Application of Pseudomonas chlororaphis and its synthetic bacterial flora in the prevention and control of corn root rot

Abstract

The present invention relates to the technical field of biocontrol bacteria, and in particular to the application of Pseudomonas chlororaphis and its synthetic bacterial flora in the prevention and treatment of corn root rot. The Pseudomonas chlororaphis RS36 of the present invention has a deposit number of CCTCC NO: M 20241983, can effectively inhibit the growth of Fusarium graminearum and Fusarium pseudoverticillium, has a good control effect on corn root rot, and is accompanied by a good growth-promoting effect, providing a new biocontrol bacterium for the prevention and treatment of corn root rot. Further, the synthetic bacterial flora composed of the above-mentioned Pseudomonas chlororaphis RS36 and the spindle-shaped lysine Bacillus RS104 separated by the present invention has a better control effect on Fusarium graminearum and Fusarium pseudoverticillium than a single strain, with a relative control effect of 41.08% and 37.78%, and the growth-promoting effect is also better than that of a single strain, and the aboveground biomass of corn can be increased by 36.15% to 65.05% compared with the diseased plants.

Description

Technical Field

[0001] The invention relates to the technical field of biocontrol bacteria, and in particular to application of Pseudomonas chlororaphis and synthetic bacterial flora thereof in preventing and controlling corn root rot. Background Art

[0002] Corn root rot is a major corn disease caused by a variety of pathogenic fungi (such as Fusarium verticillioides, Fusariumgraminearum, etc.), which poses a serious threat to corn growth and yield. The disease damages the root system, causing slow plant growth and wilting, and significantly reducing yield and stress resistance. The prevalence of corn root rot is affected by a variety of factors, including soil conditions, climate change, and agricultural management measures. Effective prevention and control measures include: implementing crop rotation to reduce pathogen accumulation, rational fertilization to enhance plant disease resistance, improving soil to improve drainage, using fungicides for chemical control, applying biological agents for biological control, and breeding disease-resistant varieties.

[0003] Specifically, crop rotation can effectively reduce the accumulation of pathogens, thereby reducing the risk of disease, but it may require adjustments to production plans and affect short-term economic benefits. Soil improvement to improve drainage has a significant effect on preventing root rot, but it involves additional cost investment. Chemical control measures such as fungicides can quickly control diseases, but long-term use may lead to increased resistance of pathogens. The breeding of disease-resistant varieties can fundamentally reduce the risk of diseases, but the development and promotion of new germplasm requires a long time and a large investment of resources. Biological control agents have good environmental protection by naturally inhibiting pathogens and reducing the use of chemical agents. Targeted research and development and optimization of biological control agents will help achieve effective disease management, thereby significantly improving the healthy growth and yield of corn. However, there is currently a lack of biological control bacteria that have good control effects on corn root rot. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of Pseudomonas chlororaphis and its synthetic bacterial flora in preventing and controlling corn root rot. The Pseudomonas chlororaphis RS36 and the synthetic bacterial flora provided by the present invention can effectively inhibit the growth of Fusarium graminearum and Fusarium verticillioides, have a good control effect on corn root rot, and provide a new biocontrol bacteria for the control of corn root rot.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a strain of Pseudomonas chlororaphis RS36. The preservation number of the Pseudomonas chlororaphis RS36 is CCTCC NO: M 20241983.

[0007] The present invention provides a synthetic bacterial community, comprising Lysinibacillus fusiformis RS104 and Pseudomonas chlororaphis RS36 described in the above technical scheme, wherein the preservation number of Lysinibacillus fusiformis RS104 is CCTCC NO: M 20241982.

[0008] The present invention provides a microbial agent, which includes the Pseudomonas chlororaphis RS36 described in the above technical solution or the synthetic bacterial flora described in the above technical solution.

[0009] Preferably, the number of viable bacteria of Pseudomonas chlororaphis RS36 or synthetic bacterial flora in the microbial agent is ≥ 1×10 8 CFU / mL.

[0010] The present invention provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps:

[0011] The Pseudomonas chlororaphis RS36 described in the above technical scheme or the synthetic bacterial flora described in the above technical scheme is cultured in a fermentation medium to obtain the microbial agent; the fermentation medium comprises: 1.5 g of citric acid, 10.0 g of glucose, 0.1 g of ferric citrate ammonium, 2.0 g of ammonium nitrate, 0.2 g of magnesium sulfate and 1 L of water.

[0012] Preferably, the culture temperature is 28-30° C. and the culture time is 5-7 d.

