CN114574544A

CN114574544A - Preparation and use methods and application of wheat stem and root rot grain inoculum

Info

Publication number: CN114574544A
Application number: CN202210274372.8A
Authority: CN
Inventors: 徐飞; 李丽娟; 宋玉立; 冯超红; 王俊美; 韩自行; 张姣姣
Original assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-06-03

Abstract

The invention relates to a preparation method, a use method and application of a wheat stem basal rot grain inoculum, and belongs to the technical field of plant pathogenic bacteria. The preparation method comprises the following steps: mixing activated fusarium pseudograminearum, sterilized grains and antibiotics to obtain a grain culture, performing tray fermentation on the grain culture for 5 days to obtain a hardened grain culture, breaking the hardened grain culture off and kneading the hardened grain culture, continuing tray fermentation, and obtaining the fusarium pseudograminearum grain inoculum after 10 days. The preparation method disclosed by the invention is space-saving, large in quantity, quick, time-saving and labor-saving. The obtained inoculum field inoculation test has obvious effect.

Description

Preparation and use methods and application of wheat stem rot grain inoculum

Technical Field

The invention relates to the technical field of plant pathogenic bacteria, in particular to a preparation method, a use method and application of a wheat stem basal rot grain inoculum.

Background

The wheat stem base rot caused by the infection of fusarium pseudograminearum is an important stem base disease in wheat production in Huang-Huai-wheat areas in China, the white spike rate in the grain filling period is 20%, and 50% yield loss can be caused. Meanwhile, fusarium pseudograminearum produces various fungaltoxins such as deoxynivalenol to pollute the straws, so that the straws lose the feeding value. The wheat stem base rot is listed in the famous book of second class crop diseases and insect pests in Henan province by the agricultural rural ministry of Henan province.

The research on the wheat stem base rot caused by fusarium pseudograminearum has the advantages of germplasm resource resistance identification, resistance material creation, resistance gene positioning and the like; the breeding and planting of disease-resistant varieties are the most economic and effective measures for preventing and controlling the diseases, wherein the link of artificial inoculation and identification in the field is very important, and thus, a large amount of inoculants are required to be prepared. In actual operation, when the quantity of materials to be identified is large, the preparation workload of the inoculum is large, and the problems of high bacterial pollution rate, low preparation speed, small quantity, complex operation and the like exist.

Traditionally, the inoculum is prepared by using a triangular flask or a polyethylene bag, and the hypha growth is inhibited and the breeding period is long due to inconvenient performance operation, poor air permeability, high humidity and the like. Therefore, the amount of the grain inoculants cultured at one time is large, and a large amount of fusarium pseudograminearum grain inoculants and a field inoculation method thereof can be cultured in a short time, so that the technical problems to be solved at present are urgently solved, and the development of a simple, convenient, easy and rapid large amount of fusarium pseudograminearum grain inoculants and a field inoculation method thereof is urgent.

Disclosure of Invention

The invention aims to provide a preparation method, a using method and application of a wheat stem basal rot grain inoculum. The preparation method provided by the invention is simple to operate, can simply, conveniently and quickly culture a large amount of fusarium pseudograminearum grain inoculants, and can solve the technical problem that the large amount of fusarium pseudograminearum grain inoculants are difficult to culture and use in fields.

The invention provides a preparation method of a wheat stem basal rot grain inoculum, which comprises the following steps:

mixing activated fusarium pseudograminearum, sterilized grains and antibiotics to obtain a grain culture, performing tray fermentation on the grain culture for 5 days to obtain a hardened grain culture, breaking and kneading the hardened grain culture, continuing tray fermentation, and obtaining a fusarium pseudograminearum grain inoculum after 10 days; the thickness that the grain culture tiled is 2 ~ 2.5 cm.

Preferably, the method of activation comprises: inoculating fusarium pseudograminearum on a PDA solid culture medium, and culturing at a constant temperature of 22-28 ℃ until the fusarium pseudograminearum overgrows the whole solid culture medium.

Preferably, the grain comprises wheat or millet grains.

Preferably, the antibiotic comprises ampicillin and streptomycin sulphate.

Preferably, during the fermentation of the tray, the tray is wrapped by tinfoil paper.

Preferably, the temperature of the tray fermentation is 22-28 ℃.

Preferably, after the fusarium pseudograminearum grain inoculum is obtained, breaking the fusarium pseudograminearum grain inoculum, drying and refrigerating.

