Application of phosphorus-solubilizing massinia sp. C22 in prevention and treatment of plant fungal diseases

Abstract

The invention discloses a novel application of massinia phosphate solubilizing C22 in prevention and treatment of fungal diseases of plants, and belongs to the technical field of agricultural biology, and particularly relates to a novel application of massinia phosphate solubilizing C22 in prevention and treatment of fungal diseases of plants. The known strain C22 has a remarkable antagonistic effect on pathogenic bacteria fusarium pseudograminearum of wheat stem rot, and the plate inhibition rate reaches 36.79%. Potting and field tests prove that wheat basal stem rot can be effectively prevented and treated by applying the C22 fungicide, the morbidity in the seedling stage is reduced by 21.64%, the morbidity in the adult-plant stage is reduced by 55.06%, the DON toxin content in plants can be remarkably reduced by 68.26%, and the pathogenic bacterium load can be remarkably reduced. According to the invention, the potential of the phosphorus-solubilizing massinia sp. C22 in the aspect of biological control is disclosed for the first time, and a new core microbial resource and a technical scheme are provided for developing a green biological pesticide for controlling wheat stem rot.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a new application of a known microorganism, particularly the application of Massilia phosphatilytica C22 in the prevention and control of plant fungal diseases, especially wheat stem rot. Background Technology

[0002] Wheat crown rot (FCR) is a globally distributed soil-borne disease, first discovered in Queensland, Australia in 1951. Currently, it is widely distributed in North America, South America, Europe, Africa, Mediterranean countries, and most wheat-producing areas in Asia. In recent years, factors such as straw return policies and frequent extreme weather events have exacerbated its spread. The pathogens (such as *Fusarium graminearum* and *Fusarium graminearum*) not only cause browning and necrosis at the base of the wheat stem, forming a "soy sauce stalk" symptom, and lead to withered white ears during the grain-filling stage, resulting in an average yield reduction of 5%–10%, with severely affected fields experiencing yield reductions exceeding 50%, but also produce toxins such as deoxynivalenol (DON) that contaminate the grains. DON toxin is one of the most toxic secondary metabolites produced by the pathogens, which can cause symptoms such as vomiting, dizziness, and fever in humans and animals, and in severe cases, endanger life.

[0003] Wheat stem base rot is widespread and causes serious damage. Various control methods have been reported, including chemical seed treatment, rational fertilization, crop rotation, and planting disease-resistant varieties. However, these methods are not highly effective in controlling the occurrence and progression of the disease. Furthermore, long-term and frequent use can lead to drug resistance in crops, gradually reducing the effectiveness of control. Therefore, there is an urgent need to find green and efficient control methods for wheat stem base rot. The rhizosphere is the interface between plant roots and soil, the area where the root system's life activities and metabolism have the most direct and intense impact on the soil. The rhizosphere is a window for efficient nutrient utilization by crops, a key micro-domain for plant-soil-microbe interactions, and a major site for the occurrence and development of soil-borne diseases. Rhizosphere microorganisms can not only effectively reduce the incidence and severity of wheat stem base rot (FCR), but also systematically activate the expression of defense-related genes in host plants, thereby significantly enhancing the overall disease resistance of the plant. In the future, screening and efficient utilization of novel biocontrol resources will become a key strategy for the control of wheat stem base rot.

[0004] In recent years, extensive research and screening have yielded a variety of microorganisms with biocontrol potential. These microorganisms can effectively inhibit the growth of *Fusarium graminearum* and reduce the severity of wheat stem rot, which is of great significance for achieving green control of this disease. Biocontrol bacteria in wheat stem rot control mainly include *Bacillus* spp. and *Trichoderma* spp. *Bacillus belesiensis* YB-185 significantly inhibits *Fusarium graminearum* mycelial growth by secreting antibacterial substances and competing for ecological niches (indoor inhibition rate 69.1%, field control efficacy 57.6%). The co-culture fermentation broth of *Trichoderma viride* HB20111 and *Trichoderma harzianum* TW21990 shows significant effects against seedling stem rot through cell wall degrading enzymes and inducing systemic resistance. *Trichoderma harzianum* seed dressing reduces the incidence of disease by 50%–65% and increases yield through direct inhibition, resistance induction, and growth promotion. Combining it with low-dose chemical fungicides can further enhance its control efficacy. Bacillus subtilis YB-05 secretes lipopeptide antibiotics and competes for ecological niches, achieving an inhibition rate of >60%. Seed treatment reduced the disease index by 50%, and when combined with shenqinmycin, the control efficacy reached 72.3%. Bacillus amyloliquefaciens inhibits pathogens through lipopeptides such as iturin, while simultaneously activating systemic resistance in wheat (e.g., upregulation of PR proteins), achieving a field control efficacy of 60%–75%. These studies indicate that biocontrol bacteria can not only effectively reduce the incidence and severity of wheat stem base rot (FCR), but also systematically activate the expression of defense-related genes in host plants, thereby significantly enhancing the overall disease resistance of plants. In the future, screening and efficient utilization of novel biocontrol resources will become a key strategy for the control of wheat stem base rot.