[0013] The present invention provides the use of Pseudomonas chlororaphis RS36 described in the above technical scheme, or the synthetic bacterial community described in the above technical scheme, or the microbial agent described in the above technical scheme, or the microbial agent prepared by the preparation method described in the above technical scheme in preventing and controlling plant fungal diseases and / or promoting plant growth, wherein the fungi include Fusarium graminearum and / or Fusarium pseudoverticillium.

[0014] Preferably, the plant is corn; and the plant disease is corn root rot.

[0015] The present invention provides a method for preventing and controlling plant fungal diseases and / or promoting plant growth, comprising: applying a microbial agent into soil planted with plants; the microbial agent is the microbial agent described in the above technical scheme or a microbial agent prepared by the preparation method described in the above technical scheme; the fungi include Fusarium graminearum and / or Fusarium pseudoverticillium.

[0016] Preferably, the amount of the microbial agent applied is ≥1×10 7 CFU / g soil.

[0017] Beneficial effects:

[0018] The present invention provides a strain of Pseudomonas chlororaphis RS36, and the deposit number of the Pseudomonas chlororaphis RS36 is CCTCC NO: M 20241983. The Pseudomonas chlororaphis RS36 provided by the present invention is a biocontrol bacterium isolated from a farmland where corn root rot occurs in Qiqihar City, Heilongjiang Province, and can effectively inhibit the growth of Fusarium graminearum and / or Fusarium pseudoverticillium, and has a good control effect on corn root rot, and the relative control effect on the pathogenic fungus Fusarium graminearum reaches 31.58%, accompanied by a good growth-promoting effect, and the aboveground biomass of corn can be increased by 30.87% compared with the diseased plant; it has beneficial biological functions such as producing IAA, solubilizing phosphorus, fixing nitrogen, and producing iron carriers, and provides a new biocontrol bacterium for the control of corn root rot.

[0019] Furthermore, the synthetic bacterial community composed of the above-mentioned Pseudomonas chlororaphis RS36 and the spindle-shaped lysine Bacillus RS104 isolated by the present invention has a better control effect on corn root rot than a single strain, and the relative control effect on Fusarium graminearum reaches 41.08%, and the growth-promoting effect is also better than a single strain, which can increase the aboveground biomass of corn by 65.05% compared with the diseased plants. The relative control effect on the pathogen Fusarium pseudoverticillium reaches 37.78%, which can increase the aboveground biomass of corn by 36.15% compared with the diseased plants.

[0020] Biological Deposit Description

[0021] Pseudomonas chlororaphis RS36, classified as Pseudomonas chlororaphis RS36, was deposited in the China Center for Type Culture Collection (CCTCC) on September 14, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University, and the deposit number is CCTCC NO: M 20241983.

[0022] Lysinibacillus fusiformis RS104, classified and named Lysinibacillus fusiformis RS104, was deposited in the China Center for Type Culture Collection (CCTCC) on September 14, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University, and the deposit number is CCTCC NO: M 20241982. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 This is the colony morphology of Pseudomonas chlororaphis RS36;

[0025] Figure 2 The colony morphology of spindle-shaped lysinibacillus RS104;

[0026] Figure 3 is the phylogenetic tree of Pseudomonas chlororaphis RS36;

[0027] Figure 4 is the phylogenetic tree of Lysinibacillus fusiformis RS104;

[0028] Figure 5 The results of the inhibition experiment of Pseudomonas chlororaphis RS36, Bacillus fusiformis RS104 and the composite bacterial agent of the two on Fusarium graminearum are shown in the figure;

[0029] Figure 6 The graph is the result of the inhibition experiment of Pseudomonas chlororaphis RS36, Bacillus fusiformis RS104 and the composite bacterial agent of the two on Fusarium verticillioides;

[0030] Figure 7 The results of the assay of the production of elongin by the bacterial solution of Pseudomonas chlororaphis RS36, the bacterial solution of Bacillus fusiformis RS104 and the composite bacterial agent of the two are shown;

[0031] Figure 8 The figure is a result of the determination of the phosphate solubilization amount of the bacterial solution of Pseudomonas chlororaphis RS36, the bacterial solution of Bacillus fusiformis RS104 and the composite bacterial agent of the two;

[0032] Fig. 9 This is a graph showing the experimental results of using a bacterial solution of Pseudomonas chlororaphis RS36, a bacterial solution of Bacillus fusiformis RS104, and a composite bacterial agent of the two to control corn root rot caused by the pathogen Fusarium graminearum;

[0033] Fig.10 This is the experimental result of using bacterial solution of Pseudomonas chlororaphis RS36, bacterial solution of Bacillus fusiformis RS104 and the composite bacterial agent of the two to control corn root rot caused by the pathogen Fusarium oxysporum. DETAILED DESCRIPTION

[0034] The invention provides a strain of Pseudomonas chlororaphis RS36. The preservation number of the Pseudomonas chlororaphis RS36 is CCTCC NO: M 20241983.

