X178 strain for inhibiting fungal diseases in grains and eliminating mycotoxins and its application

Abstract

This invention relates to the X178 strain, which inhibits fungal diseases in grains and removes mycotoxins, and its applications. The X178 strain belongs to the Nocardiaceae family (…). Nocardiopsis This fungus (sp.) is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 34027. Specifically, it exhibits inhibitory effects against grain pathogenic fungi Aspergillus flavus, Aspergillus niger, and Fusarium graminearum, and can remove two mycotoxins: aflatoxin B1 (AFB1) and / or zearalenone.

Description

Technical Field

[0001] This invention relates to the field of microorganisms, and particularly to biocontrol microorganisms. Background Technology

[0002] Grain fungi and mycotoxins are among the major causes of grain loss and quality decline. Grain fungi include Aspergillus spp. ( Aspergillus Alternaria ( ) Alternaria ), Penicillium ( Penicillium ) and Fusarium genus ( Fusarium Fungi such as aflatoxin B1 (AFB1) and zearalenone (ZEN) produced by filamentous fungi have toxic secondary metabolites that have adverse effects such as carcinogenicity, teratogenicity, nephrotoxicity, and hepatotoxicity, threatening human health.

[0003] Given the large volume and long storage period of grain, the prevention and control of contamination by grain fungi and fungal toxins is particularly important. Summary of the Invention

[0004] One of the present inventions provides a Nocardia strain ( Nocardiopsis sp.)X178 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34027.

[0005] The second invention provides a composition comprising Nocardia as described in the first invention.

[0006] In one specific embodiment, the dosage form of the composition is one of a suspension, powder, and granules.

[0007] In one specific embodiment, the dosage form of the composition is an oil suspension or a wettable powder.

[0008] The third invention provides the use of Nocardia as described in the first invention or the composition as described in any one of the second inventions in the inhibition of pathogenic fungi and / or the removal of fungal toxins.

[0009] In one specific embodiment, the pathogenic fungus is at least one of Aspergillus flavus, Aspergillus niger, and Fusarium graminearum.

[0010] In one specific embodiment, the mycotoxin is aflatoxin B1 (AFB1) and / or zearalenone.

[0011] The beneficial effects of the present invention are as follows: X178 of the present invention has an inhibitory effect on the grain pathogenic fungi Aspergillus flavus, Aspergillus niger and Fusarium graminearum, and it can remove two fungal toxins, aflatoxin B1 (AFB1) and / or zearalenone.

[0012] Strain Preservation: The strain screened in this invention is named X178. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34027, deposited on March 31, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Nocardiaceae. Nocardiopsis sp. Attached Figure Description

[0013] Figure 1 The inhibitory activity of isolate X178 against Aspergillus flavus, Aspergillus niger and Fusarium graminearum was demonstrated.

[0014] Figure 2 The phylogenetic tree of strain X178 is shown. Detailed Implementation

[0015] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0016] Unless otherwise specified, the reagents and other materials used in the embodiments of this invention can be purchased commercially.

[0017] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121°C for 20 min by high temperature and moist heat.

[0018] LB solid medium: LB liquid medium with 15 g / L agar, sterilized at 121 degrees Celsius for 20 min.

[0019] PDA solid culture medium formula: 200 g / L potato, 20 g / L glucose, 15 g / L agar, sterilized at 121 degrees Celsius for 20 min by moist heat.

[0020] CMC (sodium carboxymethyl cellulose) liquid culture medium: CMC 15 g / L, KH2PO4 1 g / L, NH4NO3 1 g / L, MgSO4·7H2O 0.5 g / L, yeast extract 1 g / L. CMC is dissolved in water at 80 degrees Celsius. After the CMC is completely dissolved in hot water, the volume is increased to 1L. The medium is then sterilized by high temperature and moist heat at 121 degrees Celsius for 20 min.

[0021] Gao's No. 1 culture medium: 20g soluble starch, 1g KNO3, 0.5g NaCl, 0.5g K2HPO4•3H2O, 0.5g MgSO4•7H2O, 0.01g FeSO4•7H2O, diluted to 1000ml with distilled water, pH 7.5, sterilized at 121°C for 20 min.

[0022] Aspergillus flavus ( Aspergillus flavus Aspergillus niger ( ) Aspergillus niger ) and Fusarium graminearum ( Fusarium gramineae Source: Isolated from stored grains by the National Food and Strategic Reserves Administration Research Institute. For details on Aspergillus flavus and Aspergillus niger strains, please refer to "Identification of Dominant Fungi in Stored Rice Based on Morphological Characteristics and Multi-Gene Sequences," authors: Qi Zhihui, Zhang Haiyang, Tian Lin, Zhuang Yuan, Li Bing, Tang Fang; Journal: Grain and Food Science and Technology; Vol. 30, No. 6, 2022: 198-209.

