The protein encoded by the rice blast HYMA gene regulates the growth and pathogenicity of rice blast

Abstract

The present application aims to solve the technical problem of providing a new choice for preventing and treating Magnaporthe oryzae.The technical solution of the present application is the application of the protein encoded by the HYMA gene of Magnaporthe oryzae in regulating the growth of Magnaporthe oryzae.The amino acid sequence of the protein encoded by the HYMA gene is shown as SEQ ID NO.2.The application discloses the application of the protein encoded by the HYMA gene in regulating the growth and development, sporulation and pathogenicity of Magnaporthe oryzae, and provides a new choice for preventing and treating Magnaporthe oryzae and the plant diseases caused by Magnaporthe oryzae.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to application of a protein encoded by a Magnaporthe oryzae HYMA gene in regulating growth and pathogenicity of Magnaporthe oryzae. BACKGROUND

[0002] Rice blast caused by Magnaporthe oryzae is commonly known as rice "cancer", and can cause rice to be lost when the disease is prevalent. Magnaporthe oryzae mainly spreads in the form of conidia by air flow, and after contacting a host plant, sequentially undergoes conidial germination, germ tube formation, appressorium differentiation, penetration peg formation to penetrate the host epidermis, and colonization and expansion of infection hyphae in host cells. The formation of conidia is crucial for the spread and formation of infection structures of Magnaporthe oryzae. At present, the prevention and control of rice blast is mainly by combining breeding of resistant varieties and rational use of fungicides. However, the breeding of resistant varieties is difficult, and the resistant rice varieties for rotation are limited; on the other hand, the fungicides for controlling rice blast are mainly preventive, and the mode of action is relatively single, or the specificity is poor, the control effect is poor, and often the fungicides are ineffective when rice blast is prevalent, so there is still a big bottleneck in the prevention and control of rice blast. Finding pathogenicity-related genes of Magnaporthe oryzae to provide ideal potential targets for the research and development of fungicides has important guiding significance for the green prevention and control of rice blast. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a new option for preventing and controlling Magnaporthe oryzae.

[0004] The technical solution of the present application is the application of a protein encoded by a Magnaporthe oryzae HYMA gene in regulating the growth of Magnaporthe oryzae; the amino acid sequence of the protein encoded by the HYMA gene is shown in SEQ ID NO. 2.

[0005] Among them, the regulation of the growth of Magnaporthe oryzae is the regulation of the growth of Magnaporthe oryzae mycelium and / or sporulation.

[0006] Specifically, the regulation is negative regulation.

[0007] In particular, the negative regulation is to inhibit or block the expression of the protein encoded by the Magnaporthe oryzae HYMA gene.

[0008] The present application also provides the application of a substance that inhibits or blocks the expression of the protein encoded by the Magnaporthe oryzae HYMA gene in inhibiting the growth of Magnaporthe oryzae; the amino acid sequence of the protein encoded by the HYMA gene is shown in SEQ ID NO. 2.

[0009] Among them, the inhibition of the growth of Magnaporthe oryzae is the inhibition of the growth of Magnaporthe oryzae mycelium and / or sporulation.

[0010] The application also provides application of the protein encoded by the Magnaporthe oryzae HYMA gene in regulating pathogenicity of Magnaporthe oryzae; the amino acid sequence of the protein encoded by the HYMA gene is shown as SEQ ID NO. 2.

[0011] Specifically, the regulation is negative regulation.

[0012] In particular, the negative regulation is inhibition of expression of the protein encoded by the Magnaporthe oryzae HYMA gene in Magnaporthe oryzae.

[0013] The application also provides application of a substance that inhibits or blocks expression of the protein encoded by the Magnaporthe oryzae HYMA gene in reducing pathogenicity of Magnaporthe oryzae; the amino acid sequence of the protein encoded by the HYMA gene is shown as SEQ ID NO. 2.

[0014] The application also provides application of a substance that knocks out the Magnaporthe oryzae HYMA gene in reducing pathogenicity of Magnaporthe oryzae; the nucleotide sequence of the HYMA gene is shown as SEQ ID NO. 1.

[0015] The application also provides application of a substance that inhibits or blocks expression of the protein encoded by the Magnaporthe oryzae HYMA gene in preventing and treating rice blast; the amino acid sequence of the protein encoded by the HYMA gene is shown as SEQ ID NO. 2.

[0016] The application also provides application of a substance that knocks out the Magnaporthe oryzae HYMA gene in preventing and treating rice blast; the nucleotide sequence of the HYMA gene is shown as SEQ ID NO. 1.

