Penicillium paniculatum complex microbial inoculant capable of antagonizing multiple soil-borne disease pathogenic bacteria and application of penicillium paniculatum complex microbial inoculant

Abstract

The invention discloses a Penicillium paniculatum complex microbial inoculant for antagonizing multiple soil-borne disease pathogenic bacteria and application thereof. The invention provides a combined bacterium, which is prepared from penicillium lanceolarium and subsaxibacter angularis K2501 (CGMCC (China General Microbiological Culture Collection Center) No.34855). The invention also provides a preparation method of the combined bacterium. The combined bacterium is prepared from the penicillium lanceolarium and the subsaxibacter angularis K2501. According to the present invention, the new species, i.e., the subsaxibacter angularis K2501, has good antibacterial activity, and can be used for preparing the antibacterial agent. According to the invention, the penicillium petici P1001 and the hypobacillusroot rot resistant K2501 are further compounded, so that an antagonistic effect on a plurality of soil-borne pathogenic bacteria is shown; and the antibacterial effect of the compound bacterial agent of the two bacteria is obviously better than that of a single bacterium. The invention has important significance for preventing and treating soil-borne diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a Penicillium crustosum complex microbial agent antagonizing multiple soil-borne disease pathogens and application thereof. BACKGROUND

[0002] Penicillium crustosum belongs to Ascomycota - Pezizomycetes-Eurotiales-Trichocomaceae-Penicilliu, a kind of filamentous fungi widely existing in natural environment, is mostly distributed in soil, water, air and the bodies of animals and plants. The polyketides, alkaloids and peptides produced by Penicillium crustosum can inhibit the growth of some fungi and bacteria through various mechanisms, such as interfering with cell wall synthesis, destroying cell membrane integrity, inhibiting protein synthesis or interfering with metabolic pathways, thus Penicillium crustosum plays an antibacterial role in agricultural soil-borne pathogens such as fungi and gram-negative bacteria.

[0003] Subsaxibacter strain belongs to Bacteroidota - Flavobacteriia - Flavobacteriales -A group of gram-negative short rods of the family Flavobacteriaceae. The type species of the genus, Subsaxibacter broadyi, was isolated from a biofilm of blue algae attached to a piece of quartzite half-buried in the ground in Antarctica in 2005 (Bowman JP, Nichols DS. Novel members of the family Flavobacteriaceae from Antarctic maritime habitats including Subsaximicrobium wynnwilliamsiigen. nov., sp. nov., Subsaximicrobium saxinquilinus sp. nov., Subsaxibacter broadyi gen. nov., sp. nov., Lacinutrix copepodicola gen. nov., sp. nov., and novel species of the genera Bizionia, Gelidibacter and Gillisia. Int J Syst Evol Microbiol 2005;55:1471-1486.); Subsaxibacter arcticus was reported in 2016 and 2018 from Arctic intertidal sand (Xu F, Zhang XY, Liu C, Shi M, Su HN, Qin QL, Chen XL, Zhang YZ, Song XY. Subsaxibacter arcticus sp. nov., isolated from Arctic intertidal sand. Int J Syst Evol Microbiol 2016;66:132-136.) and Subsaxibacter sediminis was isolated from Arctic sediment (Sharma A, Jani K, Feng GD, Karodi P, Vemuluri VR, Zhu HH, Shivaji S, Thite V, Kajale S, Rahi P, et al. Subsaxibacter sediminis sp. nov., isolated from Arctic glacial sediment and emended description of the genus Subsaxibacter.Int J Syst Evol Microbiol 2018;68:1678-1682.) So far, only the above three validly described species are included in the genus Subsaxibacter (https: / / lpsn.dsmz.de / genus / subsaxibacter). There is no report on the application of the strains of this genus.

[0004] Fusarium equiseti, Streptomyces scabies, Clavibacter cicatricum, and Erwinia carotovora subsp. carotovora are pathogenic bacteria that cause various soil-borne diseases. The use of microbial agents to inhibit pathogenic bacteria that cause soil-borne diseases is a new measure to promote the development of green agriculture. SUMMARY

[0005] The purpose of the present application is to provide a Penicillium crustosum complex microbial agent that antagonizes various soil-borne disease pathogens and its application.

[0006] In a first aspect, the present application claims a combined bacterium.

[0007] The combined bacterium claimed in the present application is composed of Penicillium crustosum and Subsaxibacter antirrotis;

[0008] The Subsaxibacter antirrotis is Subsaxibacter antirrotis K2501, which is registered in the China General Microbiological Culture Collection Center with the registration number CGMCC No. 34855.

