Epicoccum nigrum and funnel mould and application thereof in identification, diagnosis and prevention of quinoa diseases

Abstract

The invention belongs to the technical field of pathogenic microorganisms, provides Epicoccum nigrum and Afunnel mould and application thereof in identification, diagnosis and prevention and treatment of quinoa diseases, and lays a foundation for diagnosis and prevention and treatment of a new disease of quinoa spiking disease. The name of E. nigrum is LM3, the E. nigrum is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.41033, and the preservation date is January 12, 2024. The name of the infundibulifera is LM2, the infundibulifera is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is 41035, and the preservation date is January 12, 2024; the LM3 and the LM2 are pathogenic bacteria of the chenopodium quinoa willd spiking disease and can be identified through a morphological method, and the effect of identifying the bacteria through a kit extraction method is good. The pathogenic bacteria can be identified through the ITS gene sequence.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogenic microorganisms, and particularly relates to Epicoccum nigrum and Choanephora cucurbitarum and their applications in the identification, diagnosis and control of quinoa diseases. Background Art

[0002] In recent years, a new disease has been found in the quinoa planting area. After the quinoa enters the heading stage, the ears of the diseased plants turn chlorotic and yellow, and then become soft until the whole ear collapses. This disease seriously affects the yield of quinoa. At present, this new symptomatic disease has no official name, and no domestic institution has given a definite answer in strict accordance with Koch's postulates for plant disease identification. Most farmers call it "bent ear disease" or "head-drop disease".

[0003] According to the results of field observations, when the quinoa ear is infected by the pathogen, the grains at the top and outside of the ear will turn chlorotic and yellow; as the disease progresses, the yellowing area gradually expands, and the ear will turn soft and yellow from top to bottom, including the stems and leaves of the ear also showing this symptom; in the late stage of the disease, most of the grains in the ear turn yellow, the ear becomes soft, and the inner stem of the ear will bend. The present invention names this disease as ear-drop disease or collapse disease.

[0004] The reported pathogens of quinoa ears include Cladosporium cladosporioides, Trichothecium roseum, Alternaria alternata and Fusarium citri: after Cladosporium cladosporioides infects the quinoa ear, a mold layer will appear on the ear, and as the disease progresses, the color of the mold layer will deepen from olive green to dark brown, and the area of mildew will become larger and larger, eventually causing the whole ear to wither and the grains to shrivel and rot. When the humidity is high, the pathogen will also infect the young stems adjacent to the ear, and a distinct dark green to dark brown mold layer will appear on the diseased part [1]; after the latter three pathogens infect the plant, during the filling stage, a layer of light pink, grayish white or dark brown mold will appear on the grains, resulting in the discoloration, filling and deformation of quinoa grains [2].

[0005] Epicoccum nigrum Epicoccum nigrum belongs to the genus Epicoccum in the Deuteromycotina subphylum ( Epicocum Link ). Most previous studies have focused on biological control. For example, the spore suspension of Epicoccum nigrum has a good protective effect on potato late blight [4]; Epicoccum nigrum and its fermentation products can reduce the incidence of postharvest gray mold of blueberry fruits caused by Botrytis cinerea [5], etc. In recent years, there have been more and more reports on plant diseases caused by Epicoccum nigrum. It has been reported that Epicoccum nigrum can cause brown spot disease of rice ears [6], leaf spot disease of Lonicera caerulea [7] and leaf diseases of Medicago sativa [8]. Choanephora cucurbitarum Choanephoracea infundibulifera is Choanephoracea a mucoromycotan fungus in the genus, and there are no relevant reports on the diseases it causes at present.

[0006] The literature and data were searched and found that there are no related reports on the new disease of ear inversion at home and abroad, and there are no related reports that black spores and funnel mold can cause quinoa ear inversion, and scientific research in this area is equivalent to a blank state. Therefore, the present invention will be the first report that black spores and funnel mold cause quinoa ear inversion, laying a theoretical foundation for the diagnosis and prevention of quinoa ear inversion, a new disease. Summary of the invention

[0007] The invention provides Echinococcus nigromaculata and Echinococcus infundibulum and applications thereof in identification, diagnosis and prevention of quinoa diseases, laying a foundation for diagnosis and prevention of quinoa ear inversion disease, a new disease.

