Multifunctional peoriae paenibacillus and application thereof

Abstract

The invention discloses a multifunctional peoriae paenibacillus and application thereof, and relates to the technical field of microorganisms, and the key point of the technical scheme is as follows: Paenibacillus peoriae S6330 is separated from rhizosphere soil; the peoriae paenibacillus has a good inhibition effect on pathogenic bacteria, pathogenic fungi and oomycetes; meanwhile, the peoriae paenibacillus has the functions of dissolving potassium, producing protease, producing amylase, producing cellulase, producing siderophore and the like, has a growth promoting effect on various crops such as wheat, corn, watermelons, small oilseed rapes, tobaccos and the like, and can remarkably improve indexes such as chlorophyll content, plant height, fresh weight, overground part weight, underground part weight and the like of plants; in addition, the peoriae paenibacillus can be used for effectively inhibiting the tobacco bacterial wilt; the peoriae paenibacillus has no environmental pollution problem, is simple in culture condition, and has wide disease prevention and growth promotion application prospects in agricultural production.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and more specifically to a multifunctional Paenibacillus piloriformis and application thereof. Background Art

[0002] Plant diseases seriously threaten agricultural production. In the past, chemical pesticides were often used as the main measure to prevent and control plant diseases due to their low cost, broad bactericidal spectrum and rapid effect. However, chemical pesticides have problems such as residues, environmental pollution and harm to human health. With the need for green development of national agriculture, biological control has received more and more attention. Beneficial microorganisms have a wide range of sources, many types, diverse mechanisms of action and can adapt well to the environment. Therefore, they can give full play to the advantages of effective control of plant diseases, safety for humans and animals, no pollution to the environment, and long-lasting control effects. At present, many commercial products with good effects have been formed, which have gradually become one of the common means of biological control of plant diseases and an important part of comprehensive disease prevention and control, with broad application prospects.

[0003] Nitrogen, phosphorus and potassium are essential nutrients for plant growth and development, and play a key role in crop production. Traditional application of chemical fertilizers plays an important role in crop production, but relevant studies have also shown that plants only use about 35% of the large amount of fertilizers invested each year, and the remaining fertilizers may enter the groundwater system, causing harm to the environment. At the same time, with the inevitable requirement of the country's comprehensive green transformation of agriculture, the Ministry of Agriculture and Rural Affairs has begun to implement the action plan for reducing the use of chemical fertilizers. Therefore, improving the utilization rate of nitrogen, phosphorus and potassium nutrients in the soil is of great significance in future crop production and soil health quality assurance. Microorganisms happen to play an important role in soil nutrient transformation. For example, silicate bacteria can dissolve potassium in potassium feldspar for plant absorption and utilization, and bacteria with phosphorus dissolving function can convert inorganic / organic insoluble phosphorus in the soil into phosphorus that can be absorbed and utilized by plants. Not only that, some microorganisms can also synthesize and secrete metabolites that promote plant growth, such as auxin, siderophore, ACC deaminase, etc.

[0004] The plant rhizosphere is the area where the life activities and metabolism of the roots themselves have the most direct and strongest impact on the soil. Attracted by the secretions of the plant roots, microorganisms such as bacteria and fungi in the soil are attracted and enriched in the rhizosphere soil, playing an important role in the growth and development of plants. Relevant studies have shown that there is a certain interaction pattern between rhizosphere probiotics and plants, which has given a clearer direction in the process of exploring microbial strain resources - the rhizosphere. As more and more beneficial rhizosphere microorganisms are discovered and applied to farmland production, the importance of beneficial rhizosphere microorganisms is self-evident. In previous studies, the beneficial rhizosphere microorganisms that were discovered were ultimately able to form the genus of microbial preparations, most of which were Bacillus. Other beneficial microorganisms developed into microbial preparations faced problems such as difficulty in fermentation and preservation.

[0005] Paenibacillus is a newly developed genus from the classification of Bacillus. It is widely distributed and commonly found in the rhizosphere of plants. Paenibacillus strains have some common characteristics of Bacillus strains, have significant biocontrol potential, and can also produce heat-resistant and stress-resistant spores, which are beneficial to the production of biocontrol agents and colonization in the soil. Previous reports have shown that Paenibacillus pierreiro can secrete a variety of hydrolases, inhibit the growth of a variety of plant pathogenic fungi, produce a variety of antagonistic substances, and has the potential to prevent and control plant diseases. However, there are few reports on the specific application of Paenibacillus pierreiro in plant biocontrol at home and abroad.

[0006] Therefore, the present invention aims to provide a multifunctional Bacillus pilosula and its application. By exploring the specific growth-promoting and antagonistic functions of Bacillus pilosula, such as bacteria and fungi, its growth-promoting effect on plants and its inhibitory effect on plant diseases are clarified, which can provide an important resource basis for the research and development of multifunctional microbial fertilizers, and provide an important reference for the green prevention and control of plant diseases and the improvement of soil and arable land quality. Summary of the invention