[0009] The Subsaxibacter antirrotis K2501 is a gram-negative bacterium and is aerobic. After being cultured on PYG medium at 28℃ for 48 hours, the strain can form a moist, smooth, and yellowish colony. The growth tolerance range of the strain is 15-37℃, 0-3% NaCl, and pH 6.0-8.0, and the optimal growth conditions are 28℃, 0-1% NaCl, and pH 7.0.

[0010] In some embodiments, the Penicillium crustosum is Penicillium crustosum P1001, which is registered in the China General Microbiological Culture Collection Center with the registration number CGMCC No. 41996.

[0011] In some embodiments, in the combined bacterium, the CFU ratio of the Penicillium crustosum to the Subsaxibacter antirrotis is (9.9-10.1):1, such as 10:1.

[0012] In a second aspect, the present application claims a Subsaxibacter antirrotis.

[0013] The subsaxibacter antirrotis claimed by the present application is subsaxibacter antirrotis K2501, which is registered in China General Microbiological Culture Collection Center with the registration number of CGMCC No. 34855.

[0014] In a third aspect, the present application claims a fermentation broth or supernatant thereof.

[0015] The fermentation broth or supernatant thereof claimed by the present application is specifically a fermentation broth or supernatant thereof as shown in (A1) or (A2) below:

[0016] (A1) a fermentation broth obtained after fermentation culture of the combined bacteria as described in the first aspect above;

[0017] (A2) a fermentation broth obtained after fermentation culture of the subsaxibacter antirrotis as described in the second aspect above.

[0018] In a fourth aspect, the present application claims a complex microbial agent or a single microbial agent.

[0019] The complex microbial agent claimed by the present application is an agent containing the combined bacteria as described in the first aspect above.

[0020] The single microbial agent claimed by the present application is an agent containing the subsaxibacter antirrotis as described in the second aspect above.

[0021] In the complex microbial agent, the active ingredient of the complex microbial agent can be the combined bacteria, metabolites of the combined bacteria and / or culture of the combined bacteria as described above.

[0022] In the single microbial agent, the active ingredient of the single microbial agent can be the subsaxibacter antirrotis, metabolites of the subsaxibacter antirrotis and / or culture of the subsaxibacter antirrotis as described above.

[0023] The term "metabolite" refers to primary metabolites and / or secondary metabolites produced in the process of microbial metabolism. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world, through catabolism and anabolism, to generate substances and energy to maintain life activities. The product of primary metabolism is the primary metabolite, such as monosaccharide or monosaccharide derivative, nucleotide, vitamin, amino acid, fatty acid, etc. and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms synthesize some substances that have no clear function for life activities of microorganisms using primary metabolites as precursors during a certain growth period. The product of secondary metabolism is the secondary metabolite, which is mostly a compound with a relatively complex molecular structure. According to its function, it can be divided into antibiotics, hormones, alkaloids, toxins, etc.

[0024] The term "culture" refers to a collective of a population of microorganisms grown and / or propagated by inoculation and cultivation. That is, the product obtained by growing and / or propagating microorganisms, which can be a biologically pure culture of microorganisms, or which can contain some amount of culture medium, metabolites, or other components produced during cultivation. The term "culture" also includes subcultures obtained by transferring microorganisms from one culture to another, which can be a culture of a single passage, or a mixture of several passages.

[0025] In the above-mentioned complex microbial agent or single microbial agent, the active ingredient can further comprise other biological components or / and non-biological components. The other active ingredients can be determined by those skilled in the art according to the desired effect.

[0026] In the above-mentioned complex microbial agent or single microbial agent, the active ingredient can further comprise a carrier. The carrier can be a carrier commonly used in the field of pesticides and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; the plant material can be at least one of wheat flour, soybean flour and starch; the high molecular compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane and / or dodecane.

[0027] The dosage form of the above-mentioned complex microbial agent or single microbial agent can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0028] According to the need, the above-mentioned complex microbial agent or single microbial agent can further add surfactants (such as Tween 20, Tween 80, etc.), adhesives, stabilizers (such as antioxidants), pH regulators, etc.

[0029] In this paper, the metabolites can be obtained from the fermentation broth of the above-mentioned combined bacteria or the above-mentioned anti- Rhizoctonia solani. The metabolites can be sterile metabolites or bacteria-containing metabolites. The sterile metabolites (sterile fermentation filtrate) can be prepared according to the following method: culturing the above-mentioned combined bacteria or the above-mentioned anti- Rhizoctonia solani in liquid medium, filtering out the combined bacteria or the anti- Rhizoctonia solani in the liquid culture (fermentation broth), and obtaining the sterile metabolites. The bacteria-containing metabolites can be prepared according to the following method: culturing the above-mentioned combined bacteria or the above-mentioned anti- Rhizoctonia solani in liquid fermentation medium, collecting the fermentation broth containing the combined bacteria or the anti- Rhizoctonia solani and the substances secreted into the liquid medium, which is the bacteria-containing metabolites.

