阅读说明：本技术 根瘤菌klbmpgc32和大肠杆菌klbmppt020在减施氮磷肥中的应用 (Application of rhizobium KLBMPGC32 and escherichia coli KLBMPPT020 in nitrogen and phosphorus fertilizer reduction ) 是由 李欠欠 吴洁 蒋继宏 王彦苏 于 2020-12-18 设计创作，主要内容包括：本发明提供了内生细菌Rhizobium sp.KLBMPGC32和Enterobacter sp.KLBMPPT020在减施氮磷肥中的应用。通过向种植百合的土壤中喷洒Rhizobium sp.KLBMPGC32和Enterobacter sp.KLBMPPT020的混合菌液,诱导土壤-百合的解磷固氮作用,从而为百合提供部分氮磷养分,减少肥料氮磷投入。本发明菌液通过固氮、溶磷等内生菌在土壤-植株中的作用,增加植物对氮磷养分的吸收,促进植物生长,提高氮磷利用潜力,减少肥料氮磷环境损失,可替代部分氮磷肥的施用。以Rhizobium sp.KLBMPGC32和Enterobacter sp.KLBMPPT020制备的菌液作为微生物肥料,无化学肥料残留,改善土壤微生态环境、土壤养分循环,提高植物产量和抗逆能力；制备过程简单,成本低,效果好,使用方便。(The invention provides application of endophytic bacteria Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 in nitrogen and phosphorus fertilizer reduction. By spraying a mixed bacterial solution of Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTPCT020 into the soil for planting lily, the phosphorus and nitrogen dissolving and fixing effects of the soil-lily are induced, so that part of nitrogen and phosphorus nutrients are provided for the lily, and the nitrogen and phosphorus input of a fertilizer is reduced. The bacterial liquid of the invention increases the absorption of plants to nitrogen and phosphorus nutrients, promotes the growth of plants, improves the utilization potential of nitrogen and phosphorus, reduces the environmental loss of nitrogen and phosphorus of fertilizers and can replace the application of partial nitrogen and phosphorus fertilizers through the action of endophytes such as nitrogen fixation, phosphorus dissolution and the like in soil-plants. The bacterial liquid prepared from Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 is used as the microbial fertilizer, no chemical fertilizer residue is left, the soil micro-ecological environment and the soil nutrient circulation are improved, and the plant yield and the stress resistance are improved; simple preparation process, low cost, good effect and convenient use.)

1. Application of endophytic bacteria Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 in nitrogen and phosphorus fertilizer reduction.

2. The application according to claim 1, wherein the application comprises: and spraying the mixed bacterial solution of the two bacteria into the soil for planting the lily to promote the phosphate dissolving and nitrogen fixing effects of the soil-lily plants.

3. The use of claim 1, wherein the phosphorus-solubilizing and nitrogen-fixing bacterial liquid is sprayed by spraying the mixed bacterial liquid into soil at the root of the lily plant.

4. The use of two bacteria of Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 in claim 1 for preparing a biological fertilizer containing phosphorus-solubilizing and nitrogen-fixing bacteria liquid.

5. The application of the biological fertilizer of claim 4 in improving nitrogen and phosphorus absorption of lily, enhancing nitrogen and phosphorus utilization capability of soil-lily plants, and promoting phosphorus and nitrogen dissolution and fixation of a soil-lily plant system.

6. The biological fertilizer of claim 4 is used for improving the yield of lily bulbs and the nitrogen and phosphorus absorption amount.

Technical Field

The invention relates to a biological fertilizer, in particular to application of endophytic bacteria in nitrogen and phosphorus fertilizer reduction of plants.

Background

In agricultural production, the application of the chemical fertilizer in the soil can effectively supplement mineral nutrient elements required in the growth process of crops, particularly nitrogen and phosphorus are taken as first and second large nutrient elements necessary for plants, the growth, development and yield of the crops are played an important role by the application of the chemical fertilizer, but meanwhile, the unreasonable application of the chemical fertilizer also causes a series of ecological environmental problems of soil-plants-environment, such as a series of environmental problems of soil acidification, increase of agricultural greenhouse gas emission, influence on air quality and visibility, harm to human health, reduction of species diversity, promotion of eutrophication of water bodies and the like due to the large application of the nitrogen fertilizer; phosphorite is non-renewable resource, and along with the increase of phosphorus fertilizer demand, limited phosphorus fertilizer resource is being exhausted fast, and the back is executed into soil to the phosphorus fertilizer simultaneously, very easily is fixed by soil, greatly reduced its validity to the crop. The problem caused by applying a large amount of nitrogen and phosphorus fertilizers to agricultural soil seriously hinders the green sustainable development of agriculture.

