Massilia bacteria, bacterial agent, preparation method and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a *Morchella esculenta* strain, its preparation method, and its applications. The *Morchella esculenta* strain of this invention has the preservation number CCTCC NO: M 2026202, M 2026203, or M 2026201. The inoculant of this invention includes the aforementioned *Morchella esculenta*. The *Morchella esculenta* strain and its inoculant of this invention not only dissolve inorganic phosphorus and promote rapeseed growth, but also enhance rapeseed's resistance to sclerotinia stem rot by inhibiting *Sclerotinia sclerotiorum*.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Masseilles bacterium, a bacterial agent, its preparation method, and its application. Background Technology

[0002] Massilia is a genus of Gram-negative bacteria in the family Oxalicobacterceae. The genus contains 43 effective species. The bacteria are short rod-shaped with apical flagella, and are non-spore-forming. They are heterotrophic aerobic microorganisms, mainly distributed in soil, plants, water bodies and air. Some strains of Massilia have excellent phosphate-solubilizing effects.

[0003] Phosphorus plays a vital role in crops, especially for phosphorus-sensitive crops like rapeseed, where many essential physiological processes for growth and development are related to phosphorus. The primary method for supplementing crops with phosphorus is through the application of phosphate fertilizers. However, the application of phosphate fertilizers faces many problems. One of the most significant issues affecting crops like rapeseed is the low utilization rate of phosphate fertilizers, typically between 10% and 25%. This is mainly because phosphorus readily combines with iron in the soil. 3+ Al 3+ and Ca 2+ Plasma forms insoluble complexes that are immobilized, which is further exacerbated by the limited solubility of traditional fertilizers. Therefore, Massey bacteria, with their phosphorus-solubilizing properties, show great promise for the development of novel phosphate fertilizers.

[0004] Chinese invention patent application CN117844684A discloses a Massia arviella strain with phosphate-solubilizing ability and its applications. This strain (Massiliaarvi P2) has the accession number CCTCCNO: M20232231. This strain of Massiaarvi P2 has the ability to dissolve insoluble phosphates, converting them into soluble phosphorus that can be directly absorbed and utilized by plants. After 7 days of cultivation in four liquid media containing insoluble phosphates (calcium phosphate, iron phosphate, aluminum phosphate, and phosphate rock), its phosphorus-solubilizing capacity was 45.2 μg / mL. -1 5.9 μg / mL -1 6.9 μg / mL -1 4.2 μg / mL -1 Combining Massiliaarvi P2 with soil phosphorus-solubilizing capabilities is of significant value for the development of microbial fertilizers such as phosphorus-solubilizing bacterial fertilizers.

[0005] The aforementioned technical solution utilizes *Bacillus simonii* to develop phosphate-solubilizing bacterial fertilizers, providing a new approach for supplementing phosphate fertilizers in rapeseed and other crops, and promoting rapid rapeseed growth. However, rapeseed growth is also affected by many other factors, such as sclerotinia stem rot.

[0006] Sclerotinia sclerotinia, caused by Sclerotinia sclerotiorum, is a devastating fungal disease affecting rapeseed and occurs in all major rapeseed-producing areas worldwide. The disease is characterized by stem rot, resulting in "white stalks," ultimately leading to premature plant death, shriveled seeds, and severe yield losses (typically 10%–30%, but can exceed 80% in severe cases) and quality degradation (reduced oil content and poorer oil quality).

[0007] Currently, the main rapeseed varieties promoted in agricultural production generally have weak resistance to sclerotinia stem rot, and there is a lack of highly resistant or immune varieties. Moreover, rapeseed production has long relied on a single chemical agent, leading to the development of drug resistance in pathogens; in addition, the use of pesticides may harm pollinating insects (such as bees), and the price of pesticides (such as fluopyram) is relatively high.

[0008] The application of biological agents to crops not only aligns with the policy requirements of "reducing pesticide use while increasing efficiency" and ensuring the quality and safety of agricultural products, but is also environmentally friendly and relatively safe for pollinating insects such as bees. Furthermore, it provides a sustainable alternative or supplementary pathway to address the prominent problems of long-term reliance on chemical pesticides, such as resistance, pesticide residues, and environmental pollution. For crops like rapeseed, developing biological agents that can simultaneously achieve phosphate solubilization and sclerotinia stem rot inhibition has significant practical implications and promising application prospects. Summary of the Invention

[0009] In view of this, the present invention provides a Masse bacteria and an inoculant that not only dissolves inorganic phosphorus but also enhances the resistance of rapeseed to sclerotinia disease by inhibiting Sclerotinia sclerotiorum.

