CN113789287A

CN113789287A - Paenibacillus polymyxa W33, microbial seedling culture substrate thereof and application

Info

Publication number: CN113789287A
Application number: CN202111269238.0A
Authority: CN
Inventors: 吴晓青; 张新建; 周方园; 张广志; 范素素; 赵晓燕; 谢雪迎; 周红姿; 王加宁
Original assignee: Ecology Institute Of Shandong Academy Of Sciences (the Sino-Japanese Friendship Biotechnology Research Center Shandong Academy Of Sciences)
Current assignee: Ecology Institute Of Shandong Academy Of Sciences (the Sino-Japanese Friendship Biotechnology Research Center Shandong Academy Of Sciences)
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2021-12-14
Anticipated expiration: 2041-10-29
Also published as: CN113789287B

Abstract

The invention particularly relates to paenibacillus polymyxa W33, a microbial seedling substrate and application thereof, and belongs to the technical field of microbes. The Paenibacillus polymyxa (Paenibacillus polymyxa) W33 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23374, the preservation date of 2021, 09 and 08 days, and the preservation addresses are as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. The invention also provides a microbial seedling culture substrate containing the paenibacillus polymyxa W33. The paenibacillus polymyxa W33 can effectively inhibit cladosporium, and the prepared microbial seedling culture substrate can effectively prevent and control cucumber scab which is explosive and has high harmfulness in greenhouse planting. The paenibacillus polymyxa W33 provided by the invention produces the plant growth hormone IAA, and the prepared microbial seedling culture substrate can obviously promote the growth of cucumbers.

Description

Paenibacillus polymyxa W33, microbial seedling culture substrate thereof and application

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to paenibacillus polymyxa W33, a microorganism seedling culture substrate and application thereof.

Background

In the production process of facility crops, the seedling culture substrate is a basic substance for the industrialized production of seedlings. At present, the problem of continuous cropping obstacle of facility soil is getting more serious, and the demand of healthy disease-resistant seedlings is increasing continuously. Researchers add functional bacteria with the functions of biocontrol and growth promotion into the seedling raising matrix, and the prepared microbial seedling raising matrix has positive effects on disease resistance and growth promotion of seedlings. The activity durability of the functional strains in the substrate and the colonization ability of the functional strains in the target crops influence the action effect and the persistence of the microbial seedling substrate on the cultivated crops, and the components of the conventional seedling substrate need to be improved so as to fully release the effects of the functional strains.

Cucumber is an important facility vegetable variety, cucumber scab caused by Cladosporium (Cladosporium) is one of explosive diseases in a facility greenhouse planting environment, the disease period is early, the duration is long, and diseases can appear in seedlings, leaves, stalks and fruits. Once large-scale disease occurs, the yield is generally reduced by 10% -30%, and fruit malformation and spot rot are caused. At present, the scab resistant cucumber variety in the market is insufficient, and the effective field control measures are mainly performed by adopting benzimidazole pesticides, strobilurin pesticides and triazole pesticides, so that the pesticide resistance of pathogenic bacteria is easy to domesticate, and the continuous use of the pesticide causes pollution to soil, water and the like. But the research of using the biocontrol bacterium to antagonize the cladosporium cucumerinum to prevent and control the cladosporium cucumerinum is less at present.

The paenibacillus polymyxa is a gram-positive bacterium which produces spores and has nitrogen fixing capacity, has biological characteristics of growth promotion, biological control and the like, is listed as one of microorganism types which can be applied commercially by the United states Environmental Protection Agency (EPA), and is also listed as a first-class strain which is free from safety identification in agricultural rural areas of China. In patent application No. 201810939677. X. The registered microbial seedling culture substrate prepared by taking the paenibacillus polymyxa as the main active component can effectively prevent and treat the pepper phytophthora blight and has the effect of promoting the growth of pepper seedlings. At present, reports that a microbial seedling culture substrate prepared by using paenibacillus polymyxa as an active ingredient can simultaneously prevent and treat cucumber scab and promote cucumber seedling growth are not found.

Disclosure of Invention

Aiming at the defects that a substrate taking paenibacillus polymyxa as an active ingredient cannot be found in the prior art, and the substrate can be used for preventing and treating the cucumber scab and promoting the growth of cucumber seedlings, the invention provides paenibacillus polymyxa W33, a microbial seedling culture substrate and application thereof, and aims to solve the technical problems.