Endophytes refer to microorganisms that are present within plant tissues during part or all of their life cycle, and infesting host plants do not exhibit significant disease symptoms, and this endophyte is a unique cost-benefit interaction between plants and microorganisms. The endophyte can directly regulate the growth and development process of plants by secreting auxin, cytokinin, gibberellin and other phytohormones; meanwhile, the endophyte can increase the nutrient absorption of plants through the actions of fixing nitrogen, dissolving phosphorus and the like, promote the growth of host plants and improve the stress resistance of the plants. The potential of endophytes of plants is excavated, the endophytes are prepared into microbial fertilizers to promote virtuous circle of soil, the application of partial nitrogen and phosphorus fertilizers is replaced, the environmental loss risk brought by the fertilizers is reduced, and the method has important research significance for agricultural green sustainable development.

As a large native country of lily plants, China has abundant germplasm resources, and accounts for more than half of the total number of lilies all over the world. The lily bulb is sweet in taste, slightly bitter and neutral in nature, has the effects of tonifying heart and lung, regulating spleen and stomach and the like, and has high medicinal value. Due to the medicinal and edible value of lily, people increasingly demand lily, but at present, lily production in China still has many problems, wherein quality and yield of lily are affected by application of a large amount of chemical fertilizer and continuous cropping obstacles.

Therefore, the phosphorus-dissolving and nitrogen-fixing endophytic bacteria liquid is prepared to replace part of the fertilizer to be applied to lily soil, so that the yield of lily and the utilization rate of nitrogen and phosphorus are improved, fertilizer resources can be saved, and the ecological environment problem caused by the application of the nitrogen and phosphorus fertilizer is reduced.

Disclosure of Invention

In order to reduce the influence of a large amount of nitrogen and phosphorus fertilizer input on lily growth and the risk of environmental loss, the invention provides the application of two endophytes, namely Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTab.020 in phosphorus and nitrogen dissolving and fixing, which forms the first aspect of the invention.

The invention also provides application of the bacterial liquid prepared from Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTP020 in reducing nitrogen and phosphorus fertilizers, and the two prepared endophytic bacterial liquids are sprayed into the root soil of the lily plant to promote the phosphorus and nitrogen dissolving and fixing effects of the soil-lily plant, improve the yield of the lily and the potential of nitrogen and phosphorus utilization, and the like, thereby forming the second aspect of the invention.

The invention has the beneficial effects that: the bacterial liquid prepared from Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 is used for replacing part of nitrogen and phosphorus fertilizers to be applied to soil, so that the potential of utilizing nitrogen and phosphorus of soil-plants is developed, and the risk of damaging the agricultural ecological environment by the nitrogen and phosphorus fertilizers is reduced; the bacterial liquid prepared from Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTP020 has the advantages of simple preparation process, low cost, good effect and convenient use.

The phosphate solubilizing bacterium Enterobacter sp.KLBMPPTa020 is obtained by separating pinellia ternata, and is published in the literature: trypan pinellia ternate endophytic bacteria diversity and influence on accumulation of pinellia ternate secondary metabolites [ D ] Jiangsu university, 2017. the phosphate solubilizing bacteria Rhizobium sp.KLBMPGC32 of the invention are separated from garlic, screened from 28 lily bulb endophytic bacteria and 208 garlic endophytic bacteria, separated from nitrogen-fixing phosphate-solubilizing endophytes by using a JNFbN-free culture medium, a NBRIP culture medium and a Monkina organophosphorus culture medium, and sequenced by using a 16S amplicon. Identifying the obtained nitrogen-fixing phosphorus-dissolving endophyte to obtain 1 nitrogen-fixing bacterium (Rhizobium sp.). The literature is presented: li Lexi, Lidan, Zhang Liang, Wang Junjuan, Yangyou, Jiangjunhong, separation of garlic endophyte and screening and identification of antagonistic strains [ J ]. Jiangsu agricultural science, 2018,46(05):97-101.

The two phosphorus-dissolving nitrogen-fixing bacteria can be obtained by separating and screening plants in garlic and pinellia ternata planting areas in various places by a conventional method, and can also be claimed by the applicant. The applicant states that within twenty years from the filing date, the public may contact the applicant to claim the species.

