CN102031231B - Growth-promoting rhizobacteria and fungicide thereof, preparation method and application of fungicide - Google Patents

Abstract

The invention discloses a plant growth-promoting bacterium and its preparation method and application, that is, Klebsiella pneumoniae ( Klebsiella pneumoniae ) and the preparation method of the bacterial preparation, and Applications in phosphorus dissolution and nitrogen fixation. The invention has the advantages of providing a nitrogen-fixing and phosphorus-dissolving bacterium that integrates phosphorus-dissolving, nitrogen-fixing and auxin-producing growth-promoting abilities, and is prepared into a nitrogen-fixing and phosphorus-dissolving bacterial agent, which has comprehensive growth-promoting performance, high fertilizer efficiency, It is characterized by low cost, and is suitable for areas where nitrogen and phosphorus are deficient and the soil is barren. The preparation process is simple, and the efficiency of nitrogen-fixing and phosphorus-dissolving fertilizer is high, so it has a good development prospect.

Description

A plant growth-promoting bacterium and its agent, its preparation method and application

technical field

The invention relates to a plant growth-promoting bacterium agent, in particular to a plant growth-promoting bacterium and its bacterium agent, as well as its preparation method and application, belonging to the field of biological fertilizers.

Background technique

As essential mineral elements for plants, nitrogen and phosphorus are important components of various structural and functional substances such as proteins, nucleic acids, phospholipids, enzymes, and plant hormones in plant living cells. It plays an important role in the process of gene expression and regulation and cell signal transduction. The lack of nitrogen and phosphorus will inhibit the growth and development of plants, resulting in a decline in crop yield and quality. Long-term large-scale application of chemical fertilizers not only increases the cost of agricultural production, but also causes the selective accumulation of certain elements in the soil and the deterioration of soil physical and chemical properties, causing pollution to the environment. Therefore, replacing or partially replacing chemical fertilizers with microbial fertilizers is an inevitable trend to maintain the sustainable development of agriculture. Soil bacteria present in the rhizosphere of plants can promote the growth of crops by fixing nitrogen in the rhizosphere, producing auxin, and dissolving a large amount of insoluble phosphorus in the soil for plant absorption, so as to achieve the purpose of promoting growth and production.

Since it is difficult to obtain strains with a variety of high-efficiency growth-promoting effects at the same time, most of the plant growth-promoting agents for dissolving phosphorus and nitrogen-fixing plants that are currently put into production are multi-strain mixed agents, and there is antagonism between strains, and the nutritional requirements in the agent and metabolic imbalances etc.

Contents of the invention

The purpose of the present invention is to solve the above problems. The present invention provides a nitrogen-fixing and phosphorus-dissolving bacterium which integrates phosphorus-dissolving, nitrogen-fixing and auxin-producing multiple growth-promoting abilities, and is prepared as a nitrogen-fixing and phosphorus-dissolving bacterial agent with growth-promoting It has comprehensive performance, high fertilizer efficiency and low cost. It is suitable for areas where nitrogen and phosphorus are deficient and the soil is barren, and has a good development prospect.

The technical scheme of the present invention is: Klebsiella pneumoniae (Klebsiella pneumoniae), its microbial preservation unit is: China Microbial Strain Preservation Management Committee General Microbiology Center, preservation address: No. 1 courtyard, Beichen West Road, Chaoyang District, Beijing, preservation The date is August 31, 2010, and the preservation number is: CGMCC No.4128; the morphological characteristics of the Klebsiella pneumoniae are: straight bacteria, 0.5-0.8 μm in diameter, 3.2-5.0 μm in length, single distribution, with Capsule, Gram stain negative, immobile, growing on YMA medium to produce round translucent colonies and secreting a large amount of polysaccharides, which can fix nitrogen and dissolve inorganic phosphorus, and can only use glucose, sucrose, soluble starch and mannitol carbon source.

Klebsiella pneumoniae (Klebsiella pneumoniae), microorganism preservation number is: CGMCC No.4128, in the application as plant growth promoting agent.