The invention also provides a use method of the wheat stem base rot grain inoculum obtained by the preparation method in the technical scheme, which comprises the following steps:

mixing the inoculum with grains, and sowing or sowing the inoculum in an inoculation ditch; when the grains are wheat grains, the using amount of the inoculum is 8-12 kg/mu; when the grains are millet grains, the application amount of the inoculum is 4-6 kg/mu.

Preferably, water and fertilizer management is performed after sowing or sowing in the inoculation trench.

The invention also provides application of the wheat stem basal rot grain inoculum obtained by the preparation method in the technical scheme in seed selection of wheat stem basal rot resistant varieties.

The invention provides a preparation method of a wheat stem basal rot grain inoculum. The preparation method of the invention adopts tray fermentation, and the preparation method of the invention saves space, is rapid in a large amount, and saves time and labor. The obtained inoculum field inoculation test has obvious effect, the average disease index in the two-year grouting period is more than 33, the average diseased stalk rate is 69 percent, specifically, the contrast diseased stalk rate in the yellow jointing stage in Anyang city of Henan province in 2019 is 65 percent, the contrast diseased stalk rate in the grouting period is 69 percent, the disease index is 33 percent, and the white spike rate is 10 percent. The contrast disease strain rate in yellow jointing stage in Anyang city of Henan province in 2020 is 70%, the stem rate in filling stage is 69%, the disease index is 36, and the white spike rate is 11%. The field test for two years achieves the disease degree of resistance identification of the wheat in the adult plant period, the average disease index of the susceptible variety is more than 20, and the disease degree requirement of the wheat variety for stem base rot resistance identification can be met.

Drawings

FIG. 1 is a schematic view of an inoculum of Fusarium pseudograminearum kernels fermented in a tray according to the present invention;

FIG. 2 is a diagram showing the occurrence of the wheat (dwarf 58) stem rot after the inoculum provided by the invention is used.

Detailed Description

The invention mixes the activated pseudofusarium graminearum, the sterilized grains and the antibiotic to obtain the grainA pellet culture. The activation method of the fusarium pseudograminearum is not specially limited, and a conventional bacterium activation method is adopted. In the present invention, the method of activation preferably comprises: inoculating fusarium pseudograminearum on a PDA solid culture medium, and culturing at a constant temperature of 22-28 ℃, more preferably 25 ℃ until the fusarium pseudograminearum overgrows the whole solid culture medium. After the incubation at constant temperature, the present invention preferably divides the resulting culture into 0.5cm pieces using sterilized toothpicks²The fusarium pseudograminearum block is used for mixing with antibiotics and grains. Fusarium pseudograminearum is used in the present invention. In the present invention, the grain preferably comprises wheat or millet grains. Before the mixing, the grains are preferably sterilized, and after the grains are sterilized for 1 hour, the grains are preferably sterilized again for 0.5 to 1 hour after being placed for 2 days. In the present invention, the antibiotic preferably includes ampicillin and streptomycin sulfate. In the present invention, after the mixing, it is preferably stirred uniformly using a sterilized spatula.

After the grain culture is obtained, shallow tray fermentation is carried out on the grain culture, the thickness of the flat spread grain culture is 2-2.5 cm, and after 5 days, the hardened grain culture is obtained. In the present invention, it is preferable that the tray is wrapped with a tin foil paper when the tray is fermented. The invention uses a tray fermentation method, which is different from the conventional triangular flask and polyethylene bag fermentation. The shallow tray fermentation method disclosed by the invention is preferably used for culturing grains on a stainless steel tray (the length, the width and the height are 47-52 cm, the x 32-36 cm, the x 6.5-8.5 cm), and is simple, convenient, large in quantity and quick. In the present invention, the stainless steel dish is more preferably set to have a length × width × height of 47cm × 32cm × 6.5 cm. When the present invention is used for the tray fermentation using stainless steel trays having a length × width × height of 47cm × 32cm × 6.5cm, the inoculum size of activated Fusarium pseudograminearum is preferably inoculated in one dish (9cm) per stainless steel tray. In the invention, the temperature of tray fermentation is preferably 22-28 ℃, more preferably 25 ℃, the flat thickness of tray fermented grains is 2-2.5 cm, the grains can be fully fermented, and each grain is coated with hyphae.

