CN117467557A

CN117467557A - Paenibacillus strain with growth promoting and biocontrol functions and application thereof

Info

Publication number: CN117467557A
Application number: CN202310702570.4A
Authority: CN
Inventors: 王玉梅; 刘欢; 任慧; 王亚玉
Original assignee: Shenzhen Huada Everything Technology Co ltd; BGI Shenzhen Co Ltd
Current assignee: Shenzhen Huada Everything Technology Co ltd; BGI Shenzhen Co Ltd
Priority date: 2023-06-13
Filing date: 2023-06-13
Publication date: 2024-01-30
Anticipated expiration: 2043-06-13
Also published as: CN117467557B

Abstract

The application relates to the field of microorganisms, in particular to a novel paenibacillus strain with growth promoting and biocontrol functions, a culture method and application thereof, wherein the paenibacillus strain comprises a sequence represented by SEQ ID NO:1 or a complement thereof or a sequence complementary to the sequence set forth in SEQ ID NO:1 compared to at least 85% identical sequences. The application provides paenibacillus Paen552 for the first time and the application thereof in promoting the growth of crops and/or preventing and controlling plant diseases caused by fusarium oxysporum, and has important significance in improving the yield of crops and promoting the green production of agriculture.

Description

Paenibacillus strain with growth promoting and biocontrol functions and application thereof

Technical Field

The application relates to the field of microorganisms, in particular to a novel paenibacillus strain with growth promoting and biocontrol functions, and a culture method and application thereof.

Background

Early studies classified paenibacillus as bacillus. Carol Ash was analyzed by 16s rDNA sequence in 1993, and found that part of Bacillus was highly specific in sequence with other Bacillus, and thus, this part of Bacillus was isolated and Paenibacillus was newly established. Paenibacillus is widely used in various environments such as rhizosphere soil, water, food and the like. According to the International Commission on bacterial nomenclature, up to 4 months of 2023, there were a total of 382 different species in Paenibacillus (https:// lpsn. Dsmz. De /). Researches show that the Paenibacillus has rich biosynthesis gene clusters, can generate various bioactive substances (such as extracellular polysaccharide, antibacterial substances, plant hormones and the like), and can be widely applied to the fields of agriculture, industry and medicine (such as biofertilizer, biological pesticide, enzyme production, anti-infection, anti-tumor and the like).

With the world population growing and the farmland decreasing, food safety has become a focus of attention. However, excessive application of fertilizer pesticides brings about a range of environmental effects. Excessive application of chemical fertilizers can easily cause soil structural damage, soil nutrient loss, soil hardening and secondary salinization of soil environment; the unreasonable application of pesticides can cause the problems of too high pesticide residue of agricultural products and environmental pollution. The plant rhizosphere growth-promoting bacteria are beneficial microorganisms which can colonize plant rhizosphere and have promotion effects on plant growth, development and stress resistance. The development and utilization of plant rhizosphere growth promoting bacteria resources can reduce the application amount of chemical fertilizers, improve the utilization rate of chemical fertilizers, partially replace pesticide application and relieve the environmental influence caused by chemical fertilizers and pesticides.

Thus, there is a need to develop new plant growth promoting and biocontrol strains to reduce the use of fertilizers and pesticides, thereby reducing environmental pollution.

Disclosure of Invention

For this reason, the embodiments of the present application provide novel paenibacillus species with growth promoting and biocontrol functions, and a culture method and application thereof.

Embodiments of the first aspect of the present application provide a paenibacillus comprising the amino acid sequence of SEQ ID NO:1 or a complement thereof or a sequence complementary to the sequence set forth in SEQ ID NO:1 compared to at least 85% identical sequences.

In some embodiments, the Paenibacillus is Paenibacillus (Paenibacillus); preferably, the paenibacillus has the amino acid sequence as set forth in SEQ ID NO:1, a 16S rDNA sequence shown in seq id no; more preferably, the strain is Paenibacillus sp.paen552 with accession number GDMCC 63375.

An embodiment of a second aspect of the present application provides a method of culturing paenibacillus as described in any one of the embodiments of the first aspect above, the method comprising inoculating the paenibacillus in a medium for culturing, the culturing having a temperature of 20 to 40 ℃.

