A strain of Acanthamoeba that inhibits Fusarium graminearum and its application

Abstract

This invention discloses a strain of Acanthamoeba that inhibits the pathogen of Fusarium graminearum and its applications. Acanthamoeba (Acanthamoeba rhysodes NJAU-ZY1, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2025219, demonstrates in plate experiments, liquid co-culture experiments, and pot experiments that inoculation with the protozoan Acanthamoeba NJAU-ZY1 significantly inhibits the growth of Fusarium graminearum pathogen, reduces its infectivity in wheat, alleviates the inhibitory effect of Fusarium graminearum on wheat growth, reduces the risk of diseases caused by this pathogen, and thus increases wheat biomass. This provides a new strategy and resource for the biological control of Fusarium graminearum pathogen and the scientific cultivation of wheat, and has important application value for the safe production of food crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and relates to a strain of Acanthamoeba that can inhibit Fusarium graminearum pathogen and its application. Background Technology

[0002] Wheat is one of the world's most important food crops, serving as a primary food source for over one-third of the global population. However, numerous diseases threaten wheat growth, spreading widely and affecting vast areas, impacting not only the plants themselves but also global food security. Among these, Fusarium head blight, often called the "number one killer" of wheat, is a globally prevalent and highly destructive disease. It not only causes yield losses but, more seriously, its toxins contaminate grains, directly threatening human and animal health. *Fusarium graminearum*, the pathogen causing Fusarium head blight, is one of the main triggers for this disease, making its control crucial.

[0003] Traditional disease control measures involve the use of pesticides or fungicides. However, frequent field management increases labor costs and also impacts environmental and food safety. Furthermore, the long-term, high-dose application of pesticides inhibits the growth of many key soil microorganisms, reduces soil microbial diversity, and disrupts the soil's micro-ecosystem. This makes it difficult to simultaneously control diseases and achieve ecological benefits.

[0004] Protozoa are an important component of soil microorganisms. As key consumers in the soil micro-food web, they primarily feed on bacteria, fungal spores, hyphae, and other small eukaryotes. Protozoa can protect plants by directly preying on pathogens. Even if protozoa do not directly consume all pathogens, their predation behavior greatly inhibits the activity and reproduction rate of pathogens, preventing them from forming sufficient numbers to overcome the plant's defense system.

[0005] However, there are currently no reports of protozoa that can inhibit Fusarium graminearum. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a Acanthamoeba protozoan that can inhibit Fusarium graminearum pathogen.

[0007] Another object of the present invention is to provide a culture of Acanthamoeba rhysodes.

[0008] Another object of the present invention is to provide the application of the Acanthamoeba mechanical culture.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A strain of Acanthamoeba rhysodes NJAU-ZY1 that inhibits Fusarium graminearum has been deposited at the China Center for Type Culture Collection (CCTCC) on November 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:C2025219.

[0011] The culture of Acanthamoeba NJAU-ZY1 described in this invention.

[0012] Preferably, the method for preparing the culture includes inoculating the Acanthamoeba NJAU-ZY1 into PAS culture medium, adding inactivated microorganisms as food, and placing it in an incubator at 18℃-22℃ for 40h-50h.

[0013] More preferably, the inactivated microorganism is inactivated Escherichia coli.

[0014] Preferably, the PAS culture medium is prepared by mixing 5 mL of reagent 1 and 5 mL of reagent 2 in 1 L of pure water; the formula of reagent 1 is: sodium chloride 24 g / L, magnesium sulfate heptahydrate 0.8 g / L, calcium chloride hexahydrate 1.2 g / L; the formula of reagent 2 is: disodium hydrogen phosphate 28.4 g / L, potassium dihydrogen phosphate 27.2 g / L.

[0015] The application of Acanthamoeba NJAU-ZY1 in inhibiting Fusarium graminearum pathogen.

[0016] The application of the Acanthamoeba NJAU-ZY1 in the preparation of products for the prevention and control of wheat scab.

[0017] The application of the culture in inhibiting Fusarium graminearum pathogen.

[0018] The application of the culture in the preparation of products for the prevention and inhibition of wheat scab.

[0019] Beneficial effects:

[0020] This invention isolates and screens *Acanthamoeba rhysodes*, a protozoan that can inhibit *Fusarium graminearum*, the pathogen of *Fusarium graminearum*, from soil. Plate confrontation experiments showed that *Acanthamoeba rhysodes* NJAU-ZY1 significantly inhibited the mycelial growth of *Fusarium graminearum*. Liquid co-culture experiments showed that *Acanthamoeba rhysodes* NJAU-ZY1 significantly inhibited the number of spores of *Fusarium graminearum*. Pot experiments showed that *Acanthamoeba rhysodes* NJAU-ZY1 reduced the infectivity of *Fusarium graminearum* to wheat and alleviated wheat scab disease. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 Photographs showing the morphological characteristics of the vegetative body (left), reproductive cyst (middle), and dormant cyst (right) of Acanthamoeba NJAU-ZY1.