[0030] In a fifth aspect, the present application claims the following any application:

[0031] (B1) The combined bacteria as described in the first aspect above or the anti- Rhizoctonia solani bacteria as described in the second aspect above or the fermentation broth or supernatant thereof as described in the third aspect above or the complex microbial agent or single microbial agent as described in the fourth aspect above for use in preventing or treating soil-borne diseases or in the preparation of a product for preventing or treating soil-borne diseases.

[0032] (B2) The combined bacteria as described in the first aspect above or the anti- Rhizoctonia solani bacteria as described in the second aspect above or the fermentation broth or supernatant thereof as described in the third aspect above or the complex microbial agent or single microbial agent as described in the fourth aspect above for use in inhibiting soil-borne disease pathogens or in the preparation of a product for inhibiting soil-borne disease pathogens.

[0033] (B3) The combined bacteria as described in the first aspect above or the anti- Rhizoctonia solani bacteria as described in the second aspect above for use in the preparation of the fermentation broth or supernatant thereof as described in the third aspect above or the complex microbial agent or single microbial agent as described in the fourth aspect above.

[0034] In a sixth aspect, the present application claims any one of the following methods:

[0035] (C1) A method for preventing or treating soil-borne diseases, comprising: applying the combined bacteria as described in the first aspect above or the anti- Rhizoctonia solani bacteria as described in the second aspect above or the fermentation broth or supernatant thereof as described in the third aspect above or the complex microbial agent or single microbial agent as described in the fourth aspect above to a plant growth medium (such as soil) or a plant.

[0036] (C2) A method for inhibiting soil-borne disease pathogens, comprising: applying the combined bacteria as described in the first aspect above or the anti- Rhizoctonia solani bacteria as described in the second aspect above or the fermentation broth or supernatant thereof as described in the third aspect above or the complex microbial agent or single microbial agent as described in the fourth aspect above to soil-borne disease pathogens or their living environment (such as soil, plant rhizosphere, etc.).

[0037] In each of the above related aspects, the microorganism (such as the combined bacteria described above or the anti- Rhizoctonia solani bacteria described above) that has an inhibitory effect on the soil-borne disease pathogens can antagonize the corresponding pathogens.

[0038] In each of the above related aspects, the soil-borne disease pathogens can be fungi or bacteria.

[0039] In some embodiments, the bacteria are actinomycetes. In some embodiments, the bacteria are gram-positive bacteria or gram-negative bacteria.

[0040] In some embodiments, the soil-borne disease pathogen is Fusarium equiseti (a fungus), Streptomyces scabiei (an actinomycete), Clavibacter michiganensis (a gram-positive bacterium), or Erwinia carotovorum (a gram-negative bacterium). In some embodiments, the soil-borne disease pathogen is Fusarium equiseti CGMCC 3.15353, Streptomyces scabiei DSM 41658, Clavibacter michiganensis DSM 46364, or Erwinia carotovorum DSM 30168. In some embodiments, the soil-borne disease can be a disease caused by each of the above soil-borne disease pathogens.

[0041] The present application discloses a new species of Subsaxibacter antirrotis strain K2501 with good antibacterial activity. The present application further discloses a compound of Penicillium crustosum P1001 and Subsaxibacter antirrotis K2501 at a ratio of 10:1, which shows an antagonistic effect on a variety of soil-borne pathogens. Moreover, the antibacterial effect of the compound of the two bacteria is significantly stronger than that of the single bacteria. The present application has important significance for the prevention and control of soil-borne diseases.

[0042] Depositary

[0043] Classification name: Penicillium crustosum;

[0044] Reference biological material: P1001;

[0045] Preservation agency: China General Microbiological Culture Collection Center;

[0046] Preservation agency abbreviation: CGMCC;

[0047] Address: No. 3, Beichen West Road, Beijing;

[0048] Preservation date: June 11, 2025;

[0049] Preservation center registration number: CGMCC No. 41996.