[0007] The invention aims to provide a multifunctional Paenibacillus peoriae and application thereof. The invention obtains Paenibacillus peoriae S6330 by isolating it from rhizosphere soil. The Paenibacillus peoriae is effective against pathogenic bacteria such as tomato bacterial canker, rice white leaf blight, rice stripe pathogen, crucifer black rot, tomato bacterial leaf spot pathogen, oat acidophilus, watermelon acidophilus, tobacco bacterial wilt pathogen, as well as the pathogenic Fusarium graminearum of wheat stem base rot, the pathogenic Fusarium oxysporum of watermelon wilt, the pathogenic Fusarium oxysporum of rice sheath blight, the pathogenic Fusarium oxysporum of cotton wilt, the pathogenic Fusarium oxysporum of rice seedling disease, the pathogenic Fusarium wilt of cotton, the pathogenic Fusarium fusarius of rice seedling disease, the pathogenic Nicotiana tabacum root rot, the pathogenic Phytophthora nicotianae of tobacco black shank disease, and the pathogenic Fusarium sclerotiorum of rapeseed. It has a good inhibitory effect on pathogenic fungi and oomycetes such as the pathogenic Sclerotinia sclerotiorum; at the same time, the Paenibacillus pilosula has the functions of solubilizing potassium, producing protease, amylase, cellulase, and siderophore, and has a growth-promoting effect on a variety of crops such as wheat, corn, watermelon, rapeseed, and tobacco, and can significantly improve the plant's chlorophyll content, plant height, fresh weight, above-ground weight, underground weight and other indicators; in addition, the Paenibacillus pilosula can effectively inhibit tobacco bacterial wilt; the Paenibacillus pilosula has no environmental pollution problems and simple culture conditions, and has broad application prospects for disease prevention and growth promotion in agricultural production.

[0008] The above technical purpose of the present invention is achieved through the following technical scheme: a multifunctional Paenibacillus peoriae and its application, the Latin name of the Paenibacillus peoriae S6330, the preservation number is CGMCC NO.29816, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (referred to as CGMCC) on January 31, 2024, and the preservation address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), Postal Code: 100101.

[0009] The present invention is further configured as follows: the 16S rRNA gene sequence of the Paenibacillus pierrei is as shown in SEQ ID NO: 1.

[0010] The present invention also provides an application of Paenibacillus pierreiu and / or a metabolite of Paenibacillus pierreiu in potassium solution, protease production, amylase production, cellulase production and siderophore production.

[0011] The present invention also provides an application of Paenibacillus pierreuil or / and a metabolite of Paenibacillus pierreuil in tobacco disease prevention, tobacco growth promotion, watermelon growth promotion, rapeseed growth promotion, corn growth promotion and wheat growth promotion.

[0012] The present invention also provides an application of Paenibacillus pierreiu and / or metabolites of Paenibacillus pierreiu in antagonizing bacterial canker of tomatoes, white leaf blight of rice, stripe pathogen of rice, black rot of crucifer, bacterial leaf spot of tomatoes, acidophilus of oats, acidophilus of watermelon, Ralstonia solanacearum, Fusarium graminearum, Fusarium oxysporum specialized type of watermelon, Rhizoctonia solani, Fusarium oxysporum specialized type of wilt, Gibberella fujikura, Thielavia nicotianae, Phytophthora tobacco and Sclerotinia sclerotiorum.

[0013] The following are the sequences involved in the scheme of the present invention:

[0014] SEQ ID NO: 1 (16S rRNA gene sequence):

[0015]

[0016] In summary, the present invention has the following beneficial effects:

[0017] 1. The bacterial strain P. peoriae S6330 of the present invention has a round colony, neat edges, opaque, grayish white on the front, and a smooth surface; experiments have shown that the P. peoriae of the present invention has a round colony, neat edges, opaque, grayish white on the front, and a smooth surface. S6330 is a stable, efficient and broad-spectrum plant disease prevention and growth-promoting bacteria. This strain has a good inhibitory effect on pathogenic bacteria such as tomato bacterial canker, rice white leaf blight, rice stripe pathogen, crucifer black rot, tomato bacterial leaf spot, oat acidophilus, watermelon acidophilus, tobacco bacterial wilt, as well as pathogenic fungi and oomycetes such as Pseudomonas graminearum, the pathogen of wheat stem base rot, Fusarium oxysporum, the watermelon-specific type of watermelon wilt, Rhizoctonia solani, the pathogen of rice sheath blight, Fusarium oxysporum, the wilt-specific type of cotton wilt, Gibberella fujikura, the pathogen of rice seedling disease, Thielavia fuciformis, the pathogen of tobacco root black rot, Phytophthora tobacco, the pathogen of tobacco black shank, and Sclerotinia sclerotiorum, the pathogen of rapeseed sclerotinia.

[0018] 2. The strain P. peoriae S6330 of the present invention has the functions of solubilizing potassium, producing protease, amylase, cellulase, and siderophore, and can effectively improve the soil nutritional environment and promote plant growth. The results show that P. peoriae S6330 is a plant rhizosphere probiotic with broad-spectrum bactericidal activity, multi-pathway growth-promoting functions, easy to cultivate, easy to ferment, good to preserve, environmentally friendly, and harmless to humans and animals. The strain has good prospects for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a phylogenetic tree diagram constructed based on the 16SrRNA gene sequence in Example 1 of the present invention;

[0020] Figure 2 Schematic diagram of the determination of the antagonistic ability of Paenibacillus pierreiu against pathogenic bacteria in Example 2 of the present invention;

[0021] Figure 3 Schematic diagram of the determination of the ability of Paenibacillus pierreiu to antagonize pathogenic fungi and oomycetes in Example 2 of the present invention;

[0022] Figure 4 This is a schematic diagram of the potassium solubilization assay of Paenibacillus pilorei in Example 3 of the present invention;

[0023] Figure 5 is a schematic diagram of the determination of protease production by Paenibacillus pierrei in Example 4 of the present invention;

