Rhizopus strain tyr1, microbial inoculum and application thereof in increasing content of 2-acetyl-1-pyrroline in rice

Abstract

The present application belongs to the technical field of microbial inoculants, and particularly relates to a strain of Rhizopus TYR1, a microbial inoculant and application of the microbial inoculant in increasing the content of 2-acetyl-1-pyrroline (2-AP) in rice. The Rhizopus TYR1 and the microbial inoculant containing the Rhizopus TYR1 can increase the content of 2-AP, an aromatic substance, in rice grains and leaves, and the content of 2-AP is significantly increased relative to a control without the Rhizopus TYR1, thereby providing technical support for increasing the aroma concentration in rice. Meanwhile, the present application further provides a method for increasing the content of 2-acetyl-1-pyrroline in rice, which is simple in operation, low in cost, easy to manage and convenient for agricultural promotion.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a Rhizopus fungus TYR1, a microbial inoculant, and its application in increasing the content of 2-acetyl-1-pyrrolidine in rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, and its quality directly affects people's diet and lifestyle. Among the indicators, the aroma of rice is one of the key factors in evaluating its quality.

[0003] 2-Acetyl-1-pyrroline (2-AP) is a substance with a strong popcorn aroma. It is a characteristic aroma compound and major aroma contributor in rice, especially fragrant rice, and is believed to be ubiquitous in fragrant rice. 2-AP can be produced in the above-ground stems, leaves, and grains of rice throughout its entire growth cycle.

[0004] Studies have found that various factors, including rice variety and cultivation methods, can affect the accumulation level of 2-AP. However, with the frequent occurrence of extreme heat and the weakening of soil fertility, the aroma concentration of fragrant rice varieties tends to decline after years of cultivation. To increase the aroma concentration in fragrant rice, researchers have tried various measures. For example, they have explored genes that regulate the content of the aroma compound 2-AP in rice varieties, hoping to increase its content through breeding. However, this method is time-consuming and cannot solve the problem in the short term. Additionally, researchers have tried to increase the content of 2-AP in fragrant rice through cultivation methods, but this is limited by implementation difficulties and high costs. Therefore, developing a low-cost, short-term, simple, and easy-to-manage improvement method is crucial for enhancing the aroma of rice.

[0005] Rhizopus fungi are a group of fungi with wide applications, mainly in food, agriculture, environment, and biomedicine, especially in food, such as food fermentation and preservation. Although they are also used in agriculture, their applications are mainly in pest and disease control, soil improvement, and growth promotion. Rhizopus fungi with the function of improving crop quality are still rare. Summary of the Invention

[0006] The purpose of this invention is to provide a Rhizopus fungus TYR1, a microbial inoculant, and its application in increasing the content of 2-acetyl-1-pyrrolidine in rice. The Rhizopus fungus TYR1 and the microbial inoculant containing the Rhizopus fungus TYR1 can effectively increase the content of 2-acetyl-1-pyrrolidine in rice.

[0007] This invention provides a Rhizopus sp. fungus TYR1, which has the accession number CGMCC No.42112.

[0008] The present invention also provides the application of the Rhizopus fungus TYR1 described in the above technical solution in microbial fertilizers and / or microbial agents.

[0009] This invention provides a microbial inoculant, which includes the Rhizopus fungus TYR1 described in the above technical solution.

[0010] Preferably, the microbial agent includes liquid and / or solid agents.

[0011] Preferably, the liquid inoculant comprises a spore suspension of Rhizopus fungus TYR1; the solid inoculant comprises Rhizopus fungus TYR1 mycelial cake.

[0012] Preferably, the OD of the spore suspension of the Rhizopus fungus TYR1 is... 600 The diameter of the mycelium of the Rhizopus fungus TYR1 is 0.2–0.4 mm; the diameter of the mycelium cake is 4.5–5.5 mm.

[0013] The present invention also provides the application of the Rhizopus fungus TYR1 described in the above technical solution or the microbial agent described in the above technical solution in increasing the content of 2-acetyl-1-pyrrolidine in rice.