[0050] Classification name: Subsaxibacter antirrotis;

[0051] Reference biological material: K2501;

[0052] Preservation agency: China General Microbiological Culture Collection Center;

[0053] Abbreviation of preservation agency: CGMCC;

[0054] Address: No. 1, Beichen West Road, No. 3, Chaoyang District, Beijing;

[0055] Preservation date: June 11, 2025;

[0056] Preservation center registration number: CGMCC No. 34855. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 It is a colony photograph of strain P1001 cultured on PDA medium at 28℃ for 24h.

[0058] Figure 2 It is a phylogenetic tree based on ITS gene sequence, showing the phylogenetic position of strain P1001.

[0059] Figure 3 It is a phylogenetic tree based on 16S rRNA gene sequence, showing the phylogenetic position of strain K2501.

[0060] Figure 4 It is a detection effect diagram of the antibacterial activity of the fermentation supernatant of strain P1001 and / or strain K2501. Wherein, A is a detection plate containing F. equiseti CGMCC 3.15353; B is a detection plate containing S. scabiei DSM 41658; C is a detection plate containing C. cyclopia DSM 46364; D is a detection plate containing E. carotovora DSM 30168. 1 is a test paper piece soaked with the fermentation supernatant of strain P1001; 2 is a test paper piece soaked with the fermentation supernatant of strain K2501; 3 is a test paper piece soaked with the fermentation supernatant of co-fermentation of strains P1001 and K2501; 4 is a test paper piece soaked with blank fermentation supernatant; 5, 6 and 7 are test paper pieces soaked with deionized water. DETAILED DESCRIPTION

[0061] The present application will be further described in conjunction with specific embodiments. The examples given are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0062] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained commercially, unless otherwise specified.

[0063] Example 1, Isolation and identification of strains P1001 and K2501

[0064] I. Isolation of strains P1001 and K2501

[0065] Strains P1001 and K2501 of the present application are both isolated from the rhizosphere soil of Kandelia candel in Shankou Mangrove Forest, Guangxi. The specific operation of strain isolation is as follows: 2 g of soil sample is weighed, and 18 mL of sterile normal saline is used to prepare a 10 -1 soil suspension; 1 mL of the 10 -1 soil suspension is then taken and placed in 9 mL of sterile normal saline, and mixed uniformly to obtain a 10 -2 soil suspension; the soil suspension is sequentially diluted with sterile normal saline to prepare a 10 -4 dilution soil suspension. 0.2 mL of the 10 -4 dilution soil suspension is uniformly spread on a PDA culture medium plate (formula: 3 g·L -1 , glucose 20 g·L -1 , agar powder 15 g·L -1 , mixed uniformly with tap water, and then made up to 1000 mL, pH 6.0), and cultured at 28°C for 3 weeks to isolate filamentous fungi. 0.2 mL of the 10 -4 dilution soil suspension is uniformly spread on a PYG culture medium plate (formula: peptone 3 g·L -1 , yeast extract 5 g·L -1 , glycerol 10 g·L -1 , betaine 1.25 g·L -1 , sodium pyruvate 1.25 g·L -1 , agar 15 g·L -1 ; mixed uniformly with deionized water, and then made up to 1000 mL, pH 7.0), and cultured at 30°C for 3 weeks to isolate bacterial strains.

[0066] The fungal single colony is picked from the PDA culture medium, transferred to a freshly prepared PDA plate, and the colony morphology is observed. Strain P1001 is obtained by isolation and purification from the PDA culture medium.

[0067] The well-grown bacterial single colony is picked from the PYG culture medium, transferred to a freshly prepared PYG plate, and the strain is purified by the four-way streak method, and a bacterial strain numbered K2501 is obtained by isolation and purification.

[0068] For the follow-up study, the above two pure strains were preserved in liquid nitrogen and frozen at -80℃ with 20% (v / v) glycerol as a protective agent.

[0069] II. Identification of strains P1001 and K2501

[0070] 1. Identification of strain P1001

[0071] (1) Morphological observation

[0072] Morphological observation of the strain: The morphology of spores and mycelium was observed under a microscope, and reference was made to Chinese Fungi (Volume 35) - Penicillium and Bergey's Manual of Identification, Manual of Fungal Identification, for morphological observation.

[0073] Strain P1001 was cultured on PDA medium at 28℃, and the initial mycelium was white, which turned gray after 24h Figure 1 ), no soluble pigment was produced, and the conidiophores were typical broom-like and adhered to each other. The spores were dark green, round or oval, about 3 μm in diameter, and easily detached after maturation. The spore production was high on PDA medium. The front texture was fluffy, and sometimes the growth state was radial, with a green or dark green color. The back produced yellow-brown pigment. According to these phenotypic characteristics, it was speculated that strain P1001 might be a Penicillium fungus (Manual of Fungal Identification).

