Wheat stem rot inoculant as well as preparation method and application thereof

Abstract

The invention provides a wheat stem rot inoculant as well as a preparation method and application thereof, and relates to the technical field of plant pathogens. The preparation method comprises the following steps: carrying out liquid phase fermentation on an activated fusarium pseudograminearum hypha block to obtain a conidium suspension, and taking the diluted conidium suspension as a secondary strain; uniformly mixing the secondary seeds with a grain culture, performing solid-phase shallow tray fermentation to obtain a hardened grain culture, and continuously fermenting the hardened grain culture to obtain the wheat stem rot inoculant. According to the preparation method, the space is saved, and a large number of inoculums can be obtained in a short time. The inoculant obtained by the invention is suitable for field inoculation tests, has a remarkable effect, and can meet the disease degree requirements of identification of basal stem rot resistance of wheat varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant pathogens, and specifically relates to a wheat stem base rot inoculum and a preparation method and application thereof. Background Art

[0002] Wheat crown rot (Fusarium crown rot, FCR) is a soil-borne disease caused by Fusarium spp. that seriously harms wheat production. The disease can occur throughout the entire growth period of wheat. The symptoms of diseased plants are mainly browning of leaf sheaths and stems at the base of the stem, necrosis of stem nodes, resulting in plant death and whitening of the ear, which seriously affects the formation and filling of wheat grains. In addition, studies have shown that when wheat crown rot occurs seriously, the yield and quality of grains are greatly reduced, and a variety of toxins such as deoxynivalenol (DON) and nivalenol (NIV) are produced, which seriously affect the health of humans and animals.

[0003] Since stem base rot has the characteristics of strong saprophytic nature of pathogens, wide host, main disease site is the stem base, and disease can occur during the whole growth period, it is difficult to effectively cure it with traditional methods such as fungicides. Therefore, planting disease-resistant varieties has become one of the most effective and environmentally friendly measures to prevent and control the occurrence of stem base rot and reduce harm. The inoculation of pathogenic organisms is the most basic and important step in the entire disease-resistant variety screening process, and the preparation of inoculum is one of the important factors that determine the success or failure of inoculation. Especially for the field inoculation environment, there are often problems such as uneven infection of wheat stem base rot, contamination by other pathogens and unstable environmental conditions. These factors are not conducive to the stable occurrence and stable identification of the disease, and thus affect the screening of disease-resistant varieties. Therefore, it is urgent to provide a wheat stem base rot inoculum suitable for the field inoculation environment to meet the identification of wheat variety resistance to stem base rot, and then facilitate the screening of wheat stem base rot resistant germplasm resources. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a wheat stem rot inoculum and a preparation method and application thereof. The preparation method of the present invention saves space and can obtain a large number of inocula in a short time; the inoculum prepared by the present invention is more suitable for field inoculation experiments.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a wheat stem rot inoculum, comprising the following steps:

[0007] Adding the activated mycelium block of Pseudofusorium graminearum into a liquid fermentation medium and shaking and culturing for 4 to 6 days to obtain a conidia suspension;

[0008] The diluted conidia suspension is used as a secondary seed, and the secondary seed is mixed with a grain culture for fermentation to obtain a hardened grain culture;

[0009] The compacted grain culture is further fermented to obtain wheat stem base rot inoculum;

[0010] The pseudograminearum Fusarium has a preservation number of CGMCC 3.20319, named 14LY24-2, and the preservation time is February 2021. The preservation unit is the China General Microbiological Culture Collection Management Center.

[0011] Preferably, the volume ratio of the activated Fusarium graminearum mycelium block to the liquid fermentation medium is 1:10-20.

[0012] Preferably, the shaking culture temperature is 23-25°C.

[0013] Preferably, the concentration of the diluted conidia suspension is 1×10 2 ~1×10 4 Pieces / mL.

[0014] Preferably, the volume ratio of the secondary seeds to the grain culture is 1:30-38.

[0015] Preferably, the fermentation conditions are 22-28° C. and 55-85% humidity for 3-8 days.

[0016] The invention also provides a wheat stem base rot inoculum obtained by the preparation method.