[0014] The present invention also provides a method for increasing the content of 2-acetyl-1-pyrrolline in rice, comprising the steps described in I or II below:

[0015] Ⅰ: Soak rice seedlings in a microbial inoculant containing Rhizopus fungus TYR1, plant the soaked rice seedlings, and then apply the microbial inoculant containing Rhizopus fungus TYR1 to the planting land for planting management;

[0016] II: Inoculate the roots of rice seedlings with a solid microbial agent containing Rhizopus fungus TYR1, and then culture the rice seedlings inoculated with the solid microbial agent in a facility.

[0017] The Rhizopus fungus TYR1 described in either I or II has the accession number CGMCC No. 42112.

[0018] Preferably, in step I, the soaking time is 1-2 hours; the microbial agent comprises a spore suspension of Rhizopus fungus TYR1, and the OD of the Rhizopus fungus TYR1 spore suspension is... 600 The concentration is 0.2–0.4; the application rate of the microbial agent containing Rhizopus fungus TYR1 is 100–200 mL / m³. 2Or 4.5–5.5 mL / plant;

[0019] In II, the solid microbial inoculant containing Rhizopus fungus TYR1 includes Rhizopus fungus TYR1 mycelium cake, the diameter of which is 4.5 to 5.5 mm.

[0020] Preferably, increasing the content of 2-acetyl-1-pyrrolidine in rice includes increasing the content of 2-acetyl-1-pyrrolidine in rice grains and / or leaves.

[0021] Beneficial effects:

[0022] This invention provides a Rhizopus fungus TYR1, which has been bio-preserved. The preservation number of the Rhizopus fungus TYR1 is CGMCC No.42112.

[0023] Based on the aforementioned Rhizopus fungus TYR1, this invention provides the application of Rhizopus fungus TYR1 in microbial fertilizers and / or microbial inoculants, and specifically provides a microbial inoculant comprising the aforementioned Rhizopus fungus TYR1. Field trials, pot trials, and sterile plate trials have shown that the Rhizopus fungus TYR1 can increase the content of the aroma compound 2-AP in rice grains and leaves, and exhibits a significant increase compared to the control without the application of Rhizopus fungus TYR1, providing technical support for increasing the aroma concentration in rice.

[0024] Furthermore, the present invention also provides a method for increasing 2-AP in rice, which is simple to operate, low in cost, easy to manage, and convenient for agricultural promotion.

[0025] Biological Preservation Information

[0026] A fungus of the genus Rhizopus, TYR1, biologically classified as Rhizopus sp., was deposited on June 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo.42112. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 This is the phylogenetic tree of the Rhizopus fungus TYR1 strain in Example 1;

[0029] Figure 2The graph shows the results of 2-AP content in Nanjing 46 grains after treatment with 10 different fungi in the field trial in 2023 in Example 3. Different letters indicate significant differences (P < 0.05).

[0030] Figure 3 The image shows the results of 2-AP content in grains and leaves of Nanjing 46 rice after treatment with Rhizopus fungus TYR1 in a field trial in 2024, as shown in Example 3.

[0031] Figure 4 The image shows the results of 2-AP content in seeds and leaves of Nanjing 46 rice after treatment with Rhizopus fungus strain TYR1 in a pot experiment in 2024, as shown in Example 4.

[0032] Figure 5 The image shows the 2-AP content in the leaves of Nanjing 46 seedlings after treatment with Rhizopus fungus TYR1 in the 2024 aseptic seedling plate experiment in Example 5.

[0033] exist Figures 3-5 The asterisk (*) indicates a significant difference (P < 0.05). Detailed Implementation

[0034] This invention provides a Rhizopus sp. fungus TYR1, which has the accession number CGMCC No.42112.

[0035] The Rhizopus fungus TYR1 described in this invention was isolated from the rhizosphere soil of paddy rice in Liyang, Jiangsu Province, and its classification as a Rhizopus fungus was confirmed using high-throughput rhizosphere microbiome screening technology. This invention extracted DNA from single colony hyphae of the Rhizopus fungus TYR1 and sequenced it. Comparison of the obtained sequencing sequences with NCBI data revealed that it showed the highest homology (0.972) with OP269826.1_Rhizopus_sp._strain_HZ0027.