In some embodiments, the pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.6, and the temperature of the culture is 25 ℃ to 35 ℃, preferably 26 ℃ to 30 ℃, more preferably 28 ℃.

In some embodiments, the medium comprises peptone 8g/L to 12g/L, preferably 10g/L, beef extract 2g/L to 4g/L, preferably 3g/L, and sodium chloride 4g/L to 6g/L, preferably 5g/L.

Embodiments of the third aspect of the present application provide an agricultural formulation comprising paenibacillus as described in any of the embodiments of the first aspect above.

In some embodiments, the agricultural formulation further comprises an adjuvant.

In some embodiments, the adjunct comprises: organic auxiliary materials and/or inorganic auxiliary materials.

In some embodiments, the agricultural formulation is in a dosage form selected from: wettable powder, water dispersible granules, suspending agents, aqueous emulsion, granules, seed coating agents or a combination thereof.

Embodiments of the fourth aspect of the present application provide the use of a paenibacillus as described in any of the embodiments of the first aspect above or an agricultural formulation as described in any of the embodiments of the third aspect above in biological nitrogen fixation.

Embodiments of the fifth aspect of the present application propose the use of paenibacillus as described in any of the embodiments of the first aspect above or of an agricultural formulation as described in any of the embodiments of the third aspect above for inhibiting fusarium oxysporum.

Embodiments of the sixth aspect of the present application propose the use of a paenibacillus as described in any of the embodiments of the first aspect above or an agricultural formulation as described in any of the embodiments of the third aspect above to promote plant growth and/or to control plant diseases caused by fusarium oxysporum.

In some embodiments, the plant disease comprises: fusarium, maize fusarium, tomato root rot, cucumber mildew, soybean root rot and/or soybean wilt.

In some embodiments, the use comprises root irrigation treatment during the plant sowing and/or seedling stage using a paenibacillus as described in any of the embodiments of the first aspect above or an agricultural formulation as described in any of the embodiments of the third aspect above.

In some embodiments, the plants may include gramineae plants selected from corn, millet and land rice and leguminous plants selected from soybean and alfalfa, preferably.

The embodiment of the application realizes the following beneficial effects:

the application provides Paenibacillus sp.paen552 and application thereof in promoting crop growth and/or preventing and controlling plant diseases caused by fusarium oxysporum, and particularly can effectively fix nitrogen, inhibit fungus fusarium oxysporum and promote plant growth, thereby reducing application amount of chemical fertilizer and pesticide and relieving environmental influence caused by chemical fertilizer and pesticide. The novel paenibacillus strain with the functions of promoting growth and biocontrol provided by the application has important significance for improving the yield of crops. Meanwhile, the microbial fertilizer which is suitable for agricultural production is manufactured through Paenibacillus sp.Paen552, so that the application amount of the fertilizer can be reduced, and the agricultural green production is promoted.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a cluster diagram of Paen552 according to example 2 of the present application.

FIG. 2 is a plate growth of Paen552 on beef extract peptone media according to example 3 of the present application.

FIG. 3 is a plate growth plot of Paen552 on nitrogen-free media according to example 4 of the present application.

FIG. 4 is a plate growth chart of the plate counter method of example 5 of the present application for testing the bacteriostatic ability of Paen552.

Fig. 5 is a growth chart of the Huagu 12 according to example 6 of the present application.

FIG. 6 is a field growth plot of gold-inlaid jade, land-based primers 46, hua Gu, zhonghuang 13 and alfalfa according to example 7 of the present application, wherein A is gold-inlaid jade, B is Zhonghuang 13, C is Hua Gu, D is land-based primer 46, E is alfalfa.

Fig. 7 is a microscopic morphology of Paen552 according to example 3 of the present application.

Strain preservation description:

paenibacillus sp.paen552: the deposit accession number is GDMCC No.63375, and the deposit institution is: the collection of microorganism strains in Guangdong province; deposit unit address: first, the middle road 100 # college experiment building 5 in the View area of Guangzhou City in Guangdong province; the preservation time is 2023, 4 and 21 days.

Detailed Description

The invention will now be described in further detail with reference to the following specific embodiments, which are given by way of illustration only and are not intended to limit the scope of the invention. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The present application is made based on the following knowledge of the inventors:

the overapplication of fertilizers and pesticides in agricultural production brings a series of environmental problems, such as the problems of damaged soil structure, loss of soil nutrients, soil hardening, secondary salinization of soil environment, excessive pesticide residue in agricultural products, environmental pollution and the like.