[0035] The Pseudomonas chlororaphis RS36 provided by the present invention is a biocontrol bacterium isolated from a farmland where corn root rot occurs in Qiqihar City, Heilongjiang Province. The colony appears orange-red on the LB plate, with neat edges, regular circles, smooth surfaces, and opaque ( Figure 1 ), can effectively inhibit the growth of Fusarium graminearum and / or Fusarium pseudoverticillioides, with inhibition rates of 9.00% and 24.19% respectively, and has a good control effect on corn root rot, with a relative control effect of 31.58% on Fusarium graminearum, accompanied by a good growth-promoting effect, which can increase the aboveground biomass of corn by 30.87% compared with diseased plants; it has beneficial functions such as producing IAA, solubilizing phosphate, fixing nitrogen, and producing iron carriers, providing a new biocontrol bacterium for the control of corn root rot.

[0036] The present invention provides a synthetic bacterial community, comprising lysinibacillus spindle RS104 and Pseudomonas chlororaphis RS36 described in the above technical scheme, wherein the preservation number of lysinibacillus spindle RS104 is CCTCC NO: M20241982.

[0037] The spindle-shaped lysinibacillus RS104 of the present invention is a biocontrol bacterium isolated from a farmland where corn root rot occurs in Qiqihar City, Heilongjiang Province. The colony appears milky white, opaque, with complete edges, round, and moist on the LB plate ( Figure 2 ), the composite flora composed of it and Pseudomonas chlororaphis RS36 has a better antagonistic effect than a single strain, with an inhibition rate of 24.33% on Fusarium graminearum and 32.35% on Fusarium pseudoverticilli. It has a better control effect on corn root rot, and the relative control effect on the pathogen Fusarium graminearum reaches 41.08%, and the growth-promoting effect is also better than a single strain, which can increase the aboveground biomass of corn by 65.03% compared with the diseased plants; the relative control effect on the pathogen Fusarium pseudoverticilli reaches 37.78%, which can increase the aboveground biomass of corn by 36.15% compared with the diseased plants. The synthetic flora provided by the present invention provides a new solution for the effective prevention and control of corn root rot, which is of great significance for promoting plant health and increasing crop yields.

[0038] The present invention provides a microbial agent, which includes the Pseudomonas chlororaphis RS36 described in the above technical solution or the synthetic bacterial flora described in the above technical solution.

[0039] As an embodiment, the number of viable bacteria of Pseudomonas chlororaphis RS36 or the synthetic bacterial flora in the microbial agent is ≥ 1×10 8 CFU / mL.

[0040] The present invention provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps:

[0041] The Pseudomonas chlororaphis RS36 described in the above technical scheme or the synthetic bacterial flora described in the above technical scheme is cultured in a fermentation medium to obtain the microbial agent; the fermentation medium comprises: 1.5 g of citric acid, 10.0 g of glucose, 0.1 g of ferric citrate ammonium, 2.0 g of ammonium nitrate, 0.2 g of magnesium sulfate and 1 L of water.

[0042] As one embodiment, the culture temperature is 28-30°C, and the culture time is 5-7 days. As another embodiment, the culture temperature is 30°C, and the culture time is 6 days.

[0043] Based on the above advantages, the present invention provides the use of Pseudomonas chlororaphis RS36 described in the above technical scheme, or the synthetic bacterial community described in the above technical scheme, or the microbial agent described in the above technical scheme, or the microbial agent prepared by the preparation method described in the above technical scheme in preventing and controlling plant fungal diseases and / or promoting plant growth, wherein the fungi include Fusarium graminearum and / or Fusarium pseudoverticillium.

[0044] As an embodiment, the plant may be corn.

[0045] As an embodiment, the plant fungal disease is corn root rot.

[0046] Based on the above advantages, the present invention provides a method for preventing and controlling plant fungal diseases and / or promoting plant growth, comprising: applying a microbial agent into soil planted with plants; the microbial agent is the microbial agent described in the above technical scheme or a microbial agent prepared by the preparation method described in the above technical scheme; the fungi include Fusarium graminearum and / or Fusarium pseudoverticillium.

[0047] As an embodiment, the amount of the microbial agent applied is ≥1×10 7 CFU / g soil.

[0048] As an embodiment, the plant may be corn.

[0049] As an embodiment, the plant fungal disease is corn root rot.