[0023] Example 1: Isolation of strains

[0024] Selected miscellaneous grain beetle ( Tribolium confusa Twenty adult insects were starved for 24 hours and then subjected to surface sterilization treatment to obtain surface-disinfected hybrid flour beetles: they were disinfected by soaking in 75% ethanol for 3 minutes, repeated 3 times; they were disinfected by soaking in 1.2% sodium hypochlorite solution for 3 minutes, repeated 3 times; finally, they were rinsed 3 to 5 times with sterile ultrapure water to eliminate microbial interference on the insect surface.

[0025] The surface-sterilized *Triplophysa flocculus* was homogenized and then mixed with 100 μL of sterile water to prepare a stock solution. The stock solution was then serially diluted three times (10-fold) to obtain serially diluted solutions. 200 μL of each dilution was evenly spread onto LB medium and incubated upside down at 28 ± 1°C for 48 h to obtain cultures for each dilution. One culture dish constituted one replicate, and three biological replicates were established for each dilution.

[0026] Single colonies with good morphology were selected and purified on LB medium using the streak method. The purified isolates were numbered and preserved for subsequent identification and bioactivity analysis.

[0027] Example 2: Antibacterial activity of isolated strains

[0028] The isolated strain was streaked onto LB solid medium and incubated upside down at 28°C for 24 hours to activate the strain. Then, single activated colonies were picked and inoculated into LB liquid medium and incubated at 28°C with constant temperature shaking at 200 rpm until OD reached [value missing]. 600 =2.0, to obtain the culture medium of the isolated strain.

[0029] Preparation of a suspension of Aspergillus flavus and Aspergillus niger conidia: The grain fungus Aspergillus flavus (… Aspergillus yellow ) and Aspergillus niger ( Aspergillus niger The conidia were activated on PDA solid medium at 28°C, then washed with 2 ml of sterile water, and suspended in sterile water for counting. A total of 2.00 × 10⁻⁶ conidia were obtained. 6 CFU / ml conidial suspension.

[0030] Preparation of Fusarium graminearum conidium suspension: The grain fungus Fusarium graminearum (… Fusarium grasses The conidia were activated on PDA solid medium at 28°C, then cultured on CMC liquid medium until conidia were produced. The culture medium was washed with sterile water, and the conidia were suspended in sterile water and counted to obtain 2.00 × 10⁻⁶. 6 CFU / ml conidial suspension.

[0031] Antimicrobial activity assay of isolated strains: The plate confrontation method was used, with *Aspergillus flavus*, *Aspergillus niger*, and *Fusarium graminearum* as target pathogens, and the isolated strains as the test strains for antimicrobial activity. 20 μL of spore suspension of the target pathogens was inoculated onto a PDA plate, and 20 μL of culture medium (OD) of the isolated strain was added 3 cm away from the spore suspension. 600 =2.0). The control group was inoculated with 20 μL of the target pathogenic fungal spore suspension in the center of the plate, without adding the isolated strain culture medium. The plates were then placed in a constant temperature incubator at 28 degrees Celsius for 7 days. One plate was considered one replicate, and three biological replicates were set up. After the culture was completed, the colony radius of the target fungus in the control group and the experimental group was measured. The average colony radius of the control group was calculated based on the colony radius of the three replicates of the control group. The relative inhibition rate of each replicate of the control group was calculated based on formula (1), i.e., the relative inhibition rate of the control group; the relative inhibition rate of each replicate of the treatment group was calculated based on formula (2), i.e., the relative inhibition rate of the treatment group, and then the average relative inhibition rate of the treatment group was calculated based on the relative inhibition rate of each replicate of the treatment group. The relative inhibition rate and the relative inhibition rate of the treatment group were analyzed by variance using IBM SPPSStatistics 26 software.

[0032] The relative inhibition rate of the control group (%) = (average colony radius of the control group - the colony radius of each replicate in the control group) × 100 / average colony radius of the control group Formula (1).

[0033] The relative inhibition rate of the treatment group (%) = (average colony radius of the control group - the colony radius of each replicate in the treatment group) × 100 / average colony radius of the control group (Formula 2).

[0034] The results showed that isolate X178 had inhibitory effects on Aspergillus flavus, Aspergillus niger, and Fusarium graminearum, and compared with the control group... P <0.05. See the antibacterial photo of X178. Figure 1 The average relative inhibition rates of X178 were 58.54%, 70.54%, and 67.86%, respectively.

[0035] Example 3: The removal effect of isolated strain X178 on aflatoxin B1.