A method for preparing a plant growth-promoting agent, which is carried out according to the following steps: (1) Klebsiella pneumoniae (Klebsiella pneumoniae) RSN19 is inoculated in YMA solid medium, and activated at 26°C-28°C for 18-24h; (2) The activated Klebsiella pneumoniae RSN19 is inoculated in a liquid medium for fermentation and culture at a temperature of 25°C to 29°C, and the number of Klebsiella pneumoniae RSN19 bacteria in each milliliter of fermentation broth is 4 to 6? ? 10 ¹⁰ ; (3) the fermented bacterial liquid of Klebsiella pneumoniae RSN19 in the above step (2), the sterile liquid culture medium and the pH 7.0 sterile phosphate buffer solution with a concentration of 10% are 4～8:2 according to the volume ratio Mix in a ratio of ~4:1; package with aseptic aluminum foil bag or aseptic polypropylene bag to make a single-species liquid inoculum; (4) combine the liquid inoculum prepared in step (3) with aseptic peat powder The mixture is mixed according to the volume ratio of 1:4-8 to make a solid microbial agent.

The liquid culture medium of the growth-promoting agent is composed of 0.5-1 parts of KH ₂ PO ₄ , 1-2 parts of yeast powder, 5-10 parts of glucose, and 0.2-0.4 parts of MgSO ₄ according to the weight fraction ratio. , 0.05-0.1 parts of NaCl, 900-1000 parts of water, wherein the pH value of the liquid medium is 6.4-7.4.

In the step (3), the fermentation broth, the sterile liquid medium and the sterile phosphate buffer are mixed according to a volume ratio of 6:3:1.

In the step (4), the volume ratio of the liquid bacterial agent to the sterile peat powder is 1:6.

The application of a plant growth-promoting bacterium agent prepared according to the above-mentioned preparation method in nitrogen fixation and phosphorus dissolution.

The invention has the advantages of providing a nitrogen-fixing and phosphorus-dissolving bacterium that integrates phosphorus-dissolving, nitrogen-fixing and auxin-producing growth-promoting abilities, and is prepared into a nitrogen-fixing and phosphorus-dissolving bacterial agent, which has comprehensive growth-promoting performance, high fertilizer efficiency, It is characterized by low cost, and is suitable for areas where nitrogen and phosphorus are deficient and the soil is barren. The preparation process is simple, and the efficiency of nitrogen-fixing and phosphorus-dissolving fertilizer is high, so it has a good development prospect.

The bacterial agent of the present invention is a single-species plant growth-promoting bacterial agent capable of fixing nitrogen, dissolving inorganic phosphorus, and producing auxin. It solves the problems of antagonism between strains and inconsistent nutritional requirements in multi-strain agents; the strain is single, the preparation process is simple, and the requirements for production and packaging equipment are low; it can promote the growth of alfalfa, oat, and red bean grass seedlings The effect is remarkable, and the cost is lower than that of multi-strain inoculum products with the same fertilizer effect, and it is easy to popularize and apply.

Detailed ways

Preferred embodiments of the present invention are described below, and it should be understood that the preferred embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

The isolation, identification and purification of embodiment 1 Klebsiella pneumoniae (Klebsiella pneumoniae): RSN19 bacterial strain is separated from the red bean grass (Onbrychisviciaefoliacv.) root nodule of surface sterilization with nitrogen-free solid medium and PKO dissolved inorganic phosphorus medium, The purified strains conformed to the general definition of Enterobacteriaceae by morphological identification. The pure culture of the bacterial strain was identified as Klebsiella pneumoniae (Klebsiella pneumoniae) (No. 084 in 2010 Weijianzi) by the Microorganism Identification and Preservation Center of the Chinese Academy of Sciences in 2010; the bacterial strain is resistant to 80mg/L kanamycin and 200mg/L Ampicillin was tolerated. The nitrogenase activity of RSN19 strain reached 6695±174nmol C2H2d-1, the amount of dissolved phosphorus (calcium phosphate) was 15.29±0.46mg/L in 4 days, and the auxin production in 6 days was 8.38mg/L. In the sand culture test under the condition of no nitrogen and only providing calcium phosphate, the biomass of alfalfa seedlings after applying RSN19 bacterial solution for 60 days increased by 119%, the plant height increased by 53%, and the number of leaves increased by 95% compared with the blank control plants without bacterial solution. %, used for soaking seeds, can significantly accelerate the elongation of radicle. Under the condition of nitrogen and phosphorus deficiency, it has obvious growth promoting effect on plants.

