CN102911895B

CN102911895B - Phosphorus-dissolving stain PAN 4 and application of phosphorus-dissolving stain PAN 4 in promoting growth of walnuts

Info

Publication number: CN102911895B
Application number: CN201210365893.0A
Authority: CN
Inventors: 朱天辉; 李姝江; 余旋; 张丽娜; 谯天敏; 朴春根; 崔晓亮
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2012-09-27
Filing date: 2012-09-27
Publication date: 2015-02-18
Anticipated expiration: 2032-09-27
Also published as: CN102911895A

Abstract

The invention discloses a phosphorus-dissolving stain PAN 4, which is preserved in China General Microbiological Culture Collection Center of Institute of Microbiology of Chinese Academy of Sciences (CGMCC) in 3#, 1# Institute, West Beichen Road, Chaoyang District, Beijing City on 11, September, 2012, wherein the preservation number is CGMCC NO.6546; the classification name is Pseudomonas vancouverensis. The Pseudomonas vancouverensis Mohn et al PAN 4 stain in the invention has strong phosphorus-dissolving ability, effectively promotes growth of walnuts, improves rhizospheric microorganism environment, promotes growth of plants, improves photosynthetic physiological characteristic and phosphorus nutrient of the walnut seedlings, and has double functions of promoting and optimizing rhizospheric microorganism environment.

Description

Phosphorus decomposing bacterial strain PAN 4 and the application in walnut growth-promoting thereof

Technical field

The present invention relates to microorganism field, in particular Pseudomonas vancouverensis Mohn et al phosphorus decomposing bacterial strain PAN 4 and the application in walnut growth-promoting thereof.

Background technology

Phosphorus is the necessary nutritive element of growth and development of plants, and scarce phosphorus can cause crop yield obviously to reduce.Although Soil total nitrogen is higher, be present in soil mainly with indissoluble state, be difficult to be absorbed and used by plants.Produce and upper solve soil lack phosphorus problem by using phosphate fertilizer, not only can exhaust limited phosphate rock resource, also may cause environmental pollution, the destruction eubiosis.Phosphate-solubilizing bacteria (phosphate solubilizing bacteria) is that phosphorus that plant in soil can be difficult to absorb by a class is converted into can the beneficial microorganism of utilization state phosphorus, it is by activating soil phosphoric or the kind and the quantity that affect secretions from plant roots, increase the absorption of root system of plant to nutritive elements such as around K, Ca, Mg, Fe, Zn, thus promote growing of plant.At present, be object of inoculation mainly with gramineous crop in the research of phosphate solubilizing microorganism, and the impact on plant plant height and biomass is only concentrated on the research of phosphate solubilizing microorganism growgh promoting effects.The result of study of forefathers shows that inoculation phosphorus decomposing microbial inoculum all has obvious growgh promoting effects to crops such as paddy rice, corn, oat, mung bean, sugarcanes.And the growgh promoting effects using xylophyta as object of inoculation research phosphorus decomposing microbial inoculum, report is very few both at home and abroad.

Walnut is distributed widely in all over the world, is one of important commodity trees of China, because of to the effect of soil conservation and the nutrient health-care function of its fruit, more and more comes into one's own.But walnut plant strain growth is comparatively large to the demand of phosphoric, and phosphorus is the important factor of its growth of restriction, has no the report that Pseudomonasvancouverensis Mohn et al applies on walnut as phosphorus decomposing microbial inoculum at present.

Summary of the invention

Phosphorus decomposing bacterial strain PAN 4 bacterial strain of the present invention adopts dilution-plate method to be located away from Sichuan Nanchong walnut rhizosphere June 5 in 2008, through being accredited as Pseudomonas vancouverensis Mohn et a.Be preserved in China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center (CGMCC) on September 11st, 2012, address is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC NO.6546; Classification And Nomenclature is: Pseudomonas vancouverensis.

The Pseudomonas vancouverensis Mohn et al PAN4 new strains that the present invention relates to, not only dissolving P capacity is strong, and efficient growth-promoting walnut, improve rhizospheric microorganism environment, Promoting plant growth, improve Juglans photosynthetic physiological characteristics and Juglans phosphorus nourishing, there is growth-promoting and optimize root system micro-ecological environment dual-use function.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

Embodiment 1 Pseudomonas vancouverensis Mohn et al PAN4(CGMCC NO.6546) fermentation of liquid preparation:

1. primary inclined plane seed: ordinary method makes beef extract-peptone (peptone 10 g, extractum carnis 3 g, NaCl 5 g, agar 15 g, distilled water 1000 mL, pH 7.0) slant medium, cultivate at 26-28 DEG C after inoculation PAN4 and make first order seed in 24 hours.

