CN115074253A - Inoculation method for corn leukoderma - Google Patents
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Abstract
The invention provides a method for inoculating corn leukoderma, which comprises the following steps: collecting typical corn leukoderma leaves with diseases, separating and purifying pathogenic bacteria, obtaining mixed liquor of hypha segments and conidia through liquid culture, adding 20% Tween solution into the mixed liquor of the hypha segments and the conidia in a small horn mouth period of corn, carrying out spray inoculation on corn leaves, shading after inoculation, carrying out spray moisture preservation on the next day, keeping the relative humidity at more than 90%, culturing in an artificial climate incubator on the third day, illuminating for 12 hours at 20 ℃ and darkness for 12 hours at 16 ℃ every day, and alternately carrying out the steps until the corn leukoderma diseases are stable with the humidity of 90%. The invention uses the mixed solution of the mycelium segments and the conidia to carry out spray inoculation on the corn plants, has uniform morbidity, quick morbidity, obvious effect, strong operability and short pathogenicity test period, is not limited by factors such as seasons, external environment, regions and the like, and improves the success rate of the artificial inoculation of the corn leukoderma.
Description
Technical Field
The invention belongs to the technical field of corn leukoderma, and particularly relates to a corn leukoderma inoculation method.
Background
The corn white spot disease (Epicoccum spp.) is small in size, light green and scattered on the surface of leaves; the scab is round, long and rectangular and is 0.3-2.0 cm, the scab becomes white and dry along with the time, the size and the color of the front and the back of the leaf are completely consistent or almost consistent, and the scab is similar to phytotoxicity spots caused by paraquat herbicide; the edge of the lesion spot is neat and clear, and the lesion spot has or does not have a dark brown edge and has no radial or unclear extension lines or extension-shaped spots; the disease spots have the symptoms of sudden subsidence, and when the disease spots are serious, the disease spots are gradually combined into irregular large disease spots, the length of the disease spots is as long as 10 centimeters, the whole leaf is withered, and the rupture and perforation can occur in the later period. The disease can cause the deformity of corn ears and low seed yield, seriously reduces the yield and quality of corn, and has the loss rate of 10 to 70 percent. The corn white spot disease is reported to be widely distributed in tropical and subtropical plateau regions with abundant rainwater and moderate temperature in central and south america, asia and africa, has a long occurrence history in China, is mainly concentrated in southwest regions in the early period, and gradually has a north-shifting trend. But at present, the domestic research on the corn leukoderma is less.
The determination of the inoculation method has important effects on evaluating the resistance of the corn to the corn leukoderma, screening medicaments for preventing and treating the corn leukoderma, researching the occurrence and extension rule of the corn leukoderma and the like. At present, the inoculation method of the corn leukoderma is less reported, and at present, only one scratching inoculation method is adopted, wherein a blade is used for scratching corn leaves and stalks, then a conidium suspension is sprayed and inoculated on the wounded part, and the moisturizing culture promotes the attack of the inoculated part. The method can be used for inoculating the corn at a specific position, but the method has obvious defects: (1) the artificially-caused wounds have different individual difference and different injury severity, so that the disease resistance degree of the corn can cause artificial errors when the disease resistance of the corn is identified; (2) the method artificially causes the wound to be inoculated, the diffusion process of the disease scab symptom cannot be accurately and completely described due to the interference of tissue discoloration after the wound is healed, and the disease symptom is atypical; (3) the method for artificially inoculating the wounds is different from the infection process that pathogenic bacteria invade tissues in the natural environment by contacting the corn epidermis and then attaching to the surfaces of leaves and stems, so that the natural morbidity cannot be completely simulated when a control medicament for controlling the corn leukoderma is screened, the infection process of pathogenic bacteria is researched, and the like, and the test result is greatly interfered; (4) the wound inoculation is caused artificially, and the mixed bacteria can be infected through the wound in the moisture-preserving culture process, so that the test result is interfered. Therefore, although the scratching method can ensure that the corn plants generate white spot symptoms at the injured parts, the scratching method can not simulate the disease process under the natural state at all and can not meet the requirements of the identification work and the prevention and treatment work of the disease resistance of the corn.