Embodiment 2 A preparation method of a plant growth-promoting bacterium agent is carried out according to the following steps: (1) Klebsiella pneumoniae (Klebsiella pneumoniae) RSN19 is inoculated in YMA (yeast mannitol-agar) solid medium, on Activation at 26°C to 28°C for 18 to 24 hours; (2) The activated Klebsiella pneumoniae RSN19 is inoculated in a liquid medium for fermentation and culture at a temperature of 25°C to 29°C, and the number of Klebsiella pneumoniae RSN19 bacteria in each milliliter of fermentation broth is cultivated 4 to 6? ? 10 ¹⁰ ; (3) the fermentation broth of Klebsiella pneumoniae RSN19 in the above step (2), the sterile liquid medium and the pH 7.0 sterile phosphate buffer solution with a concentration of 10% are 6:3:1 by volume Mix in the ratio; Encapsulate with aseptic aluminum foil bag or aseptic polypropylene bag, make single-species liquid inoculum; (4) liquid inoculum prepared in the above step (3) and aseptic peat powder are according to volume ratio: Mixed at a ratio of 1:6 to make a solid microbial agent.

The liquid culture medium of the growth-promoting agent is composed of 0.5-1 parts of KH ₂ PO ₄ , 1-2 parts of yeast powder, 5-10 parts of glucose, and 0.2-0.4 parts of MgSO ₄ according to the weight fraction ratio. , 0.05-0.1 parts of NaCl, 900-1000 parts of water, wherein the pH value of the liquid medium is 6.4-7.4.

Embodiment 3 bacterial agent of the present invention is under the condition of controlling nitrogen and phosphorus nutrition, sand culture promoting effect 1, experimental material 1.1 test material Liquid bacterial agent of the present invention: Klebsiella pneumoniae (Klebsiella pneumoniae), its microbial preservation number For: CGMCC No.4128.

1.2 Plant materials Seeds of Medicogosativa.cv.LongDong and Medicogo.sativa.cv.Queen, Qnobrychisspp.cv.Gansu and Calibre (Avena.sativa L.Calibre) ) seeds were provided by the Key Laboratory of Grassland Ecosystem Ministry of Education, the seed purity was 98%, and the seeds were sterilized with 70% ethanol solution for 5 minutes. In order to eliminate the influence of rhizobia and nitrogen-fixing bacteria on the experimental results, the seeds were soaked in sterile water to accelerate germination, the seed coat was removed, and finally rinsed with sterile deionized water for 5 to 8 times before sowing.

1.3 Sand culture nutrient solution Hoagland's complete nutrient solution: macroelements: Ca(NO3)2 4H2O 1417mg/L; KNO3 607mg/L; MgSO4 493mg/L; (NH4)3PO4 115mg/L; trace elements: H3BO3 2.86mg /L; MnCl2·4H2O 1.81mg/L; ZnSO4·7H2O 0.22mg/L; CuSO4·5H2O 0.08mg/L; H2MoO4·H2O 0.02mg/L; FeSO450mg/L. The nutrient solution was diluted to 1/4 concentration with sterile deionized water and then adjusted to pH 7.0±0.2 with NaOH (1mol/L) or HCl (1mol/L).

Hoagland's nitrogen-free and phosphorus-free nutrient solution is based on Hoagland's complete nutrient solution, changing the KNO3607mg/L in the formula to KCl 582mg/L, and removing (NH4)3PO4 to prepare, and adjusting the pH value of the solution to 7.0± 0.1.

