A ustilaginid inoculum, a method for preparing the same and use thereof in ustilaginid inoculation

Abstract

This invention discloses an inoculum for *Aspergillus oryzae*, its preparation method, and its application in *Aspergillus oryzae* inoculation, belonging to the field of agricultural microbiology technology. This invention prepares an *Aspergillus oryzae* inoculum with germinating spores (GC) as its core through a two-step shake-flask culture combined with filtration, centrifugation, and spore germination steps, effectively solving problems such as hyphal aging, low spore activity, and asynchronous infection in existing inoculation methods. This inoculum possesses a young hyphal structure and strong infection activity, significantly improving inoculation efficiency and disease uniformity. When used for the preparation of transcriptomic, metabolomic, and other multi-omics samples, it avoids non-specific immune interference caused by the release of PAMPs due to hyphal breakage, ensuring synchronous infection processes and reliable immune responses. This invention provides a stable, efficient, and background-clean inoculation system for studying the *Aspergillus oryzae*-rice interaction mechanism and has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to an inoculum for Aspergillus oryzae, its preparation method, and its application in Aspergillus oryzae inoculation. Background Technology

[0002] Rice false smut is a widespread and significant fungal disease that has been gradually worsening in recent years, becoming one of the three major diseases affecting rice production. *Aspergillus oryzae* exhibits flower organ-specific infection characteristics, inducing the formation of false smut pellets within rice florets. The occurrence of these pellets not only hinders normal grain filling but also leads to the sterility of surrounding florets, severely impacting rice yield and quality. Furthermore, these pellets can produce various secondary metabolic toxins, threatening food safety and the healthy development of livestock farming. Therefore, elucidating the pathogenic mechanism of *Aspergillus oryzae* and the disease resistance response of rice is of great significance for ensuring stable and increased rice yields and food security.

[0003] Currently, research on rice false smut largely relies on artificial inoculation techniques to assess the pathogenicity of the pathogen and the resistance of rice varieties. However, existing inoculation methods suffer from poor stability and low efficiency, which have become key bottlenecks restricting research in this field. Existing patented technologies mainly include the following representative schemes: 1) Field spraying and soil inoculation method (such as invention patent application No. 201510836639.8 and invention patent application No. 201910776427.3): using rice false smut thick-walled spores for field spraying or applying inoculated soil, suitable for large-scale material identification, but with problems such as generally low incidence rate and large influence from differences in rice heading time, resulting in insufficient data reliability and repeatability; 2) Indoor injection inoculation method (such as application No. 2011100287). 35.1 Invention Patent): The controllability of inoculation conditions is improved by injecting conidial suspension, but the viability of the inoculum is low, the disease effect is unstable, and the equipment cost is high and limited by indoor space, making it difficult to meet the needs of large-scale resistance identification; 3) Mycelial segment and spore spraying method (such as invention patent application No. 202110896863.1): Mycelial segments and conidia that have been shaken for 5-7 days are sprayed for inoculation, but there are still problems such as low disease rate of panicles and disease rate of grains, and the inability to guarantee that each panicle is diseased, which limits its application in accurate identification.

[0004] Furthermore, with the widespread application of multi-omics technologies (such as metabolomics, Bulk-RNA-seq, and single-cell transcriptomics) in the study of rice false smut-rice interaction mechanisms, existing inoculation systems have revealed significant deficiencies in terms of sample scientific rigor and accuracy. Specifically, these deficiencies include: 1) Insufficient inoculum activity: Prolonged shaking culture (5-7 days) leads to mycelial aging and reduced conidial activity, affecting infection efficiency; 2) Distorted immune response: Broken mycelial aggregates release excessive pathogen-associated molecular patterns (PAMPs), artificially amplifying the plant's immune response and interfering with the accurate assessment of innate immune strength; 3) Asynchronous infection process: Mycelia and conidia may infect asynchronously, leading to disordered immune response timing and affecting the analysis of the dynamic immune process.

