Alcaligenes faecalis and application of disease prevention and growth promotion

Abstract

The application discloses an alcaligenes faecalis strain and a disease prevention and growth promotion application thereof. The strain has been preserved in the China General Microbiological Culture Collection Center on November 1, 2023, and the preservation number is CGMCC No. 28835, and the preservation name is alcaligenes faecalis WYH-571. The strain is applied to the field of agricultural biological control, can form a biological membrane to stably colonize in the rhizosphere of plants, and can produce IAA, iron carrier and other substances to promote the growth of plants.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biological control technology, specifically relating to a strain of alkali-producing bacteria with disease prevention and growth-promoting functions (…). Alcaligenes faecalis WYH-571 and its applications. Background Technology

[0002] Plant diseases pose a serious threat to global food production and security. The long-term use of chemical pesticides and fertilizers has exacerbated problems such as environmental pollution, pesticide resistance in bacterial strains, and imbalances in soil microecology. There is an urgent need to explore microbial products that can replace chemical pesticides and fertilizers. Although a large number of microbial products already exist, their low colonization rate and short duration of effectiveness in the field have made the discovery of broad-spectrum and highly efficient microorganisms from plant-related microbial communities such as rhizosphere and root endophytes a research hotspot for scholars both domestically and internationally. Summary of the Invention

[0003] Based on new research findings, the purpose of this invention is to provide a strain of alkali-producing bacteria with disease prevention and growth-promoting functions ( ). Alcaligenes faecalis WYH-571 and its application in agricultural production.

[0004] The fecal alkali-producing bacteria provided by this invention ( Alcaligenes faecalis WYH-571, this strain is classified and named Alcaligenes faecalis. Alcaligenes faecalis It was deposited on November 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 28835.

[0005] The above-mentioned fecal alkali-producing bacteria ( Alcaligenes faecalis The bacterial suspension, fermentation broth, or fermentation product of WYH-571 are also within the scope of protection of this invention.

[0006] The above-mentioned fecal alkali-producing bacteria ( Alcaligenes faecalis The application of WYH-571 bacterial suspension, fermentation broth, or fermentation products in promoting plant growth is also within the scope of protection of this invention.

[0007] The above-mentioned fecal alkali-producing bacteria ( Alcaligenes faecalis The application of WYH-571 bacterial suspension, fermentation broth, or fermentation products in the prevention and control of plant diseases is also within the scope of this invention. Specifically, the plant pathogen is *Fusarium wilt* (cucumber wilt). Fusarium oxysporum (Schl.) F.sp cucumerinum Owen ).

[0008] The above-mentioned fecal alkali-producing bacteria ( Alcaligenes faecalisThe application of WYH-571 bacterial suspension, fermentation broth, or fermentation products in the preparation of plant growth promoters is also within the scope of this invention. In this application, the growth-promoting properties include the production of IAA and siderophores, as well as the degradation of organophosphates.

[0009] In addition, the fecal alkali-producing bacterium WYH-571 of the present invention or its fermentation products can also be used to prepare plant biocontrol agents and organic fertilizers.

[0010] This invention relates to fecal alkali-producing bacteria ( Alcaligenes faecalis The preparation method of WYH-571 bacterial suspension and cell-free fermentation broth is as follows: The activated strain is inoculated into a 250 mL Erlenmeyer flask containing 100 mL LB liquid medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL distilled water, pH=7.2-7.4). After culturing at 30℃ for 48 h, the fermentation broth is centrifuged, and the supernatant is the cell-free fermentation broth. The bacterial cells are resuspended in an equal volume of sterile water to obtain the bacterial suspension. Attached Figure Description

[0011] Figure 1 The fecal alkali-producing bacteria provided in Example 1 of this invention ( Alcaligenes faecalis WYH-571 developmental tree.

[0012] Figure 2 It is the fecal alkali-producing bacteria in Example 2 of this invention ( Alcaligenes faecalis WYH-571 produces the standard curve of IAA.

[0013] Figure 3 It is the fecal alkali-producing bacteria in Example 2 of this invention ( Alcaligenes faecalis WYH-571's ability to produce iron carriers.

[0014] Figure 4 It is the fecal alkali-producing bacteria in Example 3 of this invention ( Alcaligenes faecalis )WYH-571 student

[0015] Biofilm formation; where (A), (B), (C), and (D) represent the biofilms of strain WYH-571 in culture media LBM, LBGM, LB, and LBG, respectively.