[0017] The present invention also provides a field inoculation method for wheat base stem rot, which comprises mixing the wheat base stem rot inoculum with grains and sowing the grains or scattering the inoculum in an inoculation furrow; when the grains are wheat grains, the amount of the inoculum used is 8.0 to 12.0 kg / mu; when the grains are millet grains, the amount of the inoculum used is 4.0 to 6.0 kg / mu.

[0018] The present invention also provides an application of the wheat base stem rot inoculum in the identification of wheat base stem rot disease.

[0019] The present invention also provides an application of the wheat stem rot inoculum in the screening of wheat stem rot resistant germplasm.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention utilizes liquid-solid biphasic fermentation to produce wheat stem base rot inoculum. The preparation method of the present invention saves space and can obtain a large number of inoculums in a short time. The preparation method of the present invention obtains a large amount of inoculum in a short time through one-time fermentation, which is 4 to 7 times that of conventional polyethylene bags, and the culture time is shortened by 6 days; the number of one-time culture operations is reduced by 4 times. In addition, the conventional polyethylene bag culture method cultivates 100 bags, contaminates 20 to 30 bags (25 bags on average), and the contamination ratio is 20 to 30% (25% on average); the preparation method of the present invention has an extremely low probability of contamination, cultivates 100 trays, and contaminates 1 to 3 trays (2 trays on average, 2% contamination rate). The present invention firstly obtains a conidia suspension by liquid fermentation, mixes the conidia suspension with a grain culture, and then ferments and cultures it. Compared with the solid shallow tray fermentation culture, the strains are directly mixed with the grain culture, which increases the inoculation area of ​​the strains and the grain culture, and allows the grain culture to be fully fermented. The fermentation culture method of the present invention uses liquid phase secondary seeds for propagation, and adopts a conidia suspension of appropriate concentration to co-culture with the grain culture, which can effectively reduce the probability of contamination and ensure the infection efficiency of the inoculum; and achieve the purpose of rapid mixing and saving fermentation time.

[0022] The inoculum obtained by the present invention is effective in field inoculation tests. In 2021, the control diseased plant rate at the jointing stage in Neihuang, Anyang City, Henan Province was 71.2%, the control diseased stem rate at maturity was 70.3%, and the disease index was 38.4. In 2022, the diseased plant rate at the jointing stage in Neihuang, Anyang City, Henan Province was 69.5%, the control diseased stem rate at maturity was 70.7%, and the disease index was 37.5. The two-year field test reached the disease degree for wheat maturity resistance identification, and the average disease index of susceptible varieties was >35, which can meet the disease degree requirements for wheat variety resistance to stem base rot identification.

[0023] Biological deposit information

[0024] Fusariumpseudograminearum, named: 14LY24-2, is deposited at: China General Microbiological Culture Collection Management Center, the address of the depository: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC 3.20319, deposit date: February 2021. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of culturing Pseudofusorium graminearum in PDA medium in the present invention;

[0026] Figure 2 This is a schematic diagram of shaking culture of conidia suspension in the present invention;

[0027] Figure 3This is a schematic diagram of using wheat grains to culture inoculum in the present invention;

[0028] Figure 4 Schematic diagram of the inoculum of different concentrations of conidia suspension after 10 days of fermentation in a solid phase shallow dish. The concentration of the conidia suspension in A is 10 / mL, the concentration of the conidia suspension in B is 100 / mL, the concentration of the conidia suspension in C is 1000 / mL, and the concentration of the conidia suspension in D is 10000 / mL.

[0029] Figure 5 To investigate the disease occurrence at the jointing stage after field inoculation;

[0030] Figure 6 To investigate the disease occurrence during the mature stage after field inoculation. DETAILED DESCRIPTION

[0031] The present invention provides a method for preparing a wheat stem rot inoculum, comprising the following steps:

[0032] Adding the activated mycelium block of Pseudofusorium graminearum into a liquid fermentation medium and shaking and culturing for 4 to 6 days to obtain a conidia suspension;

[0033] The diluted conidia suspension is used as a secondary seed, and the secondary seed is mixed with a grain culture for fermentation to obtain a hardened grain culture;

[0034] The compacted grain culture is further fermented to obtain wheat stem base rot inoculum;

[0035] The pseudograminearum Fusarium has a preservation number of CGMCC 3.20319, named 14LY24-2, and the preservation time is February 2021. The preservation unit is the China General Microbiological Culture Collection Management Center.