[0023] Figure 2 This is a phylogenetic tree of the 18S rDNA gene sequence of Acanthamoeba NJAU-ZY1 of this invention.

[0024] Figure 3 The images show plate photographs comparing the mycelial growth status of *Fusarium graminearum* in the plate confrontation experiment of Example 3 of this invention, specifically the PAS control group (left) and the *Acanthamoeba histolytica* NJAU-ZY1 treatment group (right).

[0025] Figure 4 This image shows a comparison of the number of Fusarium spores in the control group (left) and the Acanthamoeba NJAU-ZY1 treatment group (right) under an inverted microscope in the liquid co-culture experiment of Example 4 of the present invention.

[0026] Figure 5 This is a bar chart comparing the number of Fusarium graminearum spores in the CK treatment group (purple) and the Acanthamoeba NJAU-ZY1 treatment group (blue) under different co-culture times in Example 4 of the present invention.

[0027] Figure 6 These are potted plant photos comparing the growth of wheat in each treatment group during the potted plant experiment in Example 5 of this invention.

[0028] Figure 7 This is a comparative bar chart of wheat plant height (left) and dry weight (right) in each treatment group during the pot experiment in Example 5 of the present invention.

[0029] Information on the preservation of biological materials

[0030] Acanthamoebarhysodes NJAU-ZY1, classified as Acanthamoebarhysodes NJAU-ZY1, is deposited at the China Center for Type Culture Collection (CCTCC) on November 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:C2025219. Detailed Implementation

[0031] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All experiments in the following examples were performed in four replicates.

[0032] PAS culture medium: Reagent 1 (Stock solution 1): Sodium chloride 24 g / L, magnesium sulfate heptahydrate 0.8 g / L, calcium chloride hexahydrate 1.2 g / L. Reagent 2 (Stock solution 2): Disodium hydrogen phosphate 28.4 g / L, potassium dihydrogen phosphate 27.2 g / L. Mix 5 mL of Reagent 1 and 5 mL of Reagent 2 in 1 L of pure water to prepare PAS culture medium.

[0033] Example 1: Isolation and purification of protozoan NJAU-ZY1

[0034] Rhizosphere soil samples were collected from tomato fields in Shandong Province at depths ranging from 0 to 20 cm. The samples were sealed in sterile self-sealing bags and brought back to the laboratory. Protozoa were isolated using a soil dilution method. The specific method was as follows: The rhizosphere soil samples were thoroughly mixed. 1 g of soil was added to a 50 mL centrifuge tube along with 30 mL of sterile deionized water. The centrifuge tube was then placed in a shaker at 250 rpm and 20 °C for 15 min to ensure thorough mixing and release of protozoa. After removing the centrifuge tube from the shaker and allowing it to stand for 10 min, the supernatant was collected and added to a 96-well plate. Inactivated E. coli (OD=0.04) was added as food, and the plate was then incubated at 20 °C in the dark for 2 days. Protozoan growth was examined under an inverted microscope at 100×, 200×, and 400× magnification. Serial dilutions were performed, and the plates were incubated at 20 °C for 2 days. Finally, single protozoan cells were picked up using a capillary tube and transferred to a new 96-well plate to obtain a pure culture of protozoa. In PAS culture medium, the protozoan NJAU-ZY1 exhibited an active state, displaying irregular, constantly changing shapes with sharp, spiny pseudopodia on its surface. Figure 1 (Left); its reproductive cyst state is a combination of both active and cystic states. Figure 1Its dormant cyst state is a multifaceted double-layered cyst wall structure, exhibiting a static state (in the middle); Figure 1 right).

[0035] Example 2: Identification of the protozoan NJAU-ZY1

[0036] Molecular biology methods were used to identify the isolated protozoa. The PCR reaction system (50 μL system) consisted of 2 μL each primer, 25 μL of 2× Mix, and ddH2O added to bring the volume to 50 μL. The primers were universal primers: P-FLA-F: SEQ ID NO: 1 / P-FLA-R: SEQ ID NO: 2. The reaction program was: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 55 s, 50℃ annealing for 50 s, 72℃ extension for 1 min, 72℃ extension for 10 min, 35 cycles, followed by storage at 16℃. The PCR products were recovered by 1.5% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The 18S rDNA sequencing result was: SEQ ID NO: 3.

[0037] Using BLAST software, the obtained 18S rDNA gene sequences were compared with the NT (Nucleotide Sequence Database) on the NCBI website. Phylogenetic analysis of the isolated protozoa was performed using MEGA software. The sequencing results of *Acanthamoeba* NJAU-ZY1 showed the highest similarity (98.49%) to the model protozoan *Acanthamoeba rhysodes*. Therefore, the isolated protozoa NJAU-ZY1 was identified as *Acanthamoeba rhysodes*. Figure 2 A phylogenetic tree based on the 18S rDNA gene sequence is presented. This strain was deposited at the China Center for Type Culture Collection on November 11, 2025, with accession number CCTCC NO: C2025219.