Therefore, in order to maximally simulate the process of the white spot pathogen infecting corn naturally, develop a test in the aspect of disease resistance identification of corn and develop prevention and treatment work of the corn white spot disease, an inoculation method which is nondestructive and can effectively infect the corn white spot disease is established, and is a scientific and technical problem to be solved currently.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a corn leukoderma inoculation method aiming at the defects of the prior art, the method truly simulates the proper inoculation environment condition required by the corn leukoderma attack through an artificial climate culture room, the corn plant is inoculated by spraying with the mixed solution of the hypha sections and the conidia, the disease attack is uniform, the disease attack is fast, the effect is obvious, the operability is strong, the pathogenicity test period is short after inoculation, the method is not limited by factors such as seasons, external environments, regions and the like, the success rate of the corn leukoderma artificial inoculation is effectively improved, the dynamic infection change of the corn plant infected by the corn leukoderma is favorably observed, and the disease resistance identification of different corn materials is favorably realized.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for inoculating corn leukoderma comprises the following steps:
s1, collecting typical corn leukoderma leaves, separating and purifying corn leukoderma pathogenic bacteria (Epicoccum sorghinum), and performing liquid culture to obtain mixed liquid of hypha segments and conidia;
the separated and purified corn white spot pathogen is epiphora sorghum sorghi (Epicoccum sorghinum), which is reported as a corn white spot pathogen by Tangmin (2021) and Zhoujia (2021);
the separation and purification method of the mixed solution of the mycelium segments and the conidia comprises the following steps:
cutting a plurality of small blocks with the size of 3mm multiplied by 3mm at a disease-health junction, disinfecting the surface of the small blocks with 75% of alcohol solution for 30s, washing the small blocks with sterile water for 3 times, disinfecting the small blocks with 5% of NaClO solution for 60s, washing the small blocks with sterile water for 3 times, airing the small blocks, placing the small blocks on a PDA culture medium, culturing the small blocks on the dark condition at the temperature of 28 ℃ until hyphae grow out, picking the tips of the hyphae to be cultured on a fresh PDA culture medium, continuously transferring the hyphae for a plurality of times until the shapes of the bacterial colonies are consistent, picking the bacterial colonies, transferring the bacterial colonies to a PDA inclined plane, storing the bacterial colonies on the PDA inclined plane at the temperature of 4 ℃, identifying the bacterial colonies as white spot disease pathogenic bacteria by pathogenicity determination, morphology and meristem means, activating and culturing the pathogenic bacteria on the PDA culture medium for 7 days, picking up bacterial cakes with the diameter of 5mm from the edges of the bacterial colonies, taking 7 blocks, placing the blocks in a PDB culture medium containing 300mL, and culturing the bacterial strains at the temperature of 28 ℃ to obtain the bacterial strains, Performing shake culture on a shaker at 160rpm in dark for 10 days, breaking by a wall breaking machine for 40s, and filtering with 4 layers of sterilized gauze to obtain mixed solution of hypha segments and conidia;
s2, in the small trumpet period of the corn, adding 20% Tween solution into the mixed solution of the mycelium segment and the conidium obtained in the S1, and carrying out spray inoculation on the corn leaf;
s3, keeping the inoculated corn plants shaded, not spraying water for moisturizing at night after inoculation, spraying for moisturizing on the next day, keeping the relative humidity above 90% to obtain the corn plants after moisturizing treatment, and culturing the corn plants after moisturizing treatment in an artificial climate incubator on the third day until the white spot disease of the corn is stable;
the culture conditions of the artificial climate incubator are as follows: culturing at 20 deg.C for 12 hr in light every day, culturing at 16 deg.C in dark for 12 hr in light, alternately culturing in light and dark, and maintaining relative humidity above 90%.
Preferably, the spray inoculation spray in S2 is uniform, and the leaves are uniformly wetted.
Preferably, after the onset of the corn leukoderma is stabilized in S3, the severity of the onset is described according to the onset symptoms of the corn leukoderma leaves: the disease is serious, moderate, mild and not diseased, the disease plant rate and the disease leaf rate are counted, and all leaves are investigated according to the disease leaf rate.
Preferably, the volume ratio of the mixed solution of the mycelium segments and the conidium and the 20% tween solution in the S2 is 2000: 1.
compared with the prior art, the invention has the following advantages:
1. the invention can simulate the disease process of the corn leukoderma to the maximum extent under natural conditions, carry out the leukoderma resistance determination and prevention and treatment work of batch corn materials, and meet different requirements of subsequent tests.
2. The invention adopts the mixed solution of the hypha segments and the conidia to carry out spray inoculation on the undamaged corn plants, has uniform inoculation effect, quick and obvious morbidity, strong operability and short pathogenicity test period, is not limited by factors such as seasons, external environment, regions and the like, and effectively improves the success rate of the artificial inoculation of the corn leukoderma. The invention finds out the temperature condition for the onset of the corn leukoderma, is beneficial to the onset of the corn leukoderma at a lower temperature (16-20 ℃), is not limited by factors such as seasons, external environments, regions and the like, can well induce the onset of the corn leukoderma in an artificial climate chamber, and is beneficial to the research of the infection mechanism of the corn leukoderma and the identification work of the disease resistance of different corn materials.