[0024] Figure 6This is a schematic diagram of the determination of amylase production by Paenibacillus piloriformis in Example 5 of the present invention;

[0025] Figure 7 This is a schematic diagram of the determination of cellulose production by Paenibacillus pierrei in Example 6 of the present invention;

[0026] Figure 8 This is a schematic diagram of the determination of siderophore production by Paenibacillus piloriae in Example 7 of the present invention;

[0027] Fig. 9 Schematic diagram of the determination of the tobacco disease prevention ability of Paenibacillus pilorei in Example 8 of the present invention;

[0028] Fig.10 is a schematic diagram of the determination of the tobacco growth-promoting ability of Paenibacillus pilorei in Example 9 of the present invention;

[0029] Fig.11 is a schematic diagram of the determination of the growth-promoting ability of Paenibacillus piloreus on watermelon in Example 10 of the present invention;

[0030] Fig.12 is a schematic diagram of the determination of the growth-promoting ability of Paenibacillus pierreiu on rapeseed in Example 11 of the present invention;

[0031] Fig.13 is a schematic diagram of the determination of the corn growth-promoting ability of Paenibacillus pilorei in Example 12 of the present invention;

[0032] Fig.14 This is a schematic diagram of the determination of the wheat growth-promoting ability of Bacillus pierrei in Example 13 of the present invention. DETAILED DESCRIPTION

[0033] The following is combined with Figure 1-14 The present invention is described in further detail.

[0034] The strains cited in the examples of the present invention are explained as follows:

[0035] 1) Clavibacillus michiganensis subsp. michiganse, which causes bacterial canker of tomatoes (Wu Xiuqin, Yang Yingzi, Luo Jinyan, Qiu Wen, An Qianli, Li Bin. Rapid detection of bacterial fruit spot pathogens of cucurbits and bacterial canker pathogens of tomatoes using loop-mediated isothermal amplification. Acta Phytophylacica Sinica, 2018, 45(06): 1235-1241), referred to as bacterial canker pathogen of tomatoes.

[0036] 2) Xanthomonas oryzae pv. oryzae that causes rice bacterial leaf blight (Yang Yayun, Zhang Dunyu, Chen Ling, Chen Yue, Yin Fuyou, Jiang Chunmiao, Xiao Suqin, Ke Xue, Yu Tengqiong, Wang Bo, Fu Jian, Zhong Qiaofang, Chen Gongyou, Cheng Zaiquan. Identification of resistance of Yunnan medicinal wild rice to four major rice diseases. Acta Phytopathologica Sinica, 2019, 1: 101-112), referred to as Xanthomonas oryzae pv. oryzae.

[0037] 3) Xanthomonas oryzae pv. oryzicola that causes rice bacterial leaf streak disease (Ye Wenxin, Chang Zheng, Liu Qianqian, et al. Mutation analysis of the xrvA gene encoding the Xanthomonas virulence regulatory protein in rice bacterial leaf streak disease [J / OL]. Molecular Plant Breeding: 1-17[2024-07-01]), referred to as rice bacterial leaf streak disease.

[0038] 4) Xanthomonas campestris pv. carnpestris that causes black rot of crucifer (Lan Nihan, Su Deshan, Zhou Le, Qin Xia, Huang Peifang, Yang Mei, Jiang Bole. Identification of small molecule compounds that inhibit the type III secretion system of black rot of crucifer in host plants. Acta Phytopathologica Sinica, 2019, 49(02): 262-270), referred to as black rot of crucifer.

[0039] 5) Pseudomonas sringaepv.tomato (Wang Ruona, Ma Yinan, Li Junzhou, et al. Analysis of the enzymatic function of HrpH, a component of the type III secretion system of Pseudomonas syringae [J]. Acta Phytopathologica Sinica, 2022, 52(03): 334-340.), which causes bacterial leaf spot of tomato (Pseudomonas sringaepv.tomato).

[0040] 6) Acidovorax avenae, which causes bacterial fruit blotch of melon (Lin Tao, Ma Guobin, Jiang Shouyang, et al. Screening of fungicides for controlling seed-borne pathogens of bacterial fruit blotch of melon. Shanghai Journal of Agriculture, 2023, 39(03): 55-60.), also known as Acidovorax avenae.

[0041] 7) Acidovorax citrulli, which causes bacterial fruit spot of watermelon (Xu Xiulan, Song Shunhua, Zhang Zhiyong, Wu Xuehong, Geng Lihua. Research progress on important seed-borne diseases of watermelon. Plant Protection, 2016, 42(01):12-18), is referred to as Acidovorax citrulli.

[0042] 8) Ralstonia solanacearum, which causes tobacco bacterial wilt (isolated from tobacco rhizosphere soil in our laboratory), is referred to as Ralstonia solanacearum.

[0043] 9) Fusarium graminearum, which causes wheat stem rot (isolated from the base of wheat stem in our laboratory), is referred to as Fusarium graminearum.

[0044] 10) Fusarium oxysporum f.sp.niveum, which causes watermelon wilt (isolated from watermelon roots in our laboratory), is referred to as Fusarium oxysporum f.sp.niveum.

[0045] 11) Rhizoctonia solani, the fungus that causes rice sheath blight (Wei Hailei, Wang Ye, Zhang Liqun, Tang Wenhua. Identification of biocontrol strains 2P24 and CPF-10 and preliminary analysis of their biocontrol-related traits. Acta Phytopathologica Sinica, 2004, 34: 80-85), is abbreviated as Rhizoctonia solani.