[0010] To achieve the above objectives, the present invention provides a Masse mold for rapeseed cultivation, wherein the Masse mold has the preservation number CCTCC NO: M 2026202 or CCTCC NO: M 2026203 or CCTCC NO: M 2026201.

[0011] Optionally, the 16S rDNA gene sequence of the Masseilles with accession number CCTCC NO: M 2026202 is shown in SEQ ID No. 1.

[0012] The *Massilia* strain provided in this invention, with accession number CCTCC NO: M 2026202, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, opposite the First Affiliated Primary School of Wuhan University. The deposit date was January 22, 2026, and the strain is classified and named *Ms. Massilia* sp. Ms. Alternatively, its English abbreviation is *Massilia scoD23*.

[0013] Optionally, the 16S rDNA gene sequence of the *M. masei* strain with accession number CCTCC NO: M 2026203 is shown in SEQ ID No. 2.

[0014] The *Massilia orientalis* strain provided in this invention, with accession number CCTCC NO: M 2026203, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, opposite the First Affiliated Primary School of Wuhan University. The deposit date was January 22, 2026, and the strain is classified and named *M. or Massilia orientalis* Mo. Alternatively, its English name can be abbreviated as *Massilia orientalis*.

[0015] Optionally, the 16S rDNA gene sequence of the *M. masei* strain with accession number CCTCC NO: M 2026201 is shown in SEQ ID No. 3.

[0016] The *Massilia phosphatilytica* strain provided in this invention, with accession number CCTCC NO: M 2026201, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, opposite the First Affiliated Primary School of Wuhan University. The deposit date was January 22, 2026. It is classified and named *Massilia phosphatilytica* Mp., or alternatively, abbreviated as *Massilia phosphatilytica*. 。

[0017] The present invention also provides a microbial agent comprising the above-mentioned Masseilles.

[0018] Optionally, the microbial agent includes a *Bacillus simonii* bacterial solution, wherein the concentration of *Bacillus simonii* in the bacterial solution is 1.0 × 10⁻⁶. 8 -2.0×10 8 cfu / mL.

[0019] The present invention provides a method for preparing a bacterial agent, comprising the following steps: activating frozen Masseuse bacteria in a solid culture medium, then picking single clones and culturing them in a liquid culture medium, collecting the bacterial cells by centrifugation, and resuspending them in sterile water to remove the culture medium and bacterial metabolites, thereby obtaining the bacterial agent.

[0020] Optionally, the cryopreservation temperature of the *Morseella catarrhalis* strain is -70 to -85°C.

[0021] Optionally, the solid culture medium is R2A solid culture medium, and the liquid culture medium is R2A liquid culture medium.

[0022] Optionally, the relative centrifugal force of the centrifugation is 5500~6500g, and the centrifugation time is 4~6min.

[0023] This invention also provides an application of *Massezia scoparia* in phosphorus solubilization in rapeseed planting soil.

[0024] The present invention also provides an application of Masseuse bacteria in promoting plant growth.

[0025] Optionally, the plant is rapeseed.

[0026] The present invention also provides an application of *Massezia spp.* in the prevention and control of sclerotinia disease caused by *Sclerotinia sclerotiorum*.

[0027] Optionally, the plant is rapeseed.

[0028] When applying the above-mentioned *Mosaix* to phosphate solubilization, promoting plant growth, and preventing sclerotinia disease caused by *Sclerotinia sclerotiorum*, the above-mentioned preparation method is used to prepare *Mosaix* into a fungal agent.

[0029] The present invention also provides an application of *Bacillus simonii* in the preparation of phosphate fertilizer for rapeseed, wherein the fertilizer is applied by root irrigation of rapeseed.

[0030] The present invention also provides the application of *Massezia spp.* in the preparation of a pesticide for treating *Sclerotinia sclerotiorum*-induced sclerotinia rot in rapeseed, wherein the pesticide is applied by spraying it onto the rapeseed leaves.

[0031] The above-described technical solution of the present invention has at least the following beneficial effects:

[0032] The *Masses* and its inoculant provided by this invention simultaneously possess phosphorus-solubilizing properties, promote plant growth under low phosphorus conditions, and prevent plant sclerotinia disease.