[0008] The present invention is achieved by the following technical solution: a strain of Echinococcus nigromaculata ( Epicoccum nigrum ), the name of the black coccus is LM3, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.41033, the deposit date is January 12, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0009] The morphological characteristics of the strain are as follows: the colony is initially white, the aerial hyphae are short and fluffy, the hyphae turn yellow-brown in the later stage, and the back of the colony secretes red or reddish-brown pigments. The conidia produced on PDA are black and aggregated into piles. Under the microscope, the molecular spores are spherical or nearly spherical, mostly aggregated into strings, and the color is yellow-brown or dark brown. The spore size is (12.4-20.9)µm×(14.0-23.5)µm. The black-attached coccus ( Epicoccum nigrum LM3, isolated and purified from quinoa ear-turning disease, was identified through genetic testing and analysis. Epicoccum nigrum .

[0010] A fungus Choanephora infundibulifera ), characterized in that: the name of the funnel mold is LM2, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41035, the deposit date is January 12, 2024, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0011] The morphological characteristics of this strain are: it grows rapidly on PDA medium, and the colony diameter can reach 63-65mm after 1 day of culture. The hyphae are white at first and grow closely to the medium. Then the aerial hyphae multiply in large quantities, appearing short cotton wool, and the color changes to dark yellow. At the same time, a large number of black sporangia will be produced at the top. Under the microscope, it was found that the black sporangia have two forms, namely small sporangiola and sporangia.

[0012] The spore stalk of the small sporangium is smooth, transparent, unseptate, upright, and unbranched. Its top is enlarged to form a primary vesicle, which will form multiple secondary branches. The top of the branch will expand to form a secondary vesicle, and multiple single spores will form on each secondary vesicle. Microscopic observation shows that the width of the small sporangium is 48.1-481.2µm and the length is 212.4-419.9µm. The single spores on the secondary vesicle can be detached at any time after maturity. The detached single spore has a shorter cylindrical spore tube at one end, which is brown to dark brown in color. Most of them are ellipsoidal in shape, and a few are nearly spherical in shape. There are no cracks on the surface, and a few have longitudinal stripes on the surface. The measurement results are 8.3-21.9µm in width and 10.5-23.1µm in length.

[0013] Another type of sporangium can be observed with the naked eye. The sporangium stalk is unseptate, transparent, and unbranched. It is yellow in color at first, turning light brown or dark brown when mature. It is spherical in shape and has a diameter of 53.8-213.9µm. After maturity, it releases a large number of spores, which are dark brown in color, fusiform to oval in shape, with more than 10 transparent appendages at each end, 8.1-12.5µm wide and 14.1-30.0µm long.

[0014] The funnel fungus of the present invention ( Choanephora infundibulifera LM2, isolated and purified from quinoa ear-turning disease, was identified through genetic testing and analysis. Choanephora infundibulifera.

[0015] The present invention also provides the use of the Echinococcus nigromaculata and the Trichoderma infundibulum as reference substances for identifying quinoa ear inversion disease.

[0016] Furthermore, E. nigrosinus and E. fusca were used as control samples, and the concentration of spore suspension of each strain was 1×10 7 CFU / mL, the inoculation volume ratio of the two strains was 1:1, and the total volume was 10 mL.

[0017] The experimental results showed that: Escherichia cocci ( Epicoccum nigrum LM3 and Fungus ( Choanephora infundibulifera LM2 is the pathogen of quinoa ear inversion, which can be identified by morphological methods. The kit extraction method is more effective for the identification of this bacterium. The pathogen can be identified by ITS gene sequence.