[0036] In the present invention, the pseudofusarium graminearum is preferably pseudofusarium graminearum (Fusariumpseudograminearum) 14LY24-2, which has a strong pathogenicity. The present invention preferably uses the pseudofusarium graminearum as a pathogen for inoculum culture, and the prepared inoculum has a strong infection ability to wheat. The pseudofusarium graminearum described in the present invention can be purchased through conventional channels.

[0037] In the present invention, the Pseudofusorium graminearum is preferably activated to obtain activated Pseudofusorium graminearum hyphae blocks. The present invention does not specifically limit the activation method of the Pseudofusorium graminearum, and a conventional bacteria activation method can be used. In the present invention, the activation method preferably includes: inoculating Pseudofusorium graminearum on a PDA solid culture medium and culturing at a constant temperature of 22 to 28°C until the Pseudofusorium graminearum grows all over the solid culture medium. More preferably, the culture temperature is 25°C. After the constant temperature culture is completed, the present invention preferably uses a sterilized toothpick to divide the obtained culture into 0.3 to 1 cm 2 The pseudograminearum Fusarium graminearum block is used as the primary species, and is more preferably divided into 0.5-0.8 cm 2 of the pseudo-Fusarium graminearum fungus.

[0038] In the present invention, the activated Fusarium graminearum hyphae block is preferably added to a liquid fermentation medium and shaken for 4 to 6 days to obtain a conidia suspension. The specific steps are preferably as follows: the activated Fusarium graminearum hyphae block is preferably added to a liquid fermentation medium at a volume ratio of 1:10 to 20, and shaken for 4 to 6 days at 120 to 180 rpm / min and 22 to 28°C to obtain a conidia suspension by liquid phase fermentation. In the present invention, the volume ratio of the activated Fusarium graminearum hyphae block to the liquid fermentation medium is more preferably 1:15. In the present invention, the shaking conditions are more preferably 150 rpm / min, and shaking for 5 days at 23 to 25°C. In the present invention, after obtaining the conidia suspension, it is preferably diluted with sterile water, and the concentration of the diluted conidia suspension is preferably 1×10 2 ~1×10 4 / mL; more preferably 1×10 3 / mL. In the present invention, the liquid fermentation medium is preferably a mung bean water liquid medium. In the present invention, the mung bean water liquid medium is preferably a 3% mung bean soup medium, and the preparation method of the 3% mung bean soup medium is preferably to add 30g of mung beans to 1L of distilled water and boil for 10min, then filter with gauze, retain the filtrate, sterilize at 121°C for 20min, and cool for use. In the present invention, the mung bean water liquid medium can be purchased through conventional channels or prepared according to conventional methods.