Soil serves as a treasury of microorganism resources, and microorganisms among the treasury are complex in composition and various in variety, and about 2000-18000 microorganisms are contained in 1g of soil. Root microorganisms are extremely susceptible to plant hosts, soil types, nutritional conditions, and climatic factors. The choice of microorganisms by the plant host seems not to be completely random, as it appears by secreting specific small molecule nutrients, attracting some beneficial, potential plant probiotic values around it, modifying the pH of the soil, improving the quality of the soil, etc. However, it is not known how microorganisms can help plants grow to increase crop yield.

Knowing the microorganism composition around the plant, analyzing the interrelation between the microorganism, the plant growth and the environmental condition, finding the optimal microorganism composition required by the plant growth, establishing a controllable model for assisting the crop health, the efficient growth and the high yield by the microorganism, and promoting the maximization of the grain production. This requires the finding of these potential plant probiotics and functional properties by correlating the information on microbial composition and functional genes associated with large-scale plants with crop growth, yield phenotypes. The potential probiotics with application value can be purposefully screened under the guidance of the big data analysis result, and the potential can be provided for agricultural application.

According to the embodiment of the application, a large number of experiments and data analysis are performed, a novel strain Paen552 of bacillus species is separated from a soybean rhizosphere soil sample, the growth of crops can be remarkably promoted, the nitrogen fixation and bacteriostasis effects are achieved, and the application amount of pesticides and fertilizers can be reduced while the growth of plants is promoted. Further, the embodiment of the application verifies the growth promoting, nitrogen fixing and bacteria inhibiting functions of the strains through a series of experiments such as a plate nitrogen fixing and bacteria inhibiting experiment, a potting growth promoting experiment, a field experiment and the like.

An embodiment of the first aspect of the present application provides a paenibacillus comprising the amino acid sequence of SEQ ID NO:1 or a complement thereof or a sequence complementary to the sequence set forth in SEQ ID NO:1 compared to at least 85% identical sequences.

In the examples of the present application, the percentage of identity generally describes the degree to which two sequences are identical, i.e. it generally describes the percentage of nucleotides at their sequence positions that correspond to the same nucleotides of the reference sequence. In the examples herein, the "sequence of at least 85% identity" refers to a sequence having any number (inclusive) of identity between 85% and 100% as compared to the sequence set forth in SEQ ID NO. 1, e.g., may have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and countless decimal numbers of sequence identities between two adjacent integers, e.g., at least 98.57%, 99.64%, 99.7%, 99.8% or 99.9% sequence identity as compared to the sequence set forth in SEQ ID NO. 1.

In some embodiments, the Paenibacillus is Paenibacillus (Paenibacillus); preferably, the paenibacillus has the amino acid sequence as set forth in SEQ ID NO:1, a 16S rDNA sequence shown in seq id no; more preferably, the strain is Paenibacillus sp.paen552 with deposit number GDMCC No. 63375.

Paenibacillus Latin (Paenibacillus Ash, priest & Collin, 1994) cells are rod-shaped, gram-positive, negative or variable, and move with periflagella. Through the analysis of the evolutionary tree, the Paen552 proposed in the examples of the present application is a new species under Paenibacillus. Paen552 is a Paenibacillus species belonging to the genus Paenibacillus and designated Paenibacillus sp.paen552; the deposit accession number is GDMCC No.63375, and the deposit institution is: the collection of microorganism strains in Guangdong province; deposit unit address: first, the middle road 100 # college experiment building 5 in the View area of Guangzhou City in Guangdong province; the preservation time is 2023, 4 and 21 days.

In the examples of the present application, the strain Paen552 having the sequence shown in SEQ ID NO. 1 can be understood by those skilled in the art as the original strain. Strains having a genomic sequence with at least 85% identity to the sequence shown in SEQ ID No. 1 can be understood by the person skilled in the art as variant strains of strain Paen552 having the sequence shown in SEQ ID No. 1. It will be appreciated that strains such as strain Paen552 may undergo spontaneous mutation or undergo artificial culture to form variant strains, such as nucleotide deletions, nucleotide additions, or nucleotide substitutions. The "variant strain" has a highly identical gene sequence to the "strain Paen552" and very similar biological functions, and the mutated gene does not substantially affect the conserved sequence of the strain Paen552 and thus does not affect the genetic stability of the strain Paen552. More specifically, the "variant strain" is also a strain of Paen552, exhibiting the physiological activity characteristics of Paen552. The specific genus of strain 1 and all species under it are also within the scope of protection of the present application.