[0036] 1 mg of aflatoxin B1 standard (Puribang Biotechnology Co., Ltd.) was dissolved in 20 mL of chromatographic grade methanol to prepare a 50 μg / mL aflatoxin B1 stock solution, which was stored at 4°C. Meanwhile, the aflatoxin B1 stock solution was used to prepare aflatoxin B1 solutions of 1.2 μg / mL, 1.0 μg / mL, 0.8 μg / mL, 0.6 μg / mL, 0.4 μg / mL, and 0.2 μg / mL. 1 mL of each solution was added dropwise to sterile 2 mL centrifuge tubes, dried in a laminar flow hood, and then reconstituted with 1 mL of anhydrous ethanol to obtain aflatoxin B1 standard solutions of each concentration, which were used to plot standard curves. On the other hand, 20 μL of aflatoxin B1 stock solution was added dropwise to a sterile 2 mL centrifuge tube, placed in a laminar flow hood and dried. The mass of aflatoxin B1 in the centrifuge tube was 1 μg. Then, it was redissolved with 20 μL of anhydrous ethanol to prepare a 50 μg / mL aflatoxin B1 removal solution.

[0037] Streak X178 plates onto LB solid medium and incubate upside down in a 28°C biochemical incubator for 24 hours for activation. After successful activation, pick a single colony and inoculate it onto LB liquid medium, then incubate at 28°C and 200 rpm with shaking until OD reaches [value missing]. 600 =1.0. Take 1 mL of bacterial culture, centrifuge at 10000 rpm for 5 min, discard the supernatant to obtain X178 cells, wash the X178 cells with 1 mL of PBS buffer solution, repeat three times. Then add 1 mL of PBS buffer solution to resuspend the cells to obtain X178 bacterial suspension. Add 480 μL of X178 bacterial suspension to the aflatoxin B1 removal solution, incubate in a 37°C metal bath for 1 h, and finally add 500 μL of methanol to terminate the reaction. The control group is prepared by adding 480 μL of PBS buffer solution to the aflatoxin B1 removal solution. Both the experimental group and the control group were performed in triplicate.

[0038] The aflatoxin B1 content in the standard solution, experimental group, and control group was determined using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (Agilent 6545 ESI Q-TOF, Agilent Technologies). Samples were filtered through a 0.2 μmol filter membrane before spectrometry. A C18 column was used for liquid chromatography analysis, with an injection volume of 2 μL, a flow rate of 0.3 mL / min, and a column temperature of 30 °C. Mobile phase A consisted of a mixture of water, formic acid (0.1%, v / v), and ammonium formate (5 mM), while mobile phase B consisted of a mixture of methanol (99.9%, v / v) and formic acid (0.1%, v / v). The gradient elution program was as follows: Phase B was maintained at 10% for 0-1 min; from 1 to 1.5 min, Phase B increased from 10% to 45%; from 1.5 to 8.5 min, Phase B increased from 45% to 100%; from 8.5 to 9.5 min, Phase B was maintained at 100%; and from 9.5 to 10 min, Phase B decreased from 100% to 10%. The total runtime was 10 minutes. Mass spectrometry detection was performed using a positive ion mode electrospray ionization source (ESI+). The nebulizer pressure was set to 40 psig, the capillary voltage to 4.0 kV, the rupture voltage to 175 V, and the cone voltage to 65 V. The nebulization temperature was 300°C, the auxiliary gas (N2) flow rate was 5 L / min, and the in-sheath gas temperature was 325°C with a flow rate of 11 L / min. The mass spectrometer was run in full scan mode, collecting data with a mass-to-charge ratio (m / z) between 100 and 1200 at a scan frequency of 2 spectra / s. Finally, the target peak intensity was determined using Agilent MasshunterWorkstation Qualitative Analysis software.

[0039] A standard curve was plotted based on the target peak intensity and the concentration of the aflatoxin B1 standard solution. The aflatoxin B1 standard curve R... 2 >0.99 indicates a good linear relationship, meeting the requirements for quantitative analysis. Based on the standard curve, the aflatoxin B1 content corresponding to the peak intensities of the three replicates in the control group and the three replicates in the treatment group was calculated. The average aflatoxin B1 content of the control group was calculated based on the aflatoxin B1 content of the three replicates in the control group. The removal rate of each replicate in the control group was calculated based on formula (3), i.e., the removal rate of the control group; the removal rate of each replicate in the treatment group was calculated based on formula (4), i.e., the removal rate of the treatment group, and then the average removal rate of the treatment group was calculated based on the removal rate of each replicate in the treatment group. An analysis of variance was performed on the removal rates of the control group and the treatment group using IBM SPPS Statistics 26 software.

[0040] The removal rate of the control group (%) = (average toxin content of the control group - toxin content of each replicate in the control group) × 100 / average toxin content of the control group (Formula 3).

