Corn endophytic disperse pantoea X24004 and application thereof

Abstract

The invention discloses corn endophytic pantoea dispersa X24004 and an application of the corn endophytic pantoea dispersa X24004. The preservation number of the Pantoea dispersa X24004 is CGMCC (China General Microbiological Culture Collection Center) No. 34510, and the preservation number of the Pantoea dispersa X24004 is CGMCC No. 34510. The pantoea dispersa X24004 has a relatively strong antagonistic effect on a plurality of pathogenic fungi such as fusarium graminearum, fusarium verticillioides, pythium aphanidermatum stem rot pathogenic bacteria and the like, has high efficiency and broad spectrum on prevention and treatment of plant fungal diseases, and also has a promoting capability on growth of corn plants; the biological agent is a broad-spectrum biological agent, a biological bacterial fertilizer and a seed treatment agent with good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control of plant diseases, and particularly relates to an endophytic Pantoea dispersa X24004 of corn and an application thereof. Background Art

[0002] Maize stalk rot (MSR), also known as corn wilt or bacterial wilt, is caused by multiple pathogens, either singly or in combination. The infection sites are primarily located at the root and stem base of corn plants and can occur throughout the plant's growth and development period. The disease typically has an incidence rate of 10% to 15%, but can reach 80% in severe cases. Under favorable conditions, it can even cause corn yield failure, and its incidence is increasing annually. In recent years, the accumulation of pathogens in the field due to the widespread use of straw in fields, coupled with the widespread adoption of susceptible corn varieties and the widespread monoculture of these varieties, has led to maize stalk rot becoming a major disease in China's corn-growing regions, alongside other diseases such as corn leaf spot, corn smut, and corn head smut.

[0003] Studies have shown that the pathogens of corn stalk rot are affected by a variety of factors and conditions, and the types of pathogens vary significantly under different geographical environments and climatic conditions. In China, the main pathogens that cause corn stalk rot are Fusarium spp. Fusarium spp.) and Pythium spp. Pythium spp.), Fusarium graminearum ( F. graminearum ) is currently the dominant pathogen isolated and responsible for corn stalk rot. It infects the stems, stem bases, and roots of corn and other cereal crops. Corn stalk rot caused by Fusarium graminearum not only reduces corn yields but also produces a large number of toxins that threaten human and livestock health, severely impacting the development of the corn industry.

[0004] Currently, effective control measures for corn stalk rot focus on seed coating. Seed dressing agents include 3.5% fludioxonil metalaxyl suspension concentrate, 25% pyrimidine-thiram wettable powder, and 58% metalaxyl-thiram wettable powder. These agents are highly effective against corn stalk rot, reducing the disease incidence by 80%. They also have no inhibitory effect on seed emergence and seedling growth, while also promoting increased corn yields. While chemical pesticide application is currently the most commonly used pathogen control measure, this approach can lead to overuse of agrochemicals and chemical residues in agricultural products, resulting in both economic and environmental drawbacks.

[0005] Therefore, prevention and control methods are increasingly focused on the field of biological control. By exploring the use of microorganisms and their secondary metabolites for disease prevention and control, promoting plant growth, increasing crop yields, and then seeking sustainable green development, it shows that the research and development and use of biological agents is a very promising means of prevention and control. Studies have shown that microorganisms can be used for biological control of corn stalk rot. Inoculating corn fields with diseased corn can prevent and control stalk rot through various mechanisms such as fungal parasitism, inducing systemic resistance, enhancing corn rhizosphere microbial communities, and improving soil fertility; the metabolites of Trichoderma harzianum can synthesize selenium and titanium dioxide, which can then be made into nanoparticles for use in the control of Fusarium oxysporum ( F. culmorum ) prevention and control. Some Bacillus isolates can produce lytic enzymes such as glucanases, proteases or chitinases, as well as siderophores and auxins. This suggests that they may act as antagonists to inhibit Fusarium-induced corn stalk rot. Some cyclic lipopeptides produced by Methylotrophic Bacillus TA-1 can cause Fusarium graminearum ( F. graminearum ) cell walls, which then invade pathogenic bacteria and release their contents. This process could serve as an effective alternative to chemical fungicides in corn cultivation. Using microorganisms to control related diseases offers lower economic costs, greater cost-effectiveness, and greater safety. Furthermore, the broad-spectrum antifungal activity of microorganisms and their metabolites makes exploring and tapping into microbial resources a highly attractive research direction.