[0039] In the present invention, the diluted conidia suspension is mixed with the grain culture as a secondary seed for fermentation to obtain a compacted grain culture. In the present invention, the method for preparing the grain culture preferably comprises the following steps: mixing sterilized grains with antibiotics to obtain a grain culture. In the present invention, the grains preferably include wheat grains or millet grains. In the present invention, the antibiotics preferably include one or two of ampicillin and streptomycin sulfate; the 75 mg / mL ampicillin preferably accounts for 0.05% to 0.2% of the grain volume, more preferably 0.1%; the 150 mg / mL streptomycin sulfate preferably accounts for 0.1% to 0.15% of the grain volume, more preferably 0.12%. In the present invention, before the grains and the antibiotics are mixed, the grains are preferably sterilized, and after sterilization for 1 hour, preferably after being placed for 2 days, they are sterilized again for 0.5 to 1 hour. In the present invention, after obtaining the grain culture, the present invention performs shallow tray fermentation on the grain culture, the grain culture is preferably spread to a thickness of 2 to 2.5 cm, and the diluted conidia suspension is added to the grain culture as a secondary seed at a volume ratio of 1:30 to 38. After 3 to 5 days, a compacted grain culture is obtained. In the present invention, during the shallow tray fermentation, it is preferred to wrap the shallow tray with tin foil. The present invention uses a shallow tray fermentation method, which is different from the conventional triangular flask and polyethylene bag fermentation. The shallow tray fermentation of the present invention is preferably to culture grains on a stainless steel plate (length × width × height of 47.5 to 52.5 cm × 32.5 to 36.5 cm × 6.5 to 8.5 cm), which is simple and convenient, and a large number of inoculum can be quickly obtained. In the present invention, the specifications of the stainless steel plate are more preferably 47.5 cm × 32.5 cm × 6.5 cm in length × width × height. As an practicable method, when the present invention uses a stainless steel plate with a length×width×height of 47.5cm×32.5cm×6.5cm for shallow tray fermentation, the temperature of the shallow tray fermentation is preferably 22-28°C, more preferably 25°C, and the thickness of the shallow tray fermentation grains is 2-2.5cm, which can ensure that the grains are fully fermented and each grain is wrapped with mycelium. The present invention uses liquid phase secondary seeds for propagation and then ferments and cultures with grain culture, which can ensure that each grain is wrapped with mycelium, so that the grains are fully fermented and the fermentation time is saved; at the same time, adding antibiotics can effectively reduce the probability of contamination during the culture process.

[0040] In the present invention, the compacted grain culture is preferably broken and crushed and then continued to be fermented in a shallow tray. After 8 days, the grain inoculum of Pseudofus graminearum is obtained. In the present invention, the 8 days later refers to the calculation from the beginning of the shallow tray fermentation. The present invention breaks the compacted grain culture apart and crushes it so that Pseudofus graminearum can grow evenly on the grain culture. After breaking the compacted grain culture apart and crushing it, it is preferably continued to be wrapped with tin foil and fermented in a shallow tray. In the present invention, the temperature of the shallow tray fermentation is preferably 22-28°C, more preferably 25°C. In the present invention, after obtaining the grain inoculum of Pseudofus graminearum, it is preferably further included to break the grain inoculum of Pseudofus graminearum, crush it, dry it, and refrigerate it. In the present invention, the drying and refrigeration methods can select conventional methods according to actual conditions. In the present invention, the drying method is preferably to dry in the shade in a ventilated place, and the refrigeration conditions are preferably refrigerated at 4°C.

[0041] The invention also provides a wheat stem base rot inoculum obtained by the preparation method.

[0042] The present invention also provides a field inoculation method for wheat stem base rot, preferably comprising the following steps: mixing the inoculum with grains, and sowing or sowing the inoculum in the inoculation furrow; when the grains are wheat grains, the amount of inoculum used is 8.0-12.0 kg / mu; when the grains are millet grains, the amount of inoculum used is 4.0-6.0 kg / mu, and sowing is performed by sowing in the inoculation furrow or using a seed and fertilizer integrated machine. In the present invention, after sowing or sowing in the inoculation furrow, water and fertilizer management is preferably performed. In the present invention, the sowing is preferably performed using a seed and fertilizer sowing machine. The present invention has no special limitation on the water and fertilizer management, and normal watering and fertilization can be performed.

[0043] The present invention also provides an application of the wheat base stem rot inoculum in the identification of wheat base stem rot disease.

[0044] The present invention also provides an application of the wheat stem rot inoculum in the screening of wheat stem rot resistant germplasm.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0047] Example 1

[0048] A method for preparing a wheat stem rot inoculum, comprising the following specific steps:

[0049] Pseudofusorium graminearum was cultured on PDA medium at 25°C for 5 days, and then a small square of 1 cm × 1 cm was scratched with a sterilized toothpick as the primary seed. Figure 1 ; The false Fusarium graminearum is false Fusarium graminearum (Fusariumpseudograminearum) 14LY24-2.

