Alcaligenes faecalis and application thereof in disease prevention and growth promotion

Abstract

The invention discloses alcaligenes faecalis and an application thereof in disease prevention and growth promotion, the strain is preserved in the China General Microbiological Culture Collection Center on November 1, 2023, the preservation number is CGMCC No.28835, and the preservation name is alcaligenes faecalis WYH-571. The alcaligenes faecalis has the advantages that the preservation number is CGMCC No.28835, and the alcaligenes faecalis WYH-571; when being applied to the field of agricultural biological prevention and control, the strain can form a biological membrane to be stably colonized at the rhizosphere of a plant, and can generate substances such as IAA and siderophores to promote the growth of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and specifically relates to a strain of Alcaligenes faecalis WYH-571 with disease prevention and growth promotion functions and application thereof. Background Art

[0002] Plant diseases pose a serious threat to the world's food production and security. For a long time, the use of chemical pesticides and fertilizers has aggravated environmental pollution, bacterial resistance, and soil microecological imbalance. It is urgent to explore microbial products that can replace chemical pesticides and fertilizers. Although there are a large number of microbial products, due to their low colonization ability and short effective period in the field, it has become a research hotspot for scholars at home and abroad to explore broad-spectrum and highly efficient microorganisms from plant-related microbial communities such as rhizosphere and root endophytes. Summary of the invention

[0003] Based on new research findings, the purpose of the present invention is to provide a strain of Alcaligenes faecalis WYH-571 with disease prevention and growth promotion functions and its application in agricultural production.

[0004] The faecalis (Alcaligenes faecalis) WYH-571 provided by the present invention is classified and named as Alcaligenes faecalis, and was deposited on November 1, 2023 in the General Microbiology Center of China Culture Collection Committee (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), Postal Code: 100101, and the preservation number is CGMCC No. 28835.

[0005] The bacterial suspension, fermentation liquid or fermentation product of the above-mentioned Alcaligenes faecalis WYH-571 is also within the scope of protection of the present invention.

[0006] The use of the bacterial suspension or fermentation liquid or fermentation product of the above-mentioned Alcaligenes faecalis WYH-571 in promoting plant growth is also within the scope of protection of the present invention.

[0007] The application of the bacterial suspension, fermentation liquid or fermentation product of Alcaligenes faecalis WYH-571 in preventing and controlling plant diseases is also within the scope of protection of the present invention. In particular, the plant pathogen is Fusarium oxysporum (Schl.) F. sp cucumerinum Owen.

[0008] The application of the bacterial suspension or fermentation liquid or fermentation product of Alcaligenes faecalis WYH-571 in the preparation of plant growth promoters is also within the scope of protection of the present invention. In this application, the growth-promoting properties are the production of IAA and siderophores and the degradation of organic phosphorus.

[0009] In addition, the Alcaligenes faecalis WYH-571 or its fermentation product can also be used to prepare plant biocontrol agents and organic fertilizers.

[0010] The preparation method of the bacterial suspension and the bacterial body-removed fermentation liquid of Alcaligenes faecalis WYH-571 of the present invention is as follows: inoculating an activated strain with a bacterial needle in a 250-mL conical flask containing 100 mL of LB liquid culture medium (10 g of tryptone, 5 g of yeast extract powder, 10 g of NaCl, 1000 mL of distilled water, pH=7.2-7.4), culturing for 48 hours at 30°C, centrifuging the fermentation liquid, obtaining a supernatant that is the bacterial body-removed fermentation liquid, and resuspending the bacterial bodies with an equal amount of sterile water to obtain a bacterial suspension. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the developmental tree of Alcaligenes faecalis WYH-571 provided in Example 1 of the present invention.

[0012] Figure 2 This is the standard curve of IAA production by Alcaligenes faecalis WYH-571 in Example 2 of the present invention.

[0013] Figure 3 It is the ability of Alcaligenes faecalis WYH-571 in Example 2 of the present invention to produce siderophore.

[0014] Figure 4 It is the formation of biofilm of Alcaligenes faecalis WYH-571 in Example 3 of the present invention; wherein (A), (B), (C), and (D) represent the biofilm of strain WYH-571 in culture medium LBM, LBGM, LB, and LBG, respectively.

[0015] Figure 5 This is the potted plant disease prevention and growth promotion effect of Alcaligenes faecalis WYH-571 on cucumber wilt in Example 5 of the present invention. DETAILED DESCRIPTION

[0016] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0019] Example 1 Isolation, purification and molecular biological identification of strain WYH-571

[0020] 10 g wheat rhizosphere soil (from local farmland in Yangling) was weighed and added to 200 mL of 0.01 M sterile PBS buffer. The rhizosphere soil suspension was obtained by shaking at room temperature and 180 r / min for 30 min. The suspension was then diluted with sterile water to 10 -1 , 10 -2 and 10 -3 , use a spreading rod to evenly spread it on TYG medium (Tryptone Yeast extract Glucose Medium, TYG), whose composition is: 1g tryptone, 1g yeast extract, 0.5g glucose, 6.34g KCl, 1.2g NaCl, 0.25g MgSO4·7H2O, 0.13g K2HPO4, 0.22g CaCl2·2H2O, 0.17g K2SO4, 2.4g Na2SO4, 0.5g NaHCO3, 0.09g Na2CO3, 0.07g FeEDTA, 15g agar, 1000mL distilled water, PH=7.2-7.4; after constant temperature culture at 28℃ for 2d, pick a single colony for purification culture.