After obtaining the hardened grain culture, the hardened grain culture is broken off and kneaded, and then the shallow fermentation is continued, and after 10 days (the 10 days later in the invention means the calculation from the beginning of the shallow fermentation), the fusarium pseudograminearum grain inoculum is obtained. The breaking-off and breaking operation of the invention can ensure that the fusarium graminearum can grow uniformly on the grain culture. Breaking the hardened grain culture, preferably continuously wrapping with tinfoil paper, and performing shallow tray fermentation. In the invention, the temperature of the tray fermentation is preferably 22-28 ℃, and more preferably 25 ℃. In the present invention, after obtaining the fusarium pseudograminearum grain inoculum, the method preferably further comprises breaking the fusarium pseudograminearum grain inoculum, drying, and refrigerating. After this breaking-off, the drying method is preferably drying in the shade in a ventilated place, and the refrigeration condition is preferably 4 ℃.

mixing the inoculum with grains, and sowing or sowing the inoculum in an inoculation ditch; when the grains are wheat grains, the using amount of the inoculum is 8-12 kg/mu; when the grain is the millet grain, the usage amount of the inoculum is 4-6 kg/mu, and the inoculum is sowed in the inoculation ditch or sowed by using a seed manure all-in-one machine. In the present invention, after sowing or sowing in the inoculation trench, water and fertilizer management is preferably performed. In the present invention, the sowing is preferably performed using a seed and fertilizer simulcaster. The invention has no special limitation on the water and fertilizer management, and the water and fertilizer management can be realized only by normally watering and fertilizing.

The preparation, application and use of the wheat stem rot grain inoculum according to the invention will be described in further detail with reference to the following specific examples, and the technical solutions of the invention include, but are not limited to, the following examples.

Example 1

Preparation of shallow tray fermented grain inoculum

Setting the thickness of the grain culture laid on the stainless steel plate to be 1cm, 1.5cm, 2cm, 2.5cm and 3cm, wherein each treatment is repeated three times, marking lines with different scales on the inner walls of different plates before the stainless steel plate is sterilized, wrapping the plates with tinfoil paper, and sterilizing for later use.

During inoculation culture, corresponding amount of grains are poured into stainless steel plates with different scales, and 1-3 ml of 75mg/ml ampicillin and 1-3 ml of 150mg/ml streptomycin sulfate are added into every 3000-3800 cubic centimeters of grains, and respectively account for 0.05-0.15% and 0.1-0.3% of the mass of the inoculum; mixing with grains with sterilized spoon, adding cultured Fusarium pseudograminearum block with culture medium, stirring to obtain grain culture, spreading the grain culture on stainless steel plate according to the above different thicknesses, wrapping the stainless steel plate with tinfoil paper, and culturing at 25 deg.C.

After 5 days, the hardened grain culture is broken off and stirred evenly, so that fusarium pseudograminearum grows uniformly on the grain culture, and the grain culture is wrapped with tinfoil paper on a stainless steel plate and is continuously cultured at the constant temperature of 25 ℃.

After 10 days, the tinfoil paper on the stainless steel plate was removed, and the grown fusarium pseudograminearum grain inoculum was collected. The grain culture hyphae with the scale of 1cm and 1.5cm on the stainless steel plate can not grow sufficiently and stop growing due to insufficient water loss, the grain culture with the scale of 3cm on the stainless steel plate can not grow sufficiently in the grain culture due to the fact that the fusarium graminearum is paved too thick, and the hyphae on the grain culture with the scale of 2cm and 2.5cm on the stainless steel plate can grow best. FIG. 1 is a schematic diagram of an inoculum of Fusarium pseudograminearum kernels fermented on a tray.

Example 2

Plot of field effect of inoculum

Determining the field inoculation amount, mixing the inoculum with grains, and sowing or sowing in an inoculation ditch; when the grains are wheat grains, the using amount is 8-12 kg/mu; when the cereal grain is the millet grain, the use amount is 4 ~ 6 kg/mu, through sowing or use the seed manure all-in-one to sow in inoculating the ditch. The inoculation amount of the wheat grain inoculants is 8-12 kg/mu, and the inoculation amount of the broadcast sowing seed furrows of the millet grain inoculants is 4-6 kg/mu. Normally watering and fertilizing after inoculation.

In 2019, the contrast disease strain rate in yellow jointing stage in Anyang city of Henan province is 65%, the stem rate in filling stage is 69%, the disease index is 33, and the white spike rate is 10%. The yellow stem disease rate in the yellow jointing stage of Anyang city in Henan province in 2020 is 70%, the stem rate of the control disease in the filling stage is 69%, the disease index is 36, and the white spike rate is 11%. The field test for two years achieves the disease degree of resistance identification of wheat in the adult plant period.