The technical scheme of the invention is as follows:

the invention aims to provide a Paenibacillus polymyxa W33 preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23374, the preservation date of 2021, 09 and 08 days, the preservation organization address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

The Paenibacillus polymyxa W33 is separated and screened from the rhizosphere of greenhouse hot pepper, and can be cultured by adopting a conventional bacterial culture medium such as TSA. The sequence of the 16S rDNA sequence of the strain W33 was sequenced, and the Blast alignment in Genbank showed that the homology of the strain W33 with Paenibacillus polymyxa L1-9(Genbank accession number: FJ178378) reached 99.65% (Query Cover: 100%, E value: 0). According to the morphological physicochemical characteristics of the strain and the sequencing comparison analysis of 16S rDNA, the strain W33 is identified as the paenibacillus polymyxa. The inhibition rate of the strain W33 to the cladosporium is 80.31%, and the content of auxin IAA generated by the strain W33 is 32.43 mg/L.

The second purpose of the invention is to provide a fermentation liquid of Paenibacillus polymyxa W33. The paenibacillus polymyxa W33 was streaked three times on TSA medium to obtain a purified single colony; inoculating a single bacterial colony of the strain W33 to a seed culture medium, and culturing at 32-36 ℃ and 160-180 rpm for 12-16 h to obtain a paenibacillus polymyxa seed solution; adding the seed solution into a fermentation culture medium according to the weight ratio of 1:50, and culturing at 32-36 ℃ and 160-180 rpm for 60-70 h to obtain the paenibacillus polymyxa W33 fermentation liquor.

The seed culture medium comprises the following components: 5g/L of sucrose, 5g/L of glucose, 7g/L of yeast powder, 2g/L of peptone and CaCl₂ 0.25g/L，MgSO₄·7H₂O 0.25g/L，K₂HPO₄1g/L, pH 7.0, autoclave sterilization at 115 ℃ for 30 min.

The fermentation medium comprises the following components: 15g/L of soluble starch, 5g/L of sucrose, 5g/L of yeast powder, 20g/L of peptone and CaCl₂ 1.5g/L，K₂HPO₄ 1g/L，MgSO₄·7H₂O 1g/L，(NH₄)₂SO₄ 2g/L，NaCl 0.5g/L。Sterilizing with high pressure steam at 121 deg.C for 20min at pH 7.0.

The viable count of the paenibacillus polymyxa W33 in the fermentation liquor is more than or equal to 5 multiplied by 10⁹CFU/mL。

The third purpose of the invention is to provide a microbial seedling substrate containing paenibacillus polymyxa W33, wherein the seedling substrate contains a paenibacillus polymyxa W33 fermentation liquid and a seedling substrate auxiliary material mixture. The weight ratio of the paenibacillus polymyxa W33 fermentation liquor to the seedling substrate auxiliary material is 1: 14-17. The viable count of the paenibacillus polymyxa W33 in the matrix is more than or equal to 3 multiplied by 10⁸CFU/matrix weight g. The organic matter content of the substrate is more than or equal to 25 percent, and the total nutrient is (N + P)₂O₅+K₂O) content is 2-5%, moisture (free water) is less than or equal to 35%, pH is 6-7, and EC is less than or equal to 2 ms/cm.

The seedling substrate auxiliary material mixture comprises the following components in parts by weight: 2-3 parts of pepper straw leavening, 1-2 parts of eggplant straw leavening, 1-2 parts of tomato straw leavening, 3-6 parts of vermiculite (with the diameter of 3-6 mm), 3-6 parts of perlite (with the diameter of 3-6 mm) and 0.5-1 part of inorganic salt solution.

The pepper straw fermented product, the eggplant straw fermented product and the tomato straw fermented product are trichoderma fermented products of pepper straw, eggplant straw and tomato straw, and the preparation method comprises the following steps: mechanically pulverizing Capsici fructus, fructus Solani Melongenae or fructus Lycopersici Esculenti into short stalk of about 1cm, and cutting into 10 pieces⁹Mixing cfu/kg of the fermentation liquid PDB with the PDB fermentation liquid of Trichoderma africanum Ta97 (Trichoderma africanum Ta97 is disclosed in the patent with the application number of 202011309961.2), fermenting at normal temperature for 30 days, sterilizing at high pressure, airing, and crushing to the diameter of 3-6 mm to obtain the straw fermentation product.

The inorganic salt solution comprises the following components: (NH)₄)₃PO₄ 3g/L，MgSO₄·7H₂O 0.5g/L。

The application of the microbial seedling culture substrate containing the paenibacillus polymyxa W33 in preventing the cucumber scab is provided.

