CN108060088B - Culture medium for magnaporthe grisea culture and spore production and preparation method thereof - Google Patents

Abstract

The invention provides a culture medium for rice blast bacterium culture and spore production and a preparation method thereof. The technical scheme is characterized in that the components of the culture medium are innovatively designed by an experimental method, and the influence of various compound components on the growth of rice blast germs is investigated on the basis of the growth rate, the spore yield and the like. Experimental results show that the culture medium obtained by mixing the rice straw leachate and the PDA culture medium according to equal volumes has a remarkable growth promoting effect on rice blast germs, and meanwhile, the spore production efficiency is remarkably improved. The culture medium has good culture effect on rice blast germs, has outstanding spore yield, is easy to obtain materials, is simple and convenient to prepare, and has good use effect.

Description

Culture medium for magnaporthe grisea culture and spore production and preparation method thereof

Technical Field

The invention relates to the technical field of microbial culture, in particular to a culture medium for rice blast bacterium culture and spore production and a preparation method thereof.

Background

The rice blast is the first disease of rice, and the pathogenic microorganism is botrytis cinerea (pyricus blazea grisea) which belongs to deuteromycetes; it is known as Oesophagostomum grisea (Magnaporthe grisea) in sexual generation. In the experimental work such as the research of disease resistance of rice varieties, the identification of physiological microspecies of rice blast germs and the like, the culture of strains and the acquisition of conidia are necessary technical links, so that the culture effect and the spore production efficiency of the rice blast germs have direct influence on the experimental work.

In the prior art, the culture medium for the rice blast bacteria culture and spore production is generally obtained by directly mixing sorghum, rice straws, barley, wheat and other components, and the culture medium is complex to prepare, slow in growth of the bacteria, unsatisfactory in spore production effect and easy to be polluted by other bacteria. Although some researchers use oatmeal culture medium, rice bran culture medium, coarse flour culture medium, corn flour culture medium, yeast starch culture medium and the like for spore production experiments, the technical problems of unsatisfactory spore production effect, difficult acquisition of materials and the like still exist generally.

Disclosure of Invention

The invention aims to provide a culture medium for rice blast fungus culture and spore production and a preparation method thereof aiming at the technical defects of the prior art, and aims to solve the technical problems of poor culture effect and low spore production efficiency of the conventional culture medium on the rice blast fungus in the prior art.

In order to achieve the technical purpose, the invention adopts the following technical scheme

A culture medium for culturing Pyricularia oryzae and producing spore is prepared by mixing equal volume of rice straw leachate and PDA culture medium; wherein the rice straw leachate is a soaking solution obtained by mixing rice straws and water according to the proportion of 3:100(g: mL) and soaking for 16-24 h; the PDA culture medium comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose and 1000 parts of water.

Preferably, the PDA culture medium further comprises 18 parts by weight of agar.

Preferably, the Pyricularia oryzae is Pyricularia grisea subsp ZB 13.

Meanwhile, the invention provides a preparation method of the culture medium for magnaporthe grisea culture and sporulation, which comprises the following steps:

1) mixing rice straws with water according to a ratio of 3:100(g: mL), and soaking for 16-24h to obtain rice straw leachate;

2) mixing 200 parts by weight of potatoes, 20 parts by weight of glucose and 1000 parts by weight of water, boiling for 15 minutes, filtering by using gauze, and taking filtrate;

3) mixing the rice straw leachate obtained in the step 1) and the filtrate obtained in the step 2) according to a volume ratio of 1:1, adding agar which is 18 per thousand of the weight of the water used in the step 2) into the mixture, and heating to dissolve the agar.

In the above technical solution, the Magnaporthe grisea is pyrrulospora grisea or tricholoma grisea.

On the basis, the invention provides the application of the culture medium for rice blast fungus sporulation, which comprises the following steps: selecting rice blast fungus strains, inoculating the rice blast fungus strains on a culture medium flat plate, culturing at the constant temperature of 28 ℃, after hyphae on the flat plate grow fully (about 7d), moving the flat plate to an indoor tissue culture frame, and culturing for 3d by black light illumination at the room temperature of 25-28 ℃; after the spores are produced sufficiently, the spores are washed by 15mL of distilled water in each dish, and the number of the spores is calculated under a 100-time microscope.