[0018] The strain Echinococcus nigromaculata of the present invention ( Epicoccum nigrum)LM3, strain number LM23-1G1, referred to as LM3, has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.41033, the deposit date is January 12, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0019] The funnel fungus Choanephora infundibulifera )LM2, strain number LM23-1Y2, referred to as LM2, has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNo.41035, the deposit date is January 12, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The field symptoms of quinoa ear stump disease are shown in the figure: A shows the symptoms of ear stump disease in the field; B shows the symptoms of ear; Figure 2 The growth status of the isolate on PDA medium; in the figure: A is strain LM1; B is strain LM2; C is strain LM3; D is strain LM4; Figure 3 This is a pathogenicity experiment of quinoa; in the figure: A is the control; B is 10 days after the combination of LM2 and LM3 is inoculated; C is 16 days after the combination of LM2 and LM3 is inoculated; D is 15 days after the single strain LM2 is inoculated; E is 20 days after the single strain LM3 is inoculated; Figure 4 In the figure: A is the control; B is the diseased ear; C is the diseased ear neck; D is the diseased kernel; Figure 5 The morphological characteristics of the colony, sporangium and spores of strain LM2; in the figure: A is the growth state of strain LM2 on PDA medium; B and C are small sporangia; D is the conidia of small sporangia; E and F are sporangia; H is the conidia of sporangium; Figure 6 The morphological characteristics of the colony and spores of strain LM3; in the figure: A is the morphological characteristics of the colony, sporangium and spores of strain LM3; B is the spore mass; C is the spore; Figure 7 For strains C. infundibulifera Phylogenetic tree of (LM2-1, LM2-2, LM2-3 and LM2-4); Figure 8 For strains Epicoccum nigrum Phylogenetic tree of (LM3-1, LM3-2, LM3-3, LM3-4, and LM3-5); Fig. 9 The growth status of strains LM2 and LM3 on different culture media; in the figure: A, B, C, and D are the growth status of strain LM2 in PDA, OA, CMA, and Czapek's medium, respectively; E, F, G, and H are the growth status of strain LM3 in PDA, OA, CMA, and Czapek's medium, respectively. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0023] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] 1. Materials and Methods 1. Test culture medium: potato dextrose agar medium (Beijing Solebow Technology Co., Ltd.); Test instruments: The main instruments are Nikon optical microscope.

[0026] Collection of diseased plants: The collection location is 38.25°N, 111.87°E, 1152.8 meters above sea level, in a quinoa greenhouse in Jingle County, Xinzhou City, Shanxi Province.

[0027] 2. Isolation and purification of pathogens: tissue separation method is used. Cut a 5mm × 5mm tissue block at the junction of the diseased and healthy parts, disinfect the lesion with 70% alcohol, then rinse it three times with sterilized distilled water, place it in PDA culture medium, inoculate 2-4 pieces in each culture dish, and culture it at 25°C. Select colonies with good growth and no contamination. When the hyphae length reaches 2cm, use an inoculation hook to pick up the mycelium at the edge of the colony, and then transfer it to the center of the PDA culture medium again. Then perform multiple separation and purification until the colonies grow uniformly and no foreign bacteria appear. Observe the colony morphology and the conidia morphology under a microscope. Select isolates with good growth and typical status for subsequent experiments.

[0028] 3. Pathogen inoculation experiment (Koch's postulates): Select "Jinli No. 3" quinoa plants with good growth, health and no disease for the experiment.

[0029] The quinoa spikes in the flowering period were tested for pathogenicity by spraying, and the control group was inoculated with sterile water. The spikes were rinsed once with 75% alcohol, and then rinsed three times with sterile water until there was no powder layer on the surface, and then dried naturally. 7 The spore suspension of the CFU / mL isolate was evenly sprayed onto the entire ear of quinoa, including the inner ear axis, and 10 mL was inoculated per strain. After inoculation, the bags were covered with moisture. When the double-strain experiment was conducted, the concentration of the spore suspension of each strain was 1×10 7 CFU / mL, the inoculation volume ratio of the two strains was 1:1, and the total volume was 10 mL.

[0030] Subsequently, the quinoa plants were placed at 25°C, 80% humidity, and 12 hours of light and 12 hours of darkness until the disease occurred. Ten quinoa plants were tested in each group. After the disease occurred, the strain was isolated and purified again from the tissue at the junction of the diseased and healthy parts according to the above method.

[0031] 4. Morphological identification: Inoculate the strains that conform to Koch's postulates into PDA medium and observe the colony characteristics and spore morphology.

[0032] 5. Molecular identification: DNA extraction, PCR amplification, phylogenetic tree construction: DNA extraction: The genomic DNA of pathogens was extracted using the CTAB method.

[0033] PCR amplification: PCR amplification was performed using primers ITS1 / 4. Primer sequences: ITS1 is shown in SEQ ID NO.1: TCCGTAGGTGAACCTGCGG; ITS4 is shown in SEQ ID NO.2: TCCTCCGCTTATTGATATGC.

[0034] Amplification system (50.0ul): genomic DNA (20ng / ul), 1.0ul 10×Buffer (containing 2.5mM Mg 2+ )5.0ul, Taq polymerase (5u / μL)1.0ul, dNTP (10mM)1.0ul, ITS1 primer (10uM)1.5ul, ITS4 primer (10uM)1.5ul, ddH 2 O 39.0 ul.