[0016] Figure 5 It is the fecal alkali-producing bacteria in Example 5 of this invention ( Alcaligenes faecalis WYH-571 has a preventive and growth-promoting effect on cucumber wilt in potted plants. Detailed Implementation

[0017] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0020] Example 1: Isolation, purification, and molecular biological identification of strain WYH-571 Weigh 10 g of wheat rhizosphere soil (from local farmland in Yangling) and add 200 mL of 0.01 M sterile PBS buffer. Shake at room temperature and 180 r / min for 30 min to obtain a rhizosphere soil suspension. Dilute with sterile water in a gradient to 10. -1 10 -2 and 10 -3 The solution was evenly spread on TYG (Tryptone Yeast Extract Glucose Medium) using a spreader. The composition of TYG was: 1 g tryptone, 1 g yeast extract, 0.5 g glucose, 6.34 g KCl, 1.2 g NaCl, and MgSO4. 7H2O 0.25 g, K2HPO4 0.13 g, CaCl2 0.22 g of 2H2O, 0.17 g of K2SO4, 2.4 g of Na2SO4, 0.5 g of NaHCO3, 0.09 g of Na2CO3, 0.07 g of FeEDTA, 15 g of agar, 1000 mL of distilled water, pH 7.2-7.4; after incubation at 28℃ for 2 days, single colonies were picked for purification culture.

[0021] DNA extraction was performed according to the procedure of the column-based bacterial DNA extraction kit from Shanghai Sangon Biotech (Shanghai) Co., Ltd. Universal bacterial primer pairs were used: F27 5'-AGA GTT TGA TCC TGG CTC AGG-3', P1541 5'-AAG GAG GTG GTG ATC CAG CCG CA-3'. Reaction conditions were: 94 ℃ denaturation for 45 s, 50 ℃ annealing for 45 s, 72 ℃ extension for 75 s, 30 cycles in a 50 μL reaction volume. PCR products were detected by agarose gel electrophoresis and sequenced. The obtained 16S rDNA full-length sequence was compared with 16S rDNA sequences obtained from databases such as Genbank, and a phylogenetic tree was constructed using the Mega 6.0 software package.

[0022] The strain WYH-571 was identified using molecular biological methods. The 16S rDNA sequence of strain WYH-571 is shown in SIQ ID NO.1, and a total of 1374 bases were determined in the 16S rDNA of strain WYH-571.

[0023] The 16S rDNA fragment obtained from the genomic DNA of the strain was amplified and compared with 16S rDNA sequences obtained from databases such as Genbank, and the accession number was obtained as WYH-571 (OR574216.1). Phylogenetic tree construction: Ten type strains were selected for phylogenetic analysis, and the constructed phylogenetic tree is shown below. Figure 1 As shown.

[0024] Example 2: Determination of growth-promoting indicators for strain WYH-571 First, the strain stored at -80 ℃ was streaked onto LB medium and activated by incubation at 28 ℃ for 24 h. Then, the activated strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium, with 1 mL of L-tryptophan added to achieve a final concentration of 100 µg / mL. The flask was incubated at 28 ℃ and 180 r / min for 5 days, with two biological replicates per strain. Eight mL of the fermentation broth was centrifuged at 8000 r / min and 4 ℃, and the supernatant was used for quantitative determination of IAA content. For IAA content quantification, 60 µL of supernatant was transferred to a 2 mL centrifuge tube, and 120 µL of Salkowski reagent was added. Colorimetric development was performed in the dark, and the absorbance of each treatment at 530 nm was measured using a UV spectrophotometer. Meanwhile, standard curves of IAA content versus absorbance were prepared using serially diluted IAA solutions (50, 10, 5, 2.5, 1.25, 0.625, and 0 µg / mL), and the IAA production capacity of the strain was evaluated based on the standard curves.

[0025] The activated strain was inoculated using a spot inoculation method on an inorganic phosphorus medium ((NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, Ca3(PO4)2 25 g, FeSO4·7H2O 0.03 g, MnSO4·H2O 0.03 g, glucose 10 g, pH 7.4-7.6) and cultured at 28 ℃ for 5 days. The inorganic phosphorus solubilizing ability of the strain was evaluated based on the presence and size of a clear zone around the strain.

[0026] The activated strain was inoculated using a spot inoculation method onto DF medium (containing 2.0 g glucose, 2.0 g gluconic acid, 2.0 g citric acid, 10 mL of trace element solution (CaCl2 200 mg, FeSO4·7H2O 200 mg, H3BO3 15 mg, ZnSO4·7H2O 20 mg, Na2MoO4 10 mg, KI 10 mg, NaBr 10 mg, MnCl2 10 mg, COCl2 5 mg, CuCl2 5 mg, AlCl3 2 mg, NiSO4 2 mg, 1000 mL deionized water) with 3 mM ACC as the sole nitrogen source, followed by 990 mL of deionized water. DF culture with 0.2% (w / v) (NH4)2SO4 as the sole nitrogen source served as a positive control, and DF medium served as a negative control. The inoculated plates were incubated at 28 ℃ for 5 days, and the ACC production capacity of the strain was comprehensively evaluated based on the growth status of the strain on the three media.