The application of the microbial seedling culture substrate containing the paenibacillus polymyxa W33 in promoting the growth of cucumbers.

The invention has the beneficial effects that:

the paenibacillus polymyxa W33 can effectively inhibit cladosporium, and the prepared microbial seedling culture substrate can effectively prevent and control cucumber scab which is easy to explode and has high harmfulness in greenhouse planting of cucumbers. The paenibacillus polymyxa W33 provided by the invention produces the plant growth hormone IAA, and the prepared microbial seedling culture substrate can obviously promote the growth of cucumbers.

The microbial seedling raising substrate provided by the invention replaces the traditional peat with the vegetable straw fermentation product in the greenhouse, and has the advantages of two, one, direct abandonment of a large amount of vegetable straws in the main production areas of greenhouse vegetables in China, such as the places with longevity and the like, and pollution and resource waste of agricultural places. Peat is an important land resource, is a good place for living and breeding rare animals and plants, plays an important role in natural balance weight, and straw fermentation products are a good substitute for peat. The invention can efficiently utilize the waste vegetable straws in the vegetable production area of the facility, and has promotion effect on environmental protection; secondly, the paenibacillus polymyxa W33 is collected from the greenhouse, naturally adapts to the humus nutrient content of the greenhouse cultivated crops, has good digestibility to the vegetable straw fermentation product, so that the bacillus polymyxa can stably survive in the substrate product for a long time, the number of viable bacteria is more than 90% of the original bacteria amount in 180 days, and the bacillus polymyxa has good colonization effect on the cucumber rhizosphere and disease prevention and growth promotion effects.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a colony map of TSA plate culture in example 1 of the present invention.

FIG. 2 is a graph comparing the effect of planting four seedling substrates on the degree of scab of cucumber in example 9 of the present invention.

FIG. 3 is a comparative graph showing the effect of planting four seedling substrates on cucumber growth in example 10 of the present invention.

In the figure, 1-sample to be tested, 2-control sample 1, 3-control sample 2, 4-control sample 3.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The invention provides a Paenibacillus polymyxa W33 strain which has been preserved in China general microbiological culture Collection center (CGMCC) in 2021, 9, 8 days, and has the following address: no. 3 of Xilu No. 1 of Beijing, Chaoyang, with the preservation number of CGMCC No. 23374. The Paenibacillus polymyxa W33 is separated and screened from the rhizosphere of greenhouse hot pepper, and can be cultured by adopting a conventional bacterial culture medium such as TSA. The method comprises the following specific steps:

1.1 Strain isolation

Collecting fresh root systems of hot peppers in a greenhouse, and cleaning the fresh root systems with sterile water until no macroscopic soil particles are attached. The roots were transferred to a new 50mL Falcon centrifuge tube, 30mL of sterilized 1 × PBS (pH 7) was added, and shaken at 180r/min at room temperature for 15 min. PBS wash was repeated 3 times. Taking out the cleaned root system, placing the root system on sterilized filter paper, and sucking PBS on the surface of the root system. The root system was cut into about 2mm pieces and mixed, and 1g of root tissue was weighed out and transferred to a 50mL centrifuge tube. 10mL of 10mM sterilized MgCl was added in sterile conditions₂The solution was resuspended and then ground with a sterile pestle until homogeneous. Diluting with sterile water in sterile test tube to 10 degree gradient²、10³、10⁴、10⁵、10⁶、10⁷And 10⁸From each gradient, 100 μ L was pipetted and spread evenly on TSA plates. The plate is inversely cultured in a microbial incubator at 30 ℃ for 1-3 days, and the growth condition of the bacterial colony is observedThe method is described. And (4) streaking the differential colonies on a new TSA plate according to the morphological characteristics of the colonies to obtain single colonies, and repeating the streaking operation for three times to purify the strain.

1.2 screening of biocontrol and growth promoting characteristics of strains

Cladosporium is separated and purified from cucumber fruits and identified as Cladosporium sp. Through a classical confrontation plate test, strains with obvious inhibition effect on cladosporium, wherein the inhibition rate of the strain W33 on cladosporium is 80.31 +/-1.35%. In addition, strains with growth-promoting potential were selected by measuring the IAA content by the classical Salkowski colorimetric method, wherein the IAA content of auxin produced by strain W33 was 32.43. + -. 5.24 mg/L.

