CN112608849A - Rice false smut germ separation method - Google Patents
Rice false smut germ separation method Download PDFInfo
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- CN112608849A CN112608849A CN202011436739.9A CN202011436739A CN112608849A CN 112608849 A CN112608849 A CN 112608849A CN 202011436739 A CN202011436739 A CN 202011436739A CN 112608849 A CN112608849 A CN 112608849A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses a separation method of rice false smut germs, which comprises the following steps: (1) collecting rice koji balls of rice with rice koji diseases, and keeping fresh for later use; (2) removing chlamydospore powder on the surface of the rice koji until yellow spore powder is exposed; (3) selecting yellow spore powder, streaking and inoculating to a culture medium; (4) the strain on the culture medium is cultured and purified to obtain the separated ustilaginoidea virens, and the scheme of the invention can realize the quick and efficient separation of the ustilaginoidea virens and provide theoretical reference for researching the anti-susceptibility evaluation of different rice varieties on the ustilaginoidea virens and the formulation of field prevention and control strategies.
Description
Technical Field
The invention relates to the field of microbial culture, in particular to a rice false smut germ separation method.
Background
Rice false smut (Rice face smut) is a fungal disease caused by Ustilaginoidaviruses (Villosporavirids, a sexual state). False smut, also known as smut, millet blossom and powdery mildew, was first discovered and reported in India by Cooke et al in 1878. The disease is widely generated in rice producing areas of more than 40 countries and regions of Asia, Africa and south America. In the past 80 years of the last century, rice false smut in China occurs but is not a main disease. After the 20 th century and 80 s, with the change of the rice cultivation mode, the excessive application of chemical fertilizers and the large-area popularization of high-yield rice and hybrid rice, the occurrence of false smut in China is increasingly serious, and the false smut is combined with rice blast and sheath blight and called new three diseases of rice. The occurrence of false smut not only affects the yield and quality of rice, but also produces various toxins, which are harmful to human and livestock, and seriously affect food safety.
The main symptoms of the rice false smut disease are caused by rice false smut pathogen infecting young ear flower device before the opening of rice, and rice false smut ball with dense hypha inside and powdery chlamydospore outside is formed at the ear part of rice in the flowering period. Therefore, rice koji is a main material for isolating rice koji germs. Because the rice false twist ball is rich in nutrient content, the surface layer of the rice false twist ball is exposed and has pores, and the rice false twist ball is easy to be polluted by mixed bacteria; and the green smut germ grows very slowly under the condition of artificial culture, thereby increasing the separation difficulty of the germ.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a rice false smut germ separation method which can simply and efficiently separate rice false smut germs.
According to the embodiment of the invention, the method for separating the ustilaginoidea virens comprises the following steps:
s1, collecting rice koji balls with rice koji diseases, and keeping fresh for later use;
s2, removing chlamydospore powder on the surface of the rice koji ball until yellow spore powder is exposed;
s3, selecting yellow spore powder, streaking and inoculating to a culture medium;
s4, culturing and purifying the strain on the culture medium to obtain the separated ustilaginoidea virens.
According to some embodiments of the invention, the rice koji balls in step S1 are collected from field naturally-occurring plants in the late stage of rice growth.
According to some embodiments of the present invention, the rice koji balls are kept fresh at 4 to 10 ℃ in step S1.
According to some embodiments of the invention, the chlamydospore powder on the rice koji surface is scraped off in step S2 and sterilized and cooled coarse inoculating needle is used for operation.
According to some embodiments of the present invention, the yellow spore powder is picked in step S3 and is processed with a sterile inoculating needle.
According to some embodiments of the invention, the medium in step S3 is PSA plate medium containing 50 μ g/mL streptomycin.
According to some embodiments of the present invention, the operations of steps S2 and S3 are performed in a biosafety cabinet after laboratory uv sterilization.
According to some embodiments of the present invention, the culturing temperature of ustilaginoidea virens in the step S4 is 26-28 ℃.
According to some embodiments of the invention, the culturing time of the ustilaginoidea virens in the step S4 is 7-10 days.
According to some embodiments of the present invention, the purification in step S4 is performed by observing colony morphology and microscopy, and rejecting the mixed bacteria and suspected strains.
According to some embodiments of the invention, the ustilaginoidea virens strain in step S4 is stored for a short period, and is transferred to a sterile PSA slant medium for 7-10 days, and then stored at 4-10 ℃ for at least 3 months and 1 time.