[0036] The present invention also provides the application of the Rhizopus fungus TYR1 described in the above technical solution in microbial fertilizers and / or microbial agents.

[0037] This invention provides a microbial inoculant, comprising the Rhizopus fungus TYR1 described in the above-mentioned technical solution. As one embodiment, the microbial inoculant can be a liquid inoculant and / or a solid inoculant; as one embodiment, the microbial inoculant can be a liquid inoculant. As one embodiment, the liquid inoculant can be a spore suspension of Rhizopus fungus TYR1. As one embodiment, the concentration of the Rhizopus fungus TYR1 spore suspension can be OD. 600 =0.2~0.4; as one embodiment, the concentration of the spore suspension of the Rhizopus fungus TYR1 can be OD600 =0.2, OD 600 =0.3, OD 600 =0.4 or OD 600 The concentration can be any value between 0.2 and 0.4. This invention does not impose any particular limitation on the preparation conditions of the Rhizopus TYR1 spore suspension; any conventional method for preparing Rhizopus spore suspensions in the art can be used. As one embodiment, the solid inoculum can be a Rhizopus TYR1 mycelial cake. As one embodiment, the diameter of the Rhizopus TYR1 mycelial cake can be 4.5–5.5 mm; as another embodiment, the diameter of the Rhizopus TYR1 mycelial cake can be 5 mm or any diameter within the range of 4.5–5.5 mm. This invention does not impose any particular limitation on the preparation method of the Rhizopus TYR1 mycelial cake; any conventional method for preparing Rhizopus TYR1 mycelial cake in the art can be used.

[0038] The present invention also provides the application of the Rhizopus fungus TYR1 described in the above technical solution or the microbial agent described in the above technical solution in increasing the content of 2-acetyl-1-pyrrolidine in rice.

[0039] In one embodiment, the rice can be fragrant rice; this invention does not specifically limit the variety of fragrant rice, and any fragrant rice variety is applicable to the technical solution of this invention; in the embodiments of this invention, Nanjing 46 is used as an example to illustrate the technical solution of this invention, but it cannot be limited to the entire scope of protection of this invention. The microbial agent of this invention has been limited in the above technical solution and will not be repeated here. In one embodiment, increasing the content of 2-acetyl-1-pyrrolidine in rice can be to increase the content of 2-acetyl-1-pyrrolidine in rice grains and / or leaves; in another embodiment, increasing the content of 2-acetyl-1-pyrrolidine in rice can be to increase the content of 2-acetyl-1-pyrrolidine in both rice grains and leaves.

[0040] The present invention also provides a method for increasing the content of 2-acetyl-1-pyrrolline in rice, comprising the steps described in I or II below:

[0041] Ⅰ: Soak rice seedlings in a microbial inoculant containing Rhizopus fungus TYR1, plant the soaked rice seedlings, and then apply the microbial inoculant containing Rhizopus fungus TYR1 to the planting land for planting management;

[0042] II: Inoculate the roots of rice seedlings with a solid microbial agent containing Rhizopus fungus TYR1, and then culture the rice seedlings inoculated with the solid microbial agent in a facility.

[0043] The Rhizopus fungus TYR1 described in either I or II has the accession number CGMCC No. 42112.

[0044] For the method described in section I, the present invention involves soaking rice seedlings in a microbial inoculant containing the Rhizopus fungus TYR1. As one embodiment, the rice seedlings can be rice seedling nurseries; the method of nurserying is not particularly limited, and conventional nurserying methods in the art can be used. As one embodiment, the microbial inoculant can be a spore suspension of the Rhizopus fungus TYR1. As one embodiment, the concentration of the Rhizopus fungus TYR1 spore suspension can be OD. 600 =0.2~0.4; as one embodiment, the concentration of the spore suspension of the Rhizopus fungus TYR1 can be OD 600 =0.2, OD 600 =0.3, OD 600 =0.4 or OD 600 The concentration can be any value between 0.2 and 0.4. In one embodiment, the roots of the rice seedlings are soaked. In one embodiment, the soaking time can be 1 to 2 hours; in another embodiment, the soaking time can be 1 hour.