[0046] 12) Fusarium oxysporium f.sp. vasinfectum, which causes cotton wilt disease (Gao Zhimou, Cao Jun, Pan Yuemin, et al. Study on the antagonistic mechanism of Trichoderma harzianum TH-1 against cotton wilt pathogen and Verticillium wilt pathogen [J]. Cotton Science, 2007(03):168-172.), also known as Fusarium oxysporium f.sp. vasinfectum.

[0047] 13) Gibberella fujikuroi, which causes rice seedling bakan disease (Chan Zhulong, Ding Kejian, Tan Genjia. Research progress on rice seedling bakan disease [J]. Anhui Agricultural Sciences, 2002(06):880-883.), is abbreviated as Gibberella fujikuroi.

[0048] 14) Thielaviopsis basicola, which causes tobacco root black rot (Dou Yanxia. Study on pathogenicity differentiation and genetic diversity of tobacco root black rot fungus Thielaviopsis basicola [D]. Southwest University, 2012.), is abbreviated as Thielaviopsis basicola.

[0049] 15) Phytophthora nicotianae, the pathogen that causes tobacco black shank (provided by China Agricultural Microbiological Culture Collection Center), also known as Phytophthora nicotianae.

[0050] 16) Sclerotinia sclerotiorum, which causes sclerotinia rot of rapeseed (Wang Lei, Liu Yao, Ding Yijuan, et al. Research progress on sclerotinia rot of rapeseed [J]. Journal of Northwest A&F University (Natural Science Edition), 2015, 43(10): 85-93.), also known as Sclerotinia sclerotiorum.

[0051] The method for preparing the culture medium used in the embodiments of the present invention is as follows:

[0052] Luria-Bertani (LB) solid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 12 g agar, distilled water to 1000 mL, pH 7.0-7.2.

[0053] Luria-Bertani (LB) liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, distilled water to 1000 mL, pH 7.0-7.2.

[0054] King's B (KB) solid medium: 20 g of peptone No. 3, MgSO 4 7H 2 O 0.4 g, glycerol 10 mL, K 2 HPO 4 1.5 g, agar 12 g, distilled water to 1000 mL.

[0055] King's B (KB) liquid medium: 20 g of peptone No. 3, MgSO 4 7H 2 O 0.4 g, glycerol 10 mL, K 2 HPO 4 1.5 g, distilled water to 1000 mL.

[0056] NutrientAgar (NA) medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, 12 g agar, pH 7.0, distilled water to 1000 mL.

[0057] Nutrient Broth (NB) medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, pH 7.0, distilled water to 1000 mL.

[0058] PDA medium: 200 g potato, 20 g glucose, 12 g agar, and distilled water to 1000 mL.

[0059] Potassium-dissolving medium: sucrose 5 g, MgSO 4 7H 2 O 0.5 g, FeCl3 0.005 g, CaCO 3 0.1 g, sericite powder 2 g, 1.5% agar, distilled to 1000 mL.

[0060] Amylase detection medium: soluble starch 1.0 g, peptone 5.0 g, NaCl 5.0 g, beef extract 5.0 g, glucose 5.0 g, agar 15.0 g, distilled water to 1 L. Autoclave: 115°C, 20 min.

[0061] Protease assay medium: peptone 10 g, NaCl 5 g, CaCl 2 0.1 g, 100 mL of skim milk (added when the culture medium is sterilized and the temperature drops to 45°C), 12 g of agar, and distilled water to make up to 1000 mL.

[0062] Sodium carboxymethyl cellulose medium: peptone 10g, yeast powder 10g, sodium carboxymethyl cellulose 10g, KH 2 PO 4 1 g, NaCl 15 g, agar 12 g, distilled water to 1000 mL;

[0063] Chrome azurol S (CAS) blue qualitative detection medium:

[0064] 0.012 g of chrome azurol sulphonate (CAS) was dissolved in 10 mL of deionized water and mixed with 2 mL of 1 mmol / L FeCl 3 The solution was mixed to obtain solution a;

[0065] Take 0.015 g of hexadecyl trimethyl ammonium bromide (HDTMA) and dissolve it in 8 mL of deionized water to obtain solution b;

[0066] Slowly add liquid a into liquid b and mix thoroughly to obtain dye solution c;

[0067] 0.1 mol / L phosphate solution (2.427 g Na 2 HPO 4 12H 2 O, 0.5905 g NaH 2 PO 4 ·2H 2 O, 0.075gKH 2 PO 4 , 0.125 g NaCl, 0.25 g NH 4Cl, 100 mL of deionized water, diluted 10 times when used) 10 mL, piperazine diethanol sulfonic acid (pipes) 6.04 g were added into a clean conical flask containing 150 mL of distilled water, mixed and the pH was adjusted to 6.8 with 50% NaOH solution, and finally 4 g of agar powder was added to obtain medium d;

[0068] Dye solution c, culture medium d and 1mmol / L CaCl 2 、1mmol / L MgSO 4 7H 2 O, 20% glucose, 10% casamino acid sterilization (115℃, 20min). 2 0.2mL, 1mmol / L MgSO 4 7H 2 4 mL of 1% casamino acid, 6 mL of 10% casamino acid, and 2 mL of 20% glucose solution were added to the culture medium d. Then, the dye solution c was slowly added and shaken thoroughly to obtain a blue qualitative detection culture medium.