[0017] The application has the following beneficial effects: the application discloses application of the protein encoded by the HYMA gene in regulating growth and development, sporulation and pathogenicity of Magnaporthe oryzae. After the HYMA gene of Magnaporthe oryzae is knocked out, the Magnaporthe oryzae shows slow hyphal growth, black colony and no conidium production; pathogenicity experiment detection shows that the Magnaporthe oryzae HYMA gene knockout body cannot form obvious lesions on the leaf, and the hyphal mass cannot form lesions and expand on the wounded rice leaf, indicating that the HYMA gene of Magnaporthe oryzae plays an important function in regulating growth and development, sporulation and pathogenicity of Magnaporthe oryzae. Based on the foregoing experimental results, the application provides a substance that inhibits expression of the protein encoded by the Magnaporthe oryzae HYMA gene, which can regulate growth and pathogenicity of Magnaporthe oryzae by negative regulation, so as to achieve the effect of preventing and treating rice blast. It can be seen that the technical scheme of the application has important significance in prevention and treatment of rice blast, and provides a new choice for preventing and treating Magnaporthe oryzae and plant diseases caused by Magnaporthe oryzae. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 , Magnaporthe oryzae HYMA gene knockout principle model.

[0019] Figure 2 ,4 PCR detection of the hyma knock-out transformants, in which lane Marker is 5000 DNA marker; lane 1, lane 5, lane 9 and lane 13 are the HYMA identification genes amplified with the wild type control bacteria P131 genomic DNA as template; lane 2, lane 6, lane 10 and lane 14 are the HYMA identification genes amplified with the genomic DNA of knock-out transformants Ahyma-1, Ahyma-2, Ahyma-3 and Ahyma-4 as template respectively; lane 3, lane 7, lane 11 and lane 15 are the upstream fragments of the knocked-out HYMA genes amplified with the genomic DNA of knock-out transformants Ahyma-1, Ahyma-2, Ahyma-3 and Ahyma-4 as template respectively; lane 4, lane 8, lane 12 and lane 16 are the downstream fragments of the knocked-out HYMA genes amplified with the genomic DNA of knock-out transformants Ahyma-1, Ahyma-2, Ahyma-3 and Ahyma-4 as template respectively.

[0020] Figure 3 Observation of the colony morphology of knock-out transformants Ahyma-1 and Ahyma-2.

[0021] Figure 4 Determination of the colony growth diameter of knock-out transformants Ahyma-1 and Ahyma-2.

[0022] Figure 5 Determination of the pathogenicity of knock-out transformants Ahyma-1 and Ahyma-2 on rice.

[0023] Figure 6 Counting of the lesion number on the rice leaves after spraying the knock-out transformants Ahyma-1 and Ahyma-2.

[0024] Figure 7 Determination of the pathogenicity of mycelial plugs of knock-out transformants Ahyma-1 and Ahyma-2 on the wounded rice leaves.

[0025] Figure 8 Counting of the lesion length of the mycelial plugs of knock-out transformants Ahyma-1 and Ahyma-2 on the wounded rice leaves.

[0026] Figure 9 Observation of the conidiospores of knock-out transformants Ahyma-1 and Ahyma-2.

[0027] Figure 10 Counting of the conidiospore yield of knock-out transformants Ahyma-1 and Ahyma-2. DETAILED DESCRIPTION

[0028] The wild type P131 of Magnaporthe grisea used in the following examples was from the laboratory of Professor Peng Youliang, China Agricultural University; the test rice plants were Lijiang Xintuanhegu.

[0029] The medium formula used in the following examples is as follows:

[0030] Tomato oatmeal medium OTA (Oat-meal Tomato Agar medium) (1 L): 40 g oatmeal was weighed and added to 800 mL water and boiled for 30 minutes, filtered, and the filtrate was taken; the tomato was crushed and filtered, and 150 mL of the filtrate was mixed with the oatmeal filtrate, and the volume was made up to 1 L, 0.6 g CaCO3 and 1.5% agar were added. High-temperature high-pressure sterilization, storage at room temperature for standby;

[0031] Liquid complete medium CM (1 L): yeast extract 6 g, enzyme hydrolyzed casein 3 g, acid hydrolyzed casein 3 g, sucrose 10 g, high-temperature high-pressure sterilization (solid CM needs to add agar powder 16 g);

[0032] Water agar medium: 1.5% agar powder;

[0033] Cell wall regeneration medium (1 L): 1 g yeast powder, 1 g enzyme hydrolyzed casein, 342 g sucrose;

[0034] Cell wall regeneration medium (solid): 1.5% agar powder was added to the liquid cell wall regeneration medium.