The novel strain Paen552 separated by the embodiment of the application can be effectively used for improving the agronomic characters of plants when being singly used, and particularly can effectively fix nitrogen, inhibit fungi fusarium oxysporum and promote plant growth, so that the application amount of chemical fertilizers and pesticides is reduced, and the environmental influence caused by the chemical fertilizers and pesticides is relieved. The novel paenibacillus strain with the functions of promoting growth and biocontrol provided by the application has important significance for improving the yield of crops.

Since the above strains also include mutant strains of the same genus of strain Paen552, the specific genus of strain Paen552 and all species under it are also within the scope of protection of the agricultural formulation of the present application.

In some embodiments, the agricultural formulation further comprises an adjuvant, wherein the adjuvant may be organic and/or inorganic. In the embodiments of the present application, the organic matter may be other additional bacterial manure capable of promoting plant growth, such as: bacterial fertilizers for increasing soil nitrogen and crop nitrogen nutrition, such as rhizobium fertilizers, nitrogen fixing bacterial fertilizers, nitrogen fixing blue algae fertilizers and the like; bacterial manure for decomposing soil organic matters, such as organic phosphorus bacterial manure and comprehensive bacterial manure; bacterial fertilizers for decomposing soil insoluble minerals, such as phosphorus bacterial fertilizers, potassium bacterial fertilizers and mycorrhizal fungi fertilizers; bacterial fertilizers for stimulating plant growth, such as growth promoting bacterial fertilizers; bacterial fertilizers for increasing the stress resistance of crop root systems, such as antibiotic fertilizers and stress-resistant fungus fertilizers. In the embodiment of the application, the organic matter can also be an organic fertilizer required for plant growth, such as manure and the like. It is understood that the organic matters in the embodiments of the present application alone or in combination are only required to ensure the promotion effect on plant growth, and the present application is not limited thereto.

In embodiments of the present application, the inorganic substance may be an agriculturally acceptable chemical component, such as an agriculturally acceptable carrier, excipient, diluent, adjuvant, vehicle, excipient, carrier, or combination thereof, and/or an inorganic fertilizer having a concentration that does not affect the microbial activity in the agricultural formulation, as the application is not limited in this regard.

In embodiments of the present application, the dosage form of the agricultural formulation may be selected from: wettable powder, water dispersible granules, suspending agents, aqueous emulsion, granules, seed coating agents or a combination thereof. It will be appreciated that the dosage form of the agricultural formulation in the examples of the present application is not limited to this as long as it is ensured that it can be applied to plants in a certain form.

The agricultural preparation provided by the embodiment of the application can be effectively used for improving the agronomic characters of plants by using the Paen552, and particularly can effectively fix nitrogen, inhibit fungi fusarium oxysporum and promote plant growth, so that the application amount of chemical fertilizers and pesticides is reduced, and the environmental influence caused by the chemical fertilizers and pesticides is relieved. The novel paenibacillus strain with the functions of promoting growth and biocontrol provided by the application has important significance for improving the yield of crops.

Embodiments of the fourth aspect of the present application propose the use of paenibacillus as described in any of the embodiments of the first aspect above or of an agricultural formulation as described in any of the embodiments of the third aspect above for promoting crop growth and/or controlling plant diseases caused by fusarium oxysporum.

Fusarium oxysporum (F.oxysporum) is a soil-borne pathogenic fungus which is distributed worldwide and has a wide host range and can cause more than 100 plant wilts of melons, solanaceae, bananas, cotton, leguminous plants, flowers and the like, from the orders of the Stemonales (Moniliales), from the family of Oenotheraceae (Tubercularia) and from the genus Fusarium (Fusarium). The paenibacillus or the agricultural preparation containing the paenibacillus provided by the embodiment of the application can effectively inhibit fusarium oxysporum, so that fusarium wilt of melon, solanaceae, banana, cotton, leguminous plants, flowers and other plants caused by the fusarium oxysporum can be effectively prevented and controlled.