[0033] The *Bacillus simonii* and its agent provided by this invention have a wide range of applications. As a biological agent, it has minimal environmental pollution and harm, avoids path dependence caused by chemical fertilizers and pesticides, and provides a new and sustainable alternative or supplementary pathway for fertilizers and pesticides. Attached Figure Description

[0034] Figure 1 This is a diagram illustrating the phosphorus-solubilizing effect of the *Morchella esculenta* strain in Example 5 of the present invention.

[0035] Figure 2 This is a low-phosphorus growth-promoting phenotype diagram of the *Bacillus simonii* inoculant plate in Example 6 of the present invention;

[0036] Figure 3 This is a comparison chart of the plant height promotion results of low-phosphorus inoculum inoculation plates in Example 6 of the present invention;

[0037] Figure 4This is a comparison chart of the fresh weight results of low-phosphorus growth-promoting plates using *Bacillus simonii* inoculant in Example 6 of the present invention;

[0038] Figure 5 This is a comparison chart of the results of promoting the number of lateral roots on a plate with low phosphorus inoculum in Example 6 of the present invention;

[0039] Figure 6 This is a phenotypic diagram of soil low-phosphorus growth-promoting effect of Bacillus simonii inoculant in Example 6 of the present invention;

[0040] Figure 7 This is a comparison chart of the dry weight results of the soil low-phosphorus-promoting underground part of the *Morchella esculenta* inoculant in Example 6 of the present invention;

[0041] Figure 8 This is a phenotypic diagram of the field inoculation of the *Morchella esculenta* inoculant in Example 6 of the present invention;

[0042] Figure 9 This is a statistical chart showing the field inoculation results of the *Morchella muscarinii* strain in Example 6 of the present invention;

[0043] Figure 10 This is a diagram showing the effect of the *Morchella esculenta* strain on plate resistance against *Sclerotinia sclerotiorum* in Example 7 of the present invention.

[0044] Figure 11 This is a comparison diagram of the relative expansion distance of Ms. muscarinicus in Example 7 of the present invention and the control group;

[0045] Figure 12 This is a diagram illustrating the effect of detached Ms leaflets of *M. muscarinii* on resisting *Sclerotinia sclerotiorum* in Example 7 of this invention.

[0046] Figure 13 This is a phenotypic chart of resistance to Sclerotinia sclerotiorum var. m. in detached leaflets of *M. m.* in Example 7 of the present invention.

[0047] Figure 14 The figures shown in Example 7 of this invention are: growth of Sclerotinia sclerotiorum on rapeseed leaves, leaf damage area, and relative sclerotiorum biomass on rapeseed leaves.

[0048] Figure 15 The figures show the growth of Sclerotinia sclerotiorum on the leaves, the area of ​​leaf damage, and the relative biomass of sclerotia on the rapeseed leaves when Masse bacillus Ms was applied to the roots of rapeseed in Example 7 of this invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figures 1-12The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0050] Example 1

[0051] Acquisition of strains

[0052] Westar root microorganisms were isolated from plants grown in a greenhouse in low-phosphorus red soil sourced from Yunnan. The process included the following steps:

[0053] First, the Westar seeds were germinated on gauze for 5 days, then transplanted into the soil. After 20 days of growth, the rapeseed roots were removed, rinsed, and placed in a sterile 50ml centrifuge tube for washing with 1×PBS. The tube was then placed on a shaker and gently shaken. The washing was repeated 3 times to thoroughly remove the soil from the root zone.

[0054] Then, in a clean bench, excess moisture was blotted from the rapeseed roots with sterile filter paper. The root segments were cut into 2 mm pieces, and 0.02 g of root tissue was weighed and mixed with 200 µL of sterile 10 mL MgCl2 to form a homogenate. The homogenate was transferred to 25 mL of MgCl2 solution and incubated at room temperature for 20 minutes. 133 µL, 44 µL, 15 µL, and 5 µL of the incubated homogenate were then pipetted into 800 mL of 10% TSB solution and thoroughly mixed to form mixtures with dilution gradients of 6000×, 18000×, 54000×, and 162000×.

[0055] Finally, the mixture was transferred to a 96-well cell culture plate, sealed, and incubated at 28°C for five days. All wells in the 96-well plate with a 30% turbidity gradient were then transferred to a new 96-well plate for further incubation. The purified strain was obtained through repeated streak separation.

[0056] Phosphate-solubilizing strains Mp, Mo, and Ms were obtained by screening using rapeseed roots. 16S rDNA sequence similarity analysis confirmed that Ms, Mo, and Mp belong to the genus Massilia.