[0005] Massilia phosphatilytica is a known rhizosphere bacterium previously reported to have the ability to dissolve insoluble phosphorus in soil, thereby promoting plant growth. However, to date, no published literature or patents have demonstrated that Massilia phosphatilytica, particularly the C22 strain involved in this application, possesses antagonistic effects and biocontrol potential against plant fungal diseases, especially against wheat stem rot. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new use for Massilia phosphatilytica C22, namely its application in the prevention and control of plant fungal diseases.

[0007] Another objective of this invention is to provide a method and biological agent for preventing and controlling plant fungal diseases.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the invention provides the use of Massilia phosphatilytica C22 in the preparation of biological agents for the control of plant fungal diseases.

[0009] Preferably, the plant fungal disease is wheat stem rot caused by Fusarium pseudograminearum.

[0010] A second aspect of the present invention provides a method for preventing and controlling plant fungal diseases, the method comprising applying an effective amount of Massilia phosphatilytica C22, its culture or its metabolites to a plant, plant seeds or soil in which the plant grows.

[0011] Preferably, the plant fungal disease is wheat stem rot caused by Fusarium graminearum.

[0012] Preferably, the application method is root irrigation, root soaking, or seed treatment.

[0013] In a third aspect, the present invention provides a biological agent for the prevention and control of plant fungal diseases, which uses at least one of Massilia phosphatilytica C22, its culture or its metabolites as an active ingredient.

[0014] Preferably, the biological agent is a liquid bacterial agent, a solid bacterial agent, or a wettable powder.

[0015] A fourth aspect of the invention provides the use of Massilia phosphatilytica C22 in reducing the content of deoxynivalenol (DON) toxin in wheat.

[0016] In a fifth aspect, the invention provides the use of Massilia phosphatilytica C22 in reducing the content of Fusarium pseudograminearum pathogen in plants.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Discovery of new uses: For the first time, a new function of the known strain of Phosphate-lying Masseie bacterium C22 in controlling wheat stem base rot has been revealed, providing a core strain resource for the development of new biological pesticides; 2. Significant effects: Plate confrontation experiments showed that strain C22 inhibited Fusarium oxysporum by 36.79%. Pot and field trials demonstrated that it significantly reduced the disease index (incidence rate decreased by 21.64%–55.06%), and substantially reduced DON toxin content (up to 68.26%) and pathogen load (decreased by 27.82%–59.44%). 3. Multiple beneficial effects: This strain not only directly inhibits pathogens, but also promotes the recovery of plant roots, possessing the dual potential of biological control and promoting plant health; 4. Safe and environmentally friendly: This strain was isolated from the rhizosphere of wheat, which is environmentally friendly and meets the development needs of green agriculture. Attached Figure Description

[0018] Figure 1 Photograph of the colony morphology of Phospholysozyme C22.

[0019] Figure 2 Gram-stained photograph of Phospholysozyme C22 (shown as a Gram-negative bacterium).

[0020] Figure 3 Scanning electron microscopy (SEM) image of Phospholysozyme C22 (showing cell morphology as straight or slightly curved bacilli).

[0021] Figure 4 Phylogenetic tree of phosphate-solubilizing muscarinic bacteria C22 constructed based on 16S rDNA sequence.

[0022] Figure 5 Phenotypic diagram of the effect of strain C22 on the growth of wheat seedlings (CK is the control, C22 is the inoculation treatment).

[0023] Figure 6 : Plate inhibition effect of strain C22 on Fusarium oxysporum.

[0024] Figure 7 Scanning electron micrograph of the inhibitory effect of strain C22 on Fusarium pseudograss hyphae.

[0025] Figure 8 : Inhibitory effect of strain C22 on wheat stem base rot in seedling stage (Fp means only pathogen is inoculated, Fp+C22 means pathogen and C22 are co-inoculated).