[0050] To further illustrate the present invention, the application of Pseudomonas chlororaphis and its synthetic flora provided by the present invention in preventing and controlling corn root rot is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The pathogenic bacteria Fusarium graminearum and Fusarium verticillioides used in the embodiments of the present invention are both commercially available pathogenic strains. Among them, Fusarium graminearum was purchased from Mingzhoubio with a product number of BMZ097952; Fusarium verticillioides was purchased from Mingzhoubio with a product number of BMZ075254.

[0052] Example 1

[0053] Rhizosphere soil samples of maize root rot were obtained from Qiqihar City, Heilongjiang Province. 10 g of the mixed soil sample was added to a 250 mL Erlenmeyer flask containing 100 mL of sterile water, and the mixture was shaken at 180 rpm for 30 minutes and allowed to stand for 2 hours. 1 mL of the supernatant was diluted in 9 mL of sterile water to 10 -3 , 10 -4 , 10 -5 , 0.1 mL of each concentration gradient was spread on LB medium plates and cultured at 30°C for 1 to 3 days. After the colonies grew, they were further isolated, purified and identified. The pathogens Fusarium graminearum and Fusarium pseudoverticillium were used as targets to screen for antagonistic and probiotic traits, and strains with significant inhibitory effects on both target pathogens and good probiotic functions were selected, named RS36 and RS104, respectively.

[0054] Morphological characteristics of strain RS36: The colonies on the LB plate are orange-red, with neat edges, regular circles, smooth surfaces, and opaque ( Figure 1 ).

[0055] Morphological characteristics of strain RS104: The colonies on the LB plate are milky white, opaque, with complete edges, round, and moist surface ( Figure 2 ).

[0056] 16S rRNA gene sequence determination: The genomic DNA of strain RS36 and strain RS86 were used as templates, and the 16S rRNA gene sequence was amplified using universal primers 27F and 1492R. The primer sequences are as follows:

[0057] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1;

[0058] 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 2.

[0059] PCR reaction system (50 μL): primer 27F 1.5 μL, primer 1492R 1.5 μL, template DNA 1 μL, 2×Repid Taq Master Mix 25 μL, sterile water 21 μL. PCR reaction conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30s, 53℃ annealing for 30s, 72℃ extension for 30s, 30 cycles; final extension at 72℃ for 10 min. The amplified product was detected by agarose gel electrophoresis and sequenced by Beijing Qingke Biotechnology Co., Ltd. The sequencing results of strain RS36 are shown in SEQ ID No. 3, and the sequencing results of strain RS104 are shown in SEQ ID No. 4, as follows:

[0060] SEQ ID NO.3:

[0061]

[0062] SEQ ID NO.4:

[0063]

[0064] The phylogenetic tree of strain RS36 and the model strain was constructed, and it was found that the sequence homology between strain RS36 and the model strain Pseudomonaschlororaphis subsp. aurantiaca DSM 19603r was the highest, reaching 100% ( Figure 3 ). A phylogenetic tree was constructed between strain RS104 and the model strain, and it was found that the sequence homology between strain RS104 and the model strain Lysinibacillusfusiformis NBRC 15717 was the highest, reaching 99.38% ( Figure 4 Based on this, strains RS36 and RS104 were classified as Pseudomonas sp. and Lysinibacillus sp., respectively.

[0065] Example 2 Inhibitory effects of single strains and composite strains on the main pathogens of corn root rot

[0066] The plate confrontation culture method was used to test the inhibitory effects of the single strains RS36, RS104 and the composite bacterial agent of the two strains obtained in Example 1 on the pathogenic bacteria Fusarium graminearum and Fusarium pseudoverticillium. The two single strains were inoculated into LB medium for activation, and the bacterial solution was diluted to 10 7 CFU / mL, composite bacterial agent (denoted as SynCom) is a mixture of equal volumes of bacterial suspensions of a single strain to 10 7 CFU / mL; the two pathogenic fungi were inoculated into PDA medium for activation, and 60 μL of single bacterial solution and 60 μL of compound bacterial agent were evenly inoculated on the sterile filter paper on one side of the PDA medium, and cultured at 28°C for 2 days; a 5 mm diameter puncher was used to punch the bacterial blocks of the same age at the edge of the pathogenic fungal colony, and the hyphae were placed 2 cm away from the filter paper with the hyphae facing down. After constant temperature culture at 25°C for 5 days, the growth of the hyphae was evaluated and compared with the control group in which the bacterial solution was replaced by sterile water. The results are shown in Figure 5 and Figure 6 .