2. secondary liquid seed: phosphate-solubilizing bacteria substratum [glucose 10 g, (NH ₄) ₂sO ₄0.5g, NaCl 0.3 g, KCl 0.3 g, FeSO ₄7H ₂o 0.03 g, MnSO ₄h ₂o 0.03 g, MgSO ₄7H ₂o 0.3 g, KH ₂pO ₄2.0 g, distilled water 1000mL]) after autoclaving, 300mL triangular flask ratio (logical oxygen controls) bottling is contained in 75mL liquid, at sterile state inoculation PAN4 inclined-plane seed, every bottle graft kind 1 inclined-plane seed, temperature 26-28 DEG C, initial pH value 7.5,120r/min shaking culture 36h, makes PAN4 liquid seeds.

3. liquid fermenting: glucose 20 g/L, (NH ₄cl) 1.0 g/L, initial pH value is 7.5, liquid amount is 75 mL, after autoclaving, be contained in 300mL triangular flask ratio (logical oxygen controls) bottling in 75mL liquid, at sterile state inoculation PAN4 liquid seeds, inoculum size 4%, temperature 26-28 DEG C, 120r/min shaking culture 72h, makes PAN4 liquid preparation.Turbidimetry for Determination biomass (OD600 value 1.100), and Simultaneous vaccination 1 mL bacterium liquid is in PKV liquid nutrient medium (glucose 10 g, (NH ₄) ₂sO ₄0.5 g, NaCl 0.3 g, KCl 0.3 g, FeSO ₄7H ₂o 0.03 g, MnSO ₄4H ₂o 0.03 g, MgSO ₄7H ₂o 0.3 g, Ca ₃(PO ₄) ₂10 g, distilled water 1000 mlL, pH 7.0 ~ 7.5), molybdenum antimony resistance colorimetric method measures its dissolving P capacity and reaches more than 200mg/L.

(2) preparation is preserved: bottled preparation normal temperature preserves half a year (1% glycerine) or low temperature (4 degree) does not affect growth-promoting effect in 1 year half.

(3) field is used:

Walnut is pressed 100-200 times of (volume ratio) extent of dilution and is filled with root along root system breadth, every strain 200 milliliters, and spring, summer respectively once, continuous 3 years, can promote that walnut grows (plant height, biomass), improves Juglans photosynthetic physiological characteristics and Juglans phosphorus nourishing; Improve walnut rhizospheric microorganism environment.

Embodiment 2

Test and carry out at Sichuan Agricultural University's forest field gene bank in March, 2009, Juglans growing period carries out Routine Management, and the position of flowerpot of rotating at regular intervals.Test soil picks up from forest genetics base, Daxing town, Ya'an, Sichuan Province, and sampling depth is 0 ~ 20 cm, sandy loam.Soil physico-chemical property is after measured: soil pH 7.1, organic content 10.43 g/kg, total nitrogen content 0.80 g/kg, content of tatal phosphorus 0.34 g/kg, available phosphorus content 4.50 mg/kg.Cross 1 mm sieve after sample folk song is dry, and with cross river sand that 1 mm sieves with 3:1(V:V) mix after for pot experiment.After Juglans transplant survival, point different concns injects liquid bacterial agent 50 mL in above-described embodiment 1 around walnut rhizosphere with asepsis injector, repeat 6 times, with substratum (not containing bacterium) the 50 mL contrast after every basin injection sterilizing.Carry out following mensuration:

1. the mensuration of walnut plant rhizosphere soil microorganism fauna and growth

In mid-October, 2009, nursery stock measured rhizosphere soil bacterium, fungi, Population of Actinomycetes after stopping growing.Measuring method is dilution-plate method, and bacterium adopts beef-protein medium; Actinomycetes adopt improvement Gause I synthetic medium, face used time every 300 mL substratum in the substratum melted and add 3% potassium bichromate 1 mL; Fungi adopts Ma Dingshi rose bengal medium, and face used time every 100 mL substratum and add 1% streptomycin solution 0.3 mL, result represents with quantity contained by every gram of dry ground.Often process repetition 5 times.

After in mid-October, 2009, nursery stock stopped growing, measure its plant height respectively, and each process is chosen 5 strains and grown consistent normal plant, complete its root, stem, leaf 30 min respectively at 105 DEG C, dry at 75 DEG C and weigh to constant weight, calculate every strain total biomass.The results are shown in Table 1:

Table 1 phosphorus decomposing microbial inoculum is on Juglans growth and the impact of rhizospheric microorganism

Bacteria suspension concentration	Rhizospheric microorganism (probiotics)	Plant height (cm)	Biomass (g/ strain)
				Stoste	3.5 ×10 ¹⁰a	48.8a	33.92a
Dilute 100 times	3.1 ×10 ¹⁰a	48.1a	33.17a
				Dilute 200 times	2.8 ×10 ¹⁰a	47.8a	32.51a
Dilute 400 times	4.5×10 ⁸b	38.1b	25.74b
				Dilute 800 times	6.5 ×10 ⁶c	35.5c	20.03c
Dilute 1000 times	1.5×10 ⁶c	32.9d	18.02d
				CK	3.5 ×10 ⁴d	32.6.d	17.64 d

Note: the different lowercase of same column represents that between reason, difference reaches 5% conspicuous level everywhere; Lower same.