[0026] Figure 9 Statistical data on the control efficacy of strain C22 against wheat stem base rot in the seedling stage (incidence rate, DON content, Fp content).

[0027] Figure 10 Phenotypic diagram of the field control efficacy of strain C22 against stem base rot of mature wheat.

[0028] Figure 11Statistical data on the field control efficacy of strain C22 against stem rot of mature wheat (incidence rate, Fp content). Detailed Implementation

[0029] The present invention will be further described below through specific embodiments, but the present invention is not limited thereto.

[0030] Example 1: Isolation and identification of phosphate-solubilizing Marseilles C22 Isolation, purification, and identification of Massilia phosphatilytica C22 from the rhizosphere of wheat.

[0031] 1.1 Culture medium preparation LB solid medium: 10 g tryptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to a final volume of 1 L; autoclave at 121°C for 20 min.

[0032] 1.2 Isolation and purification of rhizosphere bacteria Weigh 5 g of rhizosphere soil sample from Huayu 198, a wheat stem rot resistant material, using a balance and place it in a 250 mL sterile Erlenmeyer flask containing 45 mL of sterile water. Add sterile glass beads to the flask and shake at 200 rpm / min for 30 min at 28°C. After the rhizosphere soil sample and sterile water are thoroughly mixed, let it stand for 10 min. Take 100 μL of the supernatant and perform serial dilutions to obtain soil suspensions of different dilutions. Spread 1 mL of the soil suspension onto LB agar plates. After bacteria have grown on the plates, streak them for further purification to obtain purified rhizosphere bacteria, including strain C22.

[0033] 1.3 Identification of Rhizosphere Bacteria C22 1.3.1 Morphological identification The purified rhizosphere bacterial strain C22 was streaked onto LB agar plates and incubated at 28°C for 48 h. The morphology of single colonies was then observed. The morphology of the strain was observed under a microscope, and the strain was Gram stained using a Gram staining kit (Beijing Solarbio Science & Technology Co., Ltd.). The morphology of the strain and the staining results were observed.

[0034] The results showed that the colonies of rhizosphere bacteria strain C22 were off-white, round, small, with smooth, slightly raised edges, and relatively hard. Figure 1 Gram-negative bacteria, staining red ( Figure 2 The cells are straight or slightly curved bacilli, arranged singly or in pairs. Figure 3 ).

[0035] 1.3.2 Molecular Identification Genomic DNA of rhizosphere bacteria C22 was extracted from activated bacterial culture using a bacterial genomic DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd.). Using this DNA as a template, PCR amplification was performed using the universal primers 27F / 1492R for the bacterial 16S rDNA gene.

[0036] Primer sequences: 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0037] The 16S rDNA sequences amplified by PCR were analyzed by BLAST sequence alignment using the NCBI website, and a phylogenetic tree was constructed using MEGA11 software. Figure 4 The rhizosphere bacteria C22 clustered with *Massilia phosphatilytica* into the same clade. The results indicated that strain C22 was a phosphate-lysating *Massilia phosphatilytica*, and therefore it was named *Massilia phosphatilytica C22*.

[0038] Example 2: Safety determination of phosphate-solubilizing Massey bacterium C22 on wheat seedling growth 2.1 Culture medium preparation LB liquid medium: 10 g tryptone, 5 g sodium chloride, 10 g yeast extract, add water to a final volume of 1 L; autoclave at 121°C for 20 min.

[0039] 2.2 Pot experiment to verify the effect of rhizosphere bacteria C22 on the growth of wheat seedlings. Natural field soil sieved through a 20-mesh sieve was mixed with vermiculite at a volume ratio of 2:1 and set aside. Before sowing, the seeds of Fanmai 8 wheat (a susceptible wheat variety) underwent surface disinfection treatment: soaking in 1% NaClO for 15 minutes and rinsing three times with sterile water. Thirty wheat seeds were planted in each pot, with three pots forming one treatment group. Two treatment groups, A and B, were established: A - blank control, and B - inoculated with rhizosphere bacteria C22 solution.

[0040] Rhizosphere bacteria C22 were inoculated into LB liquid medium and incubated at 28°C and 200 rpm for 20 h for later use. Once the wheat had grown to the two-leaf stage, the OD of the prepared C22 bacterial suspension was adjusted with sterile water. 600 =0.5, and inoculated into group B wheat, adding 100 mL of bacterial suspension C22 to each pot of wheat for root irrigation. Plant growth was observed 20 days after inoculation. Results are as follows. Figure 5As shown, compared with the control group, wheat plants inoculated with strain C22 grew normally without any adverse effects, indicating that the strain is safe for wheat and meets the basic requirements for use as a biocontrol agent.