[0005] The aforementioned problems not only reduce the reliability of pathogenicity assessment and rice disease resistance evaluation, but also pose difficulties in elucidating the flower organ-specific infection mechanism of *Aspergillus oryzae*. Therefore, developing an efficient, stable, and multi-omics-compatible artificial inoculation method for *Aspergillus oryzae* has become a pressing technical challenge in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an *Aspergillus oryzae* inoculum, its preparation method, and its application in *Aspergillus oryzae* inoculation, thereby addressing the problems existing in the prior art. This invention prepares an *Aspergillus oryzae* inoculum with germinating spores (GC) as its core through a two-step shake-flask culture combined with filtration, centrifugation, and spore germination steps. This effectively solves the problems of mycelial aging, low spore activity, and asynchronous infection in existing inoculation methods. This invention provides a stable, efficient, and background-clean inoculation system for studying the *Aspergillus oryzae*-rice interaction mechanism and has promising application prospects.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a method for preparing Aspergillus oryzae inoculum, comprising the following steps:

[0009] 1) The mycelial blocks of Aspergillus oryzae were cultured in PSB medium for the first time in shake flasks to obtain a mixture of mycelial balls and conidia, which was denoted as HC inoculum;

[0010] 2) Filter the HC inoculum to remove mycelial balls and obtain a filtrate containing conidia; centrifuge the filtrate containing conidia to obtain a spore precipitate;

[0011] 3) The spore precipitate is resuspended in PSB medium and cultured a second time in a shake flask until the spores germinate and produce tender mycelia, which is recorded as the rice arbuscular mycorrhizal inoculum.

[0012] Furthermore, in step 1), the temperature of the first shake flask culture is 26-30℃, the rotation speed is 160-200 rpm, and the culture time is 5-7 days; in step 3), the temperature of the second shake flask culture is 26-30℃, the rotation speed is 160-200 rpm, and the culture time is 16-20 h.

[0013] Furthermore, in step 2), the diameter of the filter pores is 22-25 μm.

[0014] Furthermore, in step 2), the centrifugation is performed at 3000-4500 rpm for 8-12 minutes.

[0015] Furthermore, in step 3), the concentration of the spores resuspended in PSB medium is adjusted to 0.8 × 10⁻⁶. 6 -1.2×10 6 per mL.

[0016] The present invention also provides an inoculum of Aspergillus rice obtained by the preparation method described above.

[0017] The present invention also provides the application of the aforementioned rice aspergillus inoculum in artificial inoculation of rice aspergillus.

[0018] The present invention also provides a method for artificial inoculation of rice aspergillus, comprising the step of inoculating rice panicles with the inoculum 3-5 days before rice heading.

[0019] Furthermore, when the purpose of inoculation is to determine the pathogenicity of rice blast fungus or the disease resistance of rice, the inoculated rice is placed in a field environment, and the inoculation effect is tested after 4 weeks; when the purpose of inoculation is for the sampling of transcriptome and metabolome materials, the inoculum is removed from the original culture medium, resuspended in sterile water, and then used for inoculation. The inoculated rice is placed in a plastic greenhouse, and the humidity is maintained at 85%-90% for 3 days after inoculation.

[0020] Furthermore, when the purpose of inoculation is to determine the pathogenicity of rice blast fungus or the disease resistance of rice, a shade net should be erected above the rice for the first 3 days after inoculation, and water should be sprayed once at noon and once in the evening.

[0021] The present invention discloses the following technical effects:

[0022] This invention successfully prepared a *Aspergillus oryzae* inoculum (GC) with homogenized germinating spores as its core through a two-step shake-flask culture combined with filtration, centrifugation, and spore germination. Traditional methods yield inoculums that are heterogeneous mixtures (HC) of mycelium and ungerminated spores, and this mixed state directly leads to uncontrollable and asynchronous infection processes. This application, through two shake-flask cultures, filtration to remove mycelium, centrifugation to purify spores, and a crucial secondary culture to spore germination, ultimately obtained a synchronized pre-germinating inoculum, fundamentally solving the problems of mycelial aging, low spore activity, and asynchronous infection in existing technologies.