[0035] Reaction conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, extension at 58°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles, total extension at 72°C for 7 min, and storage at 4°C.

[0036] ITS sequence sequencing: The PCR product was sent to Paisono for sequencing. Primer sequence: ITS1 is shown in SEQ ID NO.1; ITS4 is shown in SEQ ID NO.2.

[0037] Construction of phylogenetic tree: The sequencing results were compared by BLAST in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the phylogenetic tree was constructed using MEGA7.0.

[0038] 6. Analysis of biological characteristics of pathogens: A. Effect of temperature on mycelial growth: The strain was cultured at 25°C in the dark for 7 days. A 6-mm-diameter bacterial cake was taken from the edge of the colony and inoculated onto a PDA plate, which was placed in an incubator at temperatures of 15, 20, 25, 28, 30, and 35°C. After culturing in the dark for 7 days, the colony diameter was measured using the cross method, and each treatment was repeated 3 times.

[0039] B. Effect of pH on mycelial growth: PDA medium was sterilized and used as standby medium. When used, PDA medium was melted and cooled to about 80°C. 1 mol / L HCL and NaOH solution were used to adjust pH to 5, 6, 7, 8, 9, and 10 respectively. Pathogenic bacteria strains were inoculated on PDA plates with different pH values, cultured at 25°C in the dark for 7 days, and colony diameters were measured using the cross method. Each treatment was repeated 3 times.

[0040] C. Effects of different light conditions on mycelial growth: Methods Referring to the effect of temperature on mycelial growth, three light conditions were set: continuous light, complete darkness, and 12h / 12h light-dark alternation, and each treatment was repeated 3 times.

[0041] D. Effect of culture medium on mycelial growth: Methods: Referring to the effect of different temperatures on mycelial growth, the pathogen cakes were inoculated onto OA (oatmeal culture medium), PDA (potato culture medium), Czapek (Czapek culture medium) and CMA (corn culture medium) plates, respectively. After culturing at 25°C in the dark for 7 days, the colony diameter was measured by the cross method. Each treatment was repeated 3 times.

[0042] E. Effects of carbon and nitrogen sources on mycelial growth: Effects of different carbon sources on mycelial growth of pathogens: Methods: Referring to the effects of different culture media on mycelial growth, Czapek medium was used as the basic culture medium, and the carbon sources therein were replaced with fructose, mannitol, soluble starch and glucose in equal amounts. Each treatment was repeated 3 times.

[0043] Effects of different nitrogen sources on mycelial growth of pathogenic bacteria: Methods: Referring to the effect of culture medium on mycelial growth, Czapek medium was used as the basic medium, and the nitrogen sources therein were replaced with urea, ammonium chloride, trypsin and glutamic acid in equal amounts. Each treatment was repeated 3 times.

[0044] 2. Experimental Results 1. Disease description: In August 2024, a new ear disease was found in about 40% of the quinoa plants in the quinoa greenhouse in Jingle County.

[0045] like Figure 1 As shown, in the early stage of the disease, the grains in the ear begin to turn yellow from top to bottom and from outside to inside; as the disease progresses, more and more grains turn yellow, and the range becomes larger and larger, and the leaves and stems of the ear also turn yellow; in the late stage of the disease, the disease gradually spreads to the entire ear, and the stems inside the ear become soft and bend. In severe cases, the entire ear will eventually become hard due to water loss and eventually collapse.

[0046] 2. Isolation and purification of pathogens: A total of 12 purified fungal strains were obtained from the 5 collected diseased plants. These isolates can be divided into 4 types through morphological observation, such as Figure 2 As shown, they were numbered LM1, LM2, LM3, and LM4, with 2, 4, 5, and 1 strains, respectively, and the isolation frequencies were 16.7%, 33.3%, 41.67%, and 0.83%, respectively.

[0047] The LM1 isolate had neat colony edges on PDA medium, aerial hyphae were interwoven and flocculent, the hyphae were white to light purple, and light purple pigment was produced on the back of the colony; the LM2 isolate grew rapidly on PDA medium, and the colony diameter could reach 63-65mm after 1 day of culture. The hyphae were initially white and grew close to the medium, and then the aerial hyphae multiplied in large numbers, showing short cotton wool, and the color changed to dark yellow, and a large number of black sporangia were produced on the top; the LM3 isolate had white colonies on PDA medium, the aerial hyphae were short and flocculent, and the hyphae later turned yellow-brown, and the back of the colony secreted red or reddish-brown pigments. The conidia produced on PDA were black and gathered in piles; the LM4 isolate grew faster, with lush hyphae, black-gray hyphae, and dark black hyphae in the later stage, and secreted black pigments.