[0057] The Ms strain gene sequence SEQ ID NO.1 is as follows:

[0058] TGCAGTCGAACGGCAGCGCGGGGCAACCTGGCGGCGAGTGGCGAACGGGTGAGTAATATATCGGAACGTACCCAGAAGTGGGGGATAACGTAGCGAAAGTTACGCTAATACCGCATACGATCTACGGATGAAAGTGGGGGACCTTCGGGCCTCATGCTTTTGGAGCGGCCGATATCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTGTCAGGGAAGAAACGGCTCCGGCTAATATCTGGGGCTAATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAAGTCTGTCGTGAAAGCCCCGGGCTTAACCTGGGAATTGCGN (N can be interpreted as "A", "C", "G", or "T")

[0059] TGGAGACTGCAAGGCTTGAATCTGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGTCAAGATTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCTACTAGTTGTCGGGTCTTAATTGACTTGGTAACGCAGCTAACGCGTGAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCAGGAACCNNCGAGAGATTGNNGGGTGCCCGAAAGGGAGCCTGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGNGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTACATACAGAGGGCCGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAGTGTATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTTTTACCAGAAGTAGGTAGCTTAACCGCAAGGAGGG (N can be interpreted as "A", "C", "G", or "T").

[0060] The gene sequence of strain M.o, SEQ ID NO.2, is as follows:

[0061]

[0062] The gene sequence of strain Mp, SEQ ID NO.3, is as follows:

[0063]

[0064] Example 2

[0065] Preparation of microbial agents:

[0066] R2A medium: 3.2 g / L powder (Qingdao Haibo Biotechnology) Main components: tryptone 0.25 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate 0.1 g / L, sodium pyruvate 0.3 g / L, peptone 0.25 g / L, glucose 0.5 g / L.

[0067] The bacterial agent was prepared using R2A solid culture medium and R2A liquid culture medium.

[0068] R2A solid medium is a mixture obtained by adding water to the above-mentioned R2A medium, with the amount of R2A medium added being 3.2 g / L, and additional agar added, with the mass of agar being 1% of the mass of the mixture;

[0069] R2A liquid medium is made by mixing the above-mentioned R2A medium with water, and the amount of R2A medium added is 3.2 g / L.

[0070] R2A solid culture medium and R2A liquid culture medium were sterilized at 121°C for 15 minutes.

[0071] Preparation of bacterial inoculum: Ms strain was removed from a -80℃ freezer and streaked onto R2A solid medium for activation. The following day, single clones were selected and incubated in R2A liquid medium at 28℃ for 12 hours. The cells were then collected by centrifugation at 6000g for 5 minutes, and resuspended twice with sterile water to remove the culture medium and bacterial metabolites. The OD value was adjusted to 0.01 to obtain the Ms bacterial inoculum. The Ms bacterial inoculum includes a *Bacillus simonii* bacterial suspension with a concentration of 10⁻⁶. 8 cfu / mL.

[0072] Example 3

[0073] Preparation of microbial agents:

[0074] R2A medium: 3.2 g / L powder (Qingdao Haibo Biotechnology) Main components: tryptone 0.25 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate 0.1 g / L, sodium pyruvate 0.3 g / L, peptone 0.25 g / L, glucose 0.5 g / L.

[0075] The bacterial agent was prepared using R2A solid culture medium and R2A liquid culture medium.

[0076] R2A solid medium is a mixture obtained by adding water to the above-mentioned R2A medium, with the amount of R2A medium added being 3.2 g / L, and additional agar added, with the mass of agar being 1% of the mass of the mixture;

[0077] R2A liquid medium is made by mixing the above-mentioned R2A medium with water, and the amount of R2A medium added is 3.2 g / L.

[0078] R2A solid culture medium and R2A liquid culture medium were sterilized at 121°C for 15 minutes.

[0079] The *Mo* strain was removed from a -80°C freezer and activated by streaking in R2A solid medium. The following day, single clones were selected and cultured in R2A liquid medium at 28°C for 12 hours. The cells were then collected by centrifugation at 6000g for 5 minutes, and resuspended twice in sterile water to remove the culture medium and bacterial metabolites. The OD value was adjusted to 0.01 to obtain the *Mo* inoculum. The *Mo* inoculum consisted of *Bacillus simonii* bacterial suspension, with a concentration of 10-1 of *Bacillus simonii*. 8 cfu / mL.

[0080] Example 4

[0081] R2A medium: 3.2 g / L powder (Qingdao Haibo Biotechnology) Main components: tryptone 0.25 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate 0.1 g / L, sodium pyruvate 0.3 g / L, peptone 0.25 g / L, glucose 0.5 g / L.