[0023] The inoculum provided by this invention possesses a young mycelial structure and strong infectivity, significantly improving inoculation efficiency and disease uniformity. It achieves a high disease incidence rate under field conditions without additional treatment, greatly enhancing data reliability and reproducibility. Furthermore, the inoculum can be resuspended in sterile water to remove residual culture medium, effectively avoiding non-specific immune interference caused by the release of PAMPs due to mycelial breakage, ensuring synchronous infection processes, and providing clean samples for multi-omics studies such as transcriptomics and metabolomics. This invention overcomes the technical bottlenecks of low efficiency and poor stability in traditional artificial inoculation, providing a stable and efficient inoculation system for studying the *Aspergillus oryzae*-rice interaction mechanism, and has promising application prospects. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a flowchart of the spore inoculation system; where A represents the traditional HC (Hypha-Conidia) inoculum; B represents the conidia obtained by filtering the HC inoculum as photographed under a microscope; C represents the GC (Germinated Conidia) inoculum produced by spore germination as photographed under a microscope; D represents the GC inoculation process in a plastic greenhouse and the inoculation effect; E represents the GC process in the field and the inoculation effect.

[0026] Figure 2 Phenotypic comparison of HC and GC inoculum under a microscope;

[0027] Figure 3The inoculation effect of germinating spores of rice blast fungus strain PJ52 in the field is shown. Among them, A is the disease phenotype after inoculation of PJ6, TP309, ZH11 and NPB rice materials with GC inoculum and HC inoculum, respectively; B is the number of rice blast heads per panicle after inoculation of PJ6, TP309, ZH11 and NPB rice materials with GC inoculum and HC inoculum, respectively; C is the rice blast head formation rate per panicle after inoculation of PJ6, TP309, ZH11 and NPB rice materials with GC inoculum and HC inoculum, respectively.

[0028] Figure 4 The inoculation effect of spores germinating from the rice blast strain JS60-2 in the field is shown. A represents the disease phenotypes of rice materials PJ6, TP309, ZH11, and NPB after inoculation with GC and HC inoculants, respectively; B represents the number of rice blast heads per panicle after inoculation with GC and HC inoculants, respectively; and C represents the rice blast head formation rate per panicle after inoculation with GC and HC inoculants, respectively.

[0029] Figure 5 The inoculation effect of spores germinating from rice blast strain PJ52 in greenhouse is shown. Among them, A is the disease phenotype after inoculation of PJ6 rice material with GC inoculum and HC inoculum; B is the number of rice blast balls per panicle after inoculation of PJ6 rice material with GC inoculum and HC inoculum; C is the rice blast ball formation rate per panicle after inoculation of PJ6 rice material with GC inoculum and HC inoculum. Detailed Implementation

[0030] This invention provides a method for artificial inoculation of Aspergillus oryzae. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] This invention provides an inoculum for Aspergillus rice, a preparation method thereof, and its application in Aspergillus rice inoculation. Using the method of this invention, the pathogenicity of Aspergillus rice or the resistance of rice varieties can be identified efficiently and in large quantities; it can also provide more scientific and reliable experimental samples for omics.

[0032] The inoculation method includes the following steps:

[0033] 1) The mycelial blocks of Aspergillus oryzae were cultured in PSB medium for the first time in shake flasks to obtain a mixture of mycelial balls and conidia, which was denoted as HC inoculum;

[0034] 2) Filter the HC inoculum to remove mycelial balls and obtain a filtrate containing conidia; centrifuge the filtrate containing conidia to obtain a spore precipitate;

[0035] 3) The spore precipitate is resuspended in PSB medium and cultured a second time in a shake flask until the spores germinate and produce tender mycelia, which is recorded as the rice arbuscular mycorrhizal inoculum.

[0036] The mycelial blocks of Aspergillus rice provided by the present invention are cultured on PSA medium until a uniform mycelial layer forms on the surface and a large number of conidia are produced. At this time, a pale yellow spore layer can be seen on the surface of the medium.

[0037] In this invention, optionally, the temperature of the first shake flask culture is 26-30℃, the rotation speed is 160-200 rpm, and the culture time is 5-7 days; in a specific embodiment of this invention, the temperature of the shake flask culture can be selected as 28℃, the shaking speed can be selected as 180 rpm, and the culture time can be selected as 6 days.

[0038] The above parameters ensure that the culture medium is shaken sufficiently to provide adequate oxygen for mycelial growth and avoid local hypoxia that could lead to a decrease in mycelial vitality.

[0039] In this invention, the mixture of mycelial balls and conidia needs to be filtered through a special filter cloth to remove mycelial balls with larger diameters; optionally, the pore size of the special filter cloth is 20~28 μm, and in a specific embodiment of this invention, the pore size is 22-25 μm.

[0040] The above-mentioned pore size ensures that the mycelial balls are intercepted on the filter cloth and that no spores remain.