The ustilaginoidea virens separation method provided by the embodiment of the invention has at least the following beneficial effects: according to the technical scheme, the rice koji balls do not need to be disinfected by alcohol and mercuric chloride, do not need to be washed by sterile water, only need to be lightly scraped to remove outer-layer chlamydospore powder until yellow or green spore powder is exposed, plate streaking is performed, single colonies with high difficulty in microscopic examination and needle point size picking are not needed for 3-4 days, the operability is strong, the simplification degree is high, and the problem of high mixed bacterium pollution rate in a direct spore knock-off separation method is solved, and the separation method has the rice koji germ separation success rate of over 93.33 percent and is stable and efficient compared with a common chlamydospore knock-off method, a flame burning method and a tissue separation method; by the scheme, the rapid and efficient separation of the ustilaginoidea virens can be realized, and theoretical reference is provided for researching the evaluation of the resistance of different rice varieties to ustilaginoidea virens and the formulation of field prevention and control strategies.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a 10-day colony morphology of a PSA plate for Ustilaginoidea virens isolated according to an embodiment of the present invention;
FIG. 2 is a 25-day colony morphology of a PSA plate for Ustilaginoidea virens obtained by isolation according to an embodiment of the present invention;
FIG. 3 is a parenchymatous conidiogram of Ustilaginoidea virens after isolation culture according to an embodiment of the present invention;
FIG. 4 is a field disease chart of late indica 98 inoculated by conidium injection according to an embodiment of the present invention;
FIG. 5 is a field plot of conidia injection inoculated R900 disease in accordance with an embodiment of the present invention;
fig. 6 is a front and back view of colony morphology of rice green smut sampled after 18 days of isolation after field injection inoculation attack according to an embodiment of the invention, wherein a is rice green smut isolated from late indica 98 of rice, and B is rice green smut isolated from R900 of rice.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
The rice green rice collection sources and the used culture medium component information in the embodiment of the invention and the comparative examples 1-3 are as follows:
collecting rice green rice: in 7 months in 2020, in a pilot plant base (Changsha county spring Huazhen, Hunan, 113.30 degrees at east longitude and 28.32 degrees at north latitude) of Huazhi biotechnology Limited company, rice green balls with natural diseases are collected in a rice field and are placed in a refrigerator for preservation for later use.
PSA culture medium: the potato juice is prepared by the following steps of weighing 200g of potatoes according to a g/L ratio, peeling and chopping the potatoes, adding single distilled water for boiling for 30min, filtering and taking supernatant, adding 20g of cane sugar and 17g of agar powder, fixing the volume to 1000ml, adding sodium hydroxide to adjust the pH value to about 7.0, subpackaging according to a certain volume, and carrying out moist heat sterilization at a high temperature of 121 ℃ for 30 min.
PSR medium: the potato chips are prepared by the following steps of peeling and chopping 200g of potatoes according to a ratio of g/L, adding single distilled water for boiling for 30min, filtering and taking supernatant, and adding 20g of cane sugar. 40g of rice straw, boiling for 20min in advance, filtering and discarding residues. Mixing, diluting to 1000ml, packaging according to a certain volume, and sterilizing at 121 deg.C for 30 min.
After sterilization, each portion of the medium was cooled to about 55 ℃ and a quantity of streptomycin (50. mu.g/mL) was added to the clean bench, shaken well and poured onto a plate for use.
PS medium: cleaning potato, peeling, cutting into small pieces, weighing 200g, adding water, boiling for 25min until completely cooked, filtering with multiple layers of gauze to remove residue, slowly adding sucrose into the filtrate, shaking, and adding water to 1000 ml. Subpackaging according to a certain volume, and performing moist heat sterilization at 121 ℃ for 30 min. And after the sterilization is finished, the culture medium is cooled and stored at room temperature for later use.
Example inner spore streaking
In an aseptic biological safety cabinet, a sterilized forceps is used for clamping the rice koji ball, and the sterilized coarse inoculating needle is adopted to scrape the chlamydospore powder on the surface layer of the rice koji ball along the same side direction until the yellow spore powder is exposed. And after the inoculating needle is sterilized, disinfected and cooled again, selecting a little yellow spore powder on the inner layer, selecting green spore powder inside the inoculating needle without the yellow spore powder, inoculating the inoculating needle on two different types of plate culture media (PSA culture medium and PSR culture medium) containing streptomycin (50 mu g/mL) by a scribing method, and placing the inoculating needle in a growth box at 27 ℃ for dark culture.
Firstly, microscopical observation and identification of Ustilaginoidea virens by using colony morphology and conidia
Colony morphology: the colony morphology of Ustilago virens in the initial culture period (7 days) of the Ustilago virens on a PSA culture medium is shown in figure 1, and the colony forms white dense hypha and gradually expands to form a round colony with a middle bulge similar to a straw hat; the colony morphology of Ustilaginoidea virens PSA medium cultured for 25 days is shown in FIG. 2, and it can be seen that the middle of the colony of Ustilaginoidea virens gradually yellows in the later stage of culture (shown in medium A), and a large amount of yellow spherical chlamydospore pile is generated around the colony of the later stage of culture by part of the strains (shown in medium B).