The invention provides a culture medium for rice blast bacterium culture and spore production and a preparation method thereof. The technical scheme is characterized in that the components of the culture medium are innovatively designed by an experimental method, and the influence of various compound components on the growth of rice blast germs is investigated on the basis of the growth rate, the spore yield and the like. The experimental result shows that the culture medium obtained by mixing the rice straw leachate and the PDA culture medium according to the equal volume has obvious growth promotion effect on rice blast germs, and the spore yield is also at a higher level; the comparison shows that the spore yield is lower when the single-component rice straw leachate or the PDA culture medium is used for culturing the rice blast germs, and the spore yield of the rice blast germs is obviously improved by the culture medium obtained by mixing the single-component rice straw leachate and the PDA culture medium according to a specific ratio. The culture medium has good culture effect on rice blast germs, has outstanding spore yield, is easy to obtain materials, is simple and convenient to prepare, and has good use effect.

Drawings

FIG. 1 is a graph showing a comparison of growth rates of the same rice blast strain on different media in an embodiment of the present invention.

FIG. 2 is a graph showing a comparison of spore yields of the same rice blast strain on different media in the embodiment of the present invention.

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

1. Materials and methods

1.1 strains

Rice blast 1-10(ZB13), pest control important laboratory preservation of agricultural hospital in Jiangxi province.

1.2 test Medium

Rice straw leachate: shearing 30g of rice straw, and soaking in 1000mL of clear water for 16-24 h;

potato leaching liquid: peeling 200g of potatoes, cutting into pieces, and soaking in 1000mL of clear water for 2 h;

rice straw corn flour culture medium: 30g of rice straw, 20g of corn flour, 18g of agar and 1000mL of clear water;

PDA culture medium: 200g of potatoes, 20g of glucose, 18g of agar and 1000mL of clear water;

oatmeal culture medium comprising 50g of oatmeal, 20g of sucrose, 16g of agar and 1000mL of water;

matching group 1: leaching rice straw leachate and potato (1:1) (adding 18g of agar per 1000 mL);

matching group 2: rice straw leachate and PDA culture medium (1: 1);

matching group 3: rice straw corn flour culture medium and potato extract (1: 1);

matching group 4: rice straw corn flour culture medium + PDA culture medium (1: 1);

matching group 5: rice straw corn flour culture medium;

matching group 6: PDA culture medium;

matching 7: rice straw extract medium (agar 18g per 1000 mL);

matching group 8: oatmeal culture medium.

1.3 sporulation method

The test strains which are purely cultured are picked up to be the same as the size of the bacterial blocks, inoculated on a culture dish (9cm) of the test 8 formula culture media and placed in a thermostat at 28 ℃ for culture. After the hyphae on the plate are fully grown (about 7 days), the plate is moved to an indoor tissue culture rack and is cultured for 3 days under the illumination of a black light lamp at the room temperature of 25-28 ℃. After sufficient spore production, the spores were washed with 15mL of distilled water per dish, and the number of spores was counted under 100-fold microscope.

1.4 comparison of sporulation yields of the same Strain on media of different formulations

After test strains 1-10 (stored in the laboratory) are activated on a PDA culture medium, a punch is used for obtaining bacterial dishes with the diameter of 5mm and the maturity of the bacterial dishes are consistent, the bacterial dishes are inoculated in the centers of culture media with different formulas, the culture media are cultured for 7d at 28 ℃, then the bacterial dishes are placed under a black light lamp for irradiation for 3d, 15mL of sterilizing water is used for washing surface hyphae, a nylon gauze is used for filtering, and then rice blast conidium suspension is obtained, the observation is carried out by a 100-fold microscope, the number of spores in one visual field is calculated, the repetition is carried out for 3 times, and the average value is taken for comparison.