[0048] 3. Pathogen inoculation experiment: The purified strains LM1, LM2, LM3 and LM4 were used alone or in combination to infect quinoa seedlings. The results are shown in Table 1.

[0049] Table 1: Pathogenicity experiments of quinoa The results showed that when single strains were inoculated, only strains LM2 and LM3 could cause yellowing of the grains, but the number was very small ( Figure 3 D, E); when the strains were inoculated in pairs, it was found that the performance of quinoa ears after inoculation with combinations 5, 6, 9 and 10 was basically the same as that after inoculation with single strains LM2 and LM3, which could cause the grains in the ears to turn yellow and soften, but the disease was mild, and the number of diseased grains was still very small, and some were even less than that after inoculation with single strains; only after infection with the combination of strains LM2 and LM3, the performance of the ears was very similar to the field symptoms.

[0050] The strains LM2 and LM3 were combined to inoculate quinoa seedlings. After 10 days of cultivation, the seeds on one tiller of the ear began to turn yellow and were 7.6 cm long ( Figure 3 B) After 16 days, the yellowed ear expanded to 21.5 cm, and the stem of the ear turned yellow and softened, and began to bend downward ( Figure 3 C) The symptoms are similar to those found in the field. Observing the ear under a microscope, the entire ear was found to be yellow and wilted ( Figure 4 B), the stems are yellow-brown, wilted and softened, and the diameter is obviously reduced ( Figure 4 C), the grains in the diseased ear are sparse, yellow-brown, significantly smaller in size, and less hard ( Figure 4 D);.

[0051] The infected ears were separated and purified again, and a total of 5 strains were obtained, of which 2 strains were consistent with the morphological characteristics of strain LM2, and 3 strains were consistent with the morphological characteristics of strain LM3. The results of pathogenicity detection combined Koch's postulates, and it was determined that isolates LM2 and LM3 were both pathogenic bacteria of ear inversion, and LM3 was relatively more pathogenic.

[0052] 4. Morphological identification of pathogens: strain LM2 grows rapidly on PDA medium. After 1 day of culture, the colony diameter can reach 63-65mm. The hyphae are white at first and grow close to the medium. Then the aerial hyphae multiply in large numbers, appearing short cotton wool and turning dark yellow in color. At the same time, a large number of black sporangia will be produced on the top ( Figure 5 A). Microscopic observation revealed that black sporangia have two forms, namely, small sporangiola and sporangia.

[0053] The spore stalk of the microsporangium is smooth, transparent, unseptate, upright, and unbranched. Its top is swollen to form a primary vesicle, which will form multiple secondary branches. The branch tops will expand to form secondary vesicles, and multiple single spores will form on each secondary vesicle. Under a microscope, the width of the microsporangium is 48.1-481.2µm and the length is 212.4-419.9µm ( Figure 5 B, C). The monospores on the secondary vesicles can detach at any time after maturity. The detached monospores have a short cylindrical spore tube at one end. The color is brown to dark brown. Most of them are ellipsoidal, and a few are nearly spherical. There are no cracks on the surface, and a few have longitudinal stripes on the surface. The measurement results are 8.3-21.9µm in width and 10.5-23.1µm in length ( Figure 5 D).

[0054] Another type of sporangium formed by strain LM2 can be observed with the naked eye. The sporangium stalk is unseptate, transparent, and unbranched. It is yellow in color at first, and turns light brown or dark brown after maturity. It is spherical in shape and has a diameter of 53.8-213.9µm ( Figure 5 EG). After maturity, it releases a large number of spores, which are dark brown in color, fusiform to oval in shape, with more than 10 transparent appendages at each end, 8.1-12.5µm in width and 14.1-30.0µm in length ( Figure 5 H).

[0055] The colonies of strain LM3 on PDA medium are initially white, with short fuzzy aerial hyphae, which turn yellow-brown in the later stage, and red or reddish-brown pigments are secreted on the back of the colonies ( Figure 6 A). Conidia produced on PDA are black and aggregated into piles. Under the microscope, the molecular spores are spherical or nearly spherical, mostly aggregated into clusters, and are yellow-brown or dark brown in color ( Figure 6B, C), spore size (12.4−20.9)µm×(14.0−23.5)µm.

