A fungus of the genus Cucurbitacum and its application

Abstract

This invention discloses a fungus of the genus *Cucurbitariaceae* and its applications, relating to the field of plant pathogen control technology. The fungus is named *Cucurbitariaceae* sp., strain number ZJU2023CU3, and its preservation number is CCTCC NO: M 20231368. The *Cucurbitariaceae* fungus ZJU2023CU3 provided by this invention exhibits inhibitory effects against *Bacillus oryzae* and *Sclerotinia serratifolia* in plate confrontation experiments; in fermentation broth experiments, the inhibition rate against *Bacillus oryzae* is as high as 47.48%, and the inhibition rate against *Sclerotinia serratifolia* is as high as 53.08%. This indicates that strain ZJU2023CU3 can effectively inhibit the growth of *Bacillus oryzae* and *Sclerotinia serratifolia*, and can be used to prepare novel, highly efficient, and broad-spectrum biocontrol agents, providing a new approach for the development of microbial applications in biological control.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen control technology, and in particular to a fungus of the genus *Cyclocarya* and its applications. Background Art

[0002] Rice blast, a globally prevalent disease, is caused by the fungus *Blastomyces oryzae*, which can damage various tissues of rice. Depending on the affected part, it can be classified into seedling blast, leaf blast, grain blast, and neck blast, with neck blast being the most damaging. Due to variations in climate, soil conditions, and rice varieties across the country, coupled with the wide host range, strong resistance, and tendency to mutate into resistant mutants, controlling rice blast has become a historically challenging problem. As one of the three major rice diseases, rice blast is characterized by rapid spread and difficulty in control, not only reducing rice yield but also affecting rice quality. The main reason for the reduced quality is that rice blast is most prevalent during periods of insufficient sunshine and abundant rainfall, which weakens photosynthesis, leading to yield reduction or quality degradation. Therefore, during the rice growing season, scientific and reasonable control measures should be adopted based on local planting conditions. Currently, the main control measures include chemical control, selection of disease-resistant rice varieties, cultivation management, and blocking of the source of infection. However, these measures all have shortcomings in certain aspects. Therefore, it is practically necessary to explore the occurrence and control strategies of rice blast.

[0003] Corn leaf blight is one of the major fungal diseases in corn-producing areas, and it is more severe in summer corn areas, generally causing yield losses of more than 15%, and in severe cases, more than 50%, seriously affecting the yield and quality of corn. Corn leaf blight is spread by air currents, can cause multiple infections, and is characterized by rapid onset and spread. High ambient temperature and humidity greatly increase susceptibility to leaf blight. The fungus primarily infects corn leaves, causing leaf spots. The disease can occur throughout the entire growth cycle of corn, but it is most prevalent during the tasseling and grain-filling stages, thus severely impacting yield. In areas where corn leaf blight occurs, it often co-occurs with other common corn diseases such as large leaf blight, therefore, integrated pest management is urgently needed.

[0004] As a relatively new method for controlling rice blast and corn leaf blight in recent years, biological control is gradually moving from the margins to the center stage. Firstly, traditional disease control methods primarily rely on chemical pesticides. While these pesticides have a powerful bactericidal effect, their high toxicity easily leads to pesticide residues in soil and water, causing irreversible and significant damage to the ecological environment. Compared to traditional control techniques, biological control offers advantages such as low cost, significant effectiveness, sustainable control, safety for humans and non-targeted organisms, no residue, and ease of coordination with other control measures, all without disrupting the environment and ecological balance. It is a crucial method for improving the safety of crop production. It can enhance the social and environmental benefits of rice and corn cultivation, making the resulting rice and corn more marketable and generating greater economic value. Therefore, biological control will become an important measure in agricultural pest and disease control. Summary of the Invention

[0005] This invention provides a fungus of the genus *Cucurbitariaceae* and its applications. The strain ZJU2023CU3 used in this application was isolated from soil at the Zijingang Campus of Zhejiang University, Hangzhou, Zhejiang Province (30.312106°N, 120.097711°E). Through sequence comparison and morphological observation, the isolated strain ZJU2023CU3 was confirmed to be a *Cucurbitariaceae* sp. fungus.