[0045] After soaking, the rice seedlings are planted, and the microbial agent containing Rhizopus fungus TYR1 is applied to the planting area before cultivation management. As one embodiment, the planting can be field planting and / or pot planting; as another embodiment, the planting can be field planting or pot planting. As one embodiment, when field planting is carried out, the application is done in the field after planting; as one embodiment, the application rate of the microbial agent containing Rhizopus fungus TYR1 is 100-200 mL / m². 2 As one embodiment, the application rate of the microbial agent containing Rhizopus fungus TYR1 is 100 mL / m³. 2 150mL / m 2 Or 200mL / m 2In one embodiment, when potted cultivation, the application involves applying the microbial agent containing Rhizopus TYR1 to the roots of rice seedlings. In one embodiment, the application rate of the microbial agent containing Rhizopus TYR1 is 4-8 mL / seedling; in another embodiment, the application rate is 5 mL / seedling. This invention does not specifically limit the planting management method; conventional planting management methods in the art can be used. This invention first soaks the rice seedlings in the microbial agent containing Rhizopus TYR1, allowing the Rhizopus TYR1 to directly adhere to the roots, avoiding interference from the soil substrate, especially the potting soil substrate. Subsequent application ensures consistent application rates. For example, when using potted cultivation, it ensures consistent application rates of the microbial agent containing Rhizopus TYR1 in each pot of rice.

[0046] In the method described in section II, the present invention inoculates the roots of rice seedlings with a solid microbial agent containing the Rhizopus TYR1 fungus, and then cultivates the rice seedlings inoculated with the solid microbial agent in a facility. As one embodiment, the solid microbial agent containing the Rhizopus TYR1 fungus can be a Rhizopus TYR1 mycelium; as one embodiment, the diameter of the Rhizopus TYR1 mycelium can be 5 mm; as one embodiment, the diameter of the Rhizopus TYR1 mycelium can be any diameter selected from 4.5 mm, 5 mm, 5.5 mm, or 4.5–5.5 mm. As one embodiment, the inoculation amount of the solid microbial agent containing the Rhizopus TYR1 fungus can be 1 piece per seedling. As one embodiment, the solid microbial agent containing the Rhizopus TYR1 fungus can be inoculated to a depth of 0.5 cm at the root of the rice seedling. As one embodiment, the facility cultivation conditions of the present invention can be: 30°C light for 14 h, 23°C darkness for 10 h, alternating cycles, with a light intensity of 400 μmol / m². 2 / s, relative humidity is 60%.

[0047] In one embodiment, for item I or II of the present invention, increasing the content of 2-acetyl-1-pyrrolidine in rice can be achieved by increasing the content of 2-acetyl-1-pyrrolidine in rice grains and / or leaves; in another embodiment, increasing the content of 2-acetyl-1-pyrrolidine in rice can be achieved by increasing the content of 2-acetyl-1-pyrrolidine in both rice grains and leaves. In one embodiment, the rice can be aromatic rice; the present invention does not specifically limit the variety of aromatic rice, and any aromatic rice variety is applicable to the technical solution of the present invention; the embodiments of the present invention use Nanjing 46 as an example to illustrate the technical solution of the present invention, but this should not be limited to the entire scope of protection of the present invention.

[0048] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] The screening and identification of Rhizopus fungus TYR1 followed these steps:

[0051] 1. Rhizosphere soil collection

[0052] Samples were collected in September 2022 from paddy rice in Liyang, Jiangsu Province, during the grain-filling stage. Specifically, the root systems of the entire rice plant were dug up, large clumps of soil were shaken off, and a large amount of residual soil was washed away with sterile water. Roots with soil attached were then cut and placed in 50 mL centrifuge tubes containing sterile 0.86% NaCl solution. Under aseptic conditions, the rice roots were removed, the soil suspension was centrifuged, and the soil precipitate was collected.