[0006] Vascular tissue serves as an efficient long-distance transport system, driven by the hydrostatic pressure gradient between roots and branches. This driving force ensures the smooth transport of solutes and signals between plant organs. Furthermore, the pores in the perforated plates between xylem units are large enough to allow bacteria to pass through. Studies have shown that some systemic bacterial colonies can spread to aboveground plant compartments via transpiration-driven xylem flow. Studies have characterized the bacterial communities inhabiting olive xylem sap using culture-dependent and -independent methods and found that Sphingomonas was the most represented genus. However, little is known about the functional relationships between xylem microbial communities and plant growth and development.

[0007] Maize (corn) plants specifically recruit a core microbiome in their xylem sap, a core microbiome that is conserved across environmental conditions and genotypes. This core microbiome contributes to plant nitrogen nutrition through biological nitrogen fixation and promotes root development. The mechanisms and functions of interactions between the xylem sap microbiome and the host await further investigation. This core microbiome may represent a promising resource for developing alternative microbial biotechnologies to improve crop performance in sustainable agriculture. Studies have shown that gammaproteobacterial OTUs in xylem sap (VE) are dominated by Enterobacteriaceae, Erwiniaceae, and Pseudomonadaceae, while Burkholderiaceae predominate in stem endosomal (SE) populations. Klebsiella and Pantoea are the dominant bacterial groups in VE. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the lack of biological control methods for the soil-borne disease of corn, Fusarium stalk rot (caused by Fusarium graminearum), and to provide a corn endophytic bacterium, Pantoea dispersa ( Pantoea dispersa X24004 (CGMCC No. 34510, deposit date: May 12, 2025, deposit code: CGMCC - General Microbiology Center, China National Committee for the Administration of Microbiological Culture Collection) and its applications. Strain X24004 was isolated from the xylem sap of maize and exhibits significant inhibitory activity against the pathogen Fusarium graminearum, displaying high-efficiency, broad-spectrum antibacterial activity.

[0009] The experimental results of the present invention show that the dispersed Pantoea X24004 has a great effect on the pathogen of corn fusarium stalk rot Fusarium graminearum ( Fusarium graminearum ) has a strong inhibitory effect on the mycelial growth of corn ear rot pathogen Fusarium spp. Fusarium verticillioides ), the pathogen of corn stalk rot, Pythium spp. Pythium arrhenomanes ) and other fungal pathogens, with inhibition rates ranging from 44.72% to 59.70%. Strain X24004 demonstrates high efficacy and broad-spectrum activity against plant fungal diseases and promotes corn plant growth, demonstrating its potential for application in the biological control of plant fungal diseases.

[0010] The first object of the present invention is to provide a dispersed Pantoea ( Pantoea dispersa )X24004, its deposit number is CGMCC No. 34510.

[0011] The second object of the present invention is to provide a biological preparation containing the Pantoea dispersae X24004, the culture of the Pantoea dispersae X24004 and / or the metabolites obtained by culturing the Pantoea dispersae X24004 as active ingredients.

[0012] Preferably, the culture of Pantoea dispersa X24004 is prepared by the following method: inoculating Pantoea dispersa X24004 into LB liquid culture medium for cultivation to obtain the culture of Pantoea dispersa X24004.

[0013] Preferably, the LB liquid medium culture is cultured at 28° C. and 150-200 rpm for 18-24 h.

[0014] The third object of the present invention is to provide a biological fertilizer containing the dispersed Pantoea X24004 and fertilizer.

[0015] The fourth object of the present invention is to provide a seed treatment agent, which contains the dispersed Pantoea X24004, fludioxonil and seed coating agent adjuvants.

[0016] The fifth object of the present invention is to provide the use of the dispersed Pantoea X24004 or the biological agent in at least one of the following (1)-(2): (1) Prevent and control plant fungal diseases; (2) Promote plant growth.