[0050] Pick 5-10 small cubes and place them in a 250mL Erlenmeyer flask containing 100mL of mung bean water medium. Pseudomonas graminearum was cultured in a mung bean water liquid medium at 150rpm / min and 25℃ to obtain a conidia suspension. The conidia suspension was diluted with sterile water to a conidia suspension of 100 / mL as a secondary seed. Figure 2 ;

[0051] Sterilized grains and antibiotics are mixed to obtain grain solid culture; the secondary seed is added to the grain culture at a volume ratio of 1:38 and solid phase shallow plate fermentation is carried out at 25°C and 70% humidity. When inoculating and culturing, 3.0 mL of 75 mg / mL ampicillin and 3.6 mL of 150 mg / mL streptomycin sulfate are added to every 3000-3800 cubic centimeters of grains, mixed with a sterilized spoon, spread on a stainless steel plate, and the stainless steel plate is wrapped with tin foil again, and cultured at a constant temperature of 25°C. Figure 3 ;

[0052] After 3 days, the compacted grain culture was broken apart and mixed evenly to allow the pseudograminearum Fusarium to grow evenly on the grain culture, and the stainless steel plate was wrapped with tin foil and continued to be cultured at a constant temperature of 25°C.

[0053] After 8 days, the tin foil on the stainless steel plate was removed and the grown Fusarium graminearum grain inoculum was collected.

[0054] Example 2

[0055] A method for preparing a wheat stem rot inoculum, comprising the following specific steps:

[0056] The Fusarium graminearum was cultured on a PDA medium at 25° C. for 3 days, and then a small square of 1 cm×1 cm was scratched with a sterilized toothpick as a primary species; the Fusarium graminearum was Fusarium graminearum (Fusariumpseudograminearum) 14LY24-2.

[0057] Pick 5-10 small cubes and place them in a 250mL Erlenmeyer flask containing 100mL of mung bean water culture medium. Shake the pseudograminearum fungus in the mung bean water liquid culture medium at 150rpm / min and 23°C to obtain a conidia suspension, and dilute the conidia suspension with sterile water to a conidia suspension of 1000 / mL as the secondary seed;

[0058] Mix sterilized grains and antibiotics to obtain grain solid culture; add secondary seeds to the grain culture at a volume ratio of 1:30 and perform solid phase shallow plate fermentation at 28°C and 80% humidity. When inoculating and culturing, add 3.0mL of 75mg / mL ampicillin and 3.6mL of 150mg / mL streptomycin sulfate to every 3000-3800 cubic centimeters of grains, mix well with a sterilized medicine spoon, spread on a stainless steel plate, wrap the stainless steel plate with tin foil again, and culture at a constant temperature of 28°C;

[0059] After 3 days, the compacted grain culture was broken apart and mixed evenly to allow the pseudograminearum Fusarium to grow evenly on the grain culture, and the stainless steel plate was wrapped with tin foil and continued to be cultured at a constant temperature of 28°C.

[0060] After 8 days, the tin foil on the stainless steel plate was removed and the grown Fusarium graminearum grain inoculum was collected.

[0061] Example 3

[0062] A method for preparing a wheat stem rot inoculum, comprising the following specific steps:

[0063] The Fusarium graminearum was cultured on a PDA medium at 25° C. for 3-5 days, and then a small square of 1 cm×1 cm was scratched with a sterilized toothpick as a primary species; the Fusarium graminearum was Fusarium graminearum (Fusariumpseudograminearum) 14LY24-2.

[0064] Pick 5-10 small cubes and place them in a 250mL Erlenmeyer flask containing 100mL of mung bean water medium. Shake the pseudograminearum fungus in the mung bean water liquid medium at 150rpm / min and 24°C to obtain a conidia suspension, and dilute the conidia suspension with sterile water to a conidia suspension of 100 / mL as the secondary seed;

[0065] Sterilized grains and antibiotics are mixed to obtain grain solid culture; secondary seeds are added to the grain culture at a volume ratio of 1:35 for solid phase shallow plate fermentation at 22°C and 60% humidity. During inoculation culture, 3.0 mL of 75 mg / mL ampicillin and 3.6 mL of 150 mg / mL streptomycin sulfate are added to every 3000-3800 cubic centimeters of grains, mixed well with a sterilized medicine spoon, spread on a stainless steel plate, wrap the stainless steel plate with tin foil again, and culture at a constant temperature of 22°C;

[0066] After 5 days, the compacted grain culture was broken apart and mixed evenly to allow the pseudograminearum Fusarium to grow evenly on the grain culture, and the stainless steel plate was wrapped with tin foil and continued to be cultured at a constant temperature of 22°C.