Drawings

FIG. 1 is a phylogenetic tree of 16S rRNA gene of azotobacter Rhizobium sp.KLBMPGC32 according to example 1 of the present invention;

FIG. 2 is a phylogenetic tree of 16S rRNA gene of phosphate solubilizing bacterium Enterobacter sp.KLBMPPTa020 according to example 1 of the present invention;

FIG. 3 is a photograph showing the coexistence/antagonism assay of Enterobacter sp.KLBMPPTa020 and Rhizobium sp.KLBMPGC32 in example 1 of the present invention;

FIG. 4 is a photograph showing the identification of nitrogen-fixing ability of two bacteria, Enterobacter sp.KLBMPPTC 020 and Rhizobium sp.KLBMPGC32, on JNFbN-free medium in example 1 of the present invention, wherein the left is the azotobacter of Rhizobium sp.KLBMPGC32, and the right is the phosphate solubilizing bacteria of Enterobacter sp.KLBMPPTC 020;

FIG. 5 is a photograph showing the identification of phosphate solubilizing ability of two bacteria, Enterobacter sp.KLBMPPT020 and Rhizobium sp.KLBMPGC32, on PVK medium in example 1 of the present invention, with the left side showing Enterobacter sp.KLBMPPT020 and the right side showing Rhizobium sp.KLBMPGC32;

FIG. 6 is a graph showing the trend of OD values of Enterobacter sp.KLBMPPTa020 and Rhizobium sp.KLBMPGC32 bacteria in example 1 of the present invention with time;

FIG. 7 is a photograph showing the harvest of lily phosphorus stems treated with different nitrogen and phosphorus fertilizers with and without bacterial liquid applied to Enterobacter sp.KLBMPPTa020 and Rhizobium sp.KLBMPGC32 in example 2 of the present invention;

FIG. 8 is a histogram of the fresh weight of lily bulbs after SPSS statistical analysis in example 2 of the present invention;

FIG. 9 is a histogram of the nitrogen uptake of lily plants after SPSS statistical analysis in example 2 of the present invention;

FIG. 10 is a histogram of phosphorus uptake of lily plants after SPSS statistical analysis in example 2 of the present invention;

FIG. 11 is a histogram of the utilization of nitrogen fertilizer after SPSS statistical analysis in example 2 of the present invention;

FIG. 12 is a bar graph showing the utilization of phosphate fertilizer after SPSS statistical analysis in example 2 of the present invention;

the specific implementation mode is as follows:

example 1: screening/identification of Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 and preparation of mixed bacterial liquid

Selecting a healthy lily conventional method to screen 28 strains of lily bulb endophytic bacteria, adding 208 strains of garlic endophytic bacteria preserved in a laboratory, and separating nitrogen-fixing and phosphorus-dissolving endophytic bacteria by using a JNFbN-free culture medium, a NBRIP culture medium and a Monkina organophosphorus culture medium. And identifying the obtained nitrogen-fixing phosphorus-solubilizing endophyte to obtain 1 strain of azotobacter sp. The phosphate solubilizing bacterium Enterobacter sp. With primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-AAGGAGGTGATCCAGCCGCA-3' PCR amplification of the 16S rDNNA gene was performed. The PCR reaction system is as follows: 12.5. mu.L of 10 XBuffer Mix (containing Taq enzyme), 0.5. mu.L of 10. mu. mol/L27F, 0.5. mu.L of 10. mu. mol/L1492R, 10.5. mu.L of ddH2O 10.5, and 1. mu.L of template DNA, wherein the PCR amplification product is detected by 0.8% agarose gel electrophoresis and then sent to Shanghai Producer company Limited for sequencing. And (3) constructing a phylogenetic tree, sending to a biological sequencing to obtain a strain 16s sequence, then carrying out BLAST sequence comparison through an NCBI website to obtain a sequence with the similarity of more than 90%, and then constructing an NJ (Neighbor-Joining) phylogenetic tree by using MEGA software. The phylogenetic tree obtained by sequencing the 16S amplicon of azotobacter Rhizobium sp.KLBMPGC32 is shown in figure 1, and the phylogenetic tree obtained by sequencing the 16S amplicon of phosphate solubilizing bacterium Enterobacter sp.KLBMPPTC 020 is shown in figure 2. Wherein the NBRIP culture medium is sterile solution, mainly comprises glucose, magnesium chloride, magnesium sulfate, potassium chloride, calcium phosphate, etc., and is sterilized, and the reagent does not contain ACC (also called 1-aminocarbonyl-1-cyclopropane carboxylic acid); monkina organophosphorus culture medium is as follows: 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.5 g of yeast extract powder, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.03 g of ferrous sulfate, 0.03 g of manganese sulfate, 0.2 g of lecithin, 1 g of calcium carbonate, 15 g of agar and pH of 7.0-7.5.