[0041] Optionally, when centrifuging the filtrate containing conidia, the centrifugation parameters are 3000-4500 rpm for 8-12 min. In a specific embodiment of the present invention, the centrifugation parameters are 4000 rpm for 10 min.

[0042] In this invention, optionally, the temperature of the second shake flask culture is 26-30℃, the rotation speed is 160-200 rpm, and the culture time is 16-20 h; in a specific embodiment of this invention, the temperature of the shake flask culture can be selected as 28℃, the shaking speed can be selected as 180 rpm, and the culture time can be selected as 18 h.

[0043] The above parameters ensure the formation of germ tubes with a mature infection structure, while also ensuring that the mycelium is young and does not age, and has strong infection activity.

[0044] Optionally, the concentration of the spores resuspended in PSB medium is adjusted to 0.8 × 10⁻⁶. 6 -1.2×10 6spores / mL. In one specific embodiment of the invention, the reselected spore concentration can be selected as 1×10⁻⁶ spores / mL. 6 per mL.

[0045] The present invention also provides a method for artificial inoculation of rice aspergillus, comprising the step of inoculating rice panicles with the inoculum obtained by the above preparation method 3-5 days before rice heading.

[0046] Optionally, when the purpose of inoculation is to determine the pathogenicity of rice aspergillus or the resistance of rice, the inoculated rice is placed in a field environment, and the inoculation effect is tested after 4 weeks; even under the condition that there is no treatment after inoculation, the number of rice blast balls and the percentage of diseased grains produced by the inoculation system are sufficient to meet the requirements for determining the pathogenicity of rice aspergillus and the resistance of rice.

[0047] When the purpose of inoculation is for sampling transcriptome and metabolome materials, the inoculum is removed from the original culture medium, resuspended in sterile water, and then used for inoculation. The inoculated rice is placed in a plastic greenhouse and kept at 85%-90% humidity for 3 days after inoculation.

[0048] Alternatively, when the purpose of inoculation is to determine the pathogenicity of rice blast fungus or the resistance of rice, a shade net should be erected over the rice for the first 3 days after inoculation, and water should be sprayed once at noon and once in the evening. This operation can significantly increase the incidence of disease.

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0051] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0052] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0053] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0054] The Aspergillus rice strains PJ52 and JS60-2 used in the embodiments of the present invention are commonly used wild-type Aspergillus rice strains in the art, provided by Yazhou Bay National Laboratory.

[0055] Example 1: Preparation of Germinating Spore Inoculum

[0056] 1) Culture of strains: Inoculate wild-type Aspergillus rice species PJ52 and JS60-2 with stable pathogenicity onto PSA medium and incubate at 28℃ for 10 days until a uniform mycelial layer forms on the surface of the medium and a large number of conidia are produced (a pale yellow spore layer is visible to the naked eye on the surface of the medium).

[0057] 2) Shaking induction: Using a sterile scalpel, cut mycelial blocks (containing conidia) with a diameter of approximately 0.5 cm, ensuring each block is the same size to guarantee uniform inoculation. Place them in 200 mL of PSB medium and shake at 28℃ and 180 rpm for 5-7 days to obtain a mixture of mycelial balls and conidia (HC). This is the inoculum for traditional artificial inoculation methods and is designated as the HC inoculum. Figure 1 A).

[0058] 3) Spore purification: Filter the mixture through a special filter cloth (pore size 22-25 μm, pre-sterilized at 121℃ for 20 min and dried) to remove larger mycelial balls (visually inspect the remaining mycelial balls on the filter cloth to ensure no large number of spores remain), and collect the filtrate containing conidia; transfer the filtrate to a centrifuge tube, centrifuge at 4000 rpm for 10 min, discard the supernatant, and obtain the conidia precipitate ( Figure 1 (B) After centrifugation, a pale yellow spore precipitate was visible at the bottom of the tube, and the supernatant was clear, indicating that the spore precipitation was complete.

[0059] 4) Spore germination: Resuspend the conidial pellet in freshly prepared PSB medium and adjust the spore concentration to 1×10⁻⁶. 6 spores / mL; cultured at 28℃ and 180 rpm for 18 hours, and observed under a microscope to confirm a spore germination rate ≥95%, forming young mycelia (Germinated Conidia, GC), which are then called GC inoculum. Figure 1 (C).