[0041] Example 3: Determination of the antibacterial activity of Massilia phosphatilytica C22 in the rhizosphere of wheat against Fusarium pseudograminearum 3.1 Culture medium preparation LB liquid medium: 10 g tryptone, 5 g sodium chloride, 10 g yeast extract, add water to a final volume of 1 L; autoclave at 121°C for 20 min.

[0042] Potato glucose solid medium (PDA): Cut 200 g of washed and peeled potatoes into small pieces and boil in water for 30 min. Filter twice through six layers of gauze, add 20 g of glucose and 15 g of agar powder to the filtrate, mix thoroughly, add distilled water to 1 L, and sterilize at 121℃ for 20 min.

[0043] 3.2 Test strains The pathogen of wheat stem rot, *Fusarium pseudograminearum*, was isolated from Caoxinzhuang Farm in Yangling Demonstration Zone, Shaanxi Province.

[0044] 3.3 Plate confrontation experiment to observe the antibacterial effect of rhizosphere bacteria C22 on pathogens The purified rhizosphere bacteria C22 were inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 20 h to obtain activated bacterial solution.

[0045] The antagonistic effect of screened rhizosphere bacteria on pathogenic fungi was determined using the plate confrontation culture method. Newly activated pathogenic fungi were inoculated into the center of a PDA plate at a 5 mm diameter mycelial cake, with 2 μL of C22 activated bacterial solution inoculated at 2.5 cm intervals to the left and right. For the blank control, 2 μL of sterile LB broth was inoculated. Each treatment was repeated three times.

[0046] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100% The antibacterial activity of strain C22 was determined using the plate control method. *Fusarium graminearum* cakes were inoculated in the center of PDA plates, and 2 μL of activated C22 bacterial solution was spot-inoculated 2.5 cm away from the cakes. Sterile LB medium was used as a control. After incubation at 28℃ for several days, the colony diameter was measured and the inhibition rate was calculated. The results showed that ( Figure 6The C22 strain significantly inhibited the mycelial growth of *Fusarium graminearum*, with an inhibition rate of 36.79%. Further observation using scanning electron microscopy revealed... Figure 7 It can be seen that strain C22 causes the pathogenic fungal hyphae to become deformed and collapse.

[0047] Example 4: Control effect of Phosphate-solubilizing Massey Bacterium C22 on wheat stem base rot in seedling stage 4.1 Culture medium preparation LB liquid medium: 10 g tryptone, 5 g sodium chloride, 10 g yeast extract, add water to a final volume of 1 L; autoclave at 121°C for 20 min.

[0048] CMC medium: CMC-Na 15 g, ammonium nitrate 1 g, yeast extract 1 g, magnesium sulfate heptahydrate 0.5 g, potassium dihydrogen phosphate 1 g, autoclaved at 121℃ for 20 min.

[0049] 4.2 Pot experiment to verify the efficacy of rhizosphere bacteria C22 against wheat stem rot in seedling stage Natural field soil sieved through a 20-mesh sieve was mixed with vermiculite at a volume ratio of 3:1 and set aside. Before sowing, the seeds of Fanmai No. 8 wheat underwent surface disinfection treatment: soaking in 1% NaClO for 15 minutes and rinsing three times with sterile water. Thirty wheat seeds were planted in each pot, with three pots forming one treatment group. Two treatment groups, A and B, were established: A-inoculated with a suspension of *Fusarium graminearum* spores (test strain), and B-inoculated with rhizosphere bacteria C22 and a suspension of *Fusarium graminearum* spores.

[0050] Rhizosphere bacteria C22 were inoculated into LB broth and incubated at 28°C and 200 rpm for 20 h for later use. The tested strain, *Fusarium graminearum*, was inoculated into CMC broth and incubated at 28°C and 200 rpm for 5 days for later use. When the wheat reached the two-leaf stage, the OD of the prepared C22 bacterial suspension was adjusted with sterile water. 600 =0.5, and inoculated into group B wheat, adding 60 mL of bacterial C22 suspension to each pot of wheat for root drenching. 3 days after inoculation, take the prepared Fusarium graminearum spore suspension, adjust the concentration to 5×105 spores / mL with sterile water, and inoculate into group B wheat, adding 60 mL of spore suspension to the roots of each wheat plant for root drenching. 21 days after inoculation, observe plant growth and disease incidence.