[0006] This invention provides a fungus of the genus Cucurbitariaceae sp., named Cucurbitariaceae sp., strain number ZJU2023CU3, and preservation number CCTCC NO: M20231368.

[0007] This invention also provides the application of the aforementioned fungi of the genus *Cochliobolus* in the preparation of plant pathogen inhibitors, wherein the plant pathogen is *Magnaporthe oryzae* or *Cochliobolusheterostrophus*.

[0008] The present invention also provides a plant pathogen inhibitor comprising a fermentation broth of the aforementioned *Cyclocarya* fungus. The volume percentage concentration of the fermentation broth is 10%–30%.

[0009] This invention also provides a method for preparing the plant pathogen inhibitor, wherein the *Cyclocarya paliurus* fungus is inoculated into a fermentation medium and cultured, and the fermentation broth after removing the fungal cells is the plant pathogen inhibitor. The culture temperature is 25℃, and the culture time is 14 days.

[0010] This invention also provides the application of the plant pathogen inhibitor in inhibiting rice blast fungus or maize leaf spot fungus. In use, the plant pathogen inhibitor is sprayed onto the plants.

[0011] The beneficial effects of this invention are:

[0012] The *Cucurbitariaceae* sp. fungus ZJU2023CU3 provided by this invention can inhibit the growth of *Oryza sativa* and *Spodoptera exigua* in plate confrontation tests; in fermentation broth tests, the inhibition rate against *Oryza sativa* is as high as 47.48%; and the inhibition rate against *Spodoptera exigua* is as high as 53.08%. This indicates that strain ZJU2023CU3 can effectively inhibit the growth of *Oryza sativa* and *Spodoptera exigua*, and can be used to prepare novel, highly efficient, and broad-spectrum biocontrol agents, providing a new approach for the development of microbial applications in biological control. Attached Figure Description

[0013] Figure 1 The image shows the colony morphology of ZJU2023CU3, a fungus of the genus *Cyclocarya*, grown on CM medium for 9 days; where A is the front view of the petri dish and B is the back view of the petri dish.

[0014] Figure 2 The images show the hyphal morphology and spore morphology of fungus ZJU2023CU3 of the genus *Cyclocarya*.

[0015] Figure 3 The image shows the results of the detection of the inhibition of rice blast fungus ZJU2023CU3 by *Cyclocarya paliurus*. In the image, A shows the inhibition of rice blast fungus ZJU2023CU3 by rice blast fungus, and B shows the growth status of rice blast fungus.

[0016] Figure 4 The graph shows the inhibitory effect of the fermentation broth of *Cyclocarya paliurus* ZJU2023CU3 on *Orychophragmus oryzae*. In this graph, A is the colony diagram showing the inhibition of *Orychophragmus oryzae* by the fermentation broth of *Cyclocarya paliurus* ZJU2023CU3, and B is the bar chart showing the relative inhibition rate of *Orychophragmus oryzae* ZJU2023CU3 on *Orychophragmus oryzae*. *** indicates p < 0.001.

[0017] Figure 5 The image shows the results of the detection of the inhibition of corn leaf blight pathogen by fungus ZJU2023CU3; where A is the inhibition of corn leaf blight pathogen by fungus ZJU2023CU3, and B is the growth status of corn leaf blight pathogen.

[0018] Figure 6The graph shows the inhibitory effect of the fermentation broth of *Cyclocarya spp.* fungus ZJU2023CU3 on *Sclerotium spp.* of corn. In this graph, A is the colony diagram of the inhibition of *Sclerotium spp.* fungus ZJU2023CU3 on *Sclerotium spp.* fungus, and B is the bar chart of the relative inhibition rate of the fermentation broth of *Cyclocarya spp.* fungus ZJU2023CU3 on *Sclerotium spp.* fungus. Detailed Implementation