[0074] (2) Identification of ITS gene sequence of the strain and construction of phylogenetic tree

[0075] Strain P1001 was transferred to PDA medium without agar and cultured at 28℃ with a rotation speed of 160 r / min for 2d, and then the mycelium was collected and subjected to repeated freezing and thawing at -20℃ and 60℃ for 3 times to break the mycelium. Genomic DNA was extracted. The ITS gene was amplified by PCR using the DNA as a template.

[0076] The PCR reaction system included: ITS1 primer 0.6 μL, ITS4 primer 0.6 μL, template 1 μL, dNTP Mix 4 μL, 2×KOD buffer 10 μL, KOD FX 0.4 μL, and ddH2O 4.4 μL.

[0077] The amplification reaction conditions were as follows: pre-denaturation at 94℃ for 5 min; 31 cycles of 98℃ for 10 s, 56℃ for 45 s, and 68℃ for 2 min; and extension at 68℃ for 5 min.

[0078] The sequence of the ITS1 primer was 5'-TCCGTAGGTGAACCTGCGG-3'; and the sequence of the ITS4 primer was 5'-TCCTCCGCTTATATGC-3'.

[0079] The PCR products of successful amplification were sent to Shanghai Biotechnology Company for sequencing.

[0080] The measured ITS gene sequence was submitted to the NCBI database for alignment analysis. The results showed that the ITS gene sequence of strain P1001 (having the nucleotide sequence shown in SEQ ID NO: 1) had 100% similarity with the Penicillium crustosum strain FRR 1669 in the NCBI database. It was confirmed that strain P1001 was a member of the Penicillium genus. The ITS gene sequences of Penicillium crustosum FRR 1669 and related strains were retrieved to construct a phylogenetic tree ( Figure 2 ), and strain P1001 and Penicillium crustosum FRR 1669 formed a stable evolutionary branch within the Penicillium genus, and the evolutionary distance between the two was zero. Combining the morphological and genomics characteristics of strain P1001, it was determined that strain P1001 was Penicillium crustosum.

[0081] After the above identification, it was determined that strain P1001 was Penicillium crustosum, which was preserved in the China General Microbiological Culture Collection Center on June 11, 2025, and the preservation number was CGMCC No. 41996. Hereinafter, strain P1001 or Penicillium crustosum P1001 is referred to as P1001.

[0082] 2. Identification of strain K2501

[0083] Strain K2501 was grown on PYG medium at 28°C, and morphological, physiological and biochemical, cell chemical and genetic level studies were conducted on K2501. Other special cases will be described.

[0084] (1) Cell morphology observation and physiological and biochemical characteristics detection of strain K2501

[0085] The growth temperature detection range of the strain K2501 is 4, 10, 15, 28, 30, 32, 35, 37, 40 and 45℃, the growth salt concentration (NaCl) detection range is 0-11% (0-11g / 100ml) of 12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11g / 100ml) concentration gradients; the growth pH detection range is 4-11 between 8 (pH4, 5, 6, 7, 8, 9, 10, 11) gradients. The physiological and biochemical functions of the strain are detected by using detection kits API 50CH, API ZYM, and BIOLOG GEN III plate and the corresponding operation method. The other physiological and biochemical characteristics of the strain, including gram staining properties, oxygen demand, contact enzyme activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, are mainly referred to the <Common Bacterial System Identification Manual> (Dong Xi-zhu, Cai Miao-ying. 2001. Common Bacterial System Identification Manual. Beijing: Science Press).

[0086] The identification results show that the strain K2501 is a gram-negative bacterium and aerobic. The strain can form a wet, smooth and yellowish colony after being cultured in PYG medium at 28℃ for 48 hours. The growth tolerance range of the strain K2501 is 15-37℃, 0-3% NaCl and pH 6.0-8.0, and the optimal growth conditions are 28℃, 0-1% NaCl and pH 7.0.

[0087] The strain K2501 is positive in oxidase and catalase tests, and can liquefy gelatin; it can utilize glucan, D-maltose, D-mannose and D-fructose, but not D-trehalose and sucrose as the sole carbon source and energy source. It is sensitive to polymyxin B (300IU) and resistant to tobramycin (10μg), streptomycin (10μg), netilmicin (30μg), tetracycline (30μg), clindamycin (2μg), novobiocin (5μg), erythromycin (15μg), vancomycin (30μg), gentamicin (10μg), penicillin G (10IU), chloramphenicol (30μg), ampicillin (10μg), cefaclor (30μg), kanamycin (30μg) and rifampicin (5μg).