[0051] The results showed that, observing the growth of the two groups of wheat plants, group A wheat plants infected with the tested strain *Fusarium graminearum* exhibited slow root development and larger lesion areas. Compared with group A wheat plants, group B wheat plants showed restored root development and were generally in a healthy root system. The results are as follows: Figure 8 and Figure 9As shown, compared with group A, wheat plants in group B had healthier root systems and fewer lesions. Data showed that the incidence rate in group B was reduced by 21.64%, the DON toxin content in the plants was significantly reduced by 68.26%, and the pathogenic fungal load Fp decreased by 27.82%.

[0052] Example 5: Field control efficacy of phosphate-solubilizing Massey bacterium C22 against stem rot in mature wheat plants. 5.1 Culture medium preparation Same as Implementation Example 4.1 5.2 Field pot experiment to verify the control efficacy of rhizosphere bacteria C22 against stem rot in mature wheat plants Natural field soil sieved through a 20-mesh sieve was mixed with vermiculite at a volume ratio of 2:1 and set aside. Before sowing, wheat seeds of Huayu 198 and Fanmai 8 underwent surface disinfection treatment: soaking in 1% NaClO for 15 minutes and rinsing three times with sterile water. Three wheat seeds were planted in each large flowerpot, with eight pots forming one treatment group. Three treatment groups were established: A - Huayu 198 disease-resistant control; B - Fanmai 8 disease-susceptible control; and C - Fanmai 8 inoculated with rhizosphere bacteria C22 and a suspension of *Fusarium oxysporum* spores.

[0053] Rhizosphere bacteria C22 were inoculated into LB liquid medium and incubated at 28°C and 200 rpm for 20 h for later use. Once the wheat had grown to the two-leaf stage, the OD of the prepared C22 bacterial suspension was adjusted with sterile water. 600 =0.5, and inoculated into group C wheat, adding 500 mL of bacterial suspension C22 to each pot of wheat for root drenching. 3 days after inoculation, the prepared *Fusarium graminearum* spore suspension was adjusted to a concentration of 5 × 10⁵ spores / mL with sterile water and inoculated into all three groups of wheat, adding 60 mL of spore suspension to the roots of each wheat plant for root drenching. Plant growth was observed until the grain-filling stage.

[0054] The results showed that the disease resistance of the three groups of wheat plants was as follows: Figure 10 and Figure 11 As shown, compared with the susceptible control group B, the wheat disease severity in group C, which was treated with C22 bacterial solution, was significantly reduced. Specifically, the disease incidence rate in group C was reduced by 55.06% and the pathogen Fp load was reduced by 59.44% compared to group B.

[0055] The above examples fully demonstrate that Phospholysozyme C22 can effectively inhibit the growth of Fusarium graminearum and has a significant and stable control effect on wheat stem base rot. At the same time, it can greatly reduce DON toxin pollution and has extremely high application value.

Claims

1. The application of a phosphate-lysating Massimophyton C22 in the preparation of a biological agent for the control of plant fungal diseases.

2. The application according to claim 1, characterized in that, The plant fungal disease mentioned is wheat stem base rot caused by Fusarium pseudograminearum.

3. A method for controlling plant fungal diseases, characterized in that, The method involves applying an effective amount of phosphate-solubilizing Massilia phosphatilytica C22, its culture, or its metabolites to plants, plant seeds, or soil in which plants grow.

4. The method according to claim 3, characterized in that, The plant fungal disease mentioned is wheat stem base rot caused by Fusarium pseudograminearum.

5. The method according to claim 3 or 4, characterized in that, The application method is root irrigation, root soaking, or seed treatment.

6. A biological agent for controlling plant fungal diseases, characterized in that, It uses at least one of Massilia phosphatilytica C22, its culture, or its metabolites as its active ingredient.

7. The biological agent according to claim 6, characterized in that, The plant fungal disease mentioned is wheat stem base rot caused by Fusarium pseudograminearum.

8. The biological agent according to claim 6, characterized in that, The biological agent is a liquid bacterial agent, a solid bacterial agent, or a wettable powder.

9. The application of Massilia phosphatilytica C22 in reducing the content of deoxynivalenol toxins in wheat.

10. Application of Massilia phosphatilytica C22 in reducing the content of Fusarium pseudograminearum pathogen in plants.

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