[0019] Example 1: Isolation of bacterial strains

[0020] (1) Soil samples were collected from the mulberry garden of Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province. The selective medium dilution plate method was used for separation. The procedure was as follows: Take five 1.5 mL centrifuge tubes and label them with 10... -2 10 -3 10 -4 10 -5 10 -6 Use a pipette to draw 0.9 mL of sterile water and add it to each centrifuge tube;

[0021] (2) Weigh 1g of sample and place it in a stoppered test tube containing 9mL of sterile water. Shake for 10min to obtain 10g of sample. -1 Soil dilution solution;

[0022] (3)10 -1 After shaking and allowing the soil dilution to stand for 2 minutes, pipette 0.1 mL of the suspension into a centrifuge tube containing 0.9 mL of sterile water. Dilute sequentially to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 Diluent;

[0023] (4) Pour 20 mL of sterilized PDA medium (with streptomycin and ampicillin added to each medium to reach 30 μg / L to inhibit bacterial growth) cooled to 50-60℃ into petri dishes; after the medium has cooled to form plates, use a pipette to aspirate 10 mL of the medium into each petri dish. -6 10 -5 10 -4 10 -3 Add 0.1 mL of the diluent suspension to a plate, and then spread each diluent evenly with a sterile spreader. Repeat 3 plates for each concentration.

[0024] (5) Invert the petri dish and incubate it in a constant temperature incubator at 25℃ for 3-5 days. Then pick a single colony of mycelium and place it on a new plate.

[0025] PDA medium: 10g glucose, 5g yeast extract, 15g agar, 1000mL distilled water.

[0026] A bacterial strain was isolated from the soil of Zhejiang University Zijingang Campus in Hangzhou, Zhejiang Province (30.312106°N, 120.097711°E) and named ZJU2023CU3.

[0027] Example 2: Identification of fungal strain morphology

[0028] After 9 days of cultivation on CM medium at 25℃ under alternating 16-hour light / 8-hour dark conditions, strain ZJU2023CU3 exhibited an average mycelial growth rate of 4.49–4.67 mm per day, and reached full coverage on 70 mm diameter plates after 14.99–15.59 days. The colonies were predominantly brownish-green to whitish on the upper surface. Pale yellow spore masses often formed near the center of the colony. Aerial hyphae were abundant and dense, spreading in a circular pattern, with irregularly shaped circular edges. Figure 1 A). The back is smooth and dark brown, with a pale yellow border around the colony. Figure 1 B). Under a microscope, its hyphae are visible; the hyphae are robust and branching, of varying thickness, and often appear swollen. The hyphae are septate and contain contents ( Figure 2 A). Conidia are mostly oval, numerous, unicellular, and measure (3.43–3.58) μm × (1.71–1.97) μm. Figure 2 B).

[0029] Based on the combined characteristics of colony morphology, the morphology of small conidia, and molecular biological identification results, strain ZJU2023CU3 was identified as a fungus belonging to the genus Cucurbitariaceae sp.

[0030] Example 3: Molecular Identification

[0031] (1) DNA extraction

[0032] 1) After culturing strain ZJU2023CU3 on CM plates at 25℃ for 7 days, the mycelia on the plates were scraped off with a toothpick and placed into a sterilized 1.5mL centrifuge tube containing 500μL of extraction buffer and an appropriate amount of quartz sand. The extraction buffer formula is: 10mM ethylenediaminetetraacetic acid, 100mM Tris-hydrochloric acid, and 1M potassium chloride.

[0033] 2) Place the centrifuge tubes in MP In a -24°C homogenizer, the mycelial tissue was broken by oscillation at 65 Hz for 2 minutes.

[0034] 3) Centrifuge at 12000 rpm for 10 min, then pipette 300 μL of the supernatant and transfer it into a brand new centrifuge tube;

[0035] 4) Add an equal volume of isopropanol to the supernatant, invert the tube to mix well, and place the centrifuge tube in a 4°C freezer for 10 minutes to precipitate DNA better.

[0036] 5) Centrifuge at 12000 rpm for 10 minutes, discard the supernatant and keep the precipitate;

[0037] 6) Add 800 μL of 70% ethanol (v / v) to dissolve the impurities, mix gently, and centrifuge at 12000 rpm for 5 min.