3. The invention simulates the proper inoculation environment condition required by the corn leukoderma, sprays and inoculates the corn plant by the mixed solution of the hypha segment and the conidium through the artificial climate culture room, has uniform and fast onset of disease, obvious effect, strong operability and short pathogenicity test period after inoculation, is not limited by factors such as seasons, external environment, regions and the like, effectively improves the success rate of the artificial inoculation of the corn leukoderma, is beneficial to observing the infection dynamic change of the corn plant infected by the corn leukoderma, and is beneficial to identifying the disease resistance of different corn materials. The corn white spot disease plant rate can reach 96.30% and the diseased leaf rate can reach 75.35%.
The present invention will be described in further detail with reference to examples.
Detailed Description
Example 1
Corn vitiligo (Epicoccum sorghinum) (Epicoccum sorghinum has been reported by Tangmin (2021), etc. and Zhouzhijia (2021), etc. as a pathogen of corn vitiligo)
The method for inoculating the corn leukoderma comprises the following steps:
s1, collecting typical corn leukoderma leaves, separating and purifying corn leukoderma pathogenic bacteria (Epicoccum sorghinum), and performing liquid culture to obtain mixed liquid of hypha segments and conidia;
the separation and purification method of the mixed solution of the mycelium segments and the conidia comprises the following steps:
cutting a plurality of small blocks with the size of 3mm multiplied by 3mm at the boundary of a disease and a health, disinfecting the surface of the small blocks with 75 percent of alcohol solution for 30s, washing the small blocks with sterile water for 3 times, disinfecting the small blocks with 5 percent of NaClO solution for 60s, washing the small blocks with the sterile water for 3 times, airing the small blocks, placing the small blocks on a PDA culture medium, culturing the small blocks under the dark condition with the temperature of 28 ℃ until hyphae grow out, picking the tips of the hyphae to be cultured on a fresh PDA culture medium, continuously transferring the hyphae for a plurality of times until the shapes of the bacterial colonies are consistent, picking the bacterial colonies, transferring the bacterial colonies to a PDA inclined plane, storing the bacterial colonies under the temperature of 4 ℃, identifying the bacterial colonies to be white spot corn disease pathogenic bacteria through pathogenicity determination, morphology and meristem means, identifying the bacterial strains to be white spot disease pathogenic bacteria, activating and culturing the bacterial strains on the PDA culture medium for 7 days, picking bacterial cakes with the diameter of 5mm from the edges of the bacterial colonies, placing the bacterial colonies in a PDB culture medium with 300mL, shaking bed with the temperature of 28 ℃ and 160rpm, performing shake culture for 10 days in dark, crushing with a wall breaking machine for 40s, and filtering with 4 layers of sterilized gauze to obtain mixed solution of mycelium segments and conidia;
s2, in the small trumpet period of the corn, adding 20% of Tween solution into the mixed solution of the mycelium segment and the conidium obtained in the step S1, carrying out spray inoculation on the corn leaves, wherein the spray is uniform, and the leaves are uniformly wetted;
s3, keeping the inoculated corn plants shaded, not spraying water for moisturizing at night after inoculation, spraying for moisturizing on the next day, keeping the relative humidity above 90% to obtain the corn plants after moisturizing treatment, and culturing the corn plants after moisturizing treatment in an artificial climate incubator on the third day until the white spot disease of the corn is stable; after the onset of the corn leukoderma is stable, the severity of the onset is described according to the onset symptoms of the corn leukoderma leaves: the disease is serious, moderate, light and not diseased, the disease plant rate and the disease leaf rate are counted, and all leaves are investigated according to the disease leaf rate;
the culture conditions of the artificial climate incubator are as follows: culturing at 20 deg.C for 12 hr in light every day, culturing at 16 deg.C in dark for 12 hr in light, alternately culturing in light and dark, and maintaining relative humidity above 90%.
In the embodiment, the test conditions of the inoculation method for the corn leukoderma are optimized:
(I) Effect of different inoculation methods
Preparation of an inoculum:
the method comprises the following steps: activating and culturing a corn leukoderma (Epicoccum sorghinum) pathogenic strain on a PDA (PDA) culture medium for 7 days, taking fungus cakes (7 blocks) with the diameter of 5mm from the edges of the colonies, placing the fungus cakes into a 500mL triangular flask filled with 300mL PDB liquid culture medium, carrying out shake culture at the temperature of 160rpm and 28 ℃ of a shaking table for 10 days, crushing the fungus cakes for 40s by using a wall breaking machine, and filtering 4 layers of sterile gauze to obtain mixed liquid of hypha segments and conidia, wherein the mixed liquid is used as an inoculum for later use.