[0017] Preferably, the plant fungal disease is corn ear rot and / or corn stalk rot. The corn stalk rot is Fusarium stalk rot. Fusarium stalk rot is corn stalk rot caused by Fusarium graminearum.

[0018] Preferably, the plant fungal disease is caused by Fusarium spp. Fusarium verticillioides ), Fusarium graminearum ( Fusarium graminearum ) and / or Pythium spp. ( Pythium arrhenomanes ) plant fungal diseases caused by.

[0019] Preferably, the plant is corn.

[0020] The sixth object of the present invention is to provide a method for preventing and controlling corn fungal diseases and promoting corn growth, comprising the following steps: applying the biological preparation to corn seeds or corn plants.

[0021] Preferably, the method of applying the biological preparation to corn seeds is to soak or coat the seeds; and the method of applying the biological preparation to corn plants is to irrigate the roots.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The dispersed Pantoea X24004 of the present invention can effectively inhibit the Fusarium graminearum) mycelium growth, inhibiting the occurrence of corn stalk rot and promoting the growth of corn plants; at the same time, it has a strong inhibitory effect on the mycelium growth of pathogenic fungi such as Fusarium spp. and Pythium spp., with a broad antibacterial spectrum, and has antibacterial activity against a variety of plant pathogenic fungi that harm corn crops; it is a broad-spectrum biological agent, biological fertilizer and seed treatment agent with good development and application prospects, and is of great significance for the biological control of corn fungal diseases.

[0023] The dispersed Pantoea X24004 of the present invention is obtained by separation and purification from the xylem tissue fluid inside corn. The strain itself is safe and non-toxic, safe to humans and animals, has good stability, is easy to store, and is environmentally friendly.

[0024] The dispersed Pantoea X24004 biological preparation of the present invention has simple culture conditions and is easy to prepare, and is convenient for large-scale promotion and application.

[0025] Preservation Instructions: The dispersed Pantoea of ​​the present invention ( Pantoea dispersa ) X24004 was deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC) on May 12, 2025, with the deposit number: CGMCC No. 34510, the deposit unit code: CGMCC-General Microbiology Center of China Culture Collection of Microorganisms, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a photo of the colony morphology of Pantoea dispersa X24004.

[0027] Figure 2 This is a Gram-stained photograph of dispersed Pantoea X24004 colonies.

[0028] Figure 3 This is a multilocus (16S rRNA, gyrB, rpoB) phylogenetic tree of Pantoea dispersa X24004.

[0029] Figure 4 It is the inhibition of Pantoea X24004 on a variety of plant pathogenic fungi; among them, A is the inhibition of Pythium spp. Pythium arrhenomanes ) (inhibition rate 44.72%), B is the inhibitory effect on Fusarium spp. ( Fusarium verticillioides ) (inhibition rate 54.04%).

[0030] Figure 5 Is to disperse Pantoea X24004 against Fusarium graminearum ( Fusarium graminearium ) was inhibited, with an inhibition rate of 59.70%.

[0031] Figure 6is the inhibition of dispersed Pantoea X24004 on the conidia germination of Fusarium graminearum; wherein, A is the conidia germination in the spore suspension of Fusarium graminearum P067 not inoculated with the activated bacterial solution of dispersed Pantoea X24004, and B is the conidia germination in the spore suspension of Fusarium graminearum P067 inoculated with the activated bacterial solution of dispersed Pantoea X24004.

[0032] Figure 7 The results show that the dispersed Pantoea X24004 has an inhibitory effect on corn stalk rot and a promoting effect on corn plant growth; A is the growth of corn plants; B is the aboveground plant height of corn plants; C is the aboveground fresh weight of corn plants; D is the incidence of stalk rot on the stems and roots of corn plants; E is the statistical result of the incidence of P067 and X24004+P067 treatments corresponding to D; CK is the blank control, X24004 is treated with the dispersed Pantoea X24004 bacterial solution, P067 is treated with the test strain Fusarium graminearum P067 spore suspension, and X24004+P067 is treated with the dispersed Pantoea X24004 bacterial solution and the test strain Fusarium graminearum P067 spore suspension.

[0033] Figure 8 It is to disperse the resistance of Pantoea X24004 to fludioxonil and fludioxonil seed coating agent.