2. Experimental method 2.1. Experimental design The experiment was carried out in April 2010 in the cultivation room of Gansu Agricultural University College of Grasslands. The cultivation medium used clean river sand, soaked in HCl for 48 hours, rinsed 6 times, and finally rinsed 3 times with distilled water, 150 ℃ drying and dry heat sterilization for 6 hours. Put 280g of dry river sand into a plastic culture cup with a diameter of 7cm and a volume of 400ml, level the sand surface, use tweezers to pick 10 plump seeds of the same size and place them evenly on the surface of the fine sand, and cover with 20g of sand. Water the liquid bacterial agent of this product (the number of viable bacteria > 106cfu/ml) diluted 100 times with sterile water to each treated seedling, add 20g/kg of calcium phosphate in the sand matrix used for cultivation, and use 1/4 Hoagland's without Nitrogen and phosphorus-free nutrient solution irrigation. For the treatment without inoculum solution, the treatment only watered with 1/4 Hoagland's nitrogen-free and phosphorus-free nutrient solution was used as the blank control (CK1); the treatment watered with 1/4 Hoagland's complete nutrient solution was used as the adequate nutrition control (CK2). Each treatment was repeated 4 times. During the treatment, the water content of each potted plant was measured by weighing method, and the water was supplemented with sterile water to 70% of the maximum water holding capacity of the sand matrix. After 50 days of treatment, the growth and morphological indexes were measured.

Determination index and method of fertilizer efficiency test Live seedling rate: after each treatment potted plant was washed and emerged, the number of live seedlings was counted, and the live seedling rate was calculated.

Plant height and tap root length: 4 seedlings were randomly selected from potted plants in each treatment to measure plant height and tap root length. Spread the cleaned plants on a glass plate, measure the natural height of the seedlings with a ruler, and then straighten the roots of the plants with tweezers to measure the length of the main root.

Single leaf area and number of leaves per plant: Take a single plant to record the number of leaves, and the area of a single leaf is weighed by tracing.

Single plant volume and single root volume: the selected seedlings were placed on filter paper, the water was blotted dry, and the plant volume and root volume were measured by the graduated cylinder drainage method.

Biomass accumulation of plants: take the whole plant in an oven, bake at 80°C until constant weight, and weigh the dry weight of a single plant with an electronic balance.

2.2 Data analysis The experimental data were statistically analyzed by LSD method with SPSS16.0 software.

3. Test results 3.1 The effect of the bacterial agent of the present invention on the survival rate of seedlings under the condition of low available phosphorus After 50 days of treatment, the application of the bacterial agent of the present invention can significantly improve the seedling survival rate of the tested crops. Longdong and Queen's alfalfa seedling survival rates are respectively increased by 32% and 28% compared with CK1, and are respectively increased by 8% and 12% compared with CK2, so that the seedling survival rates of red bean grass and oat are respectively increased by 27% to 44% compared with CK1. Table 1 The present invention Effect of inoculant on survival rate of alfalfa, oat and red bean grass seedlings (%) plant material CK1 CK2 Liquid graphics processing of the present invention Longdong clover 52% 76% 84% queen sativa 60% 68% 88% Gansu red bean grass 56% 88% 83% Oats (Calbre) 41% 85% 79% CK1 was treated with 1/4 Hoagland's nitrogen-free and phosphorus-free nutrient solution + calcium phosphate, and CK2 was treated with 1/4 Hoagland's complete nutrient solution, which were used as controls under normal nutritional conditions. The same below.

3.2 The effect of the bacterial agent of this product on the plant height and tap root length of crop seedlings After 50 days of treatment, the bacterial agent of this product increased the average plant height of Longdong alfalfa by 68.5% and 26.67% respectively compared with CK1 and CK2, and made Queen alfalfa higher than CK1 and CK2. 70.92% and 35.9%, making the plant height of oat seedlings 49.08% and 40.54% higher than CK1 and CK2, the difference was significant (P<0.05). And it can increase the plant height of Ormosia gansuensis by 21.14% and 17.35% compared with CK1 and CK2, respectively.

接种本产品的陇东苜蓿根系长度比CK1和CK2分别增高50.2％和18.98％，使皇后苜蓿根系长度比CK1和CK2高出56.23％和30.5％，使燕麦幼苗的根长高出CK1和CK2高出76.51％和53.09％。使甘肃红豆草根系长度比CK1和CK2分别提高25.69％和91.62％，差异显著(P＜0.05)。Table 2 Infection of bacterial agent of the present invention on alfalfa, oat and red bean grass seedling plant height (unit: cm)

The root length of Longdong alfalfa inoculated with this product is 50.2% and 18.98% higher than that of CK1 and CK2, the root length of queen alfalfa is 56.23% and 30.5% higher than that of CK1 and CK2, and the root length of oat seedlings is higher than that of CK1 and CK2 Out 76.51% and 53.09%. The root length of Gansu red bean grass was increased by 25.69% and 91.62% compared with CK1 and CK2, and the difference was significant (P<0.05).