[0082] The bacterial agent was prepared using R2A solid culture medium and R2A liquid culture medium.

[0083] R2A solid medium is a mixture obtained by adding water to the above-mentioned R2A medium, with the amount of R2A medium added being 3.2 g / L, and additional agar added, with the mass of agar being 1% of the mass of the mixture;

[0084] R2A liquid medium is made by mixing the above-mentioned R2A medium with water, and the amount of R2A medium added is 3.2 g / L.

[0085] R2A solid culture medium and R2A liquid culture medium were sterilized at 121°C for 15 minutes.

[0086] The *Mp* strain was removed from a -80°C freezer and activated by streaking in R2A solid medium. The following day, single clones were selected and cultured in R2A liquid medium at 28°C for 12 hours. The cells were then collected by centrifugation at 6000g for 5 minutes, and resuspended twice in sterile water to remove the culture medium and bacterial metabolites. The OD value was adjusted to 0.01 to obtain the *Mp* bacterial agent. The *Mp* bacterial agent included *Bacillus simonii* bacterial suspension, with a concentration of 10-1 *Bacillus simonii* in the suspension. 8 cfu / mL.

[0087] Example 5

[0088] Application of Marseilles inoculant in soil phosphorus dissolution.

[0089] Test of the solubility of inorganic phosphorus by *Morchella esculenta* inoculant

[0090] Inorganic phosphorus medium: 17g / L powder (Haibo Biotechnology), main components: glucose 10g / L, ammonium sulfate 0.5g / L, yeast extract 0.5g / L, sodium chloride 0.3g / L, potassium chloride 0.3g / L, magnesium sulfate 0.3g / L, ferrous sulfate 0.03g / L, manganese sulfate 0.03g / L, calcium phosphate 5.0g / L. Prepare three portions of the inorganic phosphorus medium.

[0091] Phosphate solubilization experiment: 5 μL of each of the Ms, Mo, and Mp bacterial agents prepared according to the methods described in Examples 2-4 was added to the center of different inorganic phosphorus culture media, and then inverted in an incubator at 28°C for 4-5 days. This yielded three experimental groups: the Ms group, the Mo group, and the Mp group. After the experiment, the presence of a phosphate-solubilizing zone in the center of the inorganic phosphorus culture medium was observed. For the phosphate-solubilizing activity of *Masses* strains, see [link to relevant documentation]. Figure 1 .

[0092] like Figure 1 As shown, the culture media containing inorganic phosphorus (calcium phosphate) in the Ms, Mo, and Mp groups all had obvious phosphorus-solubilizing rings in the center, indicating that the Ms, Mo, and Mp inoculants can all dissolve inorganic phosphorus, thereby meeting their own nutritional needs.

[0093] Example 6

[0094] Application of *Morchella esculenta* inoculant in promoting plant growth.

[0095] Test on the growth-promoting effect of *Mexicospermum erythrorhizon* inoculant

[0096] Seed sterilization: The rapeseed variety used was Westar. The seeds were soaked in 75% alcohol for 45 seconds, washed three times with sterile water, then thoroughly sterilized by soaking in 10% sodium hypochlorite for 5 minutes. Finally, they were washed 4-5 times with sterile distilled water. The seeds were then sown in 1 / 2 MS medium for pre-germination using sterile tweezers. Three sterilized seed samples were prepared.

[0097] Microbial agent preparation: Ms microbial agent, Mo microbial agent and Mp microbial agent prepared in Examples 2-4.

[0098] Phosphorus-deficient plant culture medium: MS-P (containing vitamins), 4328.66 mg / L, product sourced from coolaber, product model PM1011-P; the phosphorus-deficient plant culture medium is mixed with water at 4328.66 mg / L and sterilized at 121℃ for 20 min, then poured into plates in a clean bench.

[0099] 1 / 2MS medium is half the concentration of MS-P medium, i.e., it is prepared at 2164.33 mg / L.

[0100] Rapeseed plate inoculation experiment: Specific concentrations of Ms, Mo, and Mp inoculants were added to the still-solidified MS-P medium. After the medium solidified, rapeseed seeds that had pre-germinated for one day were transferred to plates, sealed with sealing film, and placed in an incubator at 22℃ with 16h light / 8h darkness for 10 days. The growth of the rapeseed was observed. A control group was also set up, without any inoculants added, and all other steps were the same as the inoculated group. This yielded four experimental results: control group (Mock), Ms group, Mo group, and Mp group.