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

[0022] The strain WYH-571 was identified by molecular biological methods. The results of the 16SrDNA base sequence determination of the strain WYH-571 are shown in SIQ ID NO.1. A total of 1374 bases of 16SrDNA of the strain WYH-571 were determined.

[0023] The fragment size was amplified from the genomic DNA of the strain. The obtained 16S rDNA full sequence was compared with the 16S rDNA sequence obtained from the Genbank database and the accession number was obtained: WYH-571 (OR574216.1). Construction of phylogenetic tree: 10 model strains were selected for phylogenetic analysis, and the constructed phylogenetic tree is shown in Figure 1 shown.

[0024] Example 2 Determination of growth-promoting indexes of strain WYH-571

[0025] First, the strain stored at -80℃ was streaked on LB medium and cultured in a 28℃ incubator for 24h for activation; then the activated strain was inoculated with a needle in a 250mL conical flask containing 100mL LB liquid medium, and 1mL L-tryptophan was added to the conical flask to make the final concentration 100μg / mL, and cultured at 28℃ and 180r / min for 5d, with 2 biological replicates for each strain; 8mL of the strain fermentation broth was taken and centrifuged at 8000r / min and 4℃, and the supernatant was aspirated for quantitative determination of the IAA content of the strain. For the quantitative determination of IAA content, 60μL of the supernatant was aspirated into a 2mL centrifuge tube, and then 120μL of Salkowski reagent was added for color development in the dark, and the absorbance of each treatment under 530nm was measured by UV spectrophotometer. At the same time, a standard curve of IAA content and absorbance value was prepared using gradiently 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 curve.

[0026] The activated strains were inoculated in an inorganic phosphate medium (NH 4 ) 2 SO 4 0.5g, NaCl 0.3g, KCl0.3g, MgSO 4 7H 2 O 0.3g, Ca 3 (PO 4 ) 2 25g, FeSO 4 7H 2 O 0.03g, MnSO 4 ·H 2 O 0.03g, glucose 10g, pH 7.4-7.6), and cultured at 28℃ for 5 days. The ability of the strain to degrade inorganic phosphorus was evaluated based on whether a transparent zone was formed around the strain and the size of the transparent zone.

[0027] The activated strain was inoculated into DF medium (2.0 g glucose, 2.0 g gluconic acid, 2.0 g citric acid, 2.0 g trace element solution (CaCl 2 200mg, FeSO 4 7H 2 O 200mg, H 3 BO 3 15mg, ZnSO 4 7H 2 O 20 mg, Na 2 MoO 4 10mg, KI 10mg, NaBr 10mg, MnCl 2 10mg, COCl 2 5mg, CuCl 2 5mg, AlCl 3 2mg, NiSO 4 2 mg, deionized water 1,000 mL) 10 mL, deionized water 990 mL) and 0.2% (w / v) (NH 4 ) 2 SO 4 DF culture as the only nitrogen source was used as the positive control, and DF culture medium was used as the negative control. The inoculated plates were cultured at 28°C for 5 days, and the ACC production capacity of the strains was comprehensively evaluated based on the growth conditions of the strains on the three culture media.

[0028] The activated strain was inoculated on chrome azure culture medium (CAS) using the dot inoculation method. The inoculated plates were cultured at 28°C for 7 days, and the ability of the strain to produce iron carriers was evaluated based on the appearance and size of the yellow halo around the colonies.

[0029] The growth-promoting indexes of the isolated strain WYH-571, such as IAA production, phosphate solubilization, ACC production and iron carrier production, were determined. Figure 2 ), strain WYH-571 was found to produce 4.28 μg / mL of IAA at 100 μg / mL; and its ability to produce siderophores was positive ( Figure 3 ); it has the ability to degrade organic phosphorus but not inorganic phosphorus, potassium, fix nitrogen and produce ACC (Table 2).

[0030] Table 2 Evaluation of growth-promoting properties of strain WYH-571

[0031]

[0032] Example 3 Biofilm formation assay of strain WYH-571

[0033] A single colony was picked and inoculated into a 250 mL conical flask containing 100 mL LB liquid medium and cultured overnight at 160 rpm / min and 30 °C. 200 μL LB, LBG (containing 1% glycerol (v / v)), LBM (containing 0.1 mM MnSO) were added to a 16-well plate. 4 ) and LBGM (containing 1% glycerol (v / v) and 0.1 mM MnSO 4 ) culture medium, take 2 μL of overnight cultured bacterial solution and gently drop it on the surface of different culture media. The 16-well plate was cultured at 30°C for 3 days, and the biofilm formation was observed and recorded.