[0034] Figure 9 The figure shows the effect of Pantoea dispersae X24004 on corn seed germination; A is a photo of corn seed germination; B is the statistical results of the root length and stem length of the germinated seedlings.

[0035] Figure 10 The synergistic effect of Pantoea dispersoids X24004 and fludioxonitrile on corn seed germination; A is a photo of corn seed germination under each treatment; B is the statistical results of root length and stem length of germinated seedlings under each treatment; CK is the blank control, X24004 is the seed soaking treatment with strain X24004 solution, Fludioxoni is the seed coating treatment with fludioxonitrile, and X+F or X24004+Fludioxoni is the simultaneous seed soaking treatment with strain X24004 solution and fludioxonitrile seed coating. DETAILED DESCRIPTION

[0036] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0037] Example 1 1. Isolation, Purification, Identification, and Antibacterial Activity Determination of Pantoea dispersa X24004 from Corn 1.1 Culture medium preparation TSB medium: 17 g trypticase, 1.5 g glucose, 3 g soytone, 2.5 g potassium dihydrogen phosphate, 5 g sodium chloride, add water to 1 L; autoclave at 121°C for 15 min.

[0038] LB medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to 1 L; autoclave at 121°C for 15 min.

[0039] PDA medium: Difco™ Potato Dextrose Agar 39 g, dilute to 1 L with water; autoclave at 121°C for 15 min.

[0040] 1.2 Isolation and purification of endophytic bacteria After disinfecting the surface of the second and third internodes at the base of the corn plant stem from bottom to top, the stem was cut off (about 30 cm from the ground), and the cross-section of the stem was covered with sterilized absorbent cotton. The opening of the sterilization bag was wrapped with sealing film and sealed. The sample was left to stand for 24 hours. The absorbent cotton soaked in xylem tissue fluid was recovered from the field and placed in a 50 mL centrifuge tube. The tube was centrifuged at 6000 g for 5 minutes, and the filtrate was collected to complete the xylem tissue fluid extraction.

[0041] Under sterile conditions, 4 mL of xylem tissue fluid was added to 36 mL of sterile water, and this step was repeated 5 times for gradient dilution. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 In the dilution gradient, 5 mL of each solution was added to 45 mL of trypticase soy broth (TSB) medium, mixed thoroughly, and transferred to a 96-well plate. Cultured at 28°C for 7 days. Three replicates were set for each dilution gradient. The optimal dilution was selected when the number of turbid wells was close to 1 / 3 of the culture plate.

[0042] At the optimal dilution, the bacteria in the turbid wells were pipetted into a new 96-well plate and numbered. 10 μL of the bacterial suspension was then streaked onto LB solid medium and incubated at 28°C for 24 h. A single colony was picked and placed into LB liquid medium and cultured overnight at 28°C with shaking at 150 rpm to obtain purified endophytic bacteria, including strain X24004. The bacterial suspension was pipetted into cryovials, an equal proportion of 50% glycerol was added, and the suspension was numbered and stored at -80°C.

[0043] 1.3 Test strains Fusarium graminearum Fusarium graminearum) strain P067, isolated from the stem base of corn plants infected with stalk rot at the Jiangcheng Experimental Base in Yunnan Province, and deposited in the Corn Disease Group of the Plant Protection Research Group of the Chinese Academy of Agricultural Sciences.

[0044] 1.4 Determination of the antibacterial activity of endophytic bacteria against Fusarium graminearum The purified endophytic bacteria were inoculated into LB liquid medium and cultured overnight at 28°C and 200 rpm to obtain an activated bacterial solution.

[0045] The antagonistic effect of the selected rhizospheric bacterial strains against the target strain was determined using the stand-off culture method. A 5 mm cake of the target strain was inoculated into the center of a PDA plate. 10 μL of activated bacterial solution was then inoculated 25 mm to the left and right of the plate. Three replicates of each activated rhizospheric bacterial solution were inoculated, and a blank control was inoculated with only the target strain. Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100.

[0046] The results showed that the endophytic bacterial strain X24004 had a significant inhibitory effect on the mycelial growth of Fusarium graminearum P067, producing obvious inhibition zones with an inhibition rate of 59.70%.