Conidium morphology: and (3) selecting a hypha block with 5-6 mm of the colony edge of the ustilaginoidea virens isolate cultured on a PSA culture medium for 25 days, inoculating the hypha block into a sterilized PS culture medium, culturing at 27 ℃ and 150rpm/min in the dark for 7 days, and obtaining a hypha spore mixed solution. After being filtered by four layers of gauze, the filtrate is taken, the characteristics of the ustilaginoidea virens thin-wall conidiospore are observed under a microscope, the ustilaginoidea virens thin-wall conidiospore is shown in figure 3, and the ustilaginoidea virens thin-wall conidiospore is colorless and transparent, has a smooth surface and is oval.
Second, the Kehh's law verifies Ustilaginoidea virens
Preparation of ustilaginoidea virens thin-wall conidium inoculum: 2 Ustilaginoidea virens separated from the PSA plate culture medium were randomly extracted and named as strain A and strain B, respectively. Separately picking hypha blocks with 5-6 mm of colony edges, inoculating the hypha blocks into a sterilized PS culture medium, culturing at 27 ℃ at 150rpm/min in the dark for 7 days. Filtering the obtained hypha spore mixed solution with four layers of gauze, collecting filtrate, centrifuging at 3000rpm for 5min, discarding supernatant, suspending and diluting with sterilized PS culture medium, and adjusting the final concentration of thin-wall conidium suspension to 105One per ml.
Late indica 98 late booting stage injection inoculation: 23 days 9 and 2020, pilot plant base of Huazhi biotechnology limited (Changsha county spring Huazhen, Hunan province, east longitude 113.30 degrees, northern latitude 28.32 degrees), late pregnancy end of rice susceptible material (one week before break), and final concentration of 105Individual/ml of conidium inoculum of strain A was subjected to spike bud injection of 1 ml. When in inoculation, the needle head of the injector is downward and forms an included angle of 45 degrees with the ear bud slightly, the injector is inoculated in the middle of the ear bud, and the inoculation liquid is injected into the ear bud from the side. Each strain was injected with 10 spikes each, marked with a hang tag, and repeated 3 times. Control was performed by injection of sterilized pure PS medium. Automatic atomizing spraying facility, set up 10 in the morning every day: 00 and 15 pm: 00 spraying once for 30 min.
R900 late booting stage injection vaccination: 30 days 9 months in 2020, pilot plant base of Huazhi biotechnology limited (Changsha county spring Huazhen, Hunan, 113.30 degrees at east longitude, 28.32 degrees at north latitude), and rice susceptible material R900 late booting stage (one week before break), the final concentration of selection was 105Individual/ml of the conidia inoculum of strain B was subjected to spike bud injection of 1 ml. When in inoculation, the needle head of the injector is downward and forms an included angle of 45 degrees with the ear bud slightly, the injector is inoculated in the middle of the ear bud, and the inoculation liquid is injected into the ear bud from the side. Each strain was injected with 10 spikes each, marked with a hang tag, and repeated 3 times. Control was performed by injection of sterilized pure PS medium. Automatic atomizing spraying facility, set up 10 in the morning every day: 00 and 15 pm: 00 spraying once for 30 min.
Investigation of disease onset: after 4 weeks, the incidence of the injections was investigated and the incidence of the spikes was counted.
The results show that: the late stage of the late indica 98 booting ear is injected with the conidium inoculum of the inoculated strain A, so that the ear disease rate can reach 0.83; the ear disease rate of the control group CK-1 without conidium can reach 0.33; the difference between the two groups is obvious (the specific data are shown in table 1), and the field pathogenesis chart of conidium injection inoculation of late indica 98 is shown in fig. 4. The conidium inoculum of the inoculated strain B is injected at the last stage of the R900 booting ear, so that the ear disease rate can reach 0.93; the control group CK-2 without conidium has the ear disease rate of 0.50, and the difference between the two groups is obvious (the specific data are shown in Table 2); the field pathogenesis chart of conidium injection inoculation R900 is shown in figure 5.
TABLE 1 occurrence of late indica 98 injection inoculated ustilaginoidea virens
t-test, 0.05 level of significance.
TABLE 2 incidence of false smut in R900 injections
t-test, 0.05 level of significance.