[0053] 2. Isolation of single fungal colonies

[0054] Weigh 10g of the soil sediment collected in step 1 and place it in a 100mL Erlenmeyer flask containing glass beads and sterile water. Shake well for 10 minutes to thoroughly mix the soil and water. Under sterile conditions, take 1mL of the soil resuspension and dilute it with sterile water to prepare soil solutions with dilutions of 0.1, 0.01, 0.001, and 0.0001. Spread the 0.01 and 0.0001 concentrations of soil solution onto PDA plates and incubate at 28℃. Subsequently, select strains for single-colony passage and isolation. Ten fungal strains were screened and named TYR1 to TYR10.

[0055] 3. Fungal genome sequencing and identification

[0056] Single colony hyphae from step 2 were scraped, and DNA was extracted using a fungal DNA extraction kit (Solepro, D2300). Using 1 μg of gDNA template, library preparation was performed according to the methods and procedures in the TruSeq DNA Sample Preparation Guide (Illumina, 15026486Rev.C). After library construction, the insert size was first checked using an Agilent 2100. Once the insert size met expectations, Q-PCR was performed using a Bio-RAD CFX 96 real-time PCR instrument with Bio-RAD KIT iQ SYBR GRN to accurately quantify the effective concentration of the library (effective concentration > 10 nM). Libraries that passed the initial screening were sequenced using paired-end sequencing (PE 150 bp) on the NovaSeq XPlus sequencing platform. Sequence similarity was compared against the NCBI database, and sequences with high homology were selected for cluster analysis using MEGA 4.0 software. The Maximum Likelihood Method (ML method) was used to construct the phylogenetic tree, with a bootstrap value set to 1000. Inconsistent base sequences were manually removed from the DNA sequences used to construct the phylogenetic tree. The phylogenetic tree results for homology comparison of the TYR1 strain genome sequence are shown below. Figure 1 It can be seen that strain TYR1 has the highest homology with OP269826.1_Rhizopus_sp._strain_HZ0027, with a similarity of 0.972. Therefore, it can be determined that this strain is a fungus of the genus Rhizopus sp.

[0057] Preparation Example

[0058] To investigate the effect of the Rhizopus fungus TYR1 screened in Example 1 on 2-AP biosynthesis in rice, three experiments were conducted using Nanjing 46 (Wuxiangjing 14 × Guandong 194, NJ46) as the material: a field trial (2 years), a pot experiment, and a sterile plate seedling experiment. This variety was provided by the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, and is widely planted in the Yangtze River Basin. Detailed information about the variety can be found at https: / / www.ricedata.cn / variety / varis / 604660.htm. Specific verification experiments are shown in Examples 2–5.

[0059] Example 2

[0060] The preparation method of TYR1 spore suspension of Rhizopus fungi is as follows:

[0061] 1. Propagation of Rhizopus fungus TYR1: Inoculate TYR1 into potato dextrose agar (PDA) slant medium and incubate at 28℃ for 3 days to activate it; transfer the slant culture into Erlenmeyer flasks containing PDA medium and incubate at 28℃ for 5 days, ensuring that the mycelia are plump and robust and the spores are growing vigorously, and then use it for later use.

[0062] 2. Preparation of spore suspension: Filter the liquid culture medium in the Erlenmeyer flask from step 1 through sterile double-layered gauze and pour it out. Add 100 mL of sterile water to the filter residue (TYR1 spores of Rhizopus). Thoroughly mix and crush the mixture using a crusher, then dilute with sterile water. Measure the absorbance (OD) of the spore suspension at 600 nm using a UV spectrophotometer. 600 Within the scope of the test, it can be used subsequently.

[0063] Example 3

[0064] The field experiment on the effect of Rhizopus fungus TYR1 (hereinafter referred to as TYR1) on increasing the content of aroma compound 2-AP in rice was conducted as follows:

[0065] 1. 2023 field trial

[0066] A field trial was conducted in June 2023 in Liyang, Jiangsu Province (31°45'N, 119°29'E). This region is characterized by years of continuous cultivation of the high-quality fragrant rice variety Nanjing 46. Preliminary physicochemical analysis of the soil in 2022 showed a pH of 6.44 and available nitrogen, phosphorus, and potassium contents of 55.23 mg·kg⁻¹. -1 17.45 mg·kg -1 and 120.08 mg·kg -1 Organic matter content: 18.94 g·kg -1 .