[0047] 1.5 Identification of endophytic bacteria X24004 1.5.1 Morphological identification The purified endophytic bacterium X24004 was streaked onto LB solid plates and incubated at 28°C for 24 h to observe the morphology of single colonies. The strain was then Gram-stained using a Gram staining kit (Beijing Solebold Technology Co., Ltd.) to observe the strain morphology and staining results.

[0048] The results showed that the colonies of endophytic bacteria X24004 were yellow, round, with smooth, moist, slightly convex surface and neat edges ( Figure 1 Gram-negative bacteria, stained red; cell morphology is straight or slightly curved ( Figure 2 ).

[0049] 1.5.2 Molecular identification Endophytic bacterium X24004 genomic DNA was extracted from activated bacterial culture using a bacterial genomic DNA extraction kit (Beijing Solaibao Technology Co., Ltd.). PCR amplification was performed using universal primers for the bacterial 16S rRNA gene, 27F / 1492R; primers for the gyrB gene, gyrB_F / R; and primers for the rpoB gene, rpoB_F / R. Primer sequences: 27F / 1492R (forward: AGAGTTTGATCCTGGCTCA; reverse: GGTTACCTTGTTACGACTT); gyrB_F / R (forward: GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA; reverse: AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT); and rpoB_F / R (forward: GGYTWYGAAGTNCGHGACGTDCA; reverse: TGACGYTGCATGTTBGMRCCCATMA).

[0050] The amplified sequences were spliced ​​using Phylosuite to obtain the 16S rRNA sequence (its nucleotide sequence is shown in SEQ ID NO.1), the gyrB sequence (its nucleotide sequence is shown in SEQ ID NO.2), and the rpoB sequence (its nucleotide sequence is shown in SEQ ID NO.3). The phylogenetic tree was constructed using MEGA 11. The endophytic bacteria X24004 and Pantoea dispersa were located in the same branch ( Figure 3 The results showed that strain X24004 was a dispersed Pantoea ( Pantoea dispersa ), hence the name Pantoea dispersa ( Pantoea dispersa )X24004.

[0051] 2. Determination of the antibacterial activity of Pantoea X24004 against various pathogens 2.1 Culture medium preparation Same as 1.1.

[0052] 2.2 Test strains Corn ear rot pathogen Fusarium spp. Fusarium verticillioides ), Fusarium graminearum, the pathogen of corn fusarium stalk rot ( Fusarium graminearum ), the pathogen of corn stalk rot, Pythium spp. Pythium arrhenomanes ), provided by the Corn Disease Research Group, Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0053] 2.3 Determination of antibacterial activity of Pantoea dispersa X24004 against various pathogens The test pathogen strain was inoculated into PDA medium for activation and plate antagonism test. The determination method was the same as 1.4.

[0054] The results showed that Pantoea dispersa X24004 had a broad-spectrum antibacterial activity (Table 1, Figure 4 、 Figure 5 ).

[0055] Table 1 Inhibitory effect of Pantoea dispersa X24004 on various pathogens 3. Determination of the Effect of Pantoea dispersa X24004 on Conidia Germination of Fusarium graminearum 3.1 Culture medium preparation Mung bean soup culture medium: Boil 10 g of mung beans in boiling water for 20 min, filter the liquid, add water to make up to 1 L, and sterilize by high-pressure steam at 121°C for 15 min.

[0056] 3.2 Test strains Same as 1.3.

[0057] 3.3 Observation of the effect of dispersed Pantoea X24004 on conidia germination of Fusarium graminearum using hydrophobic slides Pantoea X24004 was inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 24 h. The OD 600 = 1, and the activated bacterial suspension was set aside. The test strain Fusarium graminearum P067 was inoculated into a conidial medium, mung bean soup, and shaken at 25°C and 150 rpm for 7 days before use. A spore suspension of the shaken test strain Fusarium graminearum P067 was centrifuged at 8000 rpm for 10 minutes at room temperature and washed three times with sterile water. Two treatments, A and B, were set up: A was a blank control, and B was inoculated with the activated bacterial suspension of Pantoea dispersa X24004. 20 μL of the spore suspension was pipetted onto a hydrophobic glass slide for each treatment, with five replicates per group. The spores were incubated at 25°C for 3 hours, and the germination of the test strain Fusarium graminearum P067 was observed.