[0069] Example 1: Isolation and identification of Paenibacillus peoriae S6330

[0070] In this embodiment, Paenibacillus peoriae S6330 is an aerobic Gram-positive bacterium, and the metabolites of Paenibacillus peoriae S6330 are extracted from substances secreted by Paenibacillus peoriae into the culture medium.

[0071] The preparation method of the metabolite of Paenibacillus pierrei is as follows: the strain S6330 is streaked on an LB solid culture medium, and after culturing at 28°C for 2 days, a single clone colony is picked and inoculated into an LB liquid culture medium, and cultured overnight at 28°C at 220rpm to obtain a mother liquor; then, the mother liquor is transferred to an LB liquid culture medium at a ratio of 1:1000, and cultured under the same conditions for 2 days to obtain a fermentation broth, which is the metabolite of Paenibacillus pierrei S6330.

[0072] Isolation process of strain S6330: strain S6330 was isolated from plant rhizosphere soil. 2 g of rhizosphere soil sample was weighed and added to a conical flask containing 18 mL of sterile distilled water. The sample was cultured at 28 °C and 200 rpm for 1 h. After standing at room temperature for 5 min, 1 mL of the supernatant was taken and diluted to 10 by 10-fold gradient. -5 100 μL of each gradient solution was spread on an LB medium plate, and each concentration gradient was repeated 3 times. After culturing in a 28°C incubator for 24 h, the strain S6330 was obtained after purification.

[0073] Identification process of strain S6330: Strain S6330 was inoculated on LB solid medium by streaking, cultured at 28°C for 2 days, and then a trace amount of monoclonal colonies were picked as templates to amplify the 16S rRNA gene according to the 16S PCR amplification system.

[0074] 16S PCR reaction system: 30 μL, As shown below:

[0075] 2× Taq PCR Master Mix 15 μL;

[0076] 27F 1.5μL;

[0077] 1492R 1.5μL;

[0078] DNA template trace amount;

[0079] dH 2 O 12 μL;

[0080] The total PCR reaction system was 30 μL.

[0081] PCR amplification procedure: step 1: pre-denaturation at 94°C for 5 min; step 2: denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 1 min, 35 cycles; step 3: full extension at 72°C for 5 min; storage at 4°C.

[0082] According to the comparison analysis of 16SrRNA (Seq ID No.1) of S6330 obtained in EZBioCloud, the results showed that S6330 was similar to Paenibacillus peoriae DSM8320. T The similarity reached 99.65%. Based on the 16SrRNA sequence of S6330 (Seq ID No.1), the phylogenetic tree was constructed by comparison analysis in EZBioCloud ( Figure 1 ), and the strain was found to be similar to Paenibacillus peoriae DSM8320 T The strains were clustered into one branch with a close relationship, so strain S6330 was identified as Paenibacillus peoriae.

[0083] Example 2: Determination of the antibacterial spectrum of Paenibacillus peoriae S6330

[0084] In this example, the antagonistic ability of strain S6330 against 8 pathogenic bacteria and 8 pathogenic fungi and oomycetes was tested, wherein the information of the tested pathogenic bacteria, pathogenic fungi and oomycetes is shown in Table 1:

[0085] Table 1 Information of pathogenic bacteria, pathogenic fungi and oomycetes tested

[0086]

[0087]

[0088] The plate antagonism test was used to detect the antagonistic ability of strain S6330 against the above-mentioned pathogenic bacteria. The experimental method is as follows: After melting the NA / LB / KB medium with an agar content of 1%, cool it to 45°C at room temperature. Add the pathogenic bacteria that have been shaken overnight in the NA / LB / KB liquid medium to the stable growth phase to the cooled NA / LB / KB medium at a ratio of 1:100 and pour the plate. After drying, inoculate 10μL of strain S6330 culture solution (LB liquid shake culture) on the plate. After drying, invert the plate in a 28°C incubator for culture, use the plate inoculated with only pathogens as the control, and set up 3 replicates for each treatment. Observe and measure the diameter of the inhibition zone after 48 hours of culture.

[0089] The plate confrontation test was used to detect the antagonistic ability of strain S6330 against pathogenic fungi and oomycetes: a 0.7 cm diameter puncher was used to take the pathogenic fungi and oomycete cakes freshly cultured on the PDA plate at 25°C, and the cakes were picked up with an inoculation needle, and the mycelium was inoculated on the center of the fresh PDA plate with the mycelium facing down, and then 10 μL of strain S6330 culture solution (LB liquid shaking culture) was inoculated at a distance of about 2.5 cm from the cake. After drying, the plates were inverted and cultured in a 25°C incubator. The plates inoculated with only pathogens were used as controls, and 3 replicates were set for each treatment. After 5-7 days of culture, the width of the inhibition zone was observed and measured.