[0067] Divide the area into 100m sections by embedding black plastic fencing into the soil (approximately 20cm deep). 2 Square experimental plots were used, with experimental and control groups (CK) set up separately. Each experimental and control group had 3 biological replicates, and each biological replicate had 3 experimental plots. The experimental and control groups were set up as follows:

[0068] Experimental group 1 (denoted as TYR1): The roots of seedlings aged 15-20 days were soaked in the solution prepared in Example 2 at a concentration of OD. 600 After 1 hour, rice seedlings were transplanted into a TYR1 spore suspension at a concentration of 0.2 g / L, and 15 L of the spore suspension was evenly spread into the plot. Regular field management was then carried out.

[0069] Experimental group 2 (designated TYR2 to TYR10 in sequence): The roots of seedlings aged 15 to 20 days were soaked in OD concentration water. 600The seedlings were placed in a TYR2-TYR10 spore suspension at a concentration of 0.2 (the preparation method of the TYR2-TYR10 spore suspension is the same as that of the TYR1 spore suspension). After 1 hour, the seedlings were transplanted and 15 LTYR2-TYR10 spore suspension was evenly sprinkled into the seedling plots that had been soaked in the same fungal spore suspension. The seedlings were then managed in a conventional field manner.

[0070] Control group (CK): The control group was a plot of land that was not treated with spore suspension, i.e., it was soaked in sterile water and no spore suspension was applied.

[0071] Rice grain samples were collected at the maturity stage for 2-AP content determination. The determination method is as follows:

[0072] Weigh 1g of finely ground rice sample using a mortar and pestle. Add 3mL of chromatographically pure dichloromethane:ethanol (1:1, V / V) containing the internal standard 2,4,6-trimethylpyrimidine (TMP, final concentration 0.5mg / L) to a 10mL sealed vial and heat in an 80℃ water bath for 3 hours. After cooling, immediately use a 1mL sterile syringe to draw 1mL of the extract and inject it through an organic needle filter membrane (0.22μm, 13mm pore size) into a 1.5mL mass spectrometry vial. Perform gas chromatography-mass spectrometry (GC-MS) analysis immediately. 2-AP aroma content was determined using a Shimadzu GC-MS QP 2010plus gas chromatography-mass spectrometer and the TMP internal standard method. Chromatographic conditions: The column was a RESTEK Rxi-5ms, 30m in length, 0.32mm in inner diameter, and 0.25μm in film thickness. The column temperature program was: 40℃ for 1 min, then increased to 65℃ at 2℃ / min and held for 1 min; then increased to 220℃ at 10℃ / min. The carrier gas was high-purity helium (purity >99.999%). Constant pressure splitless injection was used, with an injection volume of 10μL. Mass spectrometry conditions: Electron impact (EI) ion source, ion source temperature 200℃; ionization energy 70eV; interface temperature 250℃; quadrupole temperature 150℃; full scan mode, scan mass range m / z 30-350.

[0073] The measurement results are shown in Table 1 and Figure 2 As shown.

[0074] Table 1. 2-AP content in Nanjing 46 grains after treatment with 10 different fungi in a field trial in 2023.

[0075]

[0076] From Table 1 and Figure 2It can be concluded that, compared with the control (CK), the 2-AP content in Nanjing 46 grains treated with fungi TYR1, TYR2, TYR7 and TYR10 were significantly increased, but the increase was most significant with Rhizopus fungus TYR1.

[0077] 2. 2024 field trial

[0078] The field trial was conducted using the same methods and locations as in step 1, “2023 Field Trial”, except that only experimental group 1 and control group were set up.

[0079] Rice grains and the second leaf from the top were collected at the rice maturity stage for 2-AP content determination. 1g of finely ground rice sample was quickly ground in a mortar, and 1g of leaf sample was quickly cut into 0.5cm pieces with scissors for testing. The determination procedure was the same as in step 1, "2023 Field Trial". The results are shown in Table 2 and... Figure 3 As shown.