In an embodiment of the present application, the paenibacillus as described in any one of the embodiments of the first aspect or the agricultural formulation as described in any one of the embodiments of the third aspect may be used for controlling plant diseases including: fusarium, maize fusarium, tomato root rot, cucumber mildew, soybean root rot and/or soybean wilt.

In some embodiments, the plant that biocontrol the plant using the paenibacillus or agricultural formulation comprising the same as set forth in the embodiments of the present application may specifically be a gramineous plant and/or a leguminous plant, wherein the gramineous plant includes, but is not limited to, corn, millet, land rice, etc.; leguminous plants include, but are not limited to, alfalfa and soybean, and the like.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Example 1: isolation of novel species Paen552

Collecting soybean rhizosphere soil sample, pouring the rhizosphere soil sample into a mortar, adding 2-3ml of sterile water, and directly grinding into powder to obtain grinding liquid. Respectively diluting the grinding fluid into 10 by adopting sterile water ^-2 To 10 ^-5 Is a gradient concentration of (c). The gradient dilutions were each applied to a beef extract peptone solid culture medium, while sterile water without bacterial liquid was applied as a blank. After the coating was completed, the culture was inverted at 28℃for 48-72 hours. And (3) picking single bacterial colony, amplifying and culturing in a liquid beef extract peptone culture medium, repeatedly streaking and amplifying for a plurality of times, and purifying to obtain the monoclonal bacterial strain with the number of Paen552.

Example 2: identification and evolutionary position determination of novel strain Paen552

Sequencing the 16S rDNA gene of the monoclonal strain Paen552 to obtain the 16S rDNA gene sequence of the Paen552 as follows SEQ ID NO: 1.

ACCACTTCGGCGGCTGGCTCCTTGCGGTTACCCCACCGACTTCGGGTGTTGTAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCAATTCCGACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGACCGGCTTTTTAGGATTAGCTCCACCTCGCGGCTTCGCGGCCCGTTGTACCGGCCATTGTAGTACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCACCCGAAGTGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCTCCTCTGTCCCGAAGGAAAGGTGTATCTCTACACCGGTCAGAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATACTCCACTGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAATGCTTAATGTGTTAACTTCGGCACCAAGGGTATCGAAACCCCTAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGCCCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTCATCCAGTTTCCAGTGCGATCCGGGGTTGAGCCCCGGGATTAAACACCAGACTTAAATGACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGGGCTTTCTTCTCAGGTACCGTCACCTTGAGAGCAGTTACTCTCCCAAGCGTTCTTCCCTGGCAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCATTGCTCCGTCAGGCTTTCGCCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCAGGCCCATCCTCAAGTGACAGATTGCTCCGTCTTTCCAGTTCCCTCCAGGCGAAGAAAACAAGTATTCGGTATTAGCTACCGTTTCCGGTAGTTGTCCCAAGCTTGAGGGCAGGTTGCCTACGTGTTACTCACCCGTCCGCCGCTAAGCATCAAGGAAGCAAGCTTCCTATCAACTCCGCTCGACTTGCATGTATTAGCAG (SEQ ID NO: 1) the sequences of approximately 1.4Kb of the sequenced Paen552 (i.e., SEQ ID NO: 1) were aligned in the 16S rDNA gene database of EzBioCloud. The comparison shows that the two strains with highest homology with the 16S rDNA gene of Paen552 in the database are Paenibacillus dongdonensis KUDC0114 (serial number: KF 425513) and Paenibacillus pabuli NBRC 13638 (serial number: BCNM 01000057), and the similarity is 98.39% and 97.99% (both are smaller than the new bacterium judgment standard 98.65%) respectively. Based on the 16S rDNA information, it was preliminarily determined that Paen552 is a new species belonging to the genus Paenibacillus.

Further, the phylogenetic tree was constructed using MAGA from the sequence of the proximal strain of Paen552 in the orthotopic fusion method (1000 replicates), and the results are shown in FIG. 1. From FIG. 1 it can be seen that Paen552 does not cluster with other species of bacteria, which also demonstrates that strain 1 isolated in example 1 is a new species under the genus Paenibacillus.