[0058] The results showed that after culturing at 25℃ for 3 h, the conidia of the test strain Fusarium graminearum P067 in group A germinated normally and the hyphae grew vigorously; the conidia germination of the test strain Fusarium graminearum P067 in group B was significantly inhibited ( Figure 6 ).

[0059] 4. Determination of the Growth-Promoting Ability of Pantoea dispersa X24004 on Maize Plants 4.1 Culture medium preparation Same as 1.1.

[0060] 4.2 Potted plant experiment to verify the ability of Pantoea dispersa X24004 to promote corn plant growth Mix nutrient soil and vermiculite in a volume ratio of 3:1, dry-heat sterilize at 121°C for 3 hours, and set aside. Plant three corn seeds in each pot, and each 15 pots are treated as a group. There are two treatments in total: A-blank control, B-inoculated with dispersed Pantoea X24004 bacteria. Dispersed Pantoea X24004 was inoculated into LB liquid medium and shaken at 28°C, 160 rpm, overnight. Centrifuge at 8000 rpm for 10 minutes at room temperature, wash three times with sterile water, and adjust the OD value. 600 = 1, set aside. At sowing, inoculate the corn in Group B with the bacterial suspension. Inoculate the roots of each corn plant with 10 mL of the dispersed Pantoea X24004 suspension. In Group A, irrigate the roots of each corn plant with 10 mL of sterile water. Maintain soil moisture throughout the entire growth process by irrigating with tap water. Observe plant growth 20 days after inoculation.

[0061] The results showed that: compared with the blank control of group A, the aboveground fresh weight of corn plants in group B increased by 12.77% and the aboveground plant height increased by 3.76% ( Figure 7 AC in).

[0062] 5. Determination of the efficacy of Pantoea dispersa X24004 against Fusarium stalk rot of corn 5.1 Culture medium preparation Same as 1.1 and 3.1.

[0063] 5.2 Test strains Same as 1.3.

[0064] 5.3 Potted plant experiment to verify the efficacy of Pantoea dispersa X24004 against Fusarium stalk rot of maize Mix nutrient soil and vermiculite in a volume ratio of 3:1, dry-heat sterilize at 121℃ for 3 h, and set aside. Plant 3 corn seeds in each flowerpot, and treat 15 pots as a group. There are three treatments in total: A, B, and C. A-blank control, B-inoculated with the test strain Fusarium graminearum P067 spore suspension, and C-inoculated with the dispersed Pantoea aeruginosa X24004 bacterial solution and the test strain Fusarium graminearum P067 spore suspension. Dispersed Pantoea aeruginosa X24004 was inoculated into LB liquid medium and shaken at 28℃ and 160 rpm overnight for later use; the test strain Fusarium graminearum P067 was inoculated into the spore-producing medium mung bean soup and shaken at 25℃ and 150 rpm for 7 d for later use. When sowing, take the dispersed Pantoea aeruginosa X24004 bacterial solution and centrifuge it at 8000 rpm at room temperature for 10 min, wash it three times with sterile water, and adjust the OD 600 = 1, inoculated into the corn of group C, 10 mL of dispersed Pantoea X24004 suspension was inoculated into the roots of each corn plant. Simultaneously, the spore suspension of Fusarium graminearum P067 was inoculated into the roots, and the concentration of the spore suspension was adjusted to 3×10 5The corn plants in Groups B and C were inoculated with 10 mL of spore suspension per plant. Moisturize the plants throughout the entire growth process. Plant growth and disease progression were observed 20 days after inoculation.

[0065] The results showed that: the growth of the three groups of corn plants was observed. The corn plants in group B were infected by the test strain Fusarium graminearum P067, and the stems of the plants in group A and group C were withered on the outside and rotted on the roots. Figure 7 Compared with the blank control of group A, the aboveground fresh weight of corn plants in group B decreased by 46.20%, and the aboveground plant height decreased by 29.42%; compared with the treatment of group B, the aboveground fresh weight of corn plants in group C increased by 26.09%, the aboveground plant height increased by 13.14%, and the incidence of stem base rot decreased by 24.57% ( Figure 7 E in).