[0067] The results showed that RS36 and RS104 had good inhibitory effects on the pathogenic fungi Fusarium graminearum and Fusarium pseudo-verticillium, with the inhibition rates of RS36 on Fusarium graminearum and Fusarium pseudo-verticillium being 9.00% and 24.19%, respectively, and the inhibition rates of RS104 on Fusarium graminearum and Fusarium pseudo-verticillium being 4.11% and 14.97%, respectively. The composite bacterial agent had a better antagonistic effect than a single strain, with an inhibition rate of 24.33% on Fusarium graminearum and 32.35% on Fusarium pseudo-verticillium, both of which were significantly higher than a single strain. This indicated that the composite bacterial agent in the present invention had a stronger inhibitory effect on both corn root rot pathogenic fungi.

[0068] Example 3 Determination of probiotic properties of single strains RS36 and RS104 and their composite bacterial preparations

[0069] (1) Determination of nitrogen fixation ability of strains

[0070] Under sterile conditions, strains RS36 and RS104 in Example 1 were inoculated into low-nitrogen culture medium using an inoculation loop, inverted and cultured at 28°C for 7 days, transferred three times to observe whether there was growth and record the growth status; the formula of the low-nitrogen culture medium is as follows: 0.03 g calcium nitrate, 0.15 g disodium hydrogen phosphate, 0.1 g calcium chloride, 0.12 g magnesium sulfate, 0.1 g potassium dihydrogen phosphate, 0.05 g ferric citrate, 1.0 mL trace elements, and 1000 mL distilled water; the trace element formula is: 2.86 g boric acid, 1.81 g manganese sulfate, 0.8 g copper sulfate, 0.02 g molybdic acid, and distilled water is made up to 1000 mL.

[0071] (2) ACC deaminase activity assay

[0072] A single strain was inoculated into fresh YN medium for activation. After overnight culture for about 16 hours, it was inoculated into a 50 ml conical flask at a 5% (v / v) inoculation rate. After shaking culture for 24 hours, the strain in DF culture medium was inoculated onto ADF solid medium and cultured in a 28°C incubator for 7 days to observe the growth of the colonies.

[0073] YN medium: sucrose 10.0 g, ammonium sulfate 1.0 g, potassium hydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, yeast extract 0.5 g, sodium chloride 0.1 g, calcium carbonate 0.5 g, distilled water 1 L, pH 7.2.

[0074] DF medium: 4 g potassium dihydrogen phosphate, 6 g disodium hydrogen phosphate, 0.2 g magnesium sulfate, 2 g glucose, 2 ml gluconic acid, 2 g citric acid, 2 g ammonium sulfate, 1 L distilled water, pH 7.2.

[0075] ADF medium: Add the sterilized 1-aminocyclopropanecarboxylic acid (ACC) to the DF solid medium without ammonium sulfate to a final concentration of 3 mmol / L, which is the ADF medium.

[0076] (3) Acidity

[0077] A single strain was inoculated into a conical flask containing YN liquid medium at a 1% (v / v) inoculation volume, and three replicates were set. The flask was placed on a shaker at 30°C and cultured at 150 rpm for 72 h. Centrifuged at 8000 rpm for 10 min, and the pH value of the supernatant was measured with a pH meter.

[0078] (4) Experiment on the determination of the production of elongin by strains

[0079] Activate and prepare a single strain suspension in YN liquid medium (OD 600 =1), the bacterial suspension of the composite strain was set to OD 600 =1, the bacterial suspensions of two single strains were mixed in equal volumes. The bacterial suspensions of the single strain and the composite strain were inoculated into YN medium with a tryptophan concentration of 0.5 mg / mL at a 1% (v / v) inoculation volume, cultured in a shaker at 30°C and 200 rpm for 3 days, centrifuged at 6000 rpm for 10 min, 500 µL of the supernatant was added with 25 µL of 10 mM orthophosphoric acid, and then 1 mL of Sackowki's colorimetric agent was added and mixed thoroughly, reacted in the dark for 30 min, and the absorbance at 530 nm was measured; the Sackowki's colorimetric agent was prepared as follows: 150 mL of concentrated sulfuric acid was slowly added to 250 mL of deionized water along the wall of the cup, and after cooling, 7.5 mL of 0.5 mol / L FeCl3 solution was added. Results are shown in Figure 7 .

[0080] The results showed that compared with a single strain, the amount of IAA produced by the mixed bacterial agent reached 11.44 mg / L, which was significantly higher than the amount of IAA produced by single strains RS36 (2.74 mg / L) and RS104 (7.01 mg / L).