[0088] The physiological and biochemical characteristics of the strain K2501 and the type strains of three species in the Subsaxibacter genus, Subsaxibacter sediminis ARC111, Subsaxibacter arcticus SM1214 and Subsaxibacter broadyi P7, are different, as shown in Table 1.

[0089] Table 1, differences in physiological and biochemical characteristics between strain K2501 and the type strain of the validly described species in the genus Subsaxibacter

[0090]

[0091] Note: +, positive; -, negative.

[0092] (2) Detection of cell fatty acid components of strain K2501

[0093] The cell fatty acid chemical components of strain K2501 were detected using gas chromatography (GC) technology (the method is described in Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI inc; 1990.).

[0094] The cell fatty acid components of strain K2501 of the present application are shown in Table 2. The results show that the main fatty acids of strain K2501 of the present application are iso-C 15:0 and anteiso-C 15:0 , accounting for 33.17% and 27.82% of the total content, respectively. These main components are consistent with the main fatty acid components of the strains of the genus Subsaxibacter (Xu F, Zhang XY, Liu C, Shi M, Su HN, Qin QL, Chen XL, Zhang YZ, Song XY. Subsaxibacter arcticus sp. nov., isolated from Arctic intertidal sand. Int J Syst Evol Microbiol 2016; 66: 132-136. Sharma A, Jani K, Feng GD, Karodi P, Vemuluri VR, Zhu HH, Shivaji S, Thite V, Kajale S, Rahi P, et al. Subsaxibacter sediminis sp. nov., isolated from Arctic glacial sediment and emended description of the genus Subsaxibacter. Int J Syst Evol Microbiol 2018; 68: 1678-1682.).

[0095] Table 2, cell fatty acid components of strain K2501

[0096] Fatty acids (%) K2501 C10:0 0.58 C14:0 5.62 C16:0 6.85 antesio-C 15:0 ]]> 27.82 iso-C 11:0 ]]> 5.46 iso-C 13:0 ]]> 0.79 iso-C 14:0 ]]> 3.66 iso-C 15:0 ]]> 33.17 iso-C 16:0 ]]> 4.24 iso-C 17:0 ]]> 1.09 iso C 15:1 F]]> 2.26 C 16:1 ω9c]]> 1.41 C 17:0 cyclo]]> 0.99 iso-C 11:0 3-OH]]> 1.01 iso-C 12:0 3-OH]]> 0.3 C 12:0 3-OH]]> 0.83 iso-C13:0 3-OH 1.4

[0097] (3) Determination of the phylogenetic position of strain K2501

[0098] Genomic DNA was extracted from strain K2501 and sequenced. The 16S rRNA gene sequence (with the nucleotide sequence shown in SEQ ID NO:2) was compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results showed that strain K2501 of this invention had the highest similarity to species of the genus *Sclerobacter*. This result suggests that strain K2501 may be a member of the genus *Sclerobacter*. Among the reported effective descriptive species in the genus *Sclerobacterium*, strain K2501 showed 16S rRNA gene similarities of 96.85%, 97.43%, and 97.5%, respectively. These similarity values ​​are all below the 98.65% threshold for distinguishing prokaryotic species, suggesting that strain K2501 may represent a new species in the genus *Sclerobacterium* (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351.). A phylogenetic tree was constructed using the 16S rRNA gene sequences of strain K2501, all effective species in the genus *Sclerobacterium*, and representative strains from neighboring genera of *Sclerobacterium*. Figure 3). On the phylogenetic tree, strain K2501 falls within the evolutionary branch of the genus Subsaxibacter, and clusters with the type strains of three species in the genus Subsaxibacter, Subsaxibacter sediminis ARC111, Subsaxibacter arcticus SM1214, and Subsaxibacter broadyi P7, forming a stable sub-branch. This result further supports that strain K2501 is a member of the genus Subsaxibacter.

[0099] To further clarify the phylogenetic position of the strain, the present application performed whole genome sequencing on strain K2501. The obtained genome sequence is 4.38 Mbp in length, and the genome G+C content is 36.6%. The average nucleotide similarity (ANI value) of the whole genome sequence of strain K2501 and the whole genome sequence of the close control bacteria was compared and calculated on EZbiocloud. Whole genome sequence analysis showed that the average nucleotide similarity (ANI) of strain K2501 with Subsaxibacter sediminis ARC111, Subsaxibacter arcticus SM1214, and Subsaxibacter broadyi P7 was 74.7%, 73.0%, and 70.7%, respectively, which were all far below the ANI threshold of 95% that distinguishes prokaryotic microbial gene species (Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 2017; 110: 1281-1286.). These results show that strain K2501 of the present application is a newly discovered gene species of the genus Subsaxibacter.