[0066] 6. Determination of Fludioxonil Resistance of Pantoea dispersa X24004 6.1 Culture medium preparation Preparation of LB-fludioxonil-containing medium: LB medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to 1 L; autoclave at 121°C for 15 min.

[0067] Take 0.1 g of fludioxonil stock and dissolve it in 10 mL of dimethyl sulfoxide (DMSO). The obtained fludioxonil concentration is 10 4 ppm, pipette 1 mL of this concentration solution into 9 mL of 0.02% Tween 80, and further dilute to obtain a fludioxonil concentration of 10 3 Add 400 μL, 300 μL, 200 μL, 100 μL, and 20 μL of fludioxonil solution to 20 mL of LB medium, respectively, and plate the plates to obtain LB-fludioxonil-containing medium with final concentrations of 20 ppm, 15 ppm, 10 ppm, 5 ppm, and 1 ppm, respectively.

[0068] In addition, 10 μL of fludioxonil suspension seed coating agent (trade name: Cialis, active ingredient 25 g / L) was added to 20 mL of LB medium to make the final concentration of the fludioxonil seed coating agent 12.5 ppm, thereby obtaining a medium containing the fludioxonil seed coating agent.

[0069] 6.2 Determination of Fludioxonil Resistance of Pantoea dispersa X24004 Inoculate Pantoea X24004 in LB liquid medium and shake at 28°C and 160 rpm overnight. Streak 10 μL of the activated bacterial solution onto four plots of drug-treated medium, with five replicates for each concentration gradient. For a control group, streak 10 μL of the activated bacterial solution onto drug-free LB medium and incubate overnight at 28°C. Observe colony growth.

[0070] The results showed that compared with the control group, Pantoea dispersa X24004 could grow normally on the medium containing drugs at various concentrations and the medium containing fludioxonil seed coating agent, and no phenotype of fludioxonil inhibiting growth appeared; this indicated that Pantoea dispersa X24004 could coexist with fludioxonil and had fludioxonil resistance ( Figure 8 ).

[0071] 7. Determination of the Effect of Pantoea dispersa X24004 on Corn Seed Germination 7.1 Culture medium preparation Same as 1.1.

[0072] 7.2 Germination Paper Test Observing the Effect of Dispersed Pantoea X24004 on Corn Seed Germination Soak the germination paper in sterile water for 3 h and set aside. Disperse Pantoea X24004 into LB liquid medium and shake at 28°C and 160 rpm overnight. Wash away the medium and adjust the OD value with sterile water. 600 = 1, and prepare a dispersed Pantoea X24004 bacterial solution for later use. Corn cultivar B73 seeds were surface disinfected with 75% alcohol and then rinsed thoroughly with sterile water for later use. For the treatment group, soak corn seeds in the dispersed Pantoea X24004 bacterial solution for 2 hours, while for the control group, soak corn seeds in sterile water for 2 hours. Ten corn seeds were placed neatly on germination paper at 2 / 3 the width of the paper. Another sheet of germination paper was placed on top, rolled up from one side, and placed in a ziplock bag to maintain moisture. Germination of the corn seeds was observed after 8 days.

[0073] The results showed that compared with the blank control group, the corn seeds germinated normally under the treatment of dispersed Pantoea X24004 bacterial solution, without inhibition phenotype, and had biological safety. At the same time, the root length of corn increased by 31.43% and the stem length increased by 36.61%, showing a significant growth-promoting effect ( Figure 9 ).

[0074] 8. Determination of the Effects of Pantoea dispersa X24004 and Fludioxonil on Corn Seed Germination 8.1 Culture medium preparation Same as 1.1.

