Eppendorf pipe, 20 μ l (50mg/mL) final concentrations is added and reach 2mg/ The lysozyme of mL is put into 37 DEG C of shaking tables, 200rmp/min, 1-2h；50 μ L of 20%SDS and 5 μ L (20mg/ of Proteinase K is added ML), mix and be put into 55 DEG C of shaking tables, 200rmp/min handles 1-2h；550 μ L (phenol: chloroform: isoamyl alcohol=25:24:1) is added Extracting, 12000rmp/min are centrifuged 10min, draw supernatant (in triplicate)；800 μ L dehydrated alcohols, 80 μ L 3mol/L second are added Sour sodium (pH4.8-5.2) mixes, and in 4 DEG C of precipitatings DNA, 1h, 12000rmp/min, 10min, abandons supernatant；200 μ L 70% are added Ethyl alcohol cleans tube wall 1-2 times, 12000rmp/min, is centrifuged 5min, abandons supernatant；After ethyl alcohol volatilization is dry, 50 1 × TE of μ L are added Dissolving DNA, and saved in -20 DEG C；The detection of 1% agarose gel electrophoresis.

4.3.4 raw unwrapping wire 16S rRNA gene magnification in

16S rRNA gene magnification condition: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 1min, 56 DEG C of annealing 1min, 72 DEG C are prolonged 2min, 30 circulations, 72 DEG C of overall elongation 10min.PCR product is through the raw work EZ Spin Column PCR Product in Shanghai Purification Kit UNlQ-1 pillar PCR product purification kit (SK1142-N) purifying, is carried out by operating guidance, pure Changing product send Suzhou Jin Weizhi Biotechnology Co., Ltd to be sequenced.

4.3.5 16S rRNA gene sequencing and phylogenetic tree building

Gained sequence submits the website EzBioCloud (www.ezbiocloud.net) to carry out sequence analysis after being sequenced, Selection similitude highest has delivered the 16S rRNA gene order of bacterial strain as reference sequence, is carried out using Clustal X software Multiple Sequence Alignment analysis, and determined by MEGA5.0 software with neighbouring method (Neighbor-Joining) phylogenetic tree construction The classification position of the endogeny rayungus.

5. experimental result

By detection, screens one plant of production heteroauxin ability, produces siderophore, the preferable endogeny rayungus of phosphate solubilization Bacterial strain is named as LP69.The bacterial strain is in ISP₄It is grown on culture medium, bacterium colony is closely smaller, edge is irregular, rough surface, bacterium Color is fallen in taupe, aerial hyphae physically well develops with substrate mycelium, and not chromogenic element, aerial hyphae forms flexible, spiral, top The spore chain for holding curling, is presented typical streptomycete feature (Fig. 1, Fig. 2).Bacterial strain LP69 can in 15 DEG C of -50 DEG C of temperature ranges Growth, cannot grow in pH < 4.0, can be grown in 0%-5%NaCl solution, can use D-Fructose, D-Glucose, D-MANNOSE, D- xylose, galactolipin are sole carbon source, cannot utilize L-arabinose, sucrose, gossypose, rhamnose；It can utilize L-cysteine, Serine, Valine cannot be only nitrogen source using L-Histidine and L-Methionine；It cannot make gelatin Liquefaction, can hydrolyze starch, decomposition of cellulose.

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

Bacterial strain LP69 has stronger Soluble phosphorus and generates the ability (table 1) of heteroauxin, siderophore.

1 endogeny rayungus LP69 growth-promoting capability result of table

2 Costa Rica streptomycete LP69 of example promotes tobacco growing effect experiment

1. prepared by inoculating strain spore bacteria suspension

Bacterial strain LP69 is inoculated on Gause I culture medium flat plate, is placed in 28 DEG C of incubators and cultivates 7 days, to entirely put down After plate covers with mycelia, culture medium is shredded with sterile scalpel, equipped in the seed bottle of sterile barley inoculum, 28 DEG C are cultivated 1 for access More than week, after all wheats cover with the spore of Costa Rica streptomycete LP69, wheat spore under sterile washing is taken out, so Spore suspension 10000rmp/min is centrifuged 5min afterwards, then the spore concentration of LP69 is adjusted to 10 with sterile water⁸cfu/mL.It is high Family name's No.1 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 are prepared.

2. 87 seed of tobacco cloud and mist is taken to be seeded in seedlings nursing plate, cultivated under natural conditions, watering is primary daily, as tobacco children When the plant height of seedling is 10cm, selection health grows fine and consistent tobacco seedling, carries out transfer.Spore suspension is in tobacco seedlings It is used when transplanting with root water, and carries out pouring root after tobacco seedlings transplanting training 10 days, spore suspension 20mL is poured near root, is made For the sterile water that equivalent is then added in the tobacco of blank control.

3. after pouring root 90 days, measuring Major Nutrient in plant height, Root morphology, fresh weight, dry weight, the number of blade and the soil of tobacco Constituent content.

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

Influence of the 2 inoculating strain LP69 of table to tobacco growing

5. the development degree of root system and the ability that plant absorbs nutrition are in close relations.Seen from table 3, through bacterial strain LP69 pouring root After processing, total root long, root total surface area, average root diameter, total root volume, total root hair number and the CK of tobacco root as a result, in the presence of Significant difference (P < 0.05).

Influence of the 3 inoculating strain LP69 of table to tobacco root

Note:^*Indicate significant difference (LSD, P < 0.05), similarly hereinafter.