Sampling and re-separating rice koji balls: 2 inoculated rice materials of late indica 98 and R900 are respectively taken, and rice yeast balls with partial diseases are returned to the room for inner layer spore streaking PSA plate re-separation culture. The separation results are shown in fig. 6, wherein a is Ustilago virens separated from rice material late indica 98, and B is Ustilago virens separated from R900, and it can be seen from the figure that Ustilago virens produces white dense hypha at the initial stage of culture and gradually expands to form round colonies with the middle bulge similar to a straw hat; the middle of the later bacterial colony gradually turns yellow, and a large amount of yellow spherical chlamydospore piles are generated around the bacterial colony and are typical morphological characteristics of ustilaginoidea virens.
The accuracy, the rigor and the scientificity of the rice false smut inner spore streaking separation method are verified again by the Koch's law.
Comparative example 1 chlamydospore knock-out method
In a sterile biosafety cabinet, the koji balls were held by sterilized forceps, and the spore powder was directly gently shaken off two different types of plate media (PSA medium, PSR medium) containing streptomycin (50 μ g/mL), and placed in a growth chamber at 26-28 ℃ for dark culture.
Comparative example 2 flame ignition method
In a sterile biological safety cabinet, clamping a rice koji ball by using sterile forceps, soaking the rice koji ball in 75% alcohol for 10s, repeatedly passing through an alcohol lamp by external flame, continuously turning over the rice koji ball to burn uniformly until the rice koji ball is extinguished, cutting the rice koji ball into two parts by using a sterilized scalpel, removing millet and burnt tissues, picking out inner layer bacterium block tissues by using a sterile inoculating needle, placing the inner layer bacterium block tissues on two different types of plate culture media (PSA culture medium and PSR culture medium) containing streptomycin (50 mu g/mL), and culturing in the dark in a growth box at the temperature of 26-28 ℃.
Comparative example 3 tissue isolation method
In a sterile biological safety cabinet, sterilizing the rice koji balls with alcohol for 1min, washing with sterile water for 3 times, sucking water by filter paper, breaking the rice koji balls off by using sterile tweezers, picking out a bacterium block tissue in the center of a section by using a sterile inoculating needle, placing the bacterium block tissue on two different types of plate culture media (PSA culture medium and PSR culture medium) containing streptomycin (50 mu g/mL), and culturing in a growth box at 26-28 ℃ in the dark.
Separation success rate statistical analysis
According to the colony morphology and the conidium microscopic observation, the mixed bacteria and the suspected strains are removed, and the separating success rate of the ustilaginoidea virens under each processing condition is counted.
The results of the different isolation methods for ustilaginoidea virens are shown in table 3, and it can be seen from the table that the average isolation success rate of PSA medium: inner spore streaking (93.33%) > flame burning (75.53%) > tissue separation (31.10%) > chlamydospore knock-off (21.13%); the average separation success rate of the PSR culture medium is as follows: inner spore streaking (53.33%) tissue isolation (13.33%) chlamydospore knock-off (3.30%) when flame burn (13.33%) is applied. Therefore, the success rate of separating the ustilaginoidea virens is the highest and reaches more than 93.33 percent by adopting the lining spore streaking method to inoculate the ustilaginoidea virens on a PSA plate culture medium containing 50 mu g/mL of streptomycin, and the difference of the success rate from other treatments is obvious.
TABLE 3 comparison of different isolation methods for Ustilaginoidea virens
Duncan's multiple range test, 0.05 significance level, and the difference between english letters represented by each sample indicates significant difference.
In conclusion, the separation success rate of the rice false smut germ separation method provided by the invention is up to more than 93.33%, and the separation method is stable and efficient compared with the commonly used chlamydospore knock-off method, flame burning method and tissue separation method.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (8)
1. A rice false smut germ separation method is characterized in that: the method comprises the following steps:
s1, collecting rice koji balls with rice koji diseases, and keeping fresh for later use;
s2, removing chlamydospore powder on the surface of the rice koji ball until yellow spore powder is exposed;
s3, selecting yellow spore powder, streaking and inoculating to a culture medium;
s4, culturing and purifying the strain on the culture medium to obtain the separated ustilaginoidea virens.
2. The method of claim 1, wherein: and in the step S1, the rice koji balls are collected from the natural disease plants in the field in the later growth stage of the rice.
3. The method of claim 1, wherein: and in the step S1, the rice koji balls are placed at the temperature of 4-10 ℃ for fresh-keeping.
4. The method of claim 1, wherein: the culture medium in the step S3 is a PSA plate culture medium containing 40-60 mu g/mL streptomycin.
5. The method of claim 1, wherein: the culture time of the ustilaginoidea virens in the step S4 is 7-10 days.
6. The method of claim 1, wherein: the culture temperature of the ustilaginoidea virens in step S4 is 26-28 ℃.
7. The method of claim 1, wherein: in the step S4, the