[0038] 7) Discard the supernatant, invert the centrifuge tube onto absorbent paper to evaporate excess ethanol. The white precipitate is the genomic DNA to be extracted. Add 50 μL of sterile water and dissolve at room temperature for 15 minutes. After dissolution, store the genomic DNA in a -20°C freezer.

[0039] (2) PCR amplification of fungal ribosomal ITS rDNA gene, 28S large ribosomal subunit rRNA gene (LSU), and 18S small ribosomal subunit rRNA gene (SSU).

[0040] ITS primers:

[0041] The upstream primer ITS1 sequence is: 5′-TCCGTAGGTGAACCTGCGG-3′.

[0042] The downstream primer ITS4 sequence is: 5′-TCCTCCGCTTATTGATATGC-3′;

[0043] LSU primers:

[0044] The upstream primer LSU-F sequence is: 5′-GTACCCGCTGAACTTAAGC-3′.

[0045] The downstream primer LSU-R sequence is: 5′-TCCTGAGGGAAACTTCG-3′;

[0046] SSU primers:

[0047] The upstream primer SSU-F sequence is: 5′-GTAGTCATATGCTTGTCTC-3′.

[0048] The downstream primer SSU-R sequence is: 5′-CTTCCGTCAATTCCTTTAAG-3′;

[0049] PCR amplification was performed in a 20 μL reaction system containing: 1 μL each of forward and reverse primers, 10 μL of Green Taq Mix, 2 μL of template DNA, and 6 μL of ddH2O.

[0050] PCR amplification was performed on a GE-TOUCH intelligent gradient gene amplification instrument. Reaction conditions: 95℃ pre-denaturation for 3 min, followed by 35 cycles of: 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min; and a final extension at 72℃ for 10 min.

[0051] (3) Gene sequencing and sequence analysis

[0052] The target DNA fragment was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing. After rigorous verification, the sequencing results yielded DNA fragment sequences as shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, namely the fungal ribosomal ITS rDNA gene, the 28S large ribosomal subunit rRNA gene (LSU), and the 18S small ribosomal subunit rRNA gene (SSU).

[0053] On the NCBI website, the nucleotide sequences of the ITS, 28S large ribosomal subunit rRNA gene (LSU), and 18S small ribosomal subunit rRNA gene (SSU) of strain ZJU2023CU3 were obtained and searched for and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. Sequence comparison showed that strain ZJU2023CU3 was most closely related to the *Cucurbitariaceae* sp. fungus. This result was consistent with the morphological identification results, indicating that the isolated strain ZJU2023CU3 is *Cucurbitariaceae* sp. fungus. This newly screened strain was named *Cucurbitariaceae* sp., with strain number ZJU2023CU3, and was deposited on July 24, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M 20231368.

[0054] Example 4: Inhibitory effect of strain ZJU2023CU3 on rice blast fungus

[0055] The ZJU2023CU3 strain was subjected to confrontation culture with *Magnaporthe oryzae* (USAST, https: / / www.atcc.org / products / 201236): *Magnaporthe oryzae* and *Magnaporthe oryzae* strains were inoculated onto opposite sides of a 90 mm diameter CM plate, 4.5 cm apart. After 14 days of confrontation culture on CM plates, the growth of the *Magnaporthe oryzae* colony leading edge closer to the ZJU2023CU3 strain was inhibited, and the mycelium showed significant inhibition. Figure 3 ).