[0060] Phenotypic comparison of HC and GC inoculum under a microscope Figure 2 As shown. By Figure 2 It can be clearly observed that HC inoculum contains a large number of ungerminated conidia and aged mycelia, while GC inoculum contains only young mycelia formed by the germination of conidia.

[0061] Example 2 Artificial inoculation of Aspergillus oryzae (for pathogenicity determination)

[0062] 1) Rice material preparation: Select rice varieties Pujiang 6 (PJ6, susceptible variety), Taipei 309 (TP309, moderately resistant variety), Zhonghua 11 (ZH11, moderately susceptible variety), and Nipponbare (NPB, resistant variety). Plant them according to conventional field management methods (row spacing 30 cm, plant spacing 20 cm, sufficient base fertilizer, regular watering and weeding). Wait until the rice grows to 3-5 days before heading (the panicle has formed but has not cracked, and the length of the panicle is 10-12 cm as observed by the naked eye); plant 30 plants of each variety, divided into 3 replicate groups, 10 plants in each group, to ensure the reproducibility of the experiment.

[0063] 2) Inoculum: GC and HC inoculum of Aspergillus rice PJ52 and JS60-2 prepared in Example 1.

[0064] 3) Inoculation procedure: Inoculation was carried out in the evening (17:00-18:00). Two different inoculums of Aspergillus rice, PJ52 and JS60-2, were slowly injected from the middle of the panicle until the inoculum overflowed from the top of the panicle. The inoculation amount per panicle was kept consistent to avoid affecting the disease outcome due to differences in the inoculation amount. A total of 4 treatment groups were set up: GC inoculum treatment group of Aspergillus rice PJ52, HC inoculum treatment group of Aspergillus rice PJ52, GC inoculum treatment group of Aspergillus rice JS60-2, and HC inoculum treatment group of Aspergillus rice JS60-2.

[0065] No additional treatment is required after inoculation; allow it to grow naturally with field temperature controlled at 25-30℃ and relative humidity at 50%-70%.

[0066] 4) Disease statistics: Four weeks after inoculation, the number of rice spikelets per panicle and the rate of rice spikelet formation were counted in each treatment group.

[0067] The results are as follows Figure 3 and Figure 4As shown, the inoculation efficiency of rice materials with NPB background for Aspergillus oryzae is very low. The inoculation results showed that after inoculating four materials with the GC inoculation system of two strains, the number of rice blast balls and the rate of diseased grains were much higher than those of the HC inoculation system; even under the condition of no treatment after inoculation, the number of rice blast balls and the rate of diseased grains produced by the GC inoculation system were sufficient to meet the requirements for Aspergillus oryzae pathogenicity and rice resistance determination.

[0068] 5) In the inoculation experiment for pathogenicity testing in this embodiment, if it is necessary to further increase the incidence rate, the following adjustments can be made to the light, temperature, and humidity:

[0069] For the first 3 days after inoculation, erect a shade net over the rice (50% shading rate, 1.5 m high, to ensure it does not affect rice growth). Spray water twice a day, at 12:00 noon and 17:00 in the evening, each time until the leaf surface is moist (use a sprayer to avoid excessive water flow damaging the panicles), maintaining a relative humidity of 80%-85%. Figure 1 E).

[0070] A comparison was made between the experimental group that underwent post-inoculation regulation and the natural culture group that received no post-inoculation treatment. The results showed that the disease incidence rate in the regulation group was 20%-25% higher than that in the natural culture group, indicating that post-inoculation regulation of light and humidity can effectively increase the incidence rate.

[0071] Example 3 Artificial inoculation of Aspergillus oryzae (for omics sampling)

[0072] 1) Inoculum treatment: Take 50 mL each of GC and HC inoculum of *Aspergillus rice* PJ52 prepared in Example 1, centrifuge at 4000 rpm for 10 min, and discard the supernatant; wash twice with sterile water; finally resuspend in sterile water and adjust the concentration to 1×10⁻⁶. 6 The inoculum was observed under a microscope after washing, and the young hyphae were free of obvious impurities. The supernatant was clear, indicating that the culture medium residue had been completely removed.