[0067] After 8 days, the tin foil on the stainless steel plate was removed and the grown Fusarium graminearum grain inoculum was collected.

[0068] Test Example 1

[0069] Effects of different concentrations of conidia suspension (secondary species) on inoculum preparation

[0070] The conidia suspension obtained from liquid fermentation culture was diluted with sterile water and set to four treatments of 10, 100, 1000, and 10000 conidia / mL. Each treatment had three replicates, and the same volume of grain culture was added to each treatment. The plates were wrapped with tin foil and sterilized for later use.

[0071] During inoculation, conidia suspensions of different concentrations were poured into 3800 cubic centimeters of grains, and 3.0 mL of 75 mg / mL ampicillin and 3.6 mL of 150 mg / mL streptomycin sulfate were added to every 3000-3800 cubic centimeters of grains; the suspensions were mixed evenly with the grains using a sterilized spoon, spread on a stainless steel plate, and the stainless steel plate was wrapped with tin foil again, and cultured at a constant temperature of 25°C;

[0072] After 3 days, break up the compacted grain culture and mix well to allow the pseudograminearum Fusarium to grow evenly on the grain culture. Wrap the stainless steel plate with tin foil and continue to culture at a constant temperature of 25°C.

[0073] After 8 days, the tin foil on the stainless steel plate was removed to collect the grown grain inoculum of F. graminearum. The growth of the secondary seed with a concentration of 10 conidia / mL was insufficient, while the growth of 100, 1000, and 10000 conidia / mL was sufficient, with no obvious difference in appearance. Figure 4 Schematic diagram of shallow tray fermentation of Fusarium graminearum millet inoculum.

[0074] Test Example 2

[0075] Effect of different ratios of conidia suspension (secondary species) to grain culture on inoculum preparation

[0076] The thickness of the grain culture spread on the stainless steel plate was set to 2.5 cm. A conidia suspension (secondary species) with a concentration of 100 / mL was selected and mixed with the grain culture. Five treatments with volume ratios of 1:15, 1:22, 1:30, 1:38, and 1:45 were set, with three replicates for each treatment. The plate was wrapped with tin foil and sterilized for later use.

[0077] During inoculation culture, pour corresponding amounts of grains into stainless steel plates with different scales, add 3.0 mL of 75 mg / mL ampicillin and 3.6 mL of 150 mg / mL streptomycin sulfate for every 3000 to 3800 cubic centimeters of grains; mix them with the grains with a sterilized medicine spoon, add the cultured Fusarium graminearum blocks with culture medium, mix well to obtain grain culture, spread the grain culture on the stainless steel plate according to the different thicknesses mentioned above, wrap the stainless steel plate with tin foil again, and culture it at a constant temperature of 25°C.

[0078] After 3 days, break up the compacted grain culture and mix well to allow the pseudograminearum Fusarium to grow evenly on the grain culture. Wrap the stainless steel plate with tin foil and continue to culture at a constant temperature of 25°C.

[0079] After 8 days, the tin foil on the stainless steel plate was removed to collect the grown grain inoculum of F. graminearum. When the volume ratio of the conidia suspension (secondary species) to the grain culture was 1:15 and 1:22, respectively, due to the high mycelium inoculation ratio and excessive water, it was easy to cause slow growth or mycelium cessation; when the volume ratio of the conidia suspension (secondary species) to the grain culture was 1:45, the inoculum could not grow fully due to the low inoculation mycelium density. When the volume ratio of the conidia suspension (secondary species) to the grain culture was 1:30 and 1:38, respectively, the inoculation ratio was appropriate, so that the mycelium on the grain culture grew best.