Performing antagonism detection between Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPT020 by using a cross-hatch method, activating the two strains by using an LB solid plate culture medium, putting the strains into an incubator by using a plate-hatch method, putting the incubator into an incubator at 28 ℃, culturing for 7 days, and regularly observing the bacterial growth condition of a bacterial intersection as shown in figure 3, wherein the boundary of the two strains has no obvious inhibition phenomenon, which indicates that the two strains have no antagonism and can coexist.

Identification of Nitrogen-fixing ability of Enterobacter sp.KLBMPPT020 and Rhizobium sp.KLBMPGC32 by nitrogen-free medium As shown in FIG. 4, two kinds of bacteria were grown in an incubator at 28 ℃ on a nitrogen-fixing medium for 7 days and transferred, and the bacteria could grow after more than five times of transfer, indicating that both the Enterobacter sp.KLPBMPPT020 and the Rhizobium sp.KLBMPGC32 had nitrogen-fixing ability. Wherein the nitrogen-free medium was prepared (JNFbN-free) as follows: k₂HPO₄0.5 g of CaCO₃1.0 g, MgSO_4·7H₂0.2 g of O, 10.0 g of cane sugar, 0.2 g of KCl, 7.0-7.5 of PH, 20.0 g of agar and 1000 ml of distilled water.

The phosphorus-dissolving capacity of Enterobacter sp.KLBMPPTab and Rhizobium sp.KLBMPGC32 is identified by PVK culture medium as shown in figure 5, the two bacteria are respectively inoculated on the left side and the right side of the PVK solid culture medium and cultured in an incubator at 28 ℃ for 7 days, a very obvious transparent ring is arranged around the Enterobacter sp.KLPBMPPT020 bacteria, which shows that the bacteria have very strong phosphorus-dissolving capacity, and the PVC of the phosphorus-dissolving capacity is about 5. And no transparent ring is arranged near Rhizobium sp.KLBMPGC32, which indicates that the phosphate solubilizing ability is not provided. Wherein the PVK culture medium is as follows: 10 g of glucose, 5 g of calcium phosphate, 0.5 g of ammonium sulfate, 0.2 g of sodium chloride, 1 g of magnesium sulfate heptahydrate, 0.5 g of yeast extract, 0.002 g of manganese sulfate monohydrate, 0.002 g of ferrous sulfate heptahydrate, 20 g of agar, 6 ml of bromophenol blue, 7-7.2 of PH and 1000 ml of distilled water.

The two strains are respectively inoculated into corresponding liquid culture media according to the inoculum size of 2 percent, the culture is carried out for 40 hours at the temperature of 28 ℃ and at the speed of 160r/min, sampling is carried out when culturing the 4 th, 8 th, 12 th, 16 th, 20 th, 24 th, 28 th, 32 th, 36 th and 40 th strains, 10 ml of samples are taken each time, each bottle of bacterial liquid is continuously sampled for 4 times, and the sampling is repeated for 3 times. The absorbance of the bacterial suspension at an appropriate wavelength was measured on a 722s visible spectrophotometer, and the growth curve of the bacterial cells was plotted with time as abscissa and the concentration of the bacterial cells as ordinate, as shown in FIG. 6. After the bacteria shaking is started, when the two bacteria are in about 8 hours, the growth rate of the concentration of the bacteria is the highest, the growth rate is an exponential phase, and at the moment, experiments can achieve a better effect so as to determine the inoculation time when the functional strains are combined.

Example 2: phosphorus and nitrogen dissolving and fixing effects of Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPTa020 mixed bacterial liquid on soil-lily plants

Endophytic bacteria Rhizobium sp.KLBMPGC32 and Enterobacter sp.KLBMPPT020 with phosphorus and nitrogen dissolving potentials are applied to prepare a composite endophytic bacteria liquid, and a potting test is carried out to verify whether the prepared bacteria liquid has the functions of synergism and emission reduction. The test plant is lilium tigrinum, the soil is typical moisture soil, and the physicochemical properties of the soil before the test are shown in table 1.