1.3 identification of the Strain

The strain W33 is streaked on a TSA culture medium to obtain a single colony, the color of the colony is white and semitransparent, the color is wet and smooth, gram staining is positive, and the shape of the cell is long rod under the observation of a microscope. The strain W33 can hydrolyze starch, is positive in catalase and negative in oxidase, and has physiological and biochemical characteristics such as nitric acid reduction reaction and the like.

The primer sequence is as follows:

27F：5'-AGAGTTTGATCMTGGCTCAG-3'。

1492R：5'-TACGGYTACCTTGTTACGACTT-3'。

the 16S rDNA sequencing sequence of strain W33 was as follows:

GGTGCTATACATGCAGTCGAGCGGGGTTAGTTAGAAGCTTGCTTCTAACTAACCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCCACAAGACAGGGATAACTACCGGAAACGGTAGCTAATACCCGATACATCCTTTTCCTGCATGGGAGAAGGAGGAAAGGCGGAGCAATCTGTCACTTGTGGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGCCTGGTAGAGTAACTGCTATTGAGGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTCTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGGGTCGCACTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTTTGACCGGTCTAGAGATAGGTCTTTCCTTCGGGACAGAGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGCTTAGTTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAGGCGCGAGGTGGAGCCAATCCTAGAAAAGCCGGTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGAGGTAACCGCAAGGAGCCAGCCGCCGAAGTG。

blast alignment in Genbank showed that strain W33 has 99.65% homology with Paenibacillus polymyxa L1-9(Genbank accession number: FJ178378) (Query Cover: 100%, E value: 0). According to the morphological physicochemical characteristics of the strain and the sequencing comparison analysis of 16S rDNA, the strain W33 is identified as the paenibacillus polymyxa.

Example 2

Preparation of fermentation liquor of paenibacillus polymyxa W33

The paenibacillus polymyxa W33 was streaked three times on TSA medium to obtain a purified single colony; a single colony of the strain W33 is inoculated on a seed culture medium and cultured for 12h at 35 ℃ and 180rpm to obtain a paenibacillus polymyxa seed solution. The seed culture medium comprises the following components: 5g/L of sucrose, 5g/L of glucose, 7g/L of yeast powder, 2g/L of peptone and CaCl₂ 0.25g/L，MgSO₄·7H₂O 0.25g/L，K₂HPO₄1g/L, pH 7.0, autoclave sterilization at 115 ℃ for 30 min.

Adding the prepared seed liquid into a fermentation culture medium according to the weight ratio of 1:50, and culturing at 35 ℃ and 180rpm for 70h to obtain the paenibacillus polymyxa W33 fermentation liquid. The fermentation medium comprises the following components: 15g/L of soluble starch, 5g/L of sucrose, 5g/L of yeast powder, 20g/L of peptone and CaCl₂ 1.5g/L，K₂HPO₄ 1g/L，MgSO₄·7H₂O 1g/L，(NH₄)₂SO₄2g/L, NaCl 0.5g/L, pH 7.0, autoclave sterilization at 121 ℃ for 20 min.

The detection proves that the viable count of the fermentation liquor of the paenibacillus polymyxa W33 is 5.89 multiplied by 10⁹CFU/mL。

Example 3

A microbial seedling substrate containing Paenibacillus polymyxa W33 comprises a mixture of the Paenibacillus polymyxa W33 fermentation broth prepared in example 2 and a seedling substrate auxiliary material. The weight ratio of the paenibacillus polymyxa W33 fermentation liquor to the seedling substrate auxiliary material is 1: 14.

The seedling substrate auxiliary material comprises the following components in parts by weight: 3 parts of pepper straw leavening, 1 part of eggplant straw leavening, 1 part of tomato straw leavening, 5 parts of vermiculite (with the diameter of 3-6 mm), 5 parts of perlite (with the diameter of 3-6 mm) and 0.8 part of inorganic salt solution.

The pepper straw fermented product, the eggplant straw fermented product and the tomato straw fermented product are trichoderma fermented products of pepper straw, eggplant straw and tomato straw, and the preparation method comprises the following steps: mechanically pulverizing Capsici fructus, fructus Solani Melongenae or fructus Lycopersici Esculenti into short stalk of about 1cm, and cutting into 10 pieces⁹Mixing cfu/kg of PDB fermentation liquor of Trichoderma africanum Ta97, fermenting at normal temperature for 30 days, sterilizing at high pressure, airing, and crushing to the diameter of 3-6 mm to obtain the straw fermentation product.