When the usage amount of the wheat inoculum is 8-12 kg/mu, and the usage amount of the millet inoculum is 4-6 kg/mu, the wheat variety in the field can be guaranteed to be fully attacked. And the wheat inoculum is used less than 8 kg/mu, and the millet inoculum is less than 4 kg/mu, so that the disease index of disease incidence of the field susceptible varieties can not be guaranteed to be more than 20, and the less disease incidence can not effectively distinguish the susceptible varieties.

Table 12020 years of occurrence of wheat stem base rot caused by yellow wheat with different inoculation amounts in Anyang city of Henan province

The dosage of wheat grain inoculum is kg/mu	Disease rate in jointing stage%	The diseased stem rate in the filling stage%	Index of the state of illness in the filling stage
				2	14	10	8
4	41	29	13
				8	70	69	36
12	64	70	35

TABLE 22019 occurrence of stem rot of wheat with different inoculation amounts of yellow wheat in Anyang city of Henan province in Henan province

The dosage of the millet inoculum is kg/mu	Disease rate in jointing stage%	The diseased stem rate in the filling stage%	Index of state of illness in grouting stage
				1	11	5	6
2	34	30	15
				4	70	65	30
6	65	69	33

FIG. 2 is a graph showing the onset of wheat (dwarf 58) stalk rot after inoculum application.

Example 3

Comparison of tray fermentation with conventional polyethylene bag fermentation

Fermenting in a conventional polyethylene bag, wherein each bag contains 300g of wheat and 400g of millet grains, and the inoculum preparation can be completed after fermenting in the bag for 2 weeks, wherein 300g of wheat inoculum and 400g of millet grain inoculum are prepared at one time. Kneading once every 2-4 days during fermentation, kneading and mixing for 4-7 times to make the hyphae grow uniformly.

Fermenting in a shallow tray (length is multiplied by width is multiplied by height is 47cm is multiplied by 32cm is multiplied by 6.5cm), the size is convenient to put into a conventional incubator, 2-2.5 cm of grains are loaded into each tray, 1.2-1.5 kg of wheat grains and 1.5-1.8 kg of millet grains can be loaded into each tray. And (3) fermenting for 10 days to complete the preparation of the inoculum, crushing and uniformly mixing the agglomerated inoculum once every 5 days, uniformly mixing 1.2-1.8 kg once, and uniformly mixing for 2 times. In addition, 1-3 ml of ampicillin and 1-3 ml of streptomycin sulfate (which respectively account for 0.05% -0.15% and 0.1% -0.3% of the mass of the inoculum) are added into each 3000-3800 cubic centimeter of grains, and the mixture of the antibiotics and the grains and the fusarium pseudograminearum is uniformly stirred by a sterilized medicine spoon, so that the pollution probability can be reduced.

TABLE 3 comparison of tray fermentation with conventional polyethylene bag fermentation

The one-time culture amount, the culture time, the operation times and the pollution probability of mass culture can indicate that the tray fermentation is superior to the conventional polyethylene bag fermentation method. Culturing 100 bags by a conventional method, wherein 20-30 bags are polluted, and the pollution proportion is 20-30%; the probability of pollution of tray fermentation is small, 100 trays are cultured, and 3-5 trays are polluted.

Shallow plate fermentation culture

The inoculum obtained by one-time fermentation of tray fermentation is more than 4-6 times of that of a conventional polyethylene bag, the culture time is shortened by 4 days, the operation times are reduced by 2-5 times, and the pollution probability is reduced by 17-25%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a wheat stem basal rot grain inoculum comprises the following steps:

2. The method of claim 1, wherein the activating comprises: inoculating fusarium pseudograminearum on a PDA solid culture medium, and culturing at a constant temperature of 22-28 ℃ until the fusarium pseudograminearum overgrows the whole solid culture medium.

3. The method of claim 1, wherein the grain comprises wheat grain or millet grain.

4. The method of claim 1, wherein the antibiotic comprises ampicillin and streptomycin sulfate.

5. The method of claim 1, wherein the tray is wrapped with tinfoil paper during fermentation.

6. The method according to claim 1, wherein the temperature of the tray fermentation is 22-28 ℃.

7. The method according to claim 1, wherein the step of obtaining the inoculum of fusarium pseudograminearum grains further comprises breaking the inoculum of fusarium pseudograminearum grains, drying, and refrigerating.

8. A use method of the wheat stem base rot grain inoculum obtained by the preparation method of any one of claims 1 to 7, comprising the following steps:

9. The use method according to claim 8, wherein water and fertilizer management is performed after sowing or sowing in the inoculation trench.

10. The application of the wheat stem basal rot grain inoculum obtained by the preparation method of any one of claims 1 to 7 in seed selection of wheat stem basal rot resistant varieties.