[0038] Example 3: Plate confrontation experiment of Acanthamoeba NJAU-ZY1 inhibiting the mycelial growth of Fusarium graminearum.

[0039] The experiment was set up with two treatments: (1) PAS control group: CK; (2) treatment group inoculated with Acanthamoeba NJAU-ZY1. Each treatment was replicated 4 times.

[0040] The tested pathogenic fungus for Fusarium graminearum was Fusarium graminearum PH-1, the type strain of Fusarium graminearum, hereinafter referred to as PH-1. The tested pathogen was Acanthamoeba NJAU-ZY1, from Example 2, preservation number: CCTCC NO:C2025219.

[0041] Preparation of Acanthamoeba NJAU-ZY1: Acanthamoeba NJAU-ZY1 was inoculated into liquid PAS medium, and inactivated Escherichia coli (OD=0.04) was added. The medium was then placed in a constant temperature incubator at 20℃ and incubated statically. After 48 h, 100 μL of the culture medium was aspirated and counted under an inverted microscope.

[0042] Preparation of Fusarium graminearum PH-1: Mycelial blocks were picked and inoculated into 50 mL of liquid CMC medium and cultured in a shaker at 25℃ and 180 rpm for 3 days to prepare a homogeneous spore suspension. After filtration, the spores were counted using a hemocytometer.

[0043] 90 mm plates were prepared using PDA solid culture medium, and 1 μL of a 1×10⁻⁶ solution was inoculated at the center of each plate. 6 The control group was inoculated with 1 μL of PAS culture medium at four symmetrical points on the edge of the plate, while the treatment group was inoculated with 1 μL of 1×10⁶ PAS culture medium at four symmetrical points on the edge of the plate. 5 Acanthamoeba NJAU-ZY1 cells / mL were used in 4 replicates per treatment and incubated in a fungal incubator at 25°C.

[0044] Observe the growth status of Fusarium graminearum mycelium in the plate after 72 hours. Figure 3 The NJAU-ZY1 treatment group showed a clear inhibition zone compared to the control group. This indicates that NJAU-ZY1 can significantly inhibit the growth of Fusarium graminearum pathogen.

[0045] Example 4: Liquid co-culture experiment on the inhibition of spore count of Fusarium graminearum pathogen by NJAU-ZY1

[0046] The experiment was set up with two treatments: (1) blank control group CK; (2) treatment group inoculated with NJAU-ZY1, with 4 replicates for each treatment.

[0047] The pathogenic fungus of the cereal sickle was PH-1, and the tested strain was Acanthamoeba NJAU-ZY1 from Example 2, preservation number: CCTCC NO: C2025219.

[0048] Protozoan preparation: NJAU-ZY1 cells were inoculated into six-well cell culture plates, and 5 ml of liquid PAS medium and 400 μL of inactivated E. coli (OD=0.04) were added. The plates were placed in a constant temperature incubator at 20°C and incubated statically. After 48 h, the cells were counted under an inverted microscope.

[0049] Preparation of Fusarium graminearum PH-1: Mycelial blocks were picked and inoculated into 50 mL of CMC medium and cultured in a shaker at 25℃ and 180 rpm for 3 days to prepare a homogeneous spore suspension. After filtration, the spores were counted using a hemocytometer.

[0050] The experiment used a 24-well cell culture plate system, with a final volume of 500 μL per well. The control group was prepared by adding 499 μL of PAS medium + 1 μL of 1×10⁻⁶ PAS medium per well. 6 / ml of *Fusarium graminearum* PH-1 suspension was added to each well of the treatment group, followed by 1μL of 1×10⁶ PAS medium. 6 / ml Fusarium graminearum PH-1 suspension, 1μL concentration is 1×10 5 / ml NJAU-ZY1 suspension. Place in a 25℃ constant temperature incubator, gently shake at 80rpm, and continue incubation for 5 days.