[0101] See the low-phosphorus growth-promoting phenotype diagram of *M. masei* inoculant plate for Figure 2 The statistical results of the results of promoting plant height with low phosphorus inoculum on matéifolia inoculum plates are shown in the figure below. Figure 3 The statistical results of the results of promoting the fresh weight of aboveground parts by low-phosphorus inoculum tablets are shown in the figure. Figure 4 The statistical results of the number of lateral roots promoted by low-phosphorus inoculum in culture plates are shown in the figure. Figure 5 .

[0102] like Figures 2-5 As shown, the height of rapeseed seedlings after inoculation increased significantly, with Mp, Mo, and Ms increasing by 42%, 59%, and 66%, respectively. The fresh weight of the aboveground parts of Mp and Ms increased by 28% and 21%, respectively, and the number of lateral roots of Ms and Mo increased by 43% and 132%, respectively.

[0103] Test on the growth-promoting effect of Marseille inoculant on sterilized rapeseed soil

[0104] Seed sterilization: The rapeseed variety used was Westar. The seeds were soaked in 75% alcohol for 45 seconds, washed three times with sterile water, then thoroughly sterilized by soaking in 10% sodium hypochlorite for 5 minutes. Finally, they were washed 4-5 times with sterile distilled water. The seeds were then sown in 1 / 2 MS medium for pre-germination using sterile tweezers. Three sterilized seed samples were prepared.

[0105] The tested soil: Red soil was collected from Shilin County, Kunming, Yunnan Province. The basic physicochemical properties of the soil were: pH value 7.8 ± 0.1, total potassium 18.1 g / kg, and total phosphorus 1.07 g / kg.

[0106] The 1 / 2MS medium is the same as the 1 / 2MS medium used in Example 6.

[0107] Soil treatment: After removing impurities from the soil, pass it through a 3mm sieve, pack it into high-temperature sterilization bags, sterilize twice at 121℃, and then cool and let it stand. Prepare three samples of the treated soil.

[0108] For potted plants, use 15 cm deep seedling pots with 5×10 holes; fill the sterilized soil into the seedling pots for later use.

[0109] Microbial agents: Ms microbial agent, Mo microbial agent and Mp microbial agent obtained according to the microbial agent preparation methods in Examples 2 to 4, the difference being that the OD value of the three microbial agents is 0.05.

[0110] Rapeseed soil cultivation experiment: Three pre-germinated rapeseed seeds were transferred to deep-hole trays filled with sterilized soil and placed in a culture room at 22℃ with 16 hours of light / 8 hours of darkness. Each week, 5 ml of low-phosphorus nutrient solution was added, and every other day, 5 ml of Ms, Mo, and Mp inoculants were added respectively. The control group received 5 ml of low-phosphorus nutrient solution weekly, and 5 ml of sterile water every other day. Indicators were measured after one month of cultivation. The results for the four groups were obtained: Ms group, Mo group, Mp group, and control group (Mock).

[0111] The low-phosphorus soil-promoting phenotype of *Massezie* is shown in […]. Figure 6 The statistical results of the dry weight of the underground part of *Morchella esculenta* in low-phosphorus soil are shown in the figure. Figure 7 .

[0112] Depend on Figure 6 , 7 It can be seen that inoculation of Masei bacterium Ms strain into rapeseed grown in sterilized soil significantly promoted the dry weight of the underground part of rapeseed. Compared with the control group (Mock), the dry weight of the roots of rapeseed inoculated with Mp, Mo and Ms increased by 62%, 126% and 96%, respectively.

[0113] Test on the growth-promoting effect of Mp inoculant on rapeseed field

[0114] Preparation of soil-cultured nutrient solution for nutrient deficiency: 1 / 2 MS-P medium (2.146 g / L), normal phosphorus solution (with added potassium dihydrogen phosphate 4.9 g / L), and low phosphorus solution (with added potassium dihydrogen phosphate 0.098 g / L).

[0115] Inoculum: Mp inoculum prepared according to the inoculum preparation method in Example 4, except that the OD value of the inoculum in this experiment is 0.05.

[0116] Field inoculation experiment of rapeseed: Fertilizer was applied at a rate of 26 kg urea, 37.5 kg superphosphate (normal phosphorus), 7.5 kg superphosphate (low phosphorus group), and 14 kg potassium oxide per mu (unit of land area). The experiment was divided into 4 groups: normal phosphorus group (CK group), normal phosphorus group with inoculant (CK+M group), low phosphorus group (LP group), and low phosphorus group with inoculant (LP+M group). Each group had 16 rapeseed seedlings (rapeseed variety ZS11), with 3 replicates. Inoculant was applied every 15 days, with 50 ml of inoculant per seedling each time.