[0080] Table 2. 2-AP content in grains and leaves of Nanjing 46 rice seedlings after treatment with Rhizopus fungus TYR1 in a field trial in 2024.

[0081]

[0082] From Table 2 and Figure 3 It can be concluded that, compared with the control (CK), the 2-AP content in both grains and leaves of Nanjing 46 rice treated with TYR1 was significantly increased.

[0083] Example 4

[0084] A pot experiment was conducted to investigate how the Rhizopus fungus TYR1 (hereinafter referred to as TYR1) increased the content of the aroma compound 2-AP in rice. The steps were as follows:

[0085] The study was conducted in June 2023 at the experimental base of the Jiangsu Academy of Agricultural Sciences. Uniform 10L plastic containers were prepared, with 5kg of sterilized, air-dried soil placed in each container. The soil was taken from the experimental fields within the Jiangsu Academy of Agricultural Sciences, and its physicochemical properties were as follows: pH 6.51, available nitrogen, phosphorus, and potassium content 51.42 mg / kg. -1 14.09 mg·kg -1 and 112.81 mg·kg -1 Organic matter content: 17.34 g·kg -1 .

[0086] The roots of 2-week-old seedlings were soaked in the OD prepared in Example 2. 600 After 1 hour, the seedlings were transferred to soil culture in a TYR1 spore suspension at a concentration of 0.2, and 20 mL of spore suspension per pot was applied around the roots of the seedlings. The control group (CK) was not soaked and only 20 mL of sterile water per pot was added.

[0087] The specific setup was as follows: control group (CK) and TYR1, with 4 seedlings per pot, 6 replicates (6 pots) for each treatment, regular fertilization and watering in the later stages, and random placement. Grain and second-to-last leaf samples were collected at three stages: 10 days after heading (10dAFH), 20 days after heading (20dAFH), and maturity (MS).

[0088] The content of 2-AP in the collected grains and leaves was determined using the same method as in Example 3. The results are shown in Table 3. Figure 4 ,exist Figure 4 In the table, (A) and (B) represent the 2-AP content in the grains and leaves of Nanjing 46 rice after treatment with Rhizopus fungus strain TYR1 in a pot experiment in 2024.

[0089] Table 3. 2-AP content in seeds and leaves of Nanjing 46 grape seedlings after treatment with Rhizopus fungus strain TYR1 in a pot experiment in 2024.

[0090]

[0091] From Table 3 and Figure 4 It can be concluded that the 2-AP content in grain and leaf samples at 10 days, 20 days, and maturity was significantly higher than that in the CK group. The 2-AP content in both leaves and grains was highest at 10 days after heading and relatively lower at maturity.

[0092] Example 5

[0093] A plate sterile seedling test was conducted to investigate how the Rhizopus fungus TYR1 (hereinafter referred to as TYR1) increased the content of the aroma compound 2-AP in rice. The steps were as follows:

[0094] In 2024, a plate sterile seedling experiment was conducted in a laboratory laminar flow hood. First, the husks of the Nanjing japonica seeds were removed. The dehusked seeds were washed with a 75% (v / v) ethanol solution for 2 minutes, then the seed surface was sterilized with a 2% (v / v) NaClO solution for 30 minutes. After rinsing 5-6 times with sterile water, excess moisture was absorbed. Intact, sterilized seeds were selected and transferred under sterile conditions to plates containing 1 / 2 Kimura B medium. Sterile seedlings were obtained after 7 days of cultivation.

[0095] Experimental group (designated TYR1): Prepare a new 1 / 2 Kimura B medium, cut off half of the medium, and transfer sterile seedlings of uniform growth. Two seedlings are placed on each plate, and three plates are used for each treatment as biological replicates. Using a 5mm punch, mycelia of the Rhizopus fungus TYR1 are collected from the periphery of the potato extract medium (PDA, pH 5.6). One mycelial cake is inoculated per seedling, 0.5cm from the base of the sterile seedling, and the plants are placed in an artificial climate greenhouse. The artificial climate greenhouse conditions are: 30℃ for 14 hours of light, 23℃ for 10 hours of darkness, alternating cycles, with a light intensity of 400 μmol / m². 2 / s, relative humidity is 60%.