[0090] The results showed that strain S6330 had good antagonistic effects on eight pathogenic bacteria, including tomato bacterial canker pathogen Corynebacterium michiganensis, rice white leaf blight pathogen Xanthomonas oryzae, rice leaf streak pathogen Xanthomonas oryzae, crucifer black rot pathogen Xanthomonas campestris, tomato bacterial leaf spot pathogen Pseudomonas syringae, melon bacterial fruit spot pathogen Acidophilus avenae and Acidophilus citrullus, and Ralstonia solanacearum. Figure 2 , among which: A, tomato bacterial canker pathogen Corynebacterium michiganensis; B, rice white leaf blight pathogen Xanthomonas oryzae; C, rice leaf streak pathogen Xanthomonas oryzae; D, crucifer black rot pathogen Xanthomonas campestris; E, tomato bacterial leaf spot pathogen Pseudomonas syringae; F, melon bacterial fruit spot pathogen Acidophilus avenae; G, melon bacterial fruit spot pathogen Acidophilus citrullus; H, tobacco bacterial wilt pathogen Ralstonia solanacearum). In addition, strain S6330 has a good inhibitory effect on the tested plant pathogenic fungi and oomycetes ( Figure 3, among which: A, Pseudomonas graminearum; B, Fusarium oxysporum watermelon-specific type; C, Rhizoctonia solani; D, Fusarium oxysporum wilt-specific type; E, Gibberella fujikura; F, Thielavia radicifolia; G, Phytophthora nicotianae; H, Sclerotinia sclerotiorum). The above results show that strain S6330 has the ability to resist a broad spectrum of plant pathogens and has the potential to be developed as a biocontrol agent.

[0091] Example 3: Potassium-dissolving ability experiment of Paenibacillus peoriae S6330

[0092] The activated strain S6330 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 220rpm. 10μL of the culture solution was inoculated on a potassium-dissolving medium plate, and 10μL of LB liquid culture medium was inoculated as a control treatment. Each treatment was repeated 3 times. After drying, the plate was inverted and cultured in a 28°C incubator for 7 days to observe whether oil droplets were formed. The results showed that on the plate inoculated with the culture solution of strain S6330, obvious oil droplets appeared around the inoculated colonies ( Figure 4 ), indicating that strain S6330 has the ability to solubilize potassium.

[0093] Example 4: Protease production capacity test of Paenibacillus peoriae S6330

[0094] The activated strain S6330 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 200rpm. 10μl of culture solution was spotted on the protease detection medium plate, and 10μL of LB liquid was spotted as the control treatment. Each treatment was repeated 3 times. After drying, it was inverted and cultured at 28°C for 2 days to observe whether a transparent circle appeared. The results showed that there was no change in the control plate, while the detection plate inoculated with the culture solution of strain S6330 showed an obvious hydrolysis transparent circle around the inoculated colony ( Figure 5 ), indicating that the protein in the plate was decomposed, proving that strain S6330 has the ability to produce protease.

[0095] Example 5: Amylase production capacity test of Paenibacillus peoriae S6330

[0096] The activated strain S6330 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 200rpm. 10μl of the culture solution was spotted on the amylase detection medium plate, and 10μL of LB liquid was spotted as the control treatment. Each treatment was repeated 3 times. After drying, it was inverted and cultured at 28°C for 3 days, and then an appropriate amount of Gram's iodine solution was added to cover the surface of the culture medium. After standing for 2 minutes, the Gram's iodine solution was poured out and observed for transparent circles. The results showed that there was no change in the control group plate, while the test plate inoculated with the culture solution of strain S6330 showed an obvious hydrolysis transparent circle around the inoculated colony ( Figure 6 ), indicating that the starch in the plate was decomposed, proving that strain S6330 has the ability to produce amylase.

[0097] Example 6: Paenibacillus peoriae S6330 cellulase production capacity test

[0098] The activated strain S6330 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 220rpm. 10μL of culture solution was spotted on a sodium carboxymethyl cellulose culture medium plate, and 10μL of LB liquid was spotted as a control treatment. Each treatment was repeated 3 times. After drying, it was incubated inverted in a 28°C incubator for 4 days, and then 1g / L Congo red dye was poured into the plate to cover it. It was then rinsed with 1mol / L NaCl solution to observe whether there was a transparent circle. The results showed that there was no change in the control group plate, while an obvious transparent circle appeared around the inoculated colony on the test plate inoculated with the culture solution of strain S6330 ( Figure 7 ), indicating that the cellulose in the plate was decomposed. This proves that strain S6330 has the ability to produce cellulase.

[0099] Example 7: Siderophore production ability test of Paenibacillus peoriae S6330

[0100] The activated strain S6330 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 220rpm. 10 μL of the culture solution was spotted on a CAS blue qualitative detection culture medium plate and cultured inverted in a 28°C incubator for 3 days to observe whether a yellow halo was produced around the colony. Since the siderophore competes for the iron ions chelated by EDTA in the culture medium, the culture medium changes from blue to yellow, so the appearance of a yellow halo around the colony indicates the production of siderophore. The results showed that a yellow halo appeared around the colony of strain S6330, proving that strain S6330 has the ability to produce siderophore ( Figure 8 ).

[0101] Example 8: Experiment on the Effective Control of Tobacco Bacterial Wilt by Paenibacillus peoriae S6330

[0102] Tobacco seedlings with consistent growth for four weeks were selected and set up a biocontrol group, a disease control group and a healthy control group, with 6 plants in each group and 3 replicates. 8 CFU·mL -1 After irrigating the roots with the S6330 suspension for 3 days, the roots were wounded with a sharp tool and irrigated with 50 mL of 1×10 8 CFU·mL -1 of Ralstonia solanacearum suspension; disease control group: firstly, 50mL10mMMgCl 2The roots were irrigated for 3 days, and then the roots were wounded with a sharp tool and irrigated with 50 mL 1×10 8 CFU·mL -1 The healthy control group was firstly treated with 50mL10mMMgCl 2 The roots were irrigated for 3 days, and then the roots were wounded with a sharp tool and irrigated with 50 mL of LB liquid medium. Each treatment group was placed in a greenhouse environment with a light / dark period of 16 / 8 h and a temperature of 28°C. The disease situation was observed and recorded every day. On the 17th day after the inoculation of the pathogen, the disease index and control effect ( Fig. 9 The results showed that root irrigation pretreatment with the suspension of strain S6330 could significantly reduce the occurrence and severity of tobacco bacterial wilt, with the greenhouse control efficiency reaching 83.33% ( Fig. 9 ).