[0056] 5. Molecular identification of pathogens: The total DNA of pathogenic bacteria LM2 and LM3 was extracted using a fungal DNA extraction kit, and the pathogens were amplified using primers ITS1 and ITS4. The PCR products were detected by 1.0% agarose gel electrophoresis and sequenced by Shanghai Paisono Company.

[0057] The isolated pathogenic bacteria LM2 (LM2-1, LM2-2, LM2-3 and LM2-4) were compared with Choanephora infundibulifera The maximum similarity of the two genes was above 99%. The phylogenetic tree based on the neighbor-joining method was constructed using MEGA 7.0 software. Figure 6 Combining the morphological characteristics and molecular identification results, it was determined that strain LM2 was a funnel fungus ( C. infundibulifera ), has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.41035.

[0058] Pathogen LM3 (LM3-1, LM3-2, LM3-3, LM3-4 and LM3-5) and Epicoccum nigrum The maximum similarity of the two genes was above 99%. The phylogenetic tree based on the neighbor-joining method was constructed using MEGA 7.0 software. Figure 7 As shown. Combining morphological characteristics and molecular identification results, strain LM3 was determined to be a black Escherichia coccus ( E.nigrum ), which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number of CGMCC No. 41033.

[0059] 6. Analysis of biological characteristics of pathogens A. Analysis of biological characteristics of strain LM2: Table 2 shows the growth status of strain LM2 on different culture media, and Table 3 shows the growth status of strain LM2 under different culture conditions for 3 days.

[0060] from Fig. 9 (A, B, C, D) and Table 2 show that strain LM2 grows well on PDA medium and oatmeal medium, and the mycelium of oatmeal medium is denser and produces more spores; for different carbon sources, strain LM2 grows better on the medium with mannitol as the carbon source; in the experiment with different nitrogen sources, strain LM2 grows better on the chlorotryptone medium with nitrogen source. Considering objective factors such as cost, the most suitable medium for strain LM2 is PDA medium. The results in Table 3 show that the most suitable temperature for mycelial growth and spore production of strain LM2 is 25℃, the best light culture condition is light and dark alternating culture, and the most suitable pH value is pH7.

[0061] Taking into account objective factors such as cost, the most suitable culture medium for strain LM2 is PDA medium, and the optimal culture conditions are: temperature is 25℃, alternating light and dark culture, pH 7.

[0062] Table 2: Growth status of strain LM2 on different culture media Table 3: Growth status of strain LM2 under different culture conditions (3d) B. Analysis of biological characteristics of strain LM3: Table 4 shows the growth status of strain LM3 on different culture media; Table 5 shows the growth status of strain LM3 at 7 days under different culture conditions.

[0063] from Fig. 9 (E, F, G, H) and Table 4 show that strain LM3 grows fastest in PDA medium, mycelium is denser on oatmeal medium, and spores grow slowly, about 20 days; for different carbon sources, mycelium growth of strain LM3 is better on starch medium; in experiments with different nitrogen sources, strain LM3 grows better on tryptone medium with peptone as nitrogen source. The results in Table 5 show that the most suitable temperature for mycelium growth and spore production of strain LM3 is 25℃, the best light culture condition is light culture, and the most suitable pH value is pH7-8.

[0064] Taking into account objective factors such as cost, the most suitable culture medium for strain LM3 is PDA medium, and the optimal culture conditions are: temperature is 25℃, light culture, pH 7-8.

[0065] Table 4: Growth status of strain LM3 on different culture media Table 5: Growth status of strain LM3 under different culture conditions (7d) Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

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Claims

1. A strain of Echinococcus nigromaculata ( Epicoccum nigrum ), characterized in that: The name of the black coccus is LM3, and it has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41033. The deposit date is January 12, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

2. A fungus Choanephora infundibulifera ), characterized in that: The name of the funnel mold is LM2, and it has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41035. The deposit date is January 12, 2024. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

3. Use of the Echinococcus nigromaculata described in claim 1 and the Aconitum quinoa described in claim 2 as reference substances for identifying quinoa ear inversion disease.

4. The use according to claim 2, characterized in that: Escherichia cocci and Echinococcus nigromaculata were used as control samples. The concentration of spore suspension of each strain was 1×10 7 CFU / mL, the inoculation volume ratio of the two strains was 1:1, and the total volume was 10 mL.

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