[0041] The removal rate (%) of the treatment group = (average toxin content of the control group - toxin content of each replicate in the treatment group) × 100 / average toxin content of the control group (Formula 4).

[0042] The results showed that X178's removal rate of aflatoxin B1 within 1 hour was significantly higher than that of the control group. P <0.05, X178 showed an average removal rate of 44.03% for aflatoxin B1 within 1 hour.

[0043] Example 4: The effect of strain X178 on the removal of zearalenone toxin

[0044] In Example 3, aflatoxin B1 was replaced with zearalenone toxin.

[0045] Mass spectrometry detection was performed using a negative ion mode electrospray ionization source (ESI-).

[0046] Everything else is the same as in Example 3.

[0047] A standard curve was plotted based on the target peak intensity and the concentration of the zearalenone toxin standard solution. The zearalenone toxin standard curve R... 2 >0.99 indicates a good linear relationship, meeting the requirements for quantitative analysis. Based on the standard curve, the zearalenone toxin content corresponding to the peak intensities of the three replicates in the control group and the three replicates in the treatment group was calculated. The average zearalenone toxin content of the control group was calculated based on the zearalenone toxin content of the three replicates in the control group. The removal rate of each replicate in the control group was calculated based on formula (3), i.e., the removal rate of the control group; the removal rate of each replicate in the treatment group was calculated based on formula (4), i.e., the removal rate of the treatment group, and then the average removal rate of the treatment group was calculated based on the removal rate of each replicate in the treatment group. An analysis of variance was performed on the removal rates of the control group and the treatment group using IBM SPPS Statistics 26 software.

[0048] The results showed that X178 achieved a significantly higher removal rate of zearalenone toxin within 1 hour compared to the control group. P <0.05, X178 achieved an average removal rate of 96.98% for zearalenone toxin within 1 hour.

[0049] Example 5: Classification and identification of X178

[0050] 1. Gram identification and morphological observation of X178 isolate

[0051] A drop of X178 bacterial suspension obtained from LB liquid medium was placed on a glass slide. After staining and destaining according to the Gram staining procedure, the slide was observed under an oil immersion microscope. The staining result of X178 was purple, indicating that it is a Gram-positive bacterium.

[0052] X178 was inoculated onto LB and Gao's No. 1 medium and incubated at 28 degrees Celsius for 24 hours. The colonies were radial in both cases.

[0053] X178 was inoculated onto LB agar. Using forceps, a sterile coverslip was inserted into the agar plate at approximately a 45-degree angle, perpendicular to the inoculation line. The plate was then inverted and incubated at 28 degrees Celsius for 24 hours. The coverslip was removed with forceps, one side was wiped clean with paper, and the infected side was placed on a slide. Observation under a microscope using both low and high magnification revealed that the spore chains ranged from straight to curved.

[0054] 2. Molecular identification of X178 isolates—16S rDNA identification

[0055] Total DNA was extracted from each isolate according to the bacterial DNA genomics kit procedure. Using 27F (as shown in SEQ ID No. 1) and 1492R (as shown in SEQ ID No. 2) primer pairs, and with the total DNA of isolate X178 as a template, 16S rDNA was amplified by PCR. The amplified PCR products were detected by gel electrophoresis and then sent to BOMIDE Biosciences for sequencing. The sequence of the 16S rDNA from strain X178 is shown in SEQ ID No. 3.

[0056] By comparing the full-length 16S rDNA sequence of X178 with sequences in the NCBI database, it was found that X178 is related to Nocardia aurea (…). N. flavescens ), a type of Nocardia ( N. sp. ), Luxentanyl Nocardia ( N. from Lucca The strain showed similarity exceeding 99.37% to several other strains. A phylogenetic tree was constructed using MEGA 11.0, and the phylogenetic tree for strain X178 is shown below. Figure 2 .according to Figure 2 The phylogenetic tree shows that strain X178 is related to Nocardia. N. sp. strain FK-13 and N. flavescens SA6 is the most closely related.

[0057] In summary, strain X178 is systematically classified as Nocardia. Nocardiopsis sp.

[0058] Strain X178 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34027, on March 31, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Nocardia. Nocardiopsis sp.

Claims

1. A type of Nocardia ( Nocardiopsis sp.)X178 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34027.

2. A composition comprising the Nocardiopsis sp. of claim 1.

3. The composition of claim 2, wherein, The dosage form of the composition is one of a suspension, a powder and a granule.

4. The composition of claim 2, wherein, The dosage form of the composition is an oil suspension or a wettable powder.

5. Use of the Nocardiopsis sp. of claim 1 or the composition of any one of claims 2 to 4 for inhibiting a pathogenic fungus, which is Fusarium graminearum, and / or for detoxifying a mycotoxin, which is aflatoxin B1 and / or zearalenone.

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