[0080] Test Example 3

[0081] Effect of inoculum use in the field

[0082] Determination of field inoculation amount: The grain inoculum of Pseudofusorium graminearum prepared in Example 1 was mixed with wheat grains, and sown in the inoculation furrow or using a seed-fertilizer machine for sowing. The amount of wheat inoculum used was 8-12 kg / mu.

[0083] The grain inoculum of Pseudofusorium graminearum prepared in Example 1 was mixed with millet grains and sown in the inoculation furrow or using a seed-fertilizer machine for sowing. The use amount of the millet grain inoculum was 4-6 kg / mu.

[0084] The amount of inoculum used can ensure that the disease index of susceptible wheat varieties in the field at maturity is >30, which can effectively distinguish resistant and susceptible varieties.

[0085] In 2021, the control disease rate of Neihuang in Anyang City, Henan Province was 71.2% at the jointing stage, the control disease rate of Neihuang in the mature stage was 70.3%, and the disease index was 38.4. In 2022, the control disease rate of Neihuang in Anyang City, Henan Province was 69.5% at the jointing stage, the control disease rate of Neihuang in the filling stage was 70.7%, and the disease index was 37.5. Both field trials reached the disease level for wheat resistance identification at the adult stage. Figure 5 This is the incidence of stem base rot in wheat (Bainong 207) at the jointing stage after the inoculum was used; Figure 6 This is a diagram showing the incidence of stem base rot in wheat (Bainong 207) at maturity after the inoculum was used.

[0086] Table 1 Incidence of wheat stem rot with different inoculation doses in Neihuang, Anyang City, Henan Province in 2021

[0087] Millet inoculum dosage kg / mu Disease rate of jointing plants % Diseased stem rate at maturity (%) Maturity index 1 12.1 5.8 6.6 2 34.8 32.3 16.8 4 71.2 70.3 38.4 6 66.4 68.9 35.2

[0088] Table 2 Incidence of wheat stem rot with different inoculation rates in Neihuang, Anyang City, Henan Province in 2022

[0089] Wheat grain inoculum dosage kg / mu Disease rate of jointing plants % Diseased stem rate at maturity (%) Maturity index 2 13.3 12.1 10.0 4 38.2 30.6 14.6 8 69.5 70.7 37.5 12 66.1 69.4 36.3

[0090] Test Example 4

[0091] Comparison of liquid-solid dual-phase shallow tray fermentation with solid-phase shallow tray fermentation and conventional polyethylene bag fermentation

[0092] Conventional polyethylene bag fermentation: Each bag contains 300g of wheat and 400g of millet. The inoculum can be prepared in the bag after 2 weeks of fermentation. 300g of wheat inoculum and 400g of millet inoculum can be prepared at one time. During the fermentation period, knead once every 2 to 4 days, and knead and mix 4 to 7 times in total to ensure uniform growth of mycelium.

[0093] Solid phase shallow plate fermentation: The activated Pseudograminearum Fusarium is directly mixed with the grain culture for fermentation, wherein the culture plate has a size of 47.5cm×32.5cm×6.5cm in length×width×height, and each plate can hold 1.6-1.8kg of wheat grains and 1.8-2.2kg of millet grains. The preparation of the inoculum can be completed after 10 days of fermentation, and 1.2-1.6kg of wheat inoculum and 1.5-1.8kg of millet inoculum can be prepared at one time. For details, please refer to patent CN114574544. The strain used is Pseudograminearum Fusarium 14LY24-2.

[0094] Liquid-solid two-phase shallow tray fermentation: Wheat inoculum and millet inoculum are obtained according to the inoculum preparation method of Example 1. The specification of the culture tray is length×width×height 47.5cm×32.5cm×6.5cm, and each tray can hold 1.6-1.8kg of wheat grains and 1.8-2.2kg of millet grains. The preparation of the inoculum can be completed after 8 days of fermentation. The agglomerated inoculum is crushed and mixed once every 4-5 days, 1.6-2.2kg is mixed once, and mixed twice in total. 1.6-1.8kg of wheat inoculum and 1.8-2.2kg of millet inoculum are prepared at one time.