[0073] 2) Inoculation and Environmental Control: The rice variety Pujiang 6 (PJ6, with uniform panicle development 3 days before heading) was selected and inoculated according to the procedure in Example 2. After inoculation, the rice was transferred to a simple plastic greenhouse (2.5m × 1.7m × 1.7m, 14s high-transmittance film, pre-sterilized with formalin). For the first 3 days after inoculation, a humidifier was placed inside the greenhouse to adjust the relative humidity to 85%-90%. The temperature was regulated through the greenhouse ventilation openings and maintained at 25-28℃. Temperature and humidity were recorded every 2 hours to ensure stable environmental parameters. Figure 1 D).

[0074] 3) Sample collection: 4 weeks after inoculation, husk tissue was collected (3 biological replicates were collected at each time point, 5 husks were collected from each replicate, and the samples were collected after mixing).

[0075] 4) Disease incidence statistics: The number of rice panicles and the rice panicle formation rate were counted in each treatment group.

[0076] The results are as follows Figure 5 As shown, Figure 5 A represents the phenotypic image of rice panicles collected after inoculation. The average number of rice blasts produced by GC inoculation was as high as 140, which was much higher than the average number of rice blasts produced by HC inoculation (48). Figure 5 (B); In addition, the single-ear disease grain rate of the GC inoculation system is mostly above 90%, with an average disease grain rate of 80% and a maximum of 98.04%. Figure 5 The C indicates that the vaccination efficiency of the GC vaccination system is much higher than that of the traditional HC vaccination system.

[0077] Therefore, the inoculation method of this embodiment can significantly improve inoculation efficiency. High inoculation efficiency can ensure that a sufficient number of disease samples are obtained in a short period of time. Sufficient sample size can improve the accuracy and reliability of analysis, which is crucial for metabolomics analysis.

[0078] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing Aspergillus oryzae inoculum, characterized in that, Includes the following steps: 1) The mycelial blocks of Aspergillus oryzae were cultured in PSB medium for the first time in shake flasks to obtain a mixture of mycelial balls and conidia, which was denoted as HC inoculum; 2) Filter the HC inoculum to remove mycelial balls and obtain a filtrate containing conidia; centrifuge the filtrate containing conidia to obtain a spore precipitate; 3) The spore precipitate is resuspended in PSB medium and cultured a second time in a shake flask until the spores germinate and produce young mycelia, which is recorded as the rice aspergillus inoculum; the conditions for the second shake flask culture are 28℃, 180 rpm for 18h, and the spore germination rate is ≥95%.

2. The preparation method according to claim 1, characterized in that, In step 1), the temperature of the first shake flask culture is 26-30℃, the rotation speed is 160-200 rpm, and the culture time is 5-7 days.

3. The preparation method according to claim 1, characterized in that, In step 2), the diameter of the filter pores is 22-25 μm.

4. The preparation method according to claim 1, characterized in that, In step 2), the centrifugation is performed at 3000-4500 rpm for 8-12 minutes.

5. The preparation method according to claim 1, characterized in that, In step 3), the concentration of the spores resuspended in PSB medium is adjusted to 0.8 × 10⁻⁶. 6 -1.2×10 6 per mL.

6. An inoculum of Aspergillus rice obtained by the preparation method according to any one of claims 1-5.

7. The use of the rice blast fungus inoculum according to claim 6 in the artificial inoculation of rice blast fungus.

8. A method for artificial inoculation of Aspergillus oryzae, characterized in that, The step includes inoculating rice panicles with the inoculum described in claim 6 3-5 days before rice heading.

9. The method for artificial inoculation of Aspergillus oryzae according to claim 8, characterized in that, When the purpose of inoculation is to determine the pathogenicity of rice blast fungus or the disease resistance of rice, the inoculated rice is placed in a field environment, and the inoculation effect is tested after 4 weeks. When the purpose of inoculation is for the sampling of transcriptome and metabolome materials, the inoculum is removed from the original culture medium, resuspended in sterile water, and then used for inoculation. The inoculated rice is placed in a plastic greenhouse, and the humidity is maintained at 85%-90% for 3 days after inoculation.

10. The method for artificial inoculation of Aspergillus oryzae according to claim 9, characterized in that, When the purpose of inoculation is to determine the pathogenicity of rice blast fungus or the disease resistance of rice, a shade net should be erected above the rice for the first 3 days after inoculation, and water should be sprayed once at noon and once in the evening.

Images