Therefore, by combining the 16S rDNA sequence alignment and MAGA tree construction results, paen552 was identified as a new strain of Paenibacillus and deposited at the Cantonese province microorganism strain collection at 2023, 4 and 21, under the accession number GDMCC No.63375, designated Paenibacillus sp.Paen552.

Example 3: colony characterization of novel strain Paen552

The novel strain Paen552 provided by the application is milky white, opaque, round and smooth in colony and thick in thallus on a beef extract peptone solid culture medium plate (shown in figure 2).

Microscopic observation: microscopic morphology of Paen552 (as shown in fig. 7) revealed that the bacterial cells of this strain were rod-shaped and gram-positive.

Example 4: nitrogen fixation Properties of novel Strain Paen552

Colonies of strain Paen552 were picked with an inoculating loop, streaked on Ashby nitrogen-free medium, and then placed in a biochemical incubator at a constant temperature of 30 ℃ for culturing for 48 hours, and the results were observed. The results showed that Paen552 grew well on Ashby nitrogen-free medium, confirming that the strain had better nitrogen fixation capacity (as shown in figure 3). The formulation of the Ashby nitrogen-free medium is: 10g/L mannitol, 0.2g/L KH ₂ PO ₄ ，0.2g/L MgSO ₄ ·7H ₂ O，0.2g/L NaCl，0.1g/L CaSO ₄ ·2H ₂ O，5g/L CaCO ₃ 。

Example 5: antibacterial properties of novel strain Paen552

The bacteriostatic ability of Paen552 was tested using the plate counter method. The pathogenic fungus Fusarium oxysporum is activated from the inclined plane to the center of the PDA plate, and cultured for 5-7d at 25 ℃. Colonies were grown to half the diameter of the plate and were confirmed to be target strains based on colony characteristics and microscopic characteristics for later use. Fusarium oxysporum is made into a bacterial cake with the diameter of 5mm and connected to the center of a PDA flat plate, and 5 mu LPaaen 552 bacterial liquid is sucked and connected to the edge of the flat plate. The radius of the pathogen from the proximal end of the bacteria was measured as the pathogen grew out of the plate. The results show that Paen552 has an inhibitory effect on fusarium oxysporum (as shown in fig. 4), and thus can be used for controlling plant diseases caused by fusarium oxysporum.

Example 6: potted plant growth promoting experiment of new strain Paen552

6.1 activating the strain Paen552, inoculating into beef extract peptone culture medium, and shake fermenting at 28deg.C and 180r/min for 5 days. Then, the bacterial liquid OD ₆₀₀ Adjusting to 0.5, collecting bacterial liquid, centrifuging at 5000rpm for 10min, removing supernatant, and re-suspending thallus with sterile water.

6.2 planting Hua Gu in flowerpots filled with field soil, after the seeds germinate for 5 days, pouring 5ml of the bacterial liquid prepared in the step 6.1 around the germinated plant roots in each pot, and setting 3 parallel groups for each treatment. And then irrigating once every 5 days for 3 times.

6.3 After 25 days, the plants in the pots were removed and their root length and plant height were measured. The specific measurement data are shown in Table 1, and the growth of the treated group and the control group are shown in FIG. 5.

TABLE 1 Effect of Vaccination with Paen552 on potted millet growth

As can be seen from FIG. 5, in the potted plant test, the millet after the new strain Paen552 provided by the invention is applied is more luxuriant in growth compared with the control group, the plant is generally higher, the plant is healthy and straight, and the leaves are greenish, which indicates that the new strain Paen552 provided by the invention has good promotion effect on the growth of the stalks and the leaves of the millet. Meanwhile, according to the statistical condition of millet growth data in table 1, the plant height, root length and stem thickness indexes of the millet after the Paen552 is inoculated are all obviously higher than those of a control group, and compared with the control group, the growth rates are respectively 22.18%, 79.98% and 26.23%, which shows that the Paen552 strain has obvious growth promoting and rooting promoting effects on the millet.