[0095] Table 3 Comparison of liquid-solid dual-phase shallow tray fermentation with solid-phase shallow tray fermentation and conventional polyethylene bag fermentation

[0096]

[0097] According to the results in Table 3, the present invention adopts liquid-solid biphase shallow plate fermentation to prepare the inoculum. The inoculum obtained by one-time fermentation is large in volume and short in time, which is 4 to 7 times that of conventional polyethylene bags, and the culture time is shortened by 6 days; the number of one-time culture operations is reduced by 4 times, and the amount of bacteria used is significantly reduced. In addition, the conventional polyethylene bag culture method cultivates 100 bags, and 20 to 30 bags (25 bags on average) are contaminated, and the contamination ratio is 20 to 30% (25% on average); the preparation method of the present invention has a very low probability of contamination, and 1 to 3 plates are contaminated in 100 plates (2 plates on average, 2% on average).

[0098] The present invention firstly obtains a conidia suspension by liquid fermentation, and then mixes the conidia suspension with a grain culture for fermentation. Compared with the solid shallow plate fermentation culture, the strain and the grain culture are directly mixed and cultured, which increases the inoculation area of ​​the strain and the grain culture, and allows the grain culture to be fully fermented. 2 The bacterial block was placed in a 100 mL culture bottle and cultured to obtain a conidia suspension. Generally, the conidia suspension obtained was 1×10 6 , and then dilute to obtain a 100 / mL conidia suspension for use in the preparation of a culture plate inoculum. 100 mL 1×10 6 The conidia suspension can be diluted 10,000 times and can be evenly inoculated into 10,000 plates of inoculum, which not only reduces inoculation contamination but also ensures inoculation uniformity. Solid shallow plate fermentation requires the use of two culture dishes of strains for mixing and culturing with grain culture. The culturing method of the present invention significantly reduces the amount of strains used and reduces the probability of breeding miscellaneous bacteria when the strains are mixed with grain culture.

[0099] Therefore, the fermentation culture method of the present invention uses liquid phase secondary seeds for propagation, and adopts a conidia suspension of appropriate concentration to co-culture with the grain culture, which can effectively reduce the probability of contamination and ensure the infection efficiency of the inoculum; and achieve the purpose of rapid mixing and saving fermentation time.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a wheat stem rot inoculum, characterized in that: The steps include: Adding the activated mycelium block of Pseudofusorium graminearum into a liquid fermentation medium and shaking and culturing for 4 to 6 days to obtain a conidia suspension; The diluted conidia suspension is used as a secondary seed, and the secondary seed is mixed with a grain culture for fermentation to obtain a hardened grain culture; The compacted grain culture is further fermented to obtain wheat stem base rot inoculum; The pseudograminearum Fusarium has a preservation number of CGMCC 3.20319, named 14LY24-2, and the preservation time is February 2021. The preservation unit is the China General Microbiological Culture Collection Management Center.

2. The preparation method according to claim 1, characterized in that: The volume ratio of the activated pseudograminearum mycelium block to the liquid fermentation medium is 1:10-20.

3. The preparation method according to claim 1, characterized in that: The temperature of the shaking culture is 23-25°C.

4. The preparation method according to claim 1, characterized in that: The concentration of the diluted conidia suspension is 1×10 2 ~1×10 4 Pieces / mL.

5. The preparation method according to claim 1, characterized in that: The volume ratio of the secondary seeds to the grain culture is 1:30-38.

6. The preparation method according to claim 1, characterized in that: The fermentation conditions are as follows: the fermentation is carried out at 22-28° C. and a humidity of 55-85% for 3-8 days.

7. The wheat stem rot inoculum obtained by the preparation method according to any one of claims 1 to 6.

8. A method for inoculating wheat stem base rot in the field, characterized in that: The wheat stem rot inoculum of claim 7 is mixed with grains and sown or the inoculum is spread in the inoculation furrow; when the grains are wheat grains, the inoculum usage amount is 8.0-12.0 kg / mu; when the grains are millet grains, the inoculum application amount is 4.0-6.0 kg / mu.

9. Use of the wheat base stem rot inoculum according to claim 7 in the identification of wheat base stem rot disease.

10. Use of the wheat stem rot inoculum according to claim 7 in screening wheat stem rot-resistant germplasm.

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