[0100] In summary, strain K2501 of the present application has many significant differences from existing strains of the genus Subsaxibacter, including physiological and biochemical, cell chemical, and genotypic aspects. The above data fully demonstrate that strain K2501 of the present application represents a new species of the genus Subsaxibacter, named Subsaxibacter antirrotis, which has been deposited at the China General Microbiological Culture Collection Center on June 11, 2025, with the accession number CGMCC No. 34855. Hereinafter, it is referred to as strain K2501 or Subsaxibacter antirrotis K2501.

[0101] Example 2, screening of antibacterial activity

[0102] The pathogenic bacteria of the tested soil-borne diseases are Fusarium equiseti CGMCC 3.15353 (fungi), Streptomyces scabiei DSM 41658 (actinomycetes), Clavibacter michiganensis DSM 46364 (gram-positive bacteria), and Erwinia carotovorum DSM 30168 (gram-negative bacteria).

[0103] The anti-microbial activity of the strains P1001 and / or K2501 was tested using the above-mentioned pathogenic bacteria of the tested soil-borne diseases as the test bacteria. The inhibition zone method was used for the determination, and the specific test steps are as follows:

[0104] (1) Fermentation of strains P1001 and K2501 separately

[0105] Preparation of fermentation broth: strains P1001 and K2501 were inoculated into fermentation medium (formula: glucose 5 g, malt paste 10 g, yeast paste 5 g, cottonseed cake 10 g, soluble starch 20 g, potassium dihydrogen phosphate 0.5 g, ammonium sulfate 5 g, calcium carbonate 3 g, sodium chloride 1 g, deionized water 1 L, pH 7.2), and placed on a shaking bed at a rotation speed of 180 rpm at 28°C for 4 days. The obtained culture solution was centrifuged at a rotation speed of 4000 rpm to obtain the fermentation broth supernatant of strain P1001 (labeled as "1") and the fermentation broth supernatant of strain K2501 (labeled as "2").

[0106] (2) Co-fermentation of strains P1001 and K2501

[0107] Preparation of fermentation broth: strains P1001 and K2501 were inoculated into fermentation medium (formula: glucose 5 g, malt paste 10 g, yeast paste 5 g, cottonseed cake 10 g, soluble starch 20 g, potassium dihydrogen phosphate 0.5 g, ammonium sulfate 5 g, calcium carbonate 3 g, sodium chloride 1 g, deionized water 1 L, pH 7.2) at a CFU ratio of P1001:K2501=10:1, and placed on a shaking bed at a rotation speed of 180 rpm at 28°C for 4 days. The obtained culture solution was centrifuged at a rotation speed of 4000 rpm to obtain the fermentation broth supernatant of co-fermentation of strains P1001 and K2501 (labeled as "3").

[0108] (3) Preparation of blank fermentation broth control

[0109] After sterilization of the fermentation medium (formula: glucose 5 g, malt paste 10 g, yeast paste 5 g, cottonseed cake 10 g, soluble starch 20 g, potassium dihydrogen phosphate 0.5 g, ammonium sulfate 5 g, calcium carbonate 3 g, sodium chloride 1 g, deionized water 1 L, pH 7.2), no inoculum was added, and it was placed on a shaking bed at a rotation speed of 180 rpm at 28°C for 4 days. Centrifugation at a rotation speed of 4000 rpm resulted in the blank fermentation broth (labeled as "4").

[0110] (4) Deionized water labeled as "5", "6", and "7" in this experiment were used as blank controls.

[0111] (5) Preparation of test plates

[0112] PDA medium plates containing 10 8 CFU / mL of each test strain grown to the logarithmic phase were prepared.

[0113] (6) Bacteriostatic effect test

[0114] 2 mL of the fermentation supernatant of each of the above-prepared strain P1001, strain K2501, and strain P1001+K2501 (labeled as "1", "2", and "3", respectively), 2 mL of the fermentation supernatant without inoculation of bacteria (labeled as "4"), and 2 mL of deionized water (labeled as "5", "6", and "7") were taken, respectively, and were used to soak filter paper pieces with a diameter of 6 mm; the filter paper pieces soaked with different liquids were placed on the plates prepared in the previous step and containing each test strain. The plates with the filter paper pieces placed thereon were cultured at 28°C for 24 hours, and the bacteriostatic circles were observed.