Example 7: field verification experiment of novel strain Paen552 of the invention

Activating the strain Paen552, inoculating into beef extract peptone culture medium, and shake fermenting at 28deg.C and 180r/min for 5 days to obtain Paen552 fermentation broth. When in use, the fermentation broth and tap water are mixed according to the following ratio of 1:100 ratio dilution. Gramineous crops and leguminous plants are respectively planted in a field district to verify the growth promotion effect of Paen552, the specific planted plants are corn, land rice, millet, soybean and alfalfa, and the crop varieties are golden-inlaid jade, land-leading 46, hua Gu, zhonghuang 13 and alfalfa. Inoculating during the growth and seeding of crops and during the seedling stage, and irrigating the same amount of fermentation diluent for each crop. After 35 days of growth, the crops were sampled and biomass data was counted. The specific measurement data are shown in tables 2, 3, 4 and 5, and the growth conditions of the treated group and the control group are shown in FIG. 6.

TABLE 2 Effect of Vaccination Paen552 on field growth of soybeans

Different treatments	Height of plant (mm)	Root length (mm)	Fresh weight on the ground (g)	Underground fresh weight (g)	Stem thickness (mm)
						Control group (mean.+ -. Standard deviation)	504.17±50.74	130.00±10.00	15.74±3.72	1.11±0.22	5.36±0.64
Paen552 (mean.+ -. Standard deviation)	557±26.83	175±50.74	23.57±4.09	1.86±0.51	5.87±0.37
						Growth rate	10.48％	34.62％	49.74％	68.18％	9.47％
p value	0.0457	0.0209	0.0613	0.0107	0.234
						Sign of p-value	*	*		*

TABLE 3 Effect of Paen552 inoculation on land Rice field growth

TABLE 4 Effect of Paen552 inoculation on millet field growth

Different treatments	Height of plant (mm)	Root length (mm)	Fresh weight on the ground (g)	Underground fresh weight (g)	Stem thickness (mm)
						Control group (mean.+ -. Standard deviation)	350.40±33.27	126.50±48.71	1.08±0.24	0.06±0.03	2.70±0.39
Paen552 (mean.+ -. Standard deviation)	372.00±48.72	143.00±33.18	1.22±0.42	0.07±0.02	2.66±0.40
						Growth rate	6.16％	13.04％	12.29％	8.06％	-1.33％
p value	0.893	0.336	0.63	0.91	0.89
						Sign of p-value

Table 5 effect of seed Paen552 on corn field growth

TABLE 6 Effect of Vaccination Paen552 on alfalfa field growth

Different treatments	Height of plant (mm)	Root length (mm)	Fresh weight on the ground (g)	Underground fresh weight (g)
					Control group (mean ± standard)Quasi-difference	49±6.69	25.7±2.52	20.65±5.82	2.99±0.43
Paen552 (mean.+ -. Standard deviation)	71.18±12.6	30.05±1.37	46.59±10.29	8.23±3.95
					Growth rate	45.26％	16.93％	125.63％	174.90％
p value	0.00116	0.114	0.096	0.0344
					Sign of p-value	**			*

As can be seen from FIG. 6, in the field test, the plants after the new strain Paen552 provided by the invention are more luxuriant compared with the overground and underground parts of the control group, the overground parts are generally higher, the robustness and the stiffness are high, and the leaves are dark green, which indicates that the new strain Paen552 provided by the invention has good promotion effect on the growth of stems, leaves and roots of the plants.

Tables 2-6 are measurements of the growth index of soybean, land rice, millet, corn, alfalfa, respectively, after application of the novel strain Paen552 provided by the present invention. As can be seen from table 2, the significant difference analysis shows that the strain height, root length and underground fresh weight of the soybeans can be significantly improved by applying the Paen552 strain, and the growth rates are respectively 10.48%, 34.62% and 68.18% compared with the control group, so that the Paen552 strain has obvious growth promoting and rooting promoting effects on the soybeans. As can be seen from Table 3, the significant difference analysis shows that the root length, the fresh weight on the ground and the stem thickness of the land rice can be greatly improved by applying the Paen552 strain, and the growth rates are respectively 34.86%, 61.72% and 99.24% compared with the control group, so that the Paen552 strain can be proved to be effective in promoting the root growth and the stem thickness of the land rice. As can be seen from Table 4, the application of the Paen552 strain increased the above-ground and below-ground portions of millet by a growth rate of 6.16% -13.04% compared with the control group, indicating that the Paen552 strain promoted the growth and rooting of millet to some extent. As can be seen from table 5, the significant difference analysis shows that the application of the Paen552 strain can significantly improve the plant height, the above-ground fresh weight, the below-ground fresh weight and the maximum leaf length of corn, and the growth rates are 23.02%, 72.76%, 155.86% and 32.24% respectively compared with the control group, which proves that the application of the Paen552 strain can significantly promote the growth of cornstalks, leaves and root systems. As can be seen from table 6, the significant difference analysis shows that the application of the Paen552 strain can significantly improve the plant height of alfalfa, significantly improve the underground fresh weight of alfalfa, and respectively increase the growth rate by 45.26% and 174.90% compared with the control group, which proves that the application of the Paen552 strain can significantly promote the growth of alfalfa stalks and root systems.