3.3本产品菌剂对作物幼苗单叶面积、单株叶片数和生物量积累的影响使用本产品菌剂后，两种苜蓿的叶面积比CK1提高123.33％～353.76％，比CK2提高36.73～66.63％；甘肃红豆草叶面积比其CK1和CK2的处理分别增长107.36％和42.76％；燕麦(Calibre)幼苗的叶面积比其CK1和CK2的处理分别增长87.40％和71.99％，差异均具有统计学意义(P＜0.05)。Table 3 Inoculant of the present invention is on the influence of tap root length of alfalfa, oat and red bean grass (unit: cm)

3.3 The effect of this product on the single leaf area, number of leaves per plant and biomass accumulation of crop seedlings After using this product, the leaf area of the two alfalfas increased by 123.33% to 353.76% compared with CK1, and increased by 36.73 to 66.63% compared with CK2 %; the leaf area of red bean grass in Gansu increased by 107.36% and 42.76% respectively compared with its CK1 and CK2 treatments; the leaf area of oat (Calibre) seedlings increased by 87.40% and 71.99% respectively compared with its CK1 and CK2 treatments, and the differences were statistically significant Significance (P<0.05).

本产品菌剂对植株的叶片数也有明显的促进作用，两种苜蓿的叶片数比其CK1的处理提高53.75％～26.63％；甘肃红豆草叶片数比其CK1和CK2的处理分别增长118.52％和56.69％；燕麦(Calibre)幼苗的叶片数比其CK1和CK2的处理分别增长39.95％和33.80％，差异均具有统计学意义(P＜0.05)。Table 4 Infection of the bacterial agent of the present invention on the single leaf area of alfalfa, oats and red bean grass (unit: cm ² )

The bacterial agent of this product also has a significant promotion effect on the number of leaves of the plant. The number of leaves of the two kinds of alfalfa increased by 53.75% to 26.63% compared with the treatment of CK1; 56.69%; the number of leaves of oat (Calibre) seedlings increased by 39.95% and 33.80% compared with the CK1 and CK2 treatments, respectively, and the differences were statistically significant (P<0.05).

与CK1相比，使用本产品菌剂的两种苜蓿单株干重平均增加90.18％以上。甘肃红豆草干重较CK1增加86.33％、较CK2增加26.19％。燕麦植株干重较其CK1的处理增加217.54％，差异有统计学意义。Table 5 Inoculum of the present invention is on the impact of alfalfa, oat and red bean grass single plant leaf number (unit: blade/strain)

Compared with CK1, the average dry weight of two alfalfa plants using this product increased by more than 90.18%. The dry weight of Gansu red bean grass increased by 86.33% compared with CK1 and increased by 26.19% compared with CK2. The dry weight of oat plants increased by 217.54% compared with the CK1 treatment, and the difference was statistically significant.

3.4本产品菌剂对幼苗单株体积的影响表7本发明菌剂对苜蓿、燕麦和红豆草植株体积的影响(单位：cm³/株)

施用本产品菌剂后、陇东苜蓿和皇后苜蓿的幼苗单株体积比CK1提高79.23％～131.87％，比CK2提高36.71％和59.44％，甘肃红豆草和燕麦(Calibre)接种本发明菌剂后与CK1和CK2比较，植株体积增加76.48％以上，差异有统计学意义(P＜0.05)。Table 6 Inoculum of the present invention is on the impact of alfalfa, oat and red bean grass single plant biomass dry weight (unit: mg/strain)

3.4 The impact of the bacterial agent of this product on the volume of the seedlings per plant Table 7 The impact of the microbial agent of the present invention on the plant volume of alfalfa, oats and red bean grass (unit: ^cm / strain)

After applying the bacterial agent of this product, the seedling volume of Longdong alfalfa and Queen's alfalfa increased by 79.23% to 131.87% compared with CK1, and increased by 36.71% and 59.44% compared with CK2. Compared with CK1 and CK2, the plant volume increased by more than 76.48%, and the difference was statistically significant (P<0.05).