[0056] Example 5: Detection of the inhibitory effect of fermentation broth of strain ZJU2023CU3 on rice blast fungus

[0057] ZJU2023CU3 strain was inoculated into 150 mL CM medium / 250 mL Erlenmeyer flask and cultured at 25℃ and 150 rpm for 12 days, followed by 2 days of static incubation at room temperature. The supernatant was collected by centrifugation and used as the fermentation broth. After filtration and sterilization, it was added to CM medium in a specific ratio to prepare fermentation broth-CM plates. These plates were then inoculated with rice blast fungus, and the antibacterial effect was observed after 6 days. Rice blast fungus was inoculated onto CM medium without fermentation broth, and with 10% and 30% fermentation broth (v / v) respectively, and cultured for 6 days. Three replicates were set up for each group at different concentrations. Figure 4 ).like Figure 4 As shown in Figure A, the higher the concentration of fermentation broth, the more significant the inhibitory effect on rice blast fungus. The inhibitory effect is as follows: Figure 4 As shown in Figure B, the inhibition effect reached 11.56% when 10% fermentation broth was added; and the inhibition effect reached approximately 47.48% when 30% fermentation broth was added.

[0058] Example 6: Inhibitory effect of strain ZJU2023CU3 on maize leaf spot pathogen

[0059] The ZJU2023CU3 strain was subjected to a confrontation culture with *Spodoptera exigua* (USAST, https: / / www.atcc.org / products / 48331): *Spodoptera exigua* and ZJU2023CU3 were inoculated onto opposite sides of a 90 mm diameter CM plate, 4.5 cm apart. After 6 days of confrontation culture on CM plates, the growth of the *Bombyx mori* colony front edge closer to the ZJU2023CU3 strain was inhibited, and the mycelium showed significant inhibition. Figure 5 ).

[0060] Example 7: Detection of the inhibitory effect of fermentation broth of strain ZJU2023CU3 on maize leaf spot pathogen.

[0061] ZJU2023CU3 strain was inoculated into 150 mL CM medium / 250 mL Erlenmeyer flask and cultured at 25℃ and 150 rpm for 12 days, followed by 2 days of static incubation at room temperature. The supernatant was collected by centrifugation and used as the fermentation broth. After filtration and sterilization, it was added to CM medium in a specific ratio to prepare fermentation broth-CM plates. These plates were then inoculated with *Sclerotium affine*, the causal agent of corn leaf blight, and the antibacterial effect was observed after 7 days. *Blastomyces oryzae* was inoculated into CM medium without fermentation broth, and with 10% and 30% fermentation broth (v / v), respectively, and cultured for 6 days. Three replicates were set up for each group at different concentrations. Figure 6 ).like Figure 6 As shown in Figure A, the fermentation broth exhibits a very significant inhibitory effect on corn leaf blight pathogens; the inhibitory effect is as follows: Figure 6As shown in the bar chart (B), the inhibition effect reached as high as 53.08% when 30% of the fermentation broth was added.

Claims

1. A species of *Calamus* ( ) Cucurbitariaceae sp.) fungus, named Cucurbitariaceae sp., Plant No. ZJU2023CU3, Preservation No. CCTCC NO: M 20231368.

2. The use of the *Cyclocarya* fungus of claim 1 in the preparation of plant pathogen inhibitors, wherein the plant pathogen is *Pseudomonas oryzae* (the causal agent of rice blast). Magnaporthe oryzae ) or corn leaf blight fungus ( Cochliobolus heterostrophus ).

3. A plant pathogen inhibitor, characterized in that, The fermentation broth includes the *Cyclocarya* fungus of claim 1, wherein the plant pathogen is *Oryza sativa* (*Oryza sativa*). Magnaporthe oryzae ) or corn leaf blight fungus ( Cochliobolus heterostrophus ).

4. The plant pathogen inhibitor according to claim 3, characterized in that, The volume percentage concentration of the fermentation broth is 10% to 30%.

5. The method for preparing the plant pathogen inhibitor according to claim 3 or 4, characterized in that, After inoculating the *Cyclocarya spp.* fungi of claim 1 into a fermentation medium and culturing them, the fermentation broth after removing the fungal cells is used as a plant pathogen inhibitor.

6. The method for preparing the plant pathogen inhibitor according to claim 5, characterized in that, The culture temperature was 25℃, and the culture period was 14 days.

7. The use of the plant pathogen inhibitor according to claim 3 or 4 in inhibiting rice blast fungus or maize leaf spot fungus.

8. The application according to claim 7, characterized in that, When using, spray the plant pathogen inhibitor onto the plant.

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