[0081] (5) Experiment on determination of iron carrier production by strains

[0082] The bacterial suspension of a single strain and a composite strain was prepared according to the method of step (4). The bacterial suspension was inoculated into MKB liquid culture medium (TOPBIO, MD193) at a 1% (v / v) inoculation amount. After culturing in a shaker at 30°C and 200 rpm for 48 h, the suspension was centrifuged at 10,000 rpm for 10 min. 1 mL of the supernatant was mixed with 1 mL of CAS detection solution and diluted to 15 mL. After 1 h of reaction, the absorbance value (As) was measured at 630 nm. The uninoculated MKB culture medium was used as the reference value (Ar). Siderophore activity units (SAU) = [(Ar−As) / Ar] × 100. The results are shown in Table 1.

[0083] Table 1 Siderophore activity and siderophore production capacity of single strains and combined strains

[0084] Strain number Siderophore activity (SAU) Siderophore production capacity RS36 38.69±4.06 b ++ RS104 20.44±1.26 c + RS36+RS104 50.12±1.84 a +++

[0085] Note: For every 0.2 decrease in the absorbance ratio As / Ar, a “+” is added. The more “+” there are, the stronger the iron carrier capacity is.

[0086] The results showed that the RS36 strain had a strong ability to produce siderophores, with a siderophore activity of 38.69 SAU. RS104 also had the ability to produce siderophores, which was significantly lower than RS36 and the compound bacterial agent, with a siderophore activity of 20.44. The compound bacterial agent had the strongest ability to produce siderophores, reaching 50.12, which was significantly higher than that of a single strain. The above results showed that both single strains and compound strains had growth-promoting potential.

[0087] (6) Experiment on the determination of the amount of phosphorus solubilized by the strain

[0088] According to the method of step (4), a bacterial suspension of a single strain and a bacterial suspension of a composite strain were prepared. The bacterial suspension was inoculated into NBRIP inorganic phosphorus (REBIO, T3037) and Montana organic phosphorus liquid medium (Coolaber, MM5060+Y) at a 1% (v / v) inoculum. After culturing at 30°C and 200 rpm for 7 days, the culture was centrifuged at 3000 rpm for 10 min. 1 mL of the supernatant was mixed with 2 mL of molybdenum antimony antichromogen and shaken well. The solution was placed in a 40°C water bath for 20 min. After cooling, the absorbance of the solution was measured at 820 nm, and the phosphorus content of the sample was calculated from the standard curve. The molybdenum antimony antichromogen was prepared as follows: a solution containing 0.4% ammonium molybdate and 0.5 mol / L H2SO4 was mixed with a 10% ascorbic acid solution in a volume ratio of 6:1, and stored at 4°C in the dark. The solution must be prepared on the same day. Results are shown in Table 1. Figure 8 .

[0089] The results showed that the compound bacterial agent had the highest ability to decompose organic and inorganic phosphorus, especially in terms of inorganic phosphorus. RS104 almost did not decompose inorganic phosphorus, and the effective phosphorus accumulation was only 0.047 mg / L. The effective phosphorus accumulation of RS36 was 15.09 mg / L, while the effective phosphorus accumulation of the compound bacterial agent reached 1.66 times that of RS36, which was 25.12 mg / L.

[0090] A series of preliminary qualitative determinations were made on properties such as acid production, IAA production, siderophore production, nitrogen fixation, phosphate solubilization, and ACC deaminase, and the final results are shown in Table 2.

[0091] Table 2 Results of determination of probiotic properties of different strains

[0092] Measurement indicators RS36 RS104 Acidity ＋ － Produced by IAA ＋ ＋ Siderophore production ＋ ＋ Phosphate solubilization ＋ ＋ Nitrogen fixation ＋ ＋ ACC deaminase ＋ ＋

[0093] Note: "+" indicates that the strain has this property, and "-" indicates that the strain does not have this property. Acidogenicity means that the pH of the strain is below 5.0; IAA production means that the IAA content is above 5 mg / L.

[0094] Example 4 Preparation of synthetic microbial agent

[0095] The culture medium used in the present invention is:

[0096] LB medium: peptone 10.0 g, yeast powder 5.0 g, sodium chloride 5.0 g, deionized water 1 L. Add 1.5% agar powder to the solid medium.

[0097] Fermentation medium: 1.5 g citric acid, 10.0 g glucose, 0.1 g ferric citrate ammonium, 2.0 g ammonium nitrate, 0.2 g magnesium sulfate, 1 L deionized water.

[0098] Detailed implementation steps of the present invention:

[0099] (1) Streak strains RS36 and RS104 on LB plates and culture at 30°C for 2 days.