The second method comprises the following steps: activating and culturing a corn leukoderma (Epicoccum sorghinum) pathogenic strain on a PDA culture medium for 7 days, then inoculating pathogenic bacteria on sorghum grains subjected to high-temperature sterilization (the preparation method of the sorghum grain culture medium comprises the steps of boiling the sorghum grains for 30-40 min, sterilizing the sorghum grains in a triangular flask at 121 ℃ for 1h, cooling the sorghum grains for later use), culturing for 5-7 days at 23-25 ℃, adding a small amount of sterile water into the triangular flask when the sorghum grains are fully distributed with hyphae, stirring by using a sterilized glass rod to break the hyphae, shaking uniformly, placing the sorghum grains in an environment with higher humidity at 23-25 ℃ for 5-7 days by black light irradiation, and performing microscopic examination on a large number of produced spores for later use.
Inoculation: selecting a plurality of pots of corns which are planted in an artificial climate chamber and are in a small-horn mouth period for standby application, and planting 3 corns in each pot. The experiment set up 3 treatments:
1. a spray inoculation method: directly spraying mixed solution of mycelium segments and conidia on the corn leaves, adding a little 20% Tween solution during spraying, and spraying until the leaf surfaces are wet; the volume ratio of the mixed solution of the spray mycelium segments and the conidium to the 20% Tween solution is 2000: 1 (method of the present example);
2. core-throwing inoculation method of sorghum grains with bacteria: directly throwing the prepared sorghum grains with bacteria into the corn leaf cores, and throwing about 10 grains per plant;
3. embedding sorghum grain sheath with bacteria: the sorghum grains with bacteria are directly embedded into the root of the leaf sheath of the corn, about 10 grains are embedded in each leaf sheath, and each leaf sheath is embedded.
Each of the above treatments was repeated 3 times, 3 plants per pot, for each 3 pots. The inoculated plants are kept shaded, water spraying and moisture preservation are not carried out in the evening after inoculation, spraying and moisture preservation are carried out the next day, the relative humidity is kept to be more than 90%, the plants are placed in an artificial climate room with the light temperature of 20 ℃ for 12h and the dark temperature of 16 ℃ for 12h after 1 day, and the disease occurrence condition is investigated until the disease is fully developed.
Disease investigation: after the disease is stable, the disease severity is described according to the disease symptoms of the leaf blades of the corn leukoderma, and the disease strain rate and the disease leaf rate are counted. The test results show that in the 3 inoculation methods, the spraying inoculation method has obvious disease effect, serious disease, uniform disease spot distribution and typical symptoms (consistent with field disease symptoms), the disease plant rate can reach 96.30%, and the disease leaf rate can reach 75.35%; although the core-throwing inoculation method for sorghum grain with bacteria has high disease rate and strong destructiveness, the disease symptoms and the disease parts are not representative, and the disease leaf rate only reaches 44.45%; the bacterial sorghum coleoptile embedding inoculation method is low in disease strain rate, disease leaf rate and disease symptoms (table 1). The comprehensive test results obtained: the optimal inoculation method for the corn leukoderma comprises the following steps: and (4) carrying out spray inoculation on mixed liquid of the mycelium segments and the conidia.
After the onset of the corn leukoderma is stable, the severity of the onset is described according to the onset symptoms of the corn leukoderma leaves: the disease is serious, moderate, mild and not diseased, the disease plant rate and the disease leaf rate are counted, and all leaves are investigated according to the disease leaf rate.
TABLE 1 Effect of different inoculation methods on the onset of leukoderma in maize
Note: the difference of letters after the same column of data represents significant difference (P < 0.05)
(II) different artificial climate chamber condition disease effect
The inoculation method is a mixed solution spray inoculation method of mycelium segments and conidia, and the method steps are the same as the inoculation method of the embodiment. The plant after the inoculation keeps shading, does not spray water and moisturize the inoculation evening, starts spraying and moisturizes the next day, keeps the relative humidity more than 90%, and the plant is put into different conditions artificial climate indoor cultivation 1 day later, and 3 artificial climate indoor condition treatments are set in the test:
1. performing illumination at 20 ℃ for 12h and dark treatment at 16 ℃ for 12 h; (method of the present embodiment)
2. Irradiating at 24 deg.C for 12h, and treating at 20 deg.C for 12h in dark;
3. at 28 ℃ for 12h, and at 24 ℃ for 12h, dark treatment.