[0035] Genetic transformation reagent formula:

[0036] 0.7 M sodium chloride (1 L): 40.908 g sodium chloride, high-temperature high-pressure sterilization;

[0037] STC: 1.2 M sorbitol, 10 mM Tris-Cl (pH 7.5), 50 mM anhydrous calcium chloride;

[0038] 1 M Tris 7.5 (1 L): Tris Base 121.14 g, adjust pH to 7.5 with dilute hydrochloric acid;

[0039] PTC (1 L): 60% polyethylene glycol 3350, 10 mM Tris-Cl (pH 7.5), 50 mM anhydrous calcium chloride;

[0040] Lyticase solution: 20 mg / mL cell wall lyticase (L1412, MERCK), prepared with 0.7 M sodium chloride, 0.22 µm bacterial filter sterilization.

[0041] Genomic DNA extraction reagent:

[0042] 2×CTAB solution: Dissolve 10 mL 1.0 M Tris-Cl (pH 8.0), 40 mL 0.5 M EDTA (pH 8.0), 20 g CTAB and 81.8 g sodium chloride in 800 mL ddH2O, bring the volume to 1 L, and dissolve overnight at 37°C; autoclave and store at room temperature;

[0043] 0.5 M EDTA (pH 8.0) (1 L): Dissolve 186.1 g of Na2EDTA·2H2O, adjust the pH to 8.0 with HCl, and bring the volume to 1 L.

[0044] TE / RNase: 10 mM Tris 8.0, 1 mM EDTA, 10 µg / mL RNase.

[0045] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0046] Example 1: Construction of HYMA gene knockout transformants of *Magnapordia oryzae*

[0047] The HYMA (MGG_03219) gene and its upstream and downstream 3 kb sequences were searched and downloaded from the whole genome sequence database of rice blast strain 70-15 (http: / / www.riceblast.org / ). Figure 1 Primers were designed at the indicated positions (primer information is shown in Table 1). Split-PCR was used for the first round of PCR (amplification system and procedure are shown in Table 2), with a total of three PCRs, yielding an LB 2.6 kb, hygromycin (HPT), and RB 2.2 kb fragments. The second round of PCR (amplification system and procedure are shown in Table 3) consisted of two PCRs, using the first round PCR product (LB 2.6 kb and hygromycin sequence) and the first round PCR product (RB 2.2 kb and hygromycin sequence) as templates, respectively, yielding LB+HP (LB 2.6 kb + the first half of the hygromycin sequence) and PT+RB (RB 2.2 kb + the second half of the hygromycin sequence). During PEG-mediated genetic transformation of wild-type bacteria P131 protoplasts, LB+HP and PT+RB homologously recombinated to form a fragment containing the complete hygromycin sequence, replacing the gene to be knocked out. Hygromycin-resistant transformants were obtained, and the knockout variants of the target gene were identified by PCR.

[0048] SEQ ID NO.1, nucleotide sequence of the HYMA gene:

[0049] atgtattctc taaatctttt caacagaggc agaactcgaa cgaataccat tgatttgccg

[0050] aaacaagctc gcgagaatgt actcaaattg gacaatccag cagcaaaggt aagaggcttc

[0051] ttggtctaca tgccccgagg ccccggtggt ggacgcgctt tgggctaacg gccgcaccgg

[0052] aaatcaggcc gaggagctct ccaaagtact caaccaaatg aagctcatac tccagggtac

[0053] acatggtatg tgtgctccca gactccctag cactacacgg ggctacgagc tgacacgcag

[0054] gacaccgttt ctgggtgcca aagactctga aagcaacccg gaccaagttt tccatctcat

[0055] cgaaggcatt atcaacgaag acctgttaca tcccctggcc gctaacctcc acaagctacc

[0056] gttcgaatcc cggaaggatg cccagttcat cttctcttct gtttttcgct ttcgcccagc

[0057] atcggcctca accaagagcg acccggttgc ccttggttgg gtggtcaacc aacgacccca

[0058] ggtgctgctg gaactgtgta gaggatacga ccacaaggag agtgctacgg ccgctggctc

[0059] cgtgctgagg gaggtgttga agcacgaggg tgcttgcaaa gttatactat acgatgacgg

[0060] cgaccagcca ggctccagtg ttaatggcgt ccaggcaatt gacagagata ggccacaaag

[0061] cggaactggc gttttctgga agttttttga ctggatcgac aggggatcct ttgaggtttc

[0062] tgctgatgcc tttacaactt tcagagtgag caacaacgtt caaaaagtct cgaaatcttt

[0063] tttgagccat gaccagggac taacatgcct caaggaaatt ctcacaaaac aaaaggaggt

[0064] ggtgccacac tatctccgag tcaacttcga cctcttctgc agcaagtaca acaccgtttt

[0065] agtccagtct agcagctatg tgaccaagag acagtccatc aagctgctgg gagagatatt

[0066] gttggatcgc tccaattact cggtaatgac cgagtacgta gcctcgggcg agcacctcaa

[0067] aatttgcatg aacttgttgc gtgacgaccg caagatggta cagtacgagg gttttcacgt

[0068] cttcaaggtg tttgtggcca acccaaacaa gtcgattccc gtgcaacgta ttctcatcat

[0069] gaacaaggac aagctcctca acttcctgtc ccatttcctc gaggatcgca ccgatgacga

[0070] gcaattcatc gacgaacgag aatttcttat caaacagatt cgcaatatgc ctgcttcgcc

[0071] tgtgccacct caggggaggt ga.

[0072] SEQ ID NO. 2, amino acid sequence of the protein encoded by the HYMA gene:

[0073] MYSLNLFNRG RTRTNTIDLP KQARENVLKL DNPAAKAEEL SKVLNQMKLI LQGTHDSESN

[0074] PDQVFHLIEG IINEDLLHPL AANLHKLPFE SRKDAQFIFS SVFRFRPASA STKSDPVALG

[0075] WVVNQRPQVL LELCRGYDHK ESATAAGSVL REVLKHEGAC KVILYDDGDQ PGSSVNGVQA

[0076] IDRDRPQSGT GVFWKFFDWI DRGSFEVSAD AFTTFREILT KQKEVVPHYL RVNFDLFCSK

[0077] YNTVLVQSSS YVTKRQSIKL LGEILLDRSN YSVMTEYVAS GEHLKICMNL LRDDRKMVQY

[0078] EGFHVFKVFV ANPNKSIPVQ RILIMNKDKL LNFLSHFLED RTDDEQFIDE REFLIKQIRN

[0079] MPASPVPPQG R.

[0080] Table 1 PCR amplification primer information

[0081]

[0082] Table 2 First round PCR reaction system and reaction procedure

[0083]

[0084] Note: pSilent1-1 is publicly available in the reference Nakayashiki, H., Hanada, S., Quoc, NB, Kadotani, N., Tosa, Y., Mayama, S., 2005. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet. Biol. 42, 275-283.

[0085] Table 3. Second-round PCR reaction system and procedure

[0086]

[0087] The PEG-mediated transformation of rice blast fungus protoplasts is performed as follows:

[0088] Under aseptic conditions, the mycelia of P131 cultured on tomato-oat medium for 5–7 days were broken up and transferred to CM complete liquid medium. The mycelia were incubated at 28°C for 36 h at 160 rpm. The mycelia were filtered through a sterile triple-layer lens paper funnel, rinsed with pre-cooled 0.7 M sodium chloride, and transferred to sterile 50 mL centrifuge tubes. 10 mL of 20 mg / mL cell wall lysin was added, and the tubes were incubated at 160 rpm for 3–4 h at 28°C. Ice bath treatment was then initiated until transformation was complete. The protoplasts were filtered through triple-layer sterile lens paper, slowly rinsed with 0.7 M sodium chloride, and transferred to new 50 mL sterile centrifuge tubes (30 mL / tube). The tubes were centrifuged at 4,000 rpm for 15 min at 4°C. The supernatant was discarded, the tubes were returned to ice, 5 mL of STC was added, and the mixture was gently mixed. The tubes were then centrifuged at 4,000 rpm for 15 min at 4°C. Remove the supernatant, add an appropriate amount of STC, and adjust the protoplast concentration to 4 × 10⁻⁶ using a hemocytometer. 7 / mL. Add 250 µL of protoplasts (adjusted concentration), 25 µL each of the amplified LB+HY and YG+RB fragments (previously prepared) to a 50 mL sterile centrifuge tube, for a total of 300 µL, as a reaction system. Set up three replicates of 300 µL systems. Incubate on ice for 15 min, then slowly add PTC dropwise in 2 mL increments per reaction system. After incubating on ice for 15 min, add approximately 30 mL of pre-chilled STC, centrifuge at 4,000 r / min, 4°C for 15 min. Discard the supernatant, add 3 mL of LR cell wall regeneration medium, rinse the precipitate, mix well, and incubate at 28°C for 12–13 h for cell wall regeneration. Pour the LR-cultured protoplasts into a 9 cm culture dish, add approximately 12 mL of SR melted and cooled to approximately 40°C, mix well, and after solidification, add approximately 15 mL of TOP Agar containing 250 µg / mL hygromycin. After solidification, incubate at 28°C for transformant growth. Genomic DNA was extracted from the transformants 3-4 days later for PCR identification.