[0103] Example 9: Experiment on Paenibacillus peoriae S6330 significantly promoting tobacco growth

[0104] Tobacco seeds were sown in seedling trays (10 cm high and 7 cm wide) and cultured in a greenhouse (light / dark hours 16 / 8 h, temperature 28°C). After 4 weeks of culture, tobacco seedlings with uniform growth were selected for growth promotion experiments.

[0105] Growth promotion experiment: A single colony of S6330 was selected and inoculated into LB medium. After culturing at 28°C and 200 rpm for 24 h, it was transferred to 50 mL LB medium at a 1% inoculum and continued to be cultured at 28°C and 200 rpm for 24 h. 2 Configure S6330 to a concentration of 1×10 8 CFU·mL -1 Each treatment was repeated 3 times with 50 mL of MgCl 2 The growth was observed and recorded every day. On the 30th day after root irrigation, the leaf area, fresh weight and aboveground weight of the tobacco were measured. Fig.10 As shown in the figure, tobacco treated with the suspension of strain S6330 grew vigorously, with an average leaf area of ​​46.90±7.39cm 2 , which was significantly higher than the control group with a leaf area of ​​35.94±8.28cm 2 The average fresh weight was 9.82±1.20g, significantly higher than the fresh weight of the control group (6.72±0.91g); the average aboveground weight was 8.45±1.29g, significantly higher than the aboveground weight of the control group (5.78±0.73g). Fig.10 ).

[0106] Example 10: Experiment on Paenibacillus peoriae S6330 significantly promoting watermelon growth

[0107] The watermelon seeds were surface sterilized with 2% sodium hypochlorite for 5 minutes, and the sterilized watermelon seeds were placed in a glass culture dish with a diameter of 20 cm, and the surface was covered with a layer of sterilized and soaked filter paper, and the seeds were germinated in a 28°C incubator. After about 5 days, the watermelon seeds turned white, and the watermelon seeds at this time were transplanted into a flower pot, and nutrient soil, perlite, and natural soil (1:1:2 volume ratio) were evenly mixed and added to the flower pot. After soaking with water, the watermelon seeds were evenly spread into the soil, covered with a small amount of soil, and placed in a greenhouse with a temperature of 25°C and a humidity of 65% to 75%. After about two weeks, the seedlings grew out. When the watermelon grew to the stage of two leaves and one heart, the watermelon seedlings with consistent growth were selected for growth promotion experiments.

[0108] Growth promotion experiment: A single colony of S6330 was selected and inoculated into LB medium. After culturing at 28°C and 200 rpm for 24 h, it was transferred to 50 mL LB medium at a 1% inoculum and continued to be cultured at 28°C and 200 rpm for 24 h. After the culture was completed, S6330 was prepared with distilled water to a concentration of 1×10 8 CFU·mL -1 The watermelon roots were irrigated with 20 mL of the S6330 suspension. Each treatment had 20 plants and was repeated 3 times. The distilled water was used as the control. The growth was observed and recorded every day. On the 30th day after the root irrigation, the stem diameter, plant height, root length, fresh weight, above-ground weight, and underground weight of the watermelon were measured. The results are as follows: Fig.11 As shown, watermelons treated with S6330 suspension for root irrigation grew vigorously, with an average stem diameter of 3.04±0.14cm, significantly higher than the stem diameter of the control group of 2.46±0.13cm; an average plant height of 16.21±1.71cm, significantly higher than the plant height of the control group of 13.08±0.68cm; an average root length of 24.58±5.01cm, significantly higher than the root length of the control group of 15.98±5.17cm; an average fresh weight of 3.31±0.64g, significantly higher than the fresh weight of the control group of 2.26±0.25g; an average aboveground weight of 2.91±0.46g, significantly higher than the aboveground weight of 1.98±0.26g in the control group; and an average underground weight of 0.43±0.10g, significantly higher than the underground weight of 0.19±0.04g in the control group ( Fig.11 ).

[0109] Example 11: Experiment on Paenibacillus peoriae S6330 significantly promoting the growth of rapeseed

[0110] Mix nutrient soil, perlite and natural soil (1:1:2) evenly and add them into the flowerpot. After soaking it with water, sprinkle rapeseed seeds evenly into the soil, cover it with a small amount of soil, and place it in a greenhouse with a temperature of 30°C and a humidity of 65% to 75%. After about a week, select rapeseed seedlings with consistent growth for growth promotion experiments.