TABLE 1 physicochemical Properties of the soil tested

Adopt the PVC earth pillar circle basin planting lily that diameter and height are 30cm, experimental totally 7 are handled, 4 are repeated, design according to 3X 2 factor, three different nitrogen phosphorus level and endophyte intergrowth group, non-intergrowth group: the test method comprises the following steps of preparing a nitrogen-free phosphate fertilizer control group (without B and NP), a nitrogen-free phosphate fertilizer + endophyte liquid (without B and NP), a nitrogen-phosphorus fertilizer control group (without B and normal NP), a nitrogen-phosphorus fertilizer control group (without B and 20% NP), a nitrogen-phosphorus fertilizer + endophyte liquid (with B and 20% NP), a nitrogen-phosphorus fertilizer control group (without B and 40% NP), and a nitrogen-phosphorus fertilizer + endophyte liquid (with B and 40% NP), wherein specific test treatment is shown in table 2.

TABLE 2 Lily fertilization treatment

As shown in figure 7, the lily bulbs without endophytic bacteria liquid are harvested in the same pot natural environment and under the same amount of nitrogen and phosphorus fertilizers, the lily bulbs without endophytic bacteria liquid are not uniform in size and lack, the influence of the environment such as water logging/rainfall or the stress such as diseases is calculated, and the bacteria addition is likely to inhibit the invasion of harmful pathogenic bacteria and enhance the adaptability of the lily to the change of the external environment. The results of the statistical analysis of SPASS (Tukey, p <0.01) on the fresh weight of the harvested lily bulbs show that (figure 8), the addition of the endophyte liquid (B) has the tendency of increasing the fresh weight of the lily bulbs, and particularly, the addition of B significantly increases the fresh weight of the bulbs under the comparison of 40% reduced NP fertilization treatment. In addition, the addition of the endophyte liquid (B) has the potential of improving the nitrogen and phosphorus absorption of plants (figures 9 and 10); under the condition of no NP fertilizer application, the nitrogen absorption amount of the plants is obviously increased by adding B, and the potential of improving the phosphorus absorption amount of lily plants is also realized, which shows that the nitrogen fixation and phosphorus dissolution capability of soil-plants are improved by B. Compared with other treatments, the fertilizing treatment of subtracting 40% of NP and adding endophyte liquid can stabilize and improve the yield, and meanwhile, the utilization rate of NP fertilizer is obviously increased (figure 11 and figure 12), wherein the utilization rate of nitrogen fertilizer is NUE% (N absorption in a fertilizing area N-absorption without fertilizer and B area N absorption)/N fertilizer application amount is 100%, and the utilization rate of phosphate fertilizer is PUE% ((P absorption in a fertilizing area P-absorption without fertilizer and B area P absorption)/P fertilizer application amount is 100%). It is worth noting that the utilization rate of nitrogen fertilizer (9-27%) and the utilization rate of phosphate fertilizer (9-32%) under each fertilization treatment are generally low, and due to the fact that rainfall is frequent and the rainfall is larger than that of the former year under the high temperature in summer in 2020, on one hand, the loss amount of nitrogen and phosphorus fertilizer leaching and the like is large, on the other hand, the underground part of lily is flooded, growth is not facilitated, nutrient substances are transferred to the underground bulb, and accordingly lily harvesting is performed in advance.

The results of nitrogen input (initial soil nitrogen + nitrogen application amount + nitrogen apparent mineralization, nitrogen output (crop nitrogen absorption amount, nitrogen surplus (nitrogen input-nitrogen output), and apparent nitrogen loss (nitrogen surplus-soil residual nitrogen) through the calculation of nitrogen apparent balance monitoring (table 3) show that under the same condition of no NP fertilizer application, the addition of the endophyte fertilizer (B) can improve the nitrogen absorption amount of the lily compared with the absence of the B, the apparent nitrogen loss is-100 mg/pot, which indicates that the B can possibly enhance the nitrogen fixation capacity of the soil-lily plants and is about 4mg N/kg soil; the nitrogen residue between treatments has no obvious difference, which indicates that most of nitrogen applied in the fertilization area is lost to the environment through leaching or gas state except that a few nitrogen fertilizers are absorbed by plants; in the NP fertilizers with the addition of B and the reduction of 20 percent and the addition of B and the reduction of 40 percent, compared with the normal NP/equivalent NP application, the addition of B has the tendency of improving the utilization of N; in addition, under the condition of equal N application, the apparent nitrogen loss is reduced by 2mg N/kg soil after the B is added, which shows that the B can partially replace a nitrogen fertilizer and has the potential of saving fertilizer and reducing the risk of nitrogen environmental loss; under the treatment of further reducing 40% of NP fertilizer, the addition of B can obviously improve the yield of lily bulbs and the NP absorption amount, which shows that B can more greatly exert the phosphorus-dissolving and nitrogen-fixing capabilities under the soil condition of nutrient deficiency.

TABLE 3 Nitrogen apparent balance calculation (unit: mg/pot)