[0089] Genomic DNA extraction: Pick approximately 0.1 g of transformant hyphae with a sterile toothpick, add a small, burnt steel ball, and 650 µL of 2×CTAB extraction buffer preheated to 65°C. Mix thoroughly by vortexing at 50 Hz for 1 min, then incubate at 65°C for 30 min, mixing every 5 min. After cooling to room temperature, add an equal volume of a phenol:chloroform:isopropanol (25:24:1) mixture, mix slowly, and centrifuge at 12,000 rpm for 15 min at 4°C. Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, mix thoroughly by inverting, and centrifuge at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to another centrifuge tube, add 0.6 volumes of isopropanol, precipitate at -20°C for at least 30 min, and centrifuge at 12,000 rpm for 10 min at 4°C. The precipitate was rinsed twice with 70% ethanol and once with anhydrous ethanol. It was then dried under vacuum. The precipitate was dissolved in 1 mL of TE / RNase, treated at 37°C for 30 min, and stored at -20°C for later use.

[0090] PCR identification: PCR identification was performed using the transformant genomic DNA as a template. Gene check primers were designed and named HYMA-LBCK and HYMA-RBCK, with primers approximately 2.6 kb upstream and 2.1 kb downstream of the knockout fragment. Amplification was performed using HPT-LBCK / GPI7-LBCK and HPT-RBCK / HYMA-RBCK (Table 4). When bands of the corresponding size appeared in both primers, and no band was observed in HYMA-F / HYMA-R amplification, it could be identified that the target knockout gene in the transformant had been replaced by HYG, and the transformant was a knockout variant of the target gene.

[0091] Table 4. PCR reaction system for transformant identification

[0092]

[0093] Through the above series of experiments, four HYMA gene knockout variants were finally obtained. Figure 2 These are named ∆hyma-1, ∆hyma-2, ∆hyma-3 and ∆hyma-4 respectively. ∆hyma-1 and ∆hyma-2 are selected as representatives for subsequent experimental operations.

[0094] Example 2: Observation of hyphal development in knockout transformants

[0095] Wild-type rice blast fungus P131, ∆hyma-1, and ∆hyma-2 were perforated using a scorched 0.5 cm punch. The mycelial blocks were placed upside down on tomato-oat medium, with one dish constituting one replicate. Three replicates were set up for each fungus. The medium with the inoculated mycelial blocks was placed upside down in a 28°C, 24-hour light (5000 Lx) incubator and cultured for 4 days. The colony morphology was photographed and recorded, and the colony diameter was also recorded.

[0096] The results are as follows Figure 3 and Figure 4 As shown, the mycelial growth of ∆hyma-1 and ∆hyma-2 was significantly slowed down, and the colony color became darker, indicating that the HYMA gene seriously affected the growth and development of rice blast fungus mycelium; the colony diameter was also significantly reduced.

[0097] Example 3: Pathogenicity Experiment of Knockout Transformant Conidia

[0098] Wild-type rice blast fungus P131, ∆hyma-1, and ∆hyma-2 were cultured on tomato-oat medium under light for 5 days. Under aseptic conditions, 300 μL of sterile water was added to the petri dish, and the mycelium was broken up using a smear loop. The mixture was then evenly spread onto a new, thicker tomato-oat medium plate and incubated upside down at 28°C for 48 h. Newly formed aerial mycelium was gently broken up using a smear loop under slowly flowing tap water. The plate was then inverted and allowed to dry. After drying, the plate was covered with double layers of gauze and incubated at 28°C for another 48 h. Conidia were washed off the tomato-oat medium plate with 0.25‰ Tween-20 solution and filtered into 50 mL centrifuge tubes using a funnel made of three layers of lens paper. The spore concentration of the conidia suspension was determined under a microscope using a hemocytometer, and the conidia concentration was adjusted to 3 × 10⁻⁶. 4The concentration of spores per mL was adjusted. If no spores were found, the mycelial solution obtained by washing with Tween water was used directly. The prepared spore suspension was sprayed onto the leaves of Lijiang Xintuan Black Rice seedlings that had been growing for about one month. All leaves of one seedling constituted one replicate, and six rice seedlings were sprayed with each strain. The inoculated rice leaves were placed in a dark chamber and kept warm and humid for 24 hours. Afterward, they were moved to a sunroom and treated with constant temperature and humidity for another 3-5 days. The disease incidence on the rice leaves was then observed. Diseased leaves were cut off, and the diseased leaves were scanned using double-sided tape attached to A4 paper to count the number of lesions.