Influence of the 4 inoculating strain LP69 of table to soil main nutrient elements

After bacterial strain LP69 root irrigation, the content of alkaline hydrolysis N, active potassic and available P in soil are significantly improved, is compared respectively CK improves 17.1%, 7.1%, 28.2% (table 4).

In conclusion the present invention is isolated from melia toosendan endogeny rayungus Costa Rica streptomycete (Streptomyces Costaricanus) LP69 can effectively facilitate tobacco growing.

The preparation of 3 bio-feritlizer of embodiment

1, microbial inoculum makes

1. plate strain (level-one 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), it is inoculated with Costa Rica It is cultivated 7 days at 28 DEG C after streptomycete (Streptomyces costaricanus) P69 and first order seed is made.

2. kernel culture (second level kind): being shredded the culture medium for covering with mycelia with sterile scalpel, access is equipped with sterile wheat In the seed bottle of grain kind, 28 DEG C of cultures 1 week or more after all wheats cover with the spore of Costa Rica streptomycete LP69, are taken Then spore suspension 10000rmp/min is centrifuged 5min by wheat spore under sterile washing out, then with sterile water by LP69's Spore concentration is adjusted to 10⁸cfu/mL。

3. solid state fermentation: using wheat bran as matrix (carrier), volume ratio is added by weight, corn flour 1%, beancake powder 1%, KH₂PO₄0.1%, CaCO₃0.1%, pH 7.0 adds water to adjust wheat bran 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 stationary culture 9 days Afterwards, natural air drying is taken out, is sent out after crushing for Costa Rica streptomycete (Streptomyces costaricanus) P69 solid-state Yeast-like fungi agent, microbial inoculum viable count are 2 × 10⁹cfu/g。

4. preparation saves: air drying preservation half a year does not influence its growth-promoting activity under microbial inoculum aseptic condition.

2, microbial inoculum P69 Field information

Test plant: flue-cured tobacco cloud 87.

When flue-cured tobacco transplantation of seedlings, handle by following 4 processing cured tobacco seedling: CK transplants more solito；Handle 1 Nutrition Soil Root dipping；It handles 2 microbial inoculum P69 and dilutes 10 times with Nutrition Soil；It handles 3 microbial inoculum P69 and dilutes 50 times of root dippings with Nutrition Soil；Handle 4 microbial inoculums P69 dilutes 100 times of root dippings with Nutrition Soil, and the measurement (table 5 of flue-cured tobacco economical character is carried out in the group phase of tobacco growth and Topping Stage With table 6).

Influence of the 5 microbial inoculum LP69 of table to flue-cured tobacco group phase economical character

In the group phase of cloud and mist 87, each dilution of the microbial inoculum LP69 within 100 times can effectively improve the strain of flue-cured tobacco The economical characters indexs such as the high and number of blade.

Influence of the 6 microbial inoculum LP69 of table to flue-cured tobacco Topping Stage economical character

In the Topping Stage of cloud and mist 87, each dilution of the microbial inoculum LP69 within 100 times equally can effectively improve flue-cured tobacco The economical characters index such as plant height, the number of blade, pitch and stem girth.

Claims (9)

1. one plant of Costa Rica streptomycete (Streptomyces costaricanus), deposit number is GDMCC 60071.

2. a kind of spore bacteria suspension of actinomyces, is prepared by the following steps to obtain:

Taking deposit number is Costa Rica's streptomycete bacterial strain of GDMCC 60071, is inoculated on Gause I culture medium flat plate, It is placed in 28 DEG C of incubators and cultivates 7 days, after entire plate covers with mycelia, shredded culture medium with sterile scalpel, access dress In the seed bottle for having sterile barley inoculum, 28 DEG C of cultures 1 week or more take out wheat sterile water after all wheats cover with spore Lower spore is washed, then by spore suspension with 10000 turns of speed per minute centrifugation 5 minutes, then with sterile water by spore concentration tune Section is 10⁸Cfu/ milliliters, obtain spore bacteria suspension.

3. the fertilizing method of spore bacteria suspension described in a kind of claim 2, comprising the following steps:

In plant seedlings transplanting, spore bacteria suspension is used with root water, and filled after plant shoots transplanting culture 10 days Root pours into 10-50 milliliters of spore bacteria suspension near root.

4. deposit number is the pityrosporion ovale of actinomyces described in Costa Rica streptomycete of GDMCC 60071 and/or claim 2 Suspension prepares the purposes of bio-feritlizer.

5. purposes according to claim 4, which is characterized in that the bio-feritlizer have produce heteroauxin, produce siderophore, Phosphorus is dissolved, soil nitrogen element content is improved, improve soil Determination of Potassium and/or improves the purposes of soil phosphorus element content.

6. a kind of bio-feritlizer for tobacco, which is characterized in that the Costa Rica for being GDMCC 60071 containing deposit number Streptomycete.

7. a kind of bacteria preparation contains Costa Rica streptomycete and acceptable auxiliary material that deposit number is GDMCC 60071.

8. bacteria preparation according to claim 7, which is characterized in that the bacteria preparation is solid-state microbial inoculum, the viable count of microbial inoculum For (1~5) × 10⁹Cfu/ grams.

9. bacteria preparation according to claim 7 or 8 is for producing heteroauxin, producing siderophore, dissolution phosphorus, raising Soil Nitrogen Constituent content improves soil Determination of Potassium and/or improves the purposes of soil phosphorus element content.