1.5 comparison of growth rates of the same Strain on different sporulation media

Culturing the test strains 1-10 on culture media with different formulas in a constant-temperature incubator at 28 ℃ for 7d, measuring the diameters of bacterial colonies, calculating the growth speed of the rice blast fungi, repeating for 3 times, and taking an average value for comparison.

1.6 calculation of colony growth Rate

Colony diameter (cm) ═ total colony diameter (cm) — diameter of inoculated bacteria dish (5mm)

2 results and analysis

2.1 comparison of growth rates of the same strains on media of different formulations

Activating test strains 1-10 on a PDA culture medium, obtaining bacterial dishes with the diameter of 5mm and consistent maturity by using a puncher, inoculating the bacterial dishes in the centers of culture media with different components, culturing at the constant temperature of 28 ℃ for 7d, determining the diameter result of the bacterial colonies, and comparing the growth conditions of the same strains on the culture media with different components, wherein the experimental results are shown in table 1 and figure 1.

From Table 1, FIG. 1 shows that the culture media of 8 different groups had significant differences in the culture rates of Pyricularia oryzae. The average colony diameters of test strains 1-10 cultured on culture medium matched groups 1, 2, 3, 4, 5, 6, 7 and 8 at constant temperature of 28 ℃ for 7d are respectively 7.66cm, 7.62cm, 7.38cm, 7.51cm, 6.45cm, 7.39cm, 5.27cm and 7.36cm, the strains of matched groups 1, 2 and 4 grow best without significant difference, the strains of matched groups 3, 6 and 8 culture strains of matched groups 5 and 7 culture strains of matched groups 7 are the worst.

TABLE 1 comparison of growth rates of the same strain on different matched groups of media

Note: lower case letters in the table indicate the results of the significance analysis of differences at 5% probability

2.2 comparison of sporulation yields of the same Strain on media of different formulations

Activating test strains 1-10 on a PDA culture medium, obtaining bacterial dishes with the diameter of 5mm and the maturity of the same size by using a puncher, inoculating the bacterial dishes in the centers of culture mediums with different formulas, culturing the bacterial dishes at 28 ℃ for 7 days, then placing the bacterial dishes under a black light lamp for irradiating the bacterial dishes for 3 days, washing surface hyphae in the next culture dish by using 15mL of sterilizing water, filtering the hyphae with a nylon gauze to obtain a rice blast fungus conidium suspension, observing the conidium suspension by using a 100-fold microscope, calculating the number of spores in a visual field, repeating the steps for 3 times, and taking an average value for comparison.

As can be seen from the experimental results shown in Table 2 and FIG. 2, the differences of the spore yields of the test strains 1-10 on the culture media of 8 different combinations are large, and the differences are significant. The average spore numbers of the spores produced by the culture medium matched group 1, matched group 2, matched group 3, matched group 4, matched group 5 and matched group 6 under a microscopic field are 35.3, 50.3, 43.3, 35.6, 44.6, 19.3, 22.6 and 37.3 respectively. The group 2 produced the best spores, then group 3 and group 5, then group 1, group 4 and group 8, and group 6 and group 7 produced the worst spores.

TABLE 2 comparison of sporulation yields of the same strain on different matched sets of media

Note: lower case letters in the table indicate the results of the significance analysis of differences at 5% probability

2.3 comparison of superiority of group 2 in oatmeal medium, barley grain medium, and spore production

Oatmeal culture medium and barley grain culture medium are the most commonly used rice blast fungus spore-producing culture medium at present, test strains 1-10 are respectively cultured by using a matching group 2, the oatmeal culture medium and the barley grain culture medium and spore is produced, and the whole spore production cycle length, the spore production amount and the economic cost are compared. Matching group 2, comparing sporulation quantity, preparing 50 culture plates from 1L of oat meal culture medium for culturing rice blast germs and producing spores, washing hypha and spores on all the plates with 1000mL of sterile water, filtering with double-layer gauze to obtain spore suspension, detecting the number of spores under a microscope by 100 times, recording the number of spores under a visual field, repeating for 3 times, and taking an average value. Weighing 1L of barley grains by using a container to prepare a barley grain culture medium, inoculating the barley blast fungi to be tested to produce spores, washing off the rice blast fungi spores on the barley grains by using 1000mL of the barley grains after the spores are produced, filtering by using double-layer gauze, detecting the number of the spores by using a microscope with the magnification of 100 times, recording the number of the spores in one visual field, repeating for 3 times, and taking an average value.