2. Juglans leaf photosynthesis physiological property measures

The mensuration of photosynthetic characteristics: the mensuration of leaf photosynthesis Gas exchange parameters adopts the Li-cor 6400(U.S.) portable photosynthetic determinator, the vigorous period (in early July, 2009) of plant strain growth, the functional leaf on annual shoot is selected to measure in the sunny morning.Measuring parameter has: Net Photosynthetic Rate (Pn), transpiration rate (Tr), calculates Leaf water use efficiency (WUE, the ratio of instant cleaning liquid photosynthetic rate and transpiration rate simultaneously.Measure and select to carry out in the comparatively stable period of temperature up to 11 time in the morning 9,3 strain plant replications are selected in each process, often select good strains in the field for seed and get the acrogen of compound leaf in the middle part of plant, and the Photosynthetic parameter of every sheet leaf makes 5 records, gets its mean value.In order to eliminate temporal error, between respectively processing during each replication, take random method for measuring.

The mensuration of chlorophyll content in leaf blades: the mensuration of chlorophyll content in leaf blades is with reference to Wang Bin etc. ^[217]method.Measuring for second day after photosynthetic characteristics measures.Every strain gathers the functional leaf 3 ~ 4 on 1 year raw branch.Measuring method is: with 1 cm punch tool punching, 0.5g leaf dish is put in every test tube, add 10 mL chlorophyll mixing vat liquors (80% acetone), under dark condition after lixiviate 24 h, shake up and after leaving standstill, supernatant liquor is poured in cuvette, do blank zeroing with mixing vat liquor, measure the optical density value at 663 nm, 645 nm and 470 nm places.The results are shown in Table 2:

Table 2 phosphorus decomposing microbial inoculum is on the impact of Juglans photosynthetic physiological characteristics

3. the mensuration of walnut plant root, stem, leaf phosphorus content

After in mid-October, 2009, plant stopped growing, the plant collected specimens of 5 strain normal growths is chosen in each process.Root, stem, leaf collection are pressed in every strain respectively.Root, stem, leaf use H after pulverizing respectively ₂sO ₄-H ₂o ₂disappear and boil, content of tatal phosphorus molybdenum antimony resistance colorimetric method measures.The results are shown in Table 3:

Table 3 phosphorus decomposing microbial inoculum is on the impact of Juglans phosphorus nourishing

The present invention filter out the new bacterium of Pseudomonas vancouverensis Mohn et al PAN4 can significantly improve the energy for growth of walnut, improve Juglans photosynthetic physiological characteristics and Juglans phosphorus nourishing; And can biological activity of soil be improved, significantly reducing costs, is an ideal biological fertilizer.

Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.

Claims

1. the application of phosphorus decomposing bacterial strain PAN 4 bacterial strain in walnut growth-promoting, is characterized in that, described phosphorus decomposing bacterial strain (Pseudomonas vancouverensis) PAN 4 bacterial strain, and deposit number is CGMCC NO.6546; By the liquid preparation of 100-200 times of dilution phosphorus decomposing bacterial strain PAN 4 bacterial strain, fill with root, every strain 200 milliliters along root system breadth, spring, summer respectively once, continuous 3 years, can promote that walnut grows, improve Juglans photosynthetic physiological characteristics and Juglans phosphorus nourishing; Improve walnut rhizospheric microorganism environment;

The fermentation of liquid preparation:

1. primary inclined plane seed: ordinary method makes beef extract-peptone slant medium, cultivates after inoculation PAN4 bacterial strain and makes first order seed in 24 hours at 26-28 DEG C;

2. secondary liquid seed: phosphate-solubilizing bacteria substratum is after autoclaving, the bottling of 300mL triangular flask ratio is contained in 75mL liquid, at sterile state inoculation PAN4 bacterial strain inclined-plane seed, every bottle graft kind 1 inclined-plane seed, temperature 26-28 DEG C, initial pH value 7.5,120r/min shaking culture 36h, makes PAN4 liquid seeds;

3. liquid fermenting: glucose 20g/L, (NH ₄cl) 1.0g/L, initial pH value is 7.5, and liquid amount is 75mL, after autoclaving, the bottling of 300mL triangular flask ratio is contained in 75mL liquid, at sterile state inoculation PAN4 liquid seeds, inoculum size 4%, temperature 26-28 DEG C, 120r/min shaking culture 72h, makes PAN4 liquid preparation.