Therefore, the newly separated and identified rhizosphere growth promoting strain Paen552 in the embodiment of the application has good promoting effect on the growth of various crops, and particularly, the rhizosphere growth promoting strain Paen552 mainly shows the promoting effect on the growth of crop stems, leaves and root systems, and the newly separated and identified rhizosphere growth promoting strain Paen552 and an agricultural preparation containing the same in the embodiment of the application can be effectively used for promoting the growth of various plants including gramineous crops, leguminous plants and the like besides realizing the control of plant diseases, so that the application amount of chemical fertilizers and pesticides can be effectively reduced, and the environmental influence caused by the chemical fertilizers and the pesticides is relieved. The Paen552 of the novel Paenibacillus with the functions of promoting growth and biocontrol provided by the application has important significance for improving the yield of crops, preventing and controlling plant diseases and protecting the environment.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A bacillus species comprising the amino acid sequence of SEQ ID NO:1 or a complement thereof or a sequence complementary to the sequence set forth in SEQ ID NO:1 compared to at least 85% identical sequences.

2. The Paenibacillus of claim 1, wherein the Paenibacillus is Paenibacillus (Paenibacillus); preferably, the paenibacillus has the amino acid sequence as set forth in SEQ ID NO:1, a 16S rDNA sequence shown in seq id no; more preferably, the strain is Paenibacillus sp.paen552 with accession number GDMCC 63375.

3. A method for culturing the paenibacillus as claimed in claim 1 or 2, which comprises inoculating the paenibacillus to a medium for culturing at a temperature of 20 to 40 ℃,

alternatively, the pH of the medium is 7.0 to 8.0, preferably 7.4 to 7.6, the temperature of the culture is 25℃to 35 ℃, preferably 26℃to 30 ℃, more preferably 28 ℃,

alternatively, the medium comprises peptone 8g/L to 12g/L, preferably 10g/L, beef extract 2g/L to 4g/L, preferably 3g/L, and sodium chloride 4g/L to 6g/L, preferably 5g/L.

4. An agricultural formulation comprising the Paenibacillus as claimed in claim 1 or 2,

optionally, the formulation of the agricultural formulation is selected from: wettable powder, water dispersible granules, suspending agents, aqueous emulsion, granules, seed coating agents or a combination thereof.

5. The agricultural formulation of claim 4, further comprising an adjuvant,

optionally, the auxiliary materials include: organic auxiliary materials and/or inorganic auxiliary materials.

6. Use of a paenibacillus according to claim 1 or 2 or an agricultural formulation according to claim 4 or 5 in biological nitrogen fixation.

7. Use of a paenibacillus according to claim 1 or 2 or an agricultural formulation according to claim 4 or 5 for inhibiting fusarium oxysporum.

8. Use of a paenibacillus according to claim 1 or 2 or an agricultural formulation according to claim 4 or 5 for promoting plant growth and/or controlling plant diseases caused by fusarium oxysporum.

9. The use according to claim 8, wherein the plant disease comprises: fusarium, maize fusarium, tomato root rot, cucumber mildew, soybean root rot and/or soybean wilt.

10. The use according to claim 8, characterized in that it comprises a rooting treatment during the sowing and/or seedling stage of plants using paenibacillus as defined in claim 1 or 2 or an agricultural formulation as defined in claim 4 or 5; preferably, the plant is selected from the group consisting of gramineous plants and leguminous plants, more preferably, the gramineous plants are selected from the group consisting of maize, millet and land rice, and the leguminous plants are selected from the group consisting of soybean and alfalfa.