[0100] (3) Pick up the single colonies of the above cultured strains and place them in liquid LB medium. Cultivate them in a shaking flask at 30°C and 180 rpm until the logarithmic growth phase. Dilute them to 10 7 CFU / mL, the seed solution of strains RS36 and RS104 was obtained;

[0101] (3) The seed liquid was inoculated into a 25 L fermenter at a 5% (v / v) inoculation rate for expansion culture. After reaching the logarithmic growth phase, the seed liquid was inoculated into a 500 L fermenter at a 5% (v / v) inoculation rate for continued fermentation culture. The culture medium was used as the fermentation medium. The fermentation culture temperature was 30°C, the volume ratio of sterile air to fermentation medium was 1:0.8~1.2, the stirring speed was controlled at 180~200 rpm, and the total production time of the inoculant was 5~7 days. After the fermentation, the concentration was 10 8 CFU / mL, the fermentation liquid was packaged in plastic buckets to make liquid inoculants of strains RS36 and RS104;

[0102] (4) The preparation method of the composite bacterial agent is as follows: the seed liquid of strains RS36 and RS104 is inoculated into a 25 L fermenter at a 2.5% (v / v) inoculation rate, and then inoculated into a 500 L fermenter at a 5% inoculation rate after reaching the logarithmic growth phase for further fermentation and culture; the fermentation temperature is 30°C, the volume ratio of sterile air to fermentation medium is 1:0.8~1.2, the stirring speed is controlled at 180~200 rpm, and the total production time of the bacterial agent is 5~7 days. After the fermentation is completed, the concentration is 10 8 CFU / mL, the fermented bacterial liquid was packaged in plastic barrels to make a composite bacterial agent.

[0103] Example 5 Experiment on the Control of Corn Root Rot by Single Bacteria and Compound Bacterial Agents

[0104] The susceptible corn seeds were selected as Jidan 37, which were germinated at 25℃ after sterile treatment. The seedlings with uniform growth were divided into 7 groups, namely blank control group (denoted as CK), control group 1 (denoted as Fg), control group 2 (denoted as Fv), strain RS36 treatment group (denoted as Fg+RS36), strain RS104 treatment group (denoted as Fg+RS104), compound bacterial agent treatment group 1 (denoted as Fg+RS36+RS104) and compound bacterial agent treatment group 2 (denoted as Fv+RS36+RS104). The specific treatments are as follows:

[0105] CK group: no pathogen Fusarium graminearum inoculation, no liquid bacterial agent prepared in Example 4 was applied;

[0106] Fg group: inoculated with Fusarium graminearum (denoted as Fg), the inoculation amount was 10 7 spores / g soil, without applying the liquid bacterial agent prepared in Example 4;

[0107] Fv group: inoculated with Fusarium verticillium (denoted as Fv), the inoculation amount was 10 7 spores / g soil, without applying the liquid bacterial agent prepared in Example 4;

[0108] Fg+RS36 group: inoculated with Fusarium graminearum (denoted as Fg), with an inoculation amount of 10 7 spores / g soil, and the liquid inoculum of strain RS36 prepared in Example 4 was applied at an amount of 10 7 CFU / g soil;

[0109] Fg+RS104 group: inoculated with Fusarium graminearum (denoted as Fg), with an inoculation amount of 10 7 spores / g soil, and the liquid inoculum of strain RS104 prepared in Example 4 was applied at an amount of 10 7 CFU / g soil;

[0110] Fg+RS36+RS104 group: inoculated with Fusarium graminearum (denoted as Fg), with an inoculation amount of 10 7 spores / g soil, the composite bacterial agent prepared in Example 4 was applied at an application rate of 10 7 CFU / g soil;

[0111] Fv+RS36+RS104 group: inoculated with Fusarium verticillium (denoted as Fv), with an inoculation amount of 10 7 spores / g soil, the composite bacterial agent prepared in Example 4 was applied at an application rate of 10 7 CFU / g soil.

[0112] After 3 weeks of cultivation under the conditions of 16 h daytime / 25°C and 8 h nighttime / 22°C, the 7 groups of seedlings were photographed, and the root fresh weight, aboveground fresh weight, disease index, relative protection effect and hydrogen peroxide content of the plants were measured to evaluate the disease inhibition effect. The calculation method of disease index and relative protection effect is as follows:

[0113] Disease index = (root length of diseased root / total root length) × 100%.

[0114] Relative prevention effect = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%, where the control group and treatment group are the results of treatment with the same pathogen.

[0115] The results for the pathogen Fusarium graminearum are shown in Fig. 9 The results of the pathogenic fungus Fusarium spp. Fig.10 , where different letters represent significant differences among different groups.