Four. Conclusion The bacterial agent of the present invention has a significant promoting effect on the growth of plants under the nutrient conditions of low phosphorus and low nitrogen, mainly manifested in removing the stress of plant phosphorus and nitrogen deficiency, promoting the growth of plant leaves and root systems, and promoting plant biomass accumulation.

The above test data show that the bacterial agent of the present invention has the following advantages. The bacterial agent of the present invention is a single-species plant growth-promoting bacterial agent with the ability to fix nitrogen, dissolve inorganic phosphorus and produce auxin, and can make full use of the most abundant insoluble inorganic in the soil Phosphate, while possessing multiple growth-promoting abilities, solves the problems of antagonism between strains and inconsistent nutritional requirements in multi-strain inoculants.

The strain is single, the preparation process is simple, and the requirements for production and packaging equipment are relatively low.

The growth-promoting effect on alfalfa, oat, and red bean grass seedlings is remarkable, and the cost is lower than that of multi-strain inoculum products with the same fertilizer effect, and it is easy to popularize and apply.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1. Klebsiella pneumoniae ( Klebsiella pneumoniae ) RSN19, its microorganism preservation number is: CGMCC No.4128; the morphological characteristics of said Klebsiella pneumoniae are: straight bacteria, diameter 0.5～0.8μm, length 3.2 ~5.0μm, single distribution, capsule, Gram staining negative, immobile, grow on YMA medium to produce round translucent colonies and secrete a large amount of polysaccharides, which can fix nitrogen and dissolve inorganic phosphorus. Glucose, sucrose , soluble starch and mannitol as the only carbon source. 2. The microbial preservation number of Klebsiella pneumoniae ( Klebsiella pneumoniae ) RSN19 as claimed in claim 1 is: the application of CGMCC No.4128 as a plant growth promoting agent. 3. a Klebsiella pneumoniae ( Klebsiella pneumoniae ) RSN19 microbial preservation number as claimed in claim 1 is: the preparation method of CGMCC No.4128 as plant growth-promoting bacterium agent, it is characterized in that carrying out according to the following steps: (1) Klebsiella pneumoniae ( Klebsiella pneumoniae ) RSN19 inoculation medium, activated at 26°C-28°C for 18-24h; (2) The activated Klebsiella pneumoniae RSN19 is inoculated in a liquid medium for fermentation culture at a temperature of 25°C to 29°C, and the number of Klebsiella pneumoniae RSN19 bacteria in each milliliter of fermentation broth is 4 to 6× ¹⁰¹⁰ ; (3) Mix the fermentation broth of Klebsiella pneumoniae RSN19 in the above step (2), sterile liquid culture medium and sterile phosphate buffer according to the ratio of 4 to 8:2 to 4:1 by volume, encapsulate and prepare Single-species liquid inoculum; (4) Mix the liquid bacterial agent prepared in the above step (3) with the sterile peat powder according to the volume ratio of 1:4-8 combined to make a solid microbial agent. 4. The preparation method of a plant growth-promoting bacterium according to claim 3, characterized in that the liquid culture medium of the growth-promoting bacterium consists of 0.5 to 1 part of KH ₂ P0 ₄ according to the weight fraction ratio , 1-2 parts of yeast powder, 5-10 parts of glucose, 0.2-0.4 parts of MgSO ₄ , 0.05-0.1 parts of NaCl, 900-1000 parts of water, wherein the pH value of the liquid medium is 6.4-7.4 . 5. the preparation method of a kind of plant growth-promoting bacterium agent according to claim 3 is characterized in that in described step (3), fermented bacterium liquid, aseptic liquid culture medium and aseptic phosphate buffer are 6 according to volume ratio :3:1 ratio mix. 6. The preparation method of a plant growth-promoting bacterial agent according to claim 3, characterized in that in the step (4), the volume ratio of the liquid bacterial agent to the sterile peat powder is 1:6. 7. The application of a plant growth-promoting bacterium prepared according to claim 3 in nitrogen fixation and phosphorus dissolution.