Uniformly mixing the fermentation liquor of the paenibacillus polymyxa W33 and the seedling substrate auxiliary material according to the weight ratio of 1:14 to obtain the microbial seedling substrate containing the paenibacillus polymyxa W33. The detection proves that the viable count of the paenibacillus polymyxa W33 in the seedling culture substrate is 4.10 multiplied by 10⁸CFU/matrix weight g.

Example 4

Preparation of fermentation liquor of paenibacillus polymyxa W33

The paenibacillus polymyxa W33 was streaked three times on TSA medium to obtain a purified single colony; single colonies of strain W33 were inoculated onto seed medium and cultured at 32 ℃ at 170rpmCulturing for 16h to obtain the paenibacillus polymyxa seed solution. The seed culture medium comprises the following components: 5g/L of sucrose, 5g/L of glucose, 7g/L of yeast powder, 2g/L of peptone and CaCl₂ 0.25g/L，MgSO₄·7H₂O 0.25g/L，K₂HPO₄1g/L, pH 7.0, autoclave sterilization at 115 ℃ for 30 min.

Adding the prepared seed liquid into a fermentation medium according to the weight ratio of 1:50, and culturing at 32 ℃ and 170rpm for 75h to obtain the paenibacillus polymyxa W33 fermentation liquid. The fermentation medium comprises the following components: 15g/L of soluble starch, 5g/L of sucrose, 5g/L of yeast powder, 20g/L of peptone and CaCl₂ 1.5g/L，K₂HPO₄ 1g/L，MgSO₄·7H₂O 1g/L，(NH₄)₂SO₄2g/L, NaCl 0.5g/L, pH 7.0, autoclave sterilization at 121 ℃ for 20 min.

The detection proves that the viable count of the fermentation liquor of the paenibacillus polymyxa W33 is 5.42 multiplied by 10⁹CFU/mL。

Example 5

A microbial seedling substrate containing Paenibacillus polymyxa W33 comprises a mixture of the Paenibacillus polymyxa W33 fermentation broth prepared in example 2 and a seedling substrate auxiliary material. The weight ratio of the paenibacillus polymyxa W33 fermentation liquor to the seedling substrate auxiliary material is 1: 15.

The seedling substrate auxiliary material comprises the following components in parts by weight: 2 parts of pepper straw leavening, 2 parts of eggplant straw leavening, 1 part of tomato straw leavening, 6 parts of vermiculite (with the diameter of 3-6 mm), 3 parts of perlite (with the diameter of 3-6 mm) and 1 part of inorganic salt solution.

Uniformly mixing the fermentation liquor of the paenibacillus polymyxa W33 and the seedling substrate auxiliary material according to the weight ratio of 1:15 to obtain the microbial seedling substrate containing the paenibacillus polymyxa W33. The detection proves that the viable count of the paenibacillus polymyxa W33 in the seedling culture substrate is 3.57 multiplied by 10⁸CFU/matrix weight g.

Example 6

Preparation of fermentation liquor of paenibacillus polymyxa W33

The paenibacillus polymyxa W33 was streaked three times on TSA medium to obtain a purified single colony; a single colony of the strain W33 is inoculated on a seed culture medium and cultured for 14h at 36 ℃ and 170rpm to obtain a paenibacillus polymyxa seed solution. The seed culture medium comprises the following components: 5g/L of sucrose, 5g/L of glucose, 7g/L of yeast powder, 2g/L of peptone and CaCl₂ 0.25g/L，MgSO₄·7H₂O 0.25g/L，K₂HPO₄1g/L, pH 7.0, autoclave sterilization at 115 ℃ for 30 min.

Adding the prepared seed liquid into a fermentation medium according to the weight ratio of 1:50, and culturing at 36 ℃ and 160rpm for 65h to obtain the paenibacillus polymyxa W33 fermentation liquid. The fermentation medium comprises the following components: 15g/L of soluble starch, 5g/L of sucrose, 5g/L of yeast powder, 20g/L of peptone and CaCl₂ 1.5g/L，K₂HPO₄ 1g/L，MgSO₄·7H₂O 1g/L，(NH₄)₂SO₄2g/L, NaCl 0.5g/L, pH 7.0, autoclave sterilization at 121 ℃ for 20 min.

The detection proves that the viable count of the fermentation liquor of the paenibacillus polymyxa W33 is 5.35 multiplied by 10⁹CFU/mL。

Example 7

A microbial seedling substrate containing Paenibacillus polymyxa W33 comprises a mixture of the Paenibacillus polymyxa W33 fermentation broth prepared in example 2 and a seedling substrate auxiliary material. The weight ratio of the paenibacillus polymyxa W33 fermentation liquor to the seedling substrate auxiliary material is 1: 17.