TABLE 3 comparison of spore production of Pyricularia oryzae by three different media

Note: "+" indicates the cost and the source difficulty, and more "+" indicates higher cost and more easily available raw material source.

As can be seen from Table 3, the number of days required for the whole sporulation cycle of the three media, i.e., the medium for oatmeal, and the medium for barley grain, 2, was 10 days, 12 days, and 15 days, respectively, and the average sporulation amount was 42, 32, and 35 per microscopic field. Therefore, the spore yield and the spore production period of the matched group 2 are superior to those of an oatmeal culture medium and a barley grain culture medium, the economic cost and the obtaining difficulty of the raw materials of the culture medium are integrated, and the matched group 2 is considered to be more advantageous than the current common culture medium in the spore production of the rice blast fungus culture.

3. Conclusion

In the culture medium for 8 kinds of rice blast bacteria and producing spores prepared in the experiment, the matched group 2 has the characteristics of quick growth of rice blast bacteria and large spore yield, and each index is superior to that of other prepared culture mediums.

The main formula of the matched group 2 is a compound of a matched group 6 and a matched group 7, and the culture speed of the matched group 2 on rice blast germs is equivalent to that of the matched group 6 and is far faster than that of the matched group 7; the spore yield of the rice blast fungus in the matched group 2 is respectively improved by 160.62 percent and 122.57 percent compared with that in the matched group 6 and the matched group 7, and the combination of the matched group 2 has great synergistic effect on the spore production capability compared with the original matched group culture medium.

The ingredients and preparation method of the formulation 2 in this experiment are as follows: rice straw leachate:

(1) shearing 30g of rice straw, and soaking in 1000mL of clear water for 16-24 h;

(2) PDA culture medium: 200g of potatoes, 20g of glucose, 18g of agar and 1000mL of clear water; firstly, 200g of potatoes, 20g of glucose and 1000mL of clear water are boiled for 15 minutes and filtered by gauze to prepare nutrient solution;

(3) preparing the rice straw leachate and the nutrient solution prepared in the step (2) into a mixed nutrient solution according to the ratio of 1:1, adding 18g of agar into every 1000mL of the nutrient solution, heating to dissolve, sterilizing, and pouring into a flat plate to finally finish the preparation of the culture medium flat plate.

The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.

Claims (3)

1. The application of a culture medium for promoting the growth of rice blast germs is characterized in that the culture medium is formed by mixing equal volume of rice straw leachate and a PDA culture medium; wherein the rice straw leachate is a soaking solution obtained by mixing rice straws and water according to the proportion of 3:100(g: mL) and soaking for 16-24 h; the PDA culture medium comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose and 1000 parts of water; the rice blast fungus is the rice blast fungus subspecies ZB 13.

2. The use as claimed in claim 1, wherein said PDA medium further comprises agar in an amount of 18 parts by weight.

3. The use according to claim 2, wherein the culture medium is prepared by a method comprising:

1) mixing rice straws with water according to a ratio of 3:100(g: mL), and soaking for 16-24h to obtain rice straw leachate;

2) mixing 200 parts by weight of potatoes, 20 parts by weight of glucose and 1000 parts by weight of water, boiling for 15 minutes, filtering by using gauze, and taking filtrate;

3) mixing the rice straw leachate obtained in the step 1) and the filtrate obtained in the step 2) according to a volume ratio of 1:1, adding agar which is 18 per thousand of the weight of the water used in the step 2) into the mixture, and heating to dissolve the agar.

Images