[0117] The field inoculation phenotype of *Massezie* is shown below. Figure 8 Statistics on field inoculation results of Bacillus simonii can be found in [link to relevant documentation]. Figure 9 .

[0118] like Figure 8 , 9 As shown, inoculating rapeseed with Mp strain in the field significantly increased the total fresh weight of rapeseed regardless of whether the soil was low-phosphorus or normal-phosphorus. The total fresh weight increased by 15% in normal-phosphorus soil and by 73% in low-phosphorus soil.

[0119] In summary, the Ms, Mo, and Mp inoculants provided by this invention have a significant promoting effect on rapeseed growth.

[0120] Example 7

[0121] Application of Marseilles inoculant in the control of plant sclerotinia disease.

[0122] Marseilles defense against Sclerotinia sclerotiorum plate test

[0123] The culture dishes used were 25 cm × 25 cm flat plates and 9 cm diameter round culture dishes.

[0124] PDA medium: 47 g / L powder (Haibo Biotechnology), 12.0 g / L potato extract powder, 20.0 g / L glucose. The PDA medium was mixed with water at a concentration of 47 g / L and then sterilized at 121°C for 20 min.

[0125] Assay for *Masses* antagonism against *Sclerotinia sclerotiorum*: *Sclerotinia sclerotiorum* was activated twice on PDA medium. Using a sterilized punch, viable *Sclerotinia sclerotiorum* cells were collected from the edges and transferred to the center of a fresh PDA medium. Four groups were set up: a control group (CK), and the other three groups (Ms, Mo, and Mp). After overnight incubation, *Masses* Ms, Mo, and Mp inoculum were centrifuged and collected. A suitable amount of bacterial solution was aliquoted at four symmetrical points on a circular medium. The bacterial solution was dried, sealed with sealing film, and inverted in an incubator at 22°C. The growth status of *Sclerotinia sclerotiorum* was observed. See the image showing the effect of *Masses* strains on *Sclerotinia sclerotiorum* on plate resistance. Figure 10The comparison of the relative spread distance of Ms and the control group (CK, or Mock) is shown in the figure below. Figure 11 The relative extension distance is the ratio of the distance between the closest point of *Sclerotinia sclerotiorum* to the farthest point of *Sclerotinia sclerotiorum* from the center of the circular culture medium to the distance between the farthest point of *Sclerotinia sclerotiorum* from the *Sclerotinia sclerotiorum* strain.

[0126] Depend on Figure 10 It was found that when *M. s ...

[0127] Depend on Figure 11 It can be seen that, compared with the control group, *Masses* Ms can inhibit the growth of *Sclerotinia sclerotiorum*.

[0128] Experiment on the defense of *Massezia sclerotiorum* against detached leaf fragments

[0129] Two groups were set up: a control group (Mock) where *Sclerotinia sclerotiorum* was inoculated onto rapeseed leaves only, and an experimental group (Ms) where *M. marshmallowii* was inoculated onto rapeseed leaves, followed by *Sclerotinia sclerotiorum* inoculation the next day. The effect of *M. marshmallowii* Ms on detached leaves against *Sclerotinia sclerotiorum* is shown in [the figure]. Figure 12 The statistical analysis of the resistance of detached *Massezia sclerotiorum* leaves to *Sclerotinia sclerotiorum* phenotypes is shown in [the table below]. Figure 13 .

[0130] like Figure 12 , 13 As shown, the leaf lesions inoculated with the Ms strain of Masseilles were significantly smaller than those in the control group (Mock). Further statistical analysis revealed that this difference was statistically significant, indicating that the Ms strain of Masseilles can protect rapeseed from Sclerotinia sclerotiorum infection.

[0131] Masseilles bacterium was applied to the leaves of rapeseed to control sclerotinia stem rot caused by Sclerotinia sclerotiorum:

[0132] Two groups were set up: a control group (Mock) and an experimental group (M.s_F). In the experimental group, *M.s.* was applied to the rapeseed leaves, and *Sclerotinia sclerotiorum* was inoculated onto the leaves the following day. In the control group, *M.s.* was not applied before inoculation with *Sclerotinia sclerotiorum*. The growth of *Sclerotinia sclerotiorum* on the rapeseed leaves of the control and experimental groups are shown in the figures below. Figure 14 a. The damaged area of ​​rapeseed leaves in the control group and the experimental group is shown in Figure 1. Figure 14 b, the relative sclerotium biomass on rapeseed leaves in the control group and the experimental group are shown in Figure 1. Figure 14 c.