[0034] The results of biofilm assay on strain WYH-571 showed that the strain could form biofilm in LBM medium, followed by LBGM medium, and no obvious biofilm was formed in LB and LBG medium ( Figure 4 ). Biofilm is an important form of existence for biocontrol bacteria to occupy ecological niches, which can help them resist adverse environments. Moreover, the formation of biofilm of biocontrol bacteria significantly affects their colonization in plants, which in turn directly affects the biological control effect.

[0035] Example 4 Determination of potted plant protection efficacy of strain WYH-571 against cucumber wilt

[0036] Cucumber seeds were sown in seedling trays for seedling raising. After one week of seedling raising, they were transplanted into seedling pots, with one seedling per pot. After transplanting, when the cucumbers grew to the 2-leaf and 1-heart stage, the root irrigation method was used for the prevention and efficacy test. The experimental treatments included: 1) inoculation with 25 mL of cucumber wilt and an equal amount of sterile water (FOC); 2) inoculation with 25 mL of cucumber wilt and an equal amount of bacterial suspension (10 8 CFU / mL) (WHF-571-FOC); 3) Inoculate only the bacterial suspension (10 8 CFU / mL) (WHF-571); 4) Inoculate 25mL sterile water (CK). Each treatment was repeated 4 times; one month after inoculation, the disease index, plant height, and fresh weight were counted, and the control effect was calculated based on the disease index.

[0037] Cucumber wilt disease index:

[0038] Level 0: no symptoms;

[0039] Level 1: Less than 1 / 4 of the leaves of the plant show wilting symptoms, the stem base is asymptomatic, and the plant grows normally;

[0040] Level 2: 1 / 4-1 / 2 of the leaves of the plant show wilting symptoms, and the lower 1 / 2 of the stem base turns brown, and the plant becomes dwarfed;

[0041] Level 3: More than 1 / 2 of the leaves of the plant show wilting symptoms, more than 1 / 2 of the stem base turns brown, and the plant is obviously dwarfed;

[0042] Level 4: The whole plant wilts and dies.

[0043]

[0044]

[0045] As shown in Table 3, strain WYH-571 has a significant protective effect on cucumber wilt, with a protective effect of 68.89%. Compared with the blank control, the height of cucumber plants treated with strain WYH-571 suspension (29.58 cm) was significantly higher than that of the control (25.55 cm). The fresh weight of cucumber plants treated with strain WYH-1275 suspension (10.78 g) was significantly higher than that of the blank control (8.46 g); it can be seen that strain WYH-1275 has a certain growth-promoting effect on cucumber plants (Table 4).

[0046] Table 3 Disease prevention effect of strain WYH-1275 on cucumber wilt

[0047] Strain name Disease index Prevention effect (%) Stem diameter (cm) Plant height(cm) Fresh weight(g) FOC 71.43 - 3.97±0.41b 20.97±3.12b 5.52±0.69c 571-FOC 22 68.89 4.76±0.55a 25.94±1.23a 10.30±1.47a CK - - 4.35±0.53a 25.55±1.55a 8.46±0.61b

[0048] Table 4 Growth-promoting effect of strain WYH-1275 on cucumber

[0049] Strain name Stem diameter (cm) Plant height(cm) Fresh weight(g) 571 4.99±0.39* 29.58±2.89* 10.78±1.28* CK 4.35±0.53 25.55±1.55 8.46±0.61

[0050]

[0051]

Claims

1. A strain of Alcaligenes faecalis WYH-571, whose accession number is CGMCC No.28835.

2. A fermentation product, characterized in that The fermentation product is produced by fermenting the Alcaligenes faecalis WYH-571 according to claim 1.

3. Use of the Alcaligenes faecalis WYH-571 or its fermentation product according to claim 1 for preparing an antibacterial agent for plant pathogens.

4. The use according to claim 3, characterized in that: The plant pathogen is cucumber wilt (Fusariumoxysporum(Schl.)F.sp cucumerinum Owen).

5. Use of the Alcaligenes faecalis WYH-571 or its fermentation product according to claim 1 for preparing a plant growth promoter.

6. The use according to claim 5, characterized in that: The plant growth promoter promotes plant growth by producing IAA and siderophore and degrading organic phosphorus.

7. Use of the Alcaligenes faecalis WYH-571 or its fermentation product according to claim 1 for preparing a plant growth promoter.

8. Use of the Alcaligenes faecalis WYH-571 or its fermentation product according to claim 1 for preparing plant biocontrol agents.

9. Use of the Alcaligenes faecalis WYH-571 or its fermentation product according to claim 1 for preparing organic fertilizer.

Images