[0115] The results show that the fermentation supernatant of strain P1001 has weak inhibitory activity on F. equiseti CGMCC 3.15353, strong inhibitory activity on S. scabiei DSM 41658 and E. carotovora DSM 30168, and almost no inhibitory effect on C. cyclopia DSM 46364; the fermentation supernatant of strain K2501 has weak inhibitory activity on F. equiseti CGMCC 3.15353, strong inhibitory activity on S. scabiei DSM 41658, C. cyclopia DSM 46364, and E. carotovora DSM 30168; the fermentation supernatant of the co-fermentation of strain P1001+K2501 has strong inhibitory effect on F. equiseti CGMCC 3.15353, S. scabiei DSM 41658, C. cyclopia DSM 46364, and E. carotovora DSM 30168, and the inhibitory activity of the fermentation supernatant of the co-fermentation of strain P1001+K2501 is stronger than that of the fermentation supernatant of the single-fermentation of each strain. Figure 4

[0116] In summary, it can be seen that the co-fermentation of strain P1001 and K2501 of the present application has significant antagonistic effect on the pathogenic bacteria of various soil-borne diseases, including some fungi, actinomycetes, gram-positive bacteria, and gram-negative bacteria.

[0117] ​The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A combination of bacteria, characterized in that: The combined bacteria consist of Penicillium pekinensis and Bacillus subtilis resistant to root rot; The anti-root rot subrrotis is Subsaxibacter antirrotis K2501, which has the registration number CGMCC No.34855 at the China General Microbiological Culture Collection Center.

2. The combined bacteria according to claim 1, characterized in that: The Penicillium crustosum mentioned is Penicillium crustosum P1001, which has the registration number CGMCC No.41996 at the China General Microbiological Culture Collection Center.

3. The combined bacteria according to claim 1 or 2, characterized in that: In the combined bacteria, the CFU ratio of Penicillium desiccant and Bacillus subtilis is (9.9-10.1):

1.

4. Antibacterial against *Bacillus subtilis*, characterized by: The anti-root rot subrrotis is Subsaxibacter antirrotis K2501, which has the registration number CGMCC No.34855 at the China General Microbiological Culture Collection Center.

5. A fermentation broth or its supernatant, characterized in that: The fermentation broth is either (A1) or (A2) as follows: (A1) Fermentation broth obtained by fermenting any of the combined bacteria described in claims 1-3; (A2) The fermentation broth obtained by fermenting the anti-root rot Bacillus subtilis according to claim 4.

6. A compound microbial agent or a single microbial agent, characterized in that: The compound microbial agent is a microbial agent containing any of the combined microorganisms described in claims 1-3; The single-strain agent is a bacterial agent containing the anti-root-rot bacteria described in claim 4.

7. Any of the following applications: (B1) The use of any of the combined bacteria of claims 1-3, or the anti-root rot Bacillus of claim 4, or the fermentation broth or supernatant of claim 5, or the compound bacterial agent or single bacterial agent of claim 6 in the prevention and control of soil-borne diseases or in the preparation of products for the prevention and control of soil-borne diseases. (B2) The use of any of the combined bacteria of claims 1-3, or the anti-root rot Bacillus of claim 4, or the fermentation broth or supernatant of claim 5, or the compound bacterial agent or single bacterial agent of claim 6 in inhibiting soil-borne pathogens or in the preparation of products for inhibiting soil-borne pathogens. (B3) The use of any of the combined bacteria of claims 1-3 or the anti-root rot Bacillus of claim 4 in the preparation of the fermentation broth or its supernatant of claim 5 or the compound bacterial agent or single bacterial agent of claim 6.

8. Any of the following methods: (C1) A method for preventing and controlling soil-borne diseases, comprising: Apply any of the combined bacteria described in claims 1-3, or the anti-root rot bacillus described in claim 4, or the fermentation broth or its supernatant described in claim 5, or the compound bacterial agent or single bacterial agent described in claim 6 to the plant growth medium or plants; (C2) A method for inhibiting soil-borne pathogens, comprising: applying to soil-borne pathogens or their living environment any of the combined bacteria of claims 1-3 or the anti-root rot bacillus of claim 4 or the fermentation broth or its supernatant of claim 5 or the compound bacterial agent or single bacterial agent of claim 6.

9. The application according to claim 7 or the method according to claim 8, characterized in that: The pathogens of the soil-borne diseases are fungi or bacteria; Furthermore, the bacteria are actinomycetes; and / or Furthermore, the bacteria are either Gram-positive or Gram-negative.

10. The application or method according to claim 9, characterized in that: The pathogens of the soil-borne diseases mentioned are Fusarium equisetifolium, Streptomyces scabica, Corynebacterium circumferentialis, or Erwinia carotenoides.

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