The seedling substrate auxiliary material comprises the following components in parts by weight: 2 parts of pepper straw leavening, 1 part of eggplant straw leavening, 2 parts of tomato straw leavening, 3 parts of vermiculite (with the diameter of 3-6 mm), 6 parts of perlite (with the diameter of 3-6 mm) and 0.5 part of inorganic salt solution.

Uniformly mixing the fermentation liquor of the paenibacillus polymyxa W33 and the seedling substrate auxiliary material according to the weight ratio of 1:15 to obtain the microbial seedling substrate containing the paenibacillus polymyxa W33. The detection proves that the viable count of the paenibacillus polymyxa W33 in the seedling culture substrate is 3.11 multiplied by 10⁸CFU/matrix weight g.

Example 8

Using the microbial growth substrate prepared in example 3 as an example, the activity of strain W33 after long-term storage in the substrate was tested using two different growth substrates.

8.1 seedling raising substrate

A sample to be tested: the microbial seedling substrate prepared in example 3;

control sample 1: the fermentation liquor prepared in the example 2 is uniformly mixed with the auxiliary materials of the common seedling raising substrate in a weight ratio of 1: 14. The common seedling substrate auxiliary material comprises the following components in parts by weight: 5 parts of sterile peat, 5 parts of vermiculite (the diameter is 3-6 mm), 5 parts of perlite (the diameter is 3-6 mm) and 0.8 part of inorganic salt solution; the inorganic salt solution comprises the following components: (NH)₄)₃PO₄ 3g/L，MgSO₄·7H₂O is 0.5 g/L. The detection proves that the viable count of the strain W33 in the control sample is 4.10 multiplied by 10⁸CFU/matrix weight g.

8.2 test conditions

The samples to be tested and the control samples were stored in sterile air tanks (6 replicates for each sample) and placed at room temperature 25 + -5 deg.C in a cool and ventilated place. The viable cell count of the strain W33 was measured by dilution and TSA plate method at 0d, 90d and 180d after storage, respectively.

8.3 test results

In the process of adopting the test conditions, the influence of the two auxiliary materials of the microbial seedling raising matrix on the viable count of the strain W33 is tested, and the test results are shown in the following table 1:

TABLE 1 influence of growth substrate adjuvant composition on viable count of Strain W33

As can be seen from table 1, with the microbial seedling raising substrate provided in example 2 of the present invention, the number of viable bacteria of strain W33 remained at a high level when stored for a long time, whereas the number of viable bacteria of strain W33 in the comparative sample was significantly decreased after stored for a long time.

Example 9

Using the microbial seedling substrate prepared in example 3 as an example, four different seedling substrate tests were compared to test the disease prevention effect on cucumber scab

9.1 seedling raising substrate

A sample to be tested: the microbial seedling substrate prepared in example 3.

Control sample 1: the same as in control sample 1 of example 8.

Control sample 2: the seedling substrate auxiliary materials in the embodiment 3 are adopted.

Control sample 3: the common seedling substrate auxiliary material of the control sample 1 in example 8 was used.

9.2 test conditions

Accelerating germination of cucumber (Jinyou No. 35) seeds by a warm soup seed soaking method under the dark condition of 28 ℃, putting four seedling-growing matrixes into seedling-growing bowls in advance, sowing one exposed-white seed in each seedling-growing bowl, and repeating four treatments of 6 selected seedlings after cotyledons are fully stretched. Then 1mL of Cladosporium (isolated)Purified from cucumber diseased fruit) spore suspension (1X 10)⁸CFU/mL) to the rhizosphere, and then watering the rhizosphere regularly, and recording the disease index after 14 days of cultivation. The disease condition grading standard refers to the agricultural industry standard NY/T144.38-2011 part 38 of the people's republic of China: a leaf grading method and an illness index calculation method of a bactericide for preventing and treating cucumber scab are disclosed.

9.3 test results

In the process of adopting the test conditions, the effect of the four microorganism seedling culture substrates on the control effect of the cucumber scab is tested, and the test results are shown in the following table 2 and figure 1.

TABLE 2 influence of four seedling substrates on the degree of scab disease in cucumber

As can be seen from table 2, the microbial seedling substrate provided in example 3 of the present invention effectively suppresses the occurrence of cucumber scab, compared to a seedling substrate without paenibacillus polymyxa W33. And the biocontrol effect of the paenibacillus polymyxa W33 is also obviously improved by adopting the straw fermentation product to replace peat.