[0111] Growth promotion experiment: A single colony of S6330 was selected and inoculated into LB medium. After culturing at 28°C and 200 rpm for 24 h, it was transferred to 50 mL LB medium at a 1% inoculum and continued to be cultured at 28°C and 200 rpm for 24 h. After the culture was completed, S6330 was prepared with distilled water to a concentration of 1×10 8 CFU·mL -1 30 mL of S6330 suspension was poured into the roots of rapeseed. Each treatment had 30 plants, and the treatment was repeated 3 times. The root irrigation with distilled water was used as the control. The growth was observed and recorded every day. On the 30th day after root irrigation, the chlorophyll content, leaf area, plant height, fresh weight, and aboveground weight of rapeseed were measured. The results are as follows: Fig.12 As shown in the figure, the rapeseed grown vigorously after root irrigation with S6330 bacterial suspension, with an average chlorophyll content of 42.04±1.29SPAD, significantly higher than the chlorophyll content of the control group of 39.18±0.1SPAD; the average leaf area reached 17.11±1.30cm 2 , which was significantly higher than the control group with a leaf area of ​​15.40±1.73cm 2 The average plant height was 11.48±0.58cm, significantly higher than the control group's plant height of 10.62±0.32cm; the average fresh weight was 3.34±0.36g, significantly higher than the control group's fresh weight of 2.81±0.43g; the average aboveground weight was 2.98±0.29g, significantly higher than the control group's aboveground weight of 2.43±0.35g ( Fig.12 ).

[0112] Example 12: Experiment on Paenibacillus peoriae S6330 significantly promoting corn growth

[0113] Mix nutrient soil, perlite and natural soil (1:1:2) evenly and add them to flower pots. Sow 4 corn seeds in each pot after soaking it with water. Cover it with a small amount of soil and place it in a greenhouse with a temperature of 30°C and a humidity of 65% to 75%. After about two weeks, the seedlings will grow out. Select the corn seedlings with consistent growth and conduct growth promotion experiments.

[0114] Growth promotion experiment: A single colony of S6330 was selected and inoculated into LB medium. After culturing at 28°C and 200 rpm for 24 h, it was transferred to 50 mL LB medium at a 1% inoculum and continued to be cultured at 28°C and 200 rpm for 24 h. After the culture was completed, S6330 was prepared with distilled water to a concentration of 1×108 CFU·mL -1 50 mL of S6330 suspension was poured into the roots of corn. 30 plants were treated in each treatment, and the treatment was repeated 3 times. Distilled water was used as the control. The growth was observed and recorded every day. On the 30th day after root irrigation, the plant height, root length, leaf length, stem length, fresh weight, and underground weight of corn were measured. The results are as follows: Fig.13 As shown, after root irrigation with S6330 suspension, corn grew vigorously, with an average plant height of 55.97±5.96cm, significantly higher than the plant height of 48.32±5.61cm in the control group; an average root length of 19.65±3.07cm, significantly higher than the root length of 15.87±4.25cm in the control group; an average leaf length of 39.87±4.73cm, significantly higher than the leaf length of 34.88±6.29cm in the control group; an average stem length of 15.06±1.95cm, significantly higher than the stem length of 12.38±1.41cm in the control group; an average fresh weight of 8.24±1.54g, significantly higher than the fresh weight of 6.95±1.85g in the control group; and an average underground weight of 3.42±0.78g, significantly higher than the underground weight of 2.27±0.64g in the control group ( Fig.13 ).

[0115] Example 13: Experiment on Paenibacillus peoriae S6330 significantly promoting wheat growth

[0116] Mix nutrient soil, perlite and natural soil (1:1:2) evenly and add them to flower pots. After soaking them with water, sow about 9 wheat seeds in each pot, cover them with a small amount of soil, and place them in a greenhouse with a temperature of 25°C and a humidity of 65% to 75%. After about a week, the wheat will grow, and wheat seedlings with consistent growth will be selected for growth promotion experiments.

[0117] Growth promotion experiment: A single colony of S6330 was selected and inoculated into LB medium. After culturing at 28°C and 200 rpm for 24 h, it was transferred to 50 mL LB medium at a 1% inoculum and continued to be cultured at 28°C and 200 rpm for 24 h. After the culture was completed, S6330 was prepared with distilled water to a concentration of 1×10 8 CFU·mL -1 50 mL of S6330 suspension was poured into the roots of wheat. 40 plants were treated in each treatment, and the treatment was repeated 3 times. Distilled water was used as the control. The growth was observed and recorded every day. On the 30th day after root irrigation, the fresh weight of wheat was measured. The results are as follows: Fig.14 As shown in Figure 2, wheat treated with S6330 suspension grew vigorously, with an average fresh weight of 0.75±0.05g, significantly higher than the control group's fresh weight of 0.64±0.10g ( Fig.14 ).

[0118] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A multifunctional Paenibacillus pilosulatus, characterized in that: The Latin name of the Paenibacillus peoriae is Paenibacillus peoriae S6330, and the preservation number is CGMCC NO.29816.

2. The multifunctional Paenibacillus pilosulae according to claim 1, characterized in that: The 16SrRNA gene sequence of the Bacillus pierrei is shown in SEQ ID NO:

1.

3. Use of the Paenibacillus pierreiu and / or the metabolites of Paenibacillus pierreiu according to claim 1 in potassium hydrolysis, protease production, amylase production, cellulase production and siderophore production.

4. Use of the Paenibacillus pierreuil or / and the metabolites of Paenibacillus pierreuil according to claim 1 in tobacco disease prevention, tobacco growth promotion, watermelon growth promotion, rapeseed growth promotion, corn growth promotion and wheat growth promotion.

5. Use of the Paenibacillus pierreiu according to claim 1 or / and the metabolites of Paenibacillus pierreiu in antagonizing bacterial canker of tomatoes, Xanthoceras oryzae, Pseudomonas aeruginosa, Ralstonia solani, Fusarium oxysporum, Fusarium oxysporum, Fusarium wilt, Gibberella fujikura, Thielavia oxysporum, Phytophthora nicotianae, and Sclerotinia sclerotiorum.

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