[0096] Control group (CK): Normal planting without any treatment.

[0097] Leaf samples were collected from the experimental and control groups 7 days after inoculation with the mycelium cake. The content of 2-AP in the leaves was measured using the same method as in Example 3. The results are shown in Table 4. Figure 5 As shown.

[0098] Table 4. 2-AP content in leaves of Nanjing 46 seedlings after treatment with Rhizopus fungus TYR1 in a 2024 aseptic seedling plate experiment.

[0099]

[0100] Note: In Table 4, the content of 2-AP was determined by taking a mixed sample of 2 seedlings from one plate for each replicate.

[0101] According to Table 4 and Figure 5 It can be concluded that, compared with the CK group, the 2-AP content in the leaves of Nanjing 46 was significantly increased after TYR1 treatment.

[0102] From the above examples, it can be concluded that: the present invention has for the first time isolated and identified a new Rhizopus strain TYR1 with significant aroma-enhancing function from the rhizosphere soil of fragrant rice through high-throughput rhizosphere microbial screening technology. Through experiments, it was found that TYR1 can effectively increase the content of 2-AP in rice grains and leaves, breaking through the traditional cognitive boundary that Rhizopus fungi are mostly used in the food field, especially in food fermentation, and promoting the functional leap of Rhizopus fungi from traditional fermentation industry to precision agriculture.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Rhizopus fungus that increases the content of 2-acetyl-1-pyrroline in rice ( Rhizopus sp .)TYR1, the preservation number of the Rhizopus fungus TYR1 is CGMCC No.42112.

2. The use of the Rhizopus fungus TYR1 as described in claim 1 in the preparation of microbial fertilizers and / or microbial agents.

3. A microbial inoculant, characterized in that, The microbial agent includes the Rhizopus fungus TYR1 as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The microbial agents include liquid agents and / or solid agents.

5. The microbial agent according to claim 4, characterized in that, The liquid inoculant includes a spore suspension of the fungus TYR1; the solid inoculant includes a fungal cake of the fungus TYR1.

6. The microbial agent according to claim 5, characterized in that, OD of the spore suspension of the Rhizopus fungus TYR1 600 The diameter of the mycelium of the Rhizopus TYR1 fungus is 0.2~0.4 mm; the diameter of the mycelium is 4.5~5.5 mm.

7. The use of the Rhizopus fungus TYR1 as described in claim 1 or the microbial agent as described in any one of claims 3 to 6 in increasing the content of 2-acetyl-1-pyrrolidine in rice.

8. A method for increasing the content of 2-acetyl-1-pyrrolline in rice, characterized in that, Includes the steps described in I or II below: Ⅰ: Soak rice seedlings in a microbial inoculant containing Rhizopus fungus TYR1, plant the soaked rice seedlings, and then apply the microbial inoculant containing Rhizopus fungus TYR1 to the planting land for planting management; II: Inoculate the roots of rice seedlings with a solid microbial agent containing Rhizopus fungus TYR1, and then culture the rice seedlings inoculated with the solid microbial agent in a facility. The Rhizopus fungus TYR1 described in either I or II has the accession number CGMCC No. 42112.

9. The method according to claim 8, characterized in that, In step I, the soaking time is 1-2 hours; the microbial agent includes a spore suspension of Rhizopus fungus TYR1, and the OD of the Rhizopus fungus TYR1 spore suspension is... 600 The concentration is 0.2~0.4; the application rate of the microbial agent containing Rhizopus fungus TYR1 is 100~200 mL / m³. 2 Or 4~8 mL / plant; In II, the solid microbial inoculant containing Rhizopus fungus TYR1 includes Rhizopus fungus TYR1 mycelium cake, the diameter of which is 4.5~5.5 mm.

10. The application according to claim 7 or the method according to claim 8 or 9, wherein increasing the content of 2-acetyl-1-pyrrolline in rice includes increasing the content of 2-acetyl-1-pyrrolline in rice grains and / or leaves.

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