Each treatment was repeated 3 times, 3 plants per pot, for each repeat of 3 pots. The disease occurrence condition is investigated until the disease is fully developed. The method of investigation was the same as in example 1.
The results obtained were: the plant disease rate is obviously different among treatments, the plant disease rate and the leaf disease rate are higher when the plants are irradiated at 20 ℃ for 12 hours and treated in darkness at 16 ℃ for 12 hours, and the plant disease rate and the leaf disease rate are respectively 96.30 percent and 75.35 percent; secondly, the illumination is carried out for 12h at the temperature of 24 ℃ and the dark treatment is carried out for 12h at the temperature of 20 ℃, and the diseased plant rate and the diseased leaf rate are respectively 78.95 percent and 65.47 percent; the plant disease rate and the leaf disease rate are lower when the plants are irradiated for 12 hours at 28 ℃, and the dark disease rate and the leaf disease rate are lower at 12 hours at 24 ℃, and are respectively 42.63 percent and 25.42 percent (Table 2). The temperature has larger influence on the onset of the corn leukoderma, and is favorable for the onset of the corn leukoderma when the temperature is lower (16-20 ℃).
After the onset of the corn leukoderma is stable, the severity of the onset is described according to the onset symptoms of the corn leukoderma leaves: the disease is serious, moderate, mild and not diseased, the disease plant rate and the disease leaf rate are counted, and all leaves are investigated according to the disease leaf rate.
TABLE 2 influence of different phytotron conditions on the onset of leukoderma in maize
Note: the difference of letters after the same column of data represents significant difference (P < 0.05)
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
Claims (3)
1. The inoculation method for the corn leukoderma is characterized by comprising the following steps:
s1, collecting typical corn leukoderma leaves, separating and purifying corn leukoderma pathogenic bacteria Epicoccum sorghinum, and obtaining mixed liquid of hypha segments and conidia through liquid culture;
the separation and purification method of the mixed solution of the mycelium segments and the conidia comprises the following steps:
cutting a plurality of small blocks with the size of 3mm multiplied by 3mm at a disease-health junction, disinfecting the surface of the small blocks with an alcohol solution with the mass fraction of 75% for 30s, washing the small blocks with sterile water for 3 times, disinfecting the small blocks with a NaClO solution with the mass fraction of 5% for 60s, washing the small blocks with the sterile water for 3 times, airing the small blocks, placing the small blocks on a PDA culture medium, culturing the small blocks under the dark condition with the temperature of 28 ℃ until hyphae grow out, picking the tips of the hyphae to be cultured on a fresh PDA culture medium, continuously transferring the hyphae for a plurality of times until the shapes of the bacterial colonies are consistent, picking the bacterial colonies, transferring the bacterial colonies to a PDA inclined plane, storing the bacterial colonies under the temperature of 4 ℃, identifying the bacterial strains as white spot corn disease pathogenic bacteria by means of pathogenicity measurement, morphology and meristem biology, taking the pathogenic bacterial strains to be activated and cultured on the PDA culture medium for 7 days, picking bacterial cakes with the diameter of 5mm from the edges of the bacterial colonies, taking 7 blocks, placing the blocks in a PDB culture medium with 300mL, and culturing the bacterial strains at the temperature of 28℃, Performing shake culture on a shaker at 160rpm in dark for 10 days, breaking by a wall breaking machine for 40s, and filtering with 4 layers of sterilized gauze to obtain mixed solution of hypha segments and conidia;
s2, in the small trumpet-shaped period of the corn, adding 20% of Tween solution into the mixed solution of the mycelium segments and the conidia obtained in the S1, and carrying out spray inoculation on the corn leaves;
s3, keeping the inoculated corn plants shaded, not spraying water for moisturizing at night after inoculation, spraying for moisturizing on the next day, keeping the relative humidity above 90% to obtain the corn plants after moisturizing treatment, and culturing the corn plants after moisturizing treatment in an artificial climate incubator on the third day until the white spot disease of the corn is stable;
the culture conditions of the artificial climate incubator are as follows: the culture was carried out daily at 20 ℃ for 12h in the light and then at 16 ℃ for 12h in the dark, with the light and dark alternating and the humidity maintained at 90%.
2. The method for inoculating corn leukoderma as claimed in claim 1, wherein the spraying inoculation in S2 is uniform, and the leaves are uniformly wetted.
3. The method for inoculating corn leukoderma as claimed in claim 1, wherein the volume ratio of the mycelium segments and conidium mixed solution to the 20% tween solution in S2 is 2000: 1.
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