[0133] according to Figure 14 It is known that when the Masse bacteria provided by this invention is applied to the leaves of rapeseed, it can prevent and control sclerotinia disease in rapeseed caused by Sclerotinia sclerotiorum.

[0134] Masseilles bacterium was applied to the roots of rapeseed to control sclerotinia stem rot caused by Sclerotinia sclerotiorum:

[0135] Two groups were set up: a control group (Mock) and an experimental group (M.s_R). In the experimental group, *M.s. spp.* was applied to the roots of rapeseed, and *Sclerotinia sclerotiorum* was inoculated onto the rapeseed leaves the following day. In the control group, *M.s. spp.* was not applied before inoculation with *Sclerotinia sclerotiorum*. The growth of *Sclerotinia sclerotiorum* on the rapeseed leaves of the control and experimental groups are shown in the figures below. Figure 15 a. The damaged area of ​​rapeseed leaves in the control group and the experimental group is shown in Figure 1. Figure 15 b, the relative sclerotium biomass on rapeseed leaves in the control group and the experimental group are shown in Figure 1. Figure 15 c.

[0136] according to Figure 15 It is known that when the Masse bacteria provided by this invention is applied to the roots of rapeseed, it can prevent and control sclerotinia disease in rapeseed caused by Sclerotinia sclerotiorum.

[0137] Depend on Figure 14 and 15 Comparison shows that the effect of applying the *Massezie* strain provided by this invention to the leaves of rapeseed in controlling sclerotinia stem rot is better than that applied to the roots of rapeseed.

[0138] Example 8

[0139] This invention provides an application of Bacillus simonii / Bacillus simonii agent in fertilizer preparation, which involves applying Bacillus simonii / Bacillus simonii agent to the roots of rapeseed for irrigation.

[0140] Example 9

[0141] This invention provides an application of Bacillus simonii / Bacillus simonii agent in the preparation of pesticides, wherein Bacillus simonii / Bacillus simonii agent is sprayed onto the leaves of rapeseed.

[0142] In summary, the Ms strain of Masseilles and its inoculant provided by this invention simultaneously possess phosphorus-solubilizing effects, promote plant growth, and prevent sclerotinia disease.

[0143] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of *Mosaicella* for rapeseed cultivation, characterized in that, The Massey bacteria mentioned are Massilia scoD23, Massilia orientalis, or Massilia phosphatilytica; The Massilia scoD23 has the accession number CCTCC NO: M 2026202, is deposited at the China Center for Type Culture Collection, and was deposited on January 22, 2026. The Massilia orientalis has the accession number CCTCC NO: M 2026203, is deposited at the China Center for Type Culture Collection, and was deposited on January 22, 2026. The accession number of Massilia phosphatilytica is CCTCC NO: M 2026201, the depository center is China Center for Type Culture Collection, and the deposit date is January 22, 2026.

2. A microbial agent, characterized in that, The microbial agent includes the *Masses* strain as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent includes a *Bacillus simonii* bacterial solution, wherein the concentration of *Bacillus simonii* in the bacterial solution is 1.0 × 10⁻⁶. 8 ~2×10 8 cfu / mL.

4. A method for preparing the microbial agent according to claim 2, characterized in that, Includes the following steps: The frozen *Masses* strain was activated in a solid culture medium, and then single clones were picked and cultured in a liquid culture medium. The cells were collected by centrifugation, and the culture medium and bacterial metabolites were removed by resuspending in sterile water to obtain the bacterial agent.

5. The method for preparing the microbial agent according to claim 4, characterized in that, The solid culture medium is R2A solid culture medium, and the liquid culture medium is R2A liquid culture medium.

6. The application of the *Massezia* strain according to claim 1 in phosphorus solubility in rapeseed planting soil.

7. An application of the *Massezie* strain according to claim 1 in promoting plant growth, characterized in that, The plant in question is rapeseed.

8. The application of *Mosas* according to claim 1 in the prevention and control of *Sclerotinia sclerotiorum* disease in plants, characterized in that... The plant in question is rapeseed.

9. The application of the *Morseella macrantha* according to claim 1 in the preparation of phosphate fertilizer for rapeseed.

10. The use of the *Massezia* strain according to claim 1 in the preparation of a pesticide for controlling *Sclerotinia sclerotiorum*-induced sclerotinia rot in rapeseed.

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