[0075] 8.2 Germination Paper Test Observing the Effect of Pantoea dispersa X24004 and Fludioxonil Co-treatment on Corn Seed Germination Soak the germination paper in sterile water for 3 h and set aside. Disperse Pantoea X24004 into LB liquid medium and shake at 28°C and 160 rpm overnight. Wash away the medium and adjust the OD value with sterile water. 600 =1, and strain X24004 bacterial solution was prepared for use. Corn variety B73 seeds were taken and four treatment groups were set up: (1) blank control, (2) seeds soaked in strain X24004 bacterial solution, (3) coated with fludioxonil seed coating agent, and (4) seeds soaked in strain X24004 bacterial solution + coated with fludioxonil seed coating agent. The strain X24004 bacterial solution soaking treatment was as follows: corn seeds were soaked in X24004 bacterial solution for 2 h. The control group was treated by soaking corn seeds in sterile water for 2 h. The fludioxonil seed coating agent coating treatment was as follows: 50 μL fludioxonil suspension seed coating agent (trade name: Celexa, active ingredient 25 g / L) was dissolved in 2 mL of sterile water, added to 50 g of corn seeds, shaken evenly, and blown dry in a fume hood. Place the corn seeds at 2 / 3 of the width of the germination paper, arrange 10 seeds neatly, cover them with another piece of germination paper, roll it up from one side, and put it in a ziplock bag to keep it moist. After 8 days, observe the germination of the corn seeds.

[0076] The results showed that compared with the blank control group, the three treatment groups all showed growth promotion phenotypes. The root length of corn seeds in the treatments of strain X24004 bacterial solution soaking, fludioxonil seed coating, and simultaneous treatments of strain X24004 bacterial solution soaking + fludioxonil seed coating increased by 52.75%, 82.24%, and 72.64%, respectively. The stem length increased by 25.84%, 58.61%, and 65.79%, respectively. Figure 10 ).

[0077] In summary, strain X24004 is a dispersed endophytic Pantoea of ​​corn, a Gram-negative bacterium with broad and highly effective antibacterial activity against a variety of plant pathogenic fungi. The bacterial suspension of strain X24004 can significantly inhibit the growth and expansion of Fusarium graminearum hyphae and spore germination, and has high antagonistic activity. Potted plant experiments have shown that the bacterial suspension of strain X240048 can play a role in preventing and treating Fusarium graminearum stem rot; strain X24004 can promote corn plant growth by applying it through root irrigation; strain X24004 can promote corn plant growth by using it as a seed treatment agent through seed soaking; strain X24004 can coexist with existing chemical seed dressing agents to synergistically promote corn seed germination and plant growth. The application of strain X24004 to prevent the occurrence of soil-borne diseases such as stem rot through biological control can reduce or avoid problems such as ecological and environmental pollution caused by chemical control, making it more environmentally friendly and efficient.

Claims

1. A dispersed strain of Pantoea ( Pantoea dispersa ) X24004, characterized in that, The deposit number is CGMCC No.34510.

2. A biological agent, characterized in that The invention contains the Pantoea dispersae X24004 according to claim 1, a culture of the Pantoea dispersae X24004 and / or a metabolite obtained by culturing the Pantoea dispersae X24004 as an active ingredient.

3. A biological fertilizer, characterized in that: Contains the dispersed Pantoea X24004 according to claim 1 and a fertilizer.

4. A seed treatment agent, characterized in that The invention contains the dispersed Pantoea X24004 according to claim 1, fludioxonil and seed coating agent excipients.

5. Use of the dispersed Pantoea X24004 of claim 1 or the biological preparation of claim 2 in at least one of the following (1)-(2): (1) Prevent and control plant fungal diseases; (2) Promote plant growth.

6. The use according to claim 5, characterized in that The plant fungal diseases are corn ear rot and / or corn stalk rot.

7. The use according to claim 5, characterized in that The plant fungal disease is caused by Fusarium spp. Fusarium verticillioides ), Fusarium graminearum ( Fusarium graminearum ) and / or Pythium spp. ( Pythium arrhenomanes ) plant fungal diseases caused by.

8. The use according to claim 5, characterized in that The plant is corn.

9. A method for preventing and controlling corn fungal diseases and promoting corn growth, characterized in that: The following steps are involved: Applying the biological preparation of claim 2 to corn seeds or corn plants.

10. The method according to claim 9, characterized in that For corn seeds, the application method is to soak or coat the seeds with the biological preparation; for corn plants, the application method is to irrigate the roots with the biological preparation.

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