[0116] The results showed that compared with the control of only applying pathogen Fg, single strains RS36, RS104 and compound bacteria significantly reduced the disease index of corn roots. The disease index of corn plants using RS36 and RS104 was 55.59% and 57.08%, respectively, and the relative control efficiency reached 31.58% and 29.75%, respectively. The relative control efficiency of the compound bacteria reached 41.08%. At the same time, both single strains and compound bacteria significantly reduced the accumulation of hydrogen peroxide in the aboveground part of corn (Fg: 8.38 mmol / g; CK: 5.02 mmol / g; Fg+RS36: 5.00 mmol / g; Fg+RS104: 5.01 mmol / g; Fg+RS36+RS104: 4.12 mmol / g). This also shows that single strains and compound bacteria can significantly inhibit corn root rot caused by Fusarium graminearum, and the disease inhibition effect of compound bacteria is better than that of single strains. The morbidity index caused by Fusarium oxysporum was 56.64%, and the relative prevention effect of the combined bacterial agent on the pathogen Fusarium oxysporum reached 37.78%.

[0117] At the same time, compared with the diseased plants, both single strains and compound agents promoted the growth of the aboveground part and roots of corn. In the aboveground part of corn, compared with the treatment group with only pathogen Fg (5.15g), the application of single strains RS36, RS104 and compound agents increased the aboveground biomass by 30.87%, 40.97% and 65.05%, reaching 6.74 g, 7.26 g and 8.50g, respectively. Compared with the control group without bacteria (6.42g), the RS36 and RS104 treatment groups increased the aboveground biomass by 4.98% and 13.08%, respectively, while the compound agent increased it by 32.40%. This shows that single strains RS36, RS104 and compound agents all promote the growth of the aboveground part of corn, and the compound agent has the best effect. Under the treatment of pathogen Fv, the aboveground biomass of corn was 4.73g, which was 26.32% lower than that of the blank control. After the application of the composite bacterial agent, the aboveground biomass of corn increased by 36.15% compared with the Fv treatment group.

[0118] In the corn root, compared with the pathogen Fg treatment (1.39g), the application of single bacteria RS36, RS104 and compound bacteria significantly increased the root fresh weight, reaching 1.73g, 1.92g and 2.03g respectively. Under the pathogen Fv treatment, the fresh weight of corn roots was only 0.98g. After the application of the compound bacteria, the fresh weight of corn roots increased by 71.43%.

[0119] In summary, the single strains RS36 and RS104 and their composite bacterial agents provided by the present invention have good control effects on corn root rot; and are accompanied by good growth-promoting effects and have beneficial functions such as producing IAA, solubilizing phosphate, fixing nitrogen, and producing iron carriers, providing a new way for the control of corn root rot.

[0120] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A synthetic bacterial community, characterized in that Including Bacillus fusiformis ( Lysinibacillus fusiformis ) RS104 and Pseudomonas chlororaphis ( Pseudomonas chlororaphis ) RS36, the preservation number of the spindle-shaped lysinibacillus RS104 is CCTCC NO: M 20241982, and the preservation number of the green needle Pseudomonas RS36 is CCTCC NO: M 20241983.

2. A microbial agent, characterized in that: The microbial agent comprises the synthetic bacterial flora according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The number of viable bacteria in the synthetic flora of the microbial agent is ≥ 1×10 8 CFU / mL.

4. The method for preparing the microbial agent according to claim 2 or 3, characterized in that: The following steps are involved: The synthetic bacterial community described in claim 1 is cultured in a fermentation medium to obtain the microbial agent; the fermentation medium comprises: 1.5 g citric acid, 10.0 g glucose, 0.1 g ferric citrate ammonium, 2.0 g ammonium nitrate, 0.2 g magnesium sulfate and 1 L water.

5. The preparation method according to claim 4, characterized in that: The culture temperature is 28-30°C; the culture time is 5-7 days.

6. Use of the synthetic bacterial flora according to claim 1 or the microbial agent according to claim 2 or 3 or the microbial agent prepared by the preparation method according to claim 4 or 5 in preventing and controlling plant fungal diseases and / or promoting plant growth, wherein the fungi include Fusarium graminearum ( Fusarium graminearum ) and / or Pseudomonas verticillasters ( Fusarium verticillioides ).

7. The use according to claim 6, characterized in that: The plant is corn; and the plant disease is corn root rot.

8. A method for preventing and controlling plant fungal diseases and / or promoting plant growth, characterized in that: include: The microbial agent is applied to soil planted with plants; the microbial agent is the microbial agent described in claim 2 or 3 or the microbial agent prepared by the preparation method described in claim 4 or 5; the fungi include Fusarium graminearum and / or Fusarium pseudoverticillium.

9. The method according to claim 8, characterized in that The amount of the microbial agent applied is ≥1×10 7 CFU / g soil.

10. The method according to claim 8, characterized in that The plant is corn; and the plant disease is corn root rot.

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