Example 10

Using the microbial seedling substrate prepared in example 3 as an example, four different seedling substrate tests were compared to determine the growth promoting effect of cucumber

10.1 seedling raising substrate

A sample to be tested: the microbial seedling substrate prepared in example 3.

Control sample 1: the same as in control sample 1 of example 8.

10.2 test conditions

Accelerating germination of cucumber (Jinyou No. 35) seeds by a warm soup seed soaking method under the dark condition of 28 ℃, putting four seedling-growing matrixes into seedling-growing bowls in advance, sowing one exposed-white seed in each seedling-growing bowl, and repeating four treatments of 6 selected seedlings after cotyledons are fully stretched. The subsequent watering management is conventional management.

10.3 test results

In the course of using the above test conditions, the plant height, stem thickness, dry weight of the above-ground and underground parts were measured 7 days after emergence of seedlings, and the strong seedling index (stem thickness/plant height + dry weight of underground/dry weight of above-ground) x dry weight of individual plant was calculated. The test results are shown in table 3 below and fig. 2.

TABLE 3 growth promoting effect of four seedling raising substrates on cucumber

As can be seen from table 3, the microbial seedling substrate provided in example 3 of the present invention significantly improves cucumber biomass and seedling growth index compared to a seedling substrate without paenibacillus polymyxa W33. And the growth promotion effect of the strain W33 is also obviously improved by adopting straw fermentation products to replace peat.

Example 11

Using the microbial seedling substrate prepared in example 3 as an example, the colonization effect of the strain W33 on the cucumber rhizosphere was compared by using two different seedling substrate tests.

11.1 seedling raising substrate

A sample to be tested: the microbial seedling substrate prepared in example 3.

Control sample 1: the same as in control sample 1 of example 8.

11.2 test conditions

Planting cucumber in two kinds of microbial seedling culture medium, and collecting yellow on days 10, 25, 30, 45 and 60The melon root system is placed in a centrifuge tube filled with 30mL of 1 XPBS buffer solution and washed three times for 20min each time in a shaking table at 180rpm, and fresh PBS is replaced each time. Sucking water on root surface with sterile filter paper, taking out 0.2g, grinding with sterile water, and diluting with 10 times gradient to 10⁸After doubling, the cells were plated on TSA plates and incubated at 30 ℃ until macroscopic single colonies grew, and counted. The experiment was set up in triplicate. And identifying the paenibacillus polymyxa according to the colony morphology, and identifying the strain by using the 16S rDNA gene to determine the accuracy of the paenibacillus polymyxa morphology identification.

11.3 test results

Using the above test conditions, the test results are shown in Table 4 below.

TABLE 4-number of colonizing viable bacteria of the strain W33 in two seedling substrates

As can be seen from Table 4, with the microbial seedling substrate provided by the embodiment of the invention, the viable count of the strain W33 in the cucumber growth cycle (60d) is kept at a higher level after the cucumber rhizosphere colonization by the Paenibacillus polymyxa W33, while the colonization viable count of the strain W33 in the comparative sample is reduced remarkably.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

SEQUENCE LISTING

<110> institute of ecology of Shandong province academy of sciences (Zhongri friendly center for biotechnology research of Shandong province academy of sciences)

<120> Paenibacillus polymyxa W33, and microbial seedling substrate and application thereof

<130> 2021.10.28

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 1

agagtttgat cmtggctcag 20

<210> 2

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 2

tacggytacc ttgttacgac tt 22

<210> 3

<211> 1439

<212> DNA

<213> Paenibacillus polymyxa

<400> 3

ggtgctatac atgcagtcga gcggggttag ttagaagctt gcttctaact aacctagcgg 60

cggacgggtg agtaacacgt aggcaacctg cccacaagac agggataact accggaaacg 120

gtagctaata cccgatacat ccttttcctg catgggagaa ggaggaaagg cggagcaatc 180

tgtcacttgt ggatgggcct gcggcgcatt agctagttgg tggggtaaag gcctaccaag 240

gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300

agactcctac gggaggcagc agtagggaat cttccgcaat gggcgaaagc ctgacggagc 360

aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgccag ggaagaacgc 420

ctggtagagt aactgctatt gaggtgacgg tacctgagaa gaaagccccg gctaactacg 480

tgccagcagc cgcggtaata cgtagggggc aagcgttgtc cggaattatt gggcgtaaag 540

cgcgcgcagg cggctcttta agtctggtgt ttaatcccga ggctcaactt cgggtcgcac 600

tggaaactgg ggagcttgag tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa 660

tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctgggctg taactgacgc 720

tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780

cgatgaatgc taggtgttag gggtttcgat acccttggtg ccgaagttaa cacattaagc 840

attccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg gggacccgca 900

caagcagtgg agtatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960

atccctttga ccggtctaga gataggtctt tccttcggga cagaggagac aggtggtgca 1020

tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080

tatgcttagt tgccagcagg tcaagctggg cactctaagc agactgccgg tgacaaaccg 1140

gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtac 1200

tacaatggcc ggtacaacgg gaagcgaagg cgcgaggtgg agccaatcct agaaaagccg 1260

gtctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaattg ctagtaatcg 1320

cggatcagca tgccgcggtg aatacgttcc cgggtcttgt acacaccgcc cgtcacacca 1380

cgagagttta caacacccga agtcggtgag gtaaccgcaa ggagccagcc gccgaagtg 1439

Claims

1. The Paenibacillus polymyxa W33 is characterized in that the Paenibacillus polymyxa is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23374, the preservation date of 2021, 09 and 08 days, and the preservation organization address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

2. A fermentation broth prepared using the Paenibacillus polymyxa W33 of claim 1, wherein the fermentation broth is prepared by: the paenibacillus polymyxa W33 was streaked three times on TSA medium to obtain a purified single colony; inoculating a single bacterial colony of the strain W33 to a seed culture medium, and culturing at 32-36 ℃ and 160-180 rpm for 12-16 h to obtain a paenibacillus polymyxa seed solution; adding the seed solution into a fermentation medium according to the weight ratio of 1:50, and culturing at 32-36 ℃ and 160-180 rpm for 60-70 h to obtain the paenibacillus polymyxa W33 fermentation liquor.

3. The fermentation broth of claim 2, wherein the seed medium comprises the following components: 5g/L of sucrose, 5g/L of glucose, 7g/L of yeast powder, 2g/L of peptone and CaCl₂ 0.25g/L，MgSO₄·7H₂O 0.25g/L，K₂HPO₄ 1g/L。

4. The fermentation broth of claim 2, wherein the fermentation medium comprises the following: 15g/L of soluble starch, 5g/L of sucrose, 5g/L of yeast powder, 20g/L of peptone and CaCl₂ 1.5g/L，K₂HPO₄ 1g/L，MgSO₄·7H₂O 1g/L，(NH₄)₂SO₄ 2g/L，NaCl 0.5g/L。

5. A microbial seedling substrate containing the Paenibacillus polymyxa W33 as defined in claim 1, which comprises the Paenibacillus polymyxa W33 fermentation broth as defined in claim 2 and a seedling substrate auxiliary material; the weight ratio of the paenibacillus polymyxa W33 fermentation liquor to the seedling substrate auxiliary material is 1: 14-17.

6. The microbial seedling substrate of claim 5, wherein the seedling substrate adjuvant mixture comprises the following components in parts by weight: 2-3 parts of pepper straw leavening, 1-2 parts of eggplant straw leavening, 1-2 parts of tomato straw leavening, 3-6 parts of vermiculite, 3-6 parts of perlite and 0.5-1 part of inorganic salt solution.

7. The microorganism of claim 6The substrate for seedling raising is characterized in that the pepper straw fermented product, the eggplant straw fermented product and the tomato straw fermented product are trichoderma fermented products of pepper straw, eggplant straw and tomato straw, and the specific preparation method comprises the following steps: mechanically pulverizing Capsici fructus, fructus Solani Melongenae or fructus Lycopersici Esculenti into 1cm short stalk, and cutting into 10 pieces⁹Mixing cfu/kg of PDB fermentation liquor of Trichoderma africanum Ta97, fermenting at normal temperature for 30 days, sterilizing at high pressure, airing and crushing to the diameter of 3-6 mm.

8. A microbial seedling substrate according to claim 6, wherein the inorganic salt solution comprises the following components: (NH)₄)₃PO₄ 3g/L，MgSO₄·7H₂O 0.5g/L。

9. Use of a microbial seedling substrate comprising paenibacillus polymyxa W33 as claimed in claim 5 for preventing cucumber scab.

10. Use of a microbial seedling substrate comprising paenibacillus polymyxa W33 as claimed in claim 5 for promoting cucumber growth.