[0051] The fungal spores were observed and counted under a microscope every 24 hours, and the results were as follows: Figure 4 As shown, at 24 h, the average spore count in the CK treatment group was 137 per μL, and the average spore count in the Acanthamoeba rhysodes treatment group was 66 per μL, a decrease of 51.82% compared to the control group; at 72 h, the average spore count in the CK treatment group was 367 per μL, and the average spore count in the Acanthamoeba rhysodes treatment group was 76 per μL, a decrease of 79.29% compared to the control group; at 120 h, the average spore count in the CK treatment group was 536 per μL, and the average spore count in the Acanthamoeba rhysodes treatment group was 57 per μL, a decrease of 89.37% compared to the control group. The number of spores in the CK treatment group increased continuously with the increase of co-culture time, while the number of spores in the Acanthamoeba rhysodes treatment group first increased and then decreased with the increase of Acanthamoeba rhysodes number in the field of view. Furthermore, the spores in the CK treatment group were intact, plump, and larger in size, while the spores in the Acanthamoeba rhysodes treatment group had many vacancies and were smaller in size. This indicates that the application of NJAU-ZY1 can significantly reduce the number of Fusarium graminearum spores and significantly inhibit the development of Fusarium graminearum spores. Figure 5 The number of spores of Fusarium graminearum showed significant differences among different treatment groups and changed regularly with co-culture time.

[0052] Example 5: Pot experiment on NJAU-ZY1's role in helping wheat resist infection by Fusarium graminearum.

[0053] The experiment was set up with four treatments: (1) blank control group CK; (2) inoculation with Fusarium graminearum PH-1 alone; (3) inoculation with NJAU-ZY1 alone; (4) inoculation with both Fusarium graminearum PH-1 and NJAU-ZY1. Each treatment was replicated in 4 places.

[0054] The pathogenic fungus of the cereal sickle was PH-1, and the tested strain was Acanthamoeba NJAU-ZY1 from Example 2, preservation number: CCTCC NO:C2025219.

[0055] Protozoan preparation: NJAU-ZY1 was inoculated into 75cm... 2 Add 50 ml of liquid PAS medium and 2.5 ml of inactivated E. coli (OD=0.04) to the cell culture flask. Incubate at 20°C for static culture. After 48 h, aspirate 100 μL of culture medium and count the cells under an inverted microscope.

[0056] Preparation of Fusarium graminearum PH-1: Mycelial blocks were picked and inoculated into 250 mL of liquid CMC medium and cultured in a shaker at 25℃ and 180 rpm for 3 days to prepare a homogeneous mycelial suspension. After filtration, the mycelial cells were counted using a hemocytometer.

[0057] Five wheat plants were inoculated into each pot with 300g of soil. The inoculation concentrations of NJAU-ZY1 and Fusarium graminearum PH-1 were both 1000 CFU / g. A treatment group was simultaneously inoculated with both NJAU-ZY1 and Fusarium graminearum. The NJAU-ZY1 inoculation was carried out first, and the plants were colonized in the soil for two days before being inoculated with Fusarium graminearum PH-1. After 30 days of cultivation in a greenhouse, samples were collected, and the dry weight and height of the wheat plants were measured. The growth of the potted plants is as follows. Figure 6 As shown.

[0058] Compared to the control group, NJAU-ZY1 inoculation alone had no significant effect on wheat biomass; inoculation with Fusarium graminearum PH-1 alone significantly reduced wheat biomass; and compared with PH-1 inoculation, combined inoculation with NJAU-ZY1 and PH-1 significantly alleviated pathogen stress, as evidenced by a significant increase in wheat plant height and dry weight. These results indicate that application of NJAU-ZY1 can significantly reduce the infectivity of Fusarium graminearum in wheat and alleviate its inhibitory effect on wheat growth. Figure 7 The study showed significant differences in wheat plant height and dry weight among different treatment groups.

Claims

1. A strain of Acanthamoeba that inhibits Fusarium graminearum pathogen. Acanthamoeba rhysodes NJAU-ZY1, characterized in that, It is deposited at the China Center for Type Culture Collection (CCTCC) on November 11, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:C2025219.

2. The Acanthamoeba protozoan of claim 1 Acanthamoeba rhysodes The culture of NJAU-ZY1 is characterized by, The method for preparing the culture includes using the Acanthamoeba protozoa... Acanthamoeba rhysodes NJAU-ZY1 was inoculated into PAS culture medium, and inactivated microorganisms were added as food. The culture was then placed in an incubator at 18℃-22℃ and allowed to stand for 40-50 hours. The inactivated microorganisms were inactivated *Escherichia coli*. The PAS culture medium was prepared by mixing 5 mL of reagent 1 and 5 mL of reagent 2 in 1 L of pure water to prepare the PAS culture medium. The formula for reagent 1 was: sodium chloride 24 g / L, magnesium sulfate heptahydrate 0.8 g / L, calcium chloride hexahydrate 1.2 g / L; the formula for reagent 2 was: disodium hydrogen phosphate 28.4 g / L, potassium dihydrogen phosphate 27.2 g / L.

3. The Acanthamoeba protozoan of claim 1 Acanthamoeba rhysodes NJAU-ZY1 inhibits the pathogenic fungus Fusarium graminearum (… Fusarium graminearum Applications in ).

4. The culture according to claim 2 inhibits the pathogenic fungus *Fusarium graminearum* (…). Fusarium graminearum Applications in ).

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