[0099] Figure 5 The results showed that wild-type P131 could infect rice normally, while ∆hyma-1 and ∆hyma-2 could not form obvious lesions; Figure 6 The results showed that the wild-type P131 produced significantly more lesions than the ∆hyma-1 and ∆hyma-2 gene mutants. These examples demonstrate the crucial role of the HYMA gene in the pathogenicity of rice blast fungus.

[0100] Example 4: Pathogenicity Experiment of Knockout Transformant Blocks

[0101] Wild-type rice blast fungus P131, ∆hyma-1, and ∆hyma-2 were cultured under light for 5 days on tomato-oat medium. After being scorched with a knife, the fungal blocks were cut into 0.25 cm pieces. 2 To determine the size of the mycelium, use a punch to create tiny wounds on the leaves of one-month-old Lijiang Xintuan Black Rice seedlings, being careful not to puncture or break the leaves. Use a toothpick to pick up a cut piece of mycelium, placing the mycelial side against the wound on the leaf. Secure the mycelium piece to the leaf with transparent tape. One leaf per rice seedling constitutes one replicate, and each strain is inoculated onto three rice seedlings. Place the inoculated rice seedlings in a sunroom, maintaining a constant temperature and humidity for approximately 7 days, and observe the disease development and lesion expansion on the rice leaves. Cut off diseased leaves, attach them to A4 paper with double-sided tape, and scan the diseased leaves to measure the length of the lesions.

[0102] Figure 7 The results showed that wild-type P131 mycelial blocks could normally infect damaged rice leaves and successfully spread, with the lesion length expanding to 2.16 cm ± 0.15 cm. In contrast, ∆hyma-1 and ∆hyma-2 did not show any lesions or spread. These examples demonstrate that the HYMA gene is crucial in the pathogenesis and mycelial spread of *Magnaporum oryzae*.

[0103] Example 5: Sporulation observation of knockout transformants

[0104] Wild-type rice blast fungus P131, ∆hyma-1, and ∆hyma-2 were cultured on tomato-oat medium under light for 5 days. Under aseptic conditions, 300 μL of sterile water was added to each petri dish, and the mycelium was broken up using a smear loop. The mycelium was then evenly spread onto a new, thicker tomato-oat medium plate and incubated upside down at 28°C for 48 h. Newly formed aerial mycelium was then gently broken up using a smear loop under slowly flowing tap water. The plates were then inverted and allowed to dry. After drying, the plates were covered with double layers of gauze and incubated at 28°C for another 48 h. One sporulation dish constituted one replicate, and three replicates were set up for each strain. Spores from each petri dish were washed with 30 mL of water, observed under a microscope, and counted using a hemocytometer.

[0105] Figure 9 The results showed that wild-type P131 could form conidia normally, while the solutions eluted from ∆hyma-1 and ∆hyma-2 only contained hyphae and no conidia were observed. Figure 10 The sporulation yield of wild-type P131 was 1.67 × 10⁻⁶. 6 The concentration of HYMA was 1 / mL, while ∆hyma-1 and ∆hyma-2 contained only a few conidia. These examples demonstrate that the HYMA gene is a key gene regulating conidia formation in *Magnaporum oryzae*. Deletion of this gene prevents *Magnaporum oryzae* from forming conidia normally, thus affecting its pathogenicity.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of the protein encoded by the HYMA gene of *Magnaporum oryzae* in regulating the growth of *Magnaporum oryzae*, characterized by: The amino acid sequence of the protein encoded by the HYMA gene is shown in SEQ ID NO.2; the regulation of rice blast fungus growth is achieved by inhibiting or blocking the expression of the protein encoded by the HYMA gene of rice blast fungus to inhibit the mycelial growth of rice blast fungus.

Images