[0027] The activated strain was inoculated onto chromic azure medium (CAS) using the spot inoculation method. After inoculation, the plates were incubated at 28°C for 7 days. The ability of the strain to produce siderophores was evaluated based on the presence and size of the yellow halo around the colony.

[0028] The growth-promoting indicators of the isolated strain WYH-571, including IAA production, phosphate solubilization, ACC production, and siderophore production, were determined. An IAA standard curve was constructed. Figure 2 The study determined that strain WYH-571 could produce 4.28 μg / mL of IAA at 100 µg / mL; and its siderophore production ability was positive. Figure 3 It has the ability to decompose organic phosphorus but not inorganic phosphorus, potassium, nitrogen fixation, or ACC production (Table 2).

[0029] Table 2 Evaluation of the growth-promoting characteristics of strain WYH-571

[0030] Example 3: Biofilm formation assay of strain WYH-571 Single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of LB liquid medium and incubated overnight at 160 rpm / min and 30 °C. 200 μL of LB, LBG (containing 1% glycerol (v / v)), LBM (containing 0.1 mM MnSO4), and LBGM (containing 1% glycerol (v / v) and 0.1 mM MnSO4) medium were added to each well of a 16-well plate. 2 μL of the overnight culture was gently added dropwise to the surface of each medium. The 16-well plates were then incubated statically at 30 °C for 3 days. Biofilm formation was observed and recorded.

[0031] Biofilm assays of strain WYH-571 showed that the strain could form a biofilm in LBM medium, followed by LBGM medium, while no significant biofilm formation was observed in LB and LBG media. Figure 4 Biofilms are an important ecological niche for biocontrol bacteria, helping them resist adverse environments. Moreover, the formation of biofilms significantly affects their colonization on plants, thus directly impacting the effectiveness of biological control.

[0032] Example 4: Pot control efficacy of strain WYH-571 against cucumber wilt. Cucumber seeds were sown in seedling trays for seedling cultivation. After one week, the seedlings were transplanted into individual pots, one seedling per pot. After transplanting, when the cucumbers reached the two-leaf stage, a root drenching method was used to determine the efficacy of the control. The experimental treatments included: 1) inoculation with only 25 mL of cucumber wilt and an equal volume of sterile water (FOC); 2) simultaneous inoculation with 25 mL of cucumber wilt and an equal volume of bacterial suspension (10... 8 CFU / mL (WHF-571-FOC); 3) Inoculate only with bacterial suspension (10 CFU / mL) 8 CFU / mL (WHF-571); 4) Inoculate with 25 mL of sterile water (CK). Each treatment was repeated 4 times; one month after inoculation, the disease index, plant height, and fresh weight were statistically analyzed, and the control efficacy was calculated based on the disease index.

[0033] Cucumber wilting disease index: Level 0: No symptoms; Grade 1: The leaves of less than 1 / 4 of the plant show wilting symptoms, while the base of the stem is asymptomatic and the plant grows normally. Grade 2: 1 / 4 to 1 / 2 of the plant's leaves show wilting symptoms, the lower 1 / 2 of the stem shows browning, and the plant is stunted; Grade 3: More than half of the plant's leaves show wilting symptoms, more than half of the stem base shows browning, and the plant is significantly stunted; Level 4: The entire plant withers and dies.

[0034]

[0035]

[0036] As shown in Table 3, strain WYH-571 showed significant control efficacy against cucumber wilt, with an efficacy rate of 68.89%. Compared with the blank control, the plant height of cucumber plants treated with strain WYH-571 (29.58 cm) was significantly higher than that of the control plant (25.55 cm). The fresh weight of cucumber plants treated with strain WYH-1275 (10.78 g) was significantly higher than that of the blank control (8.46 g); thus, strain WYH-1275 has a certain growth-promoting effect on cucumber plants (Table 4).

[0037] Table 3. Disease control effect of strain WYH-1275 against cucumber wilt.

[0038] Table 4. Growth-promoting effects of strain WYH-1275 on cucumber.

Claims

1. Alkaloid-producing bacteria in feces ( Alcaligenes faecalis WYH-571 is used in the preparation of antibacterial agents for plant pathogens, characterized in that... The alkali-producing bacterium WYH-571 has the accession number CGMCC No. 28835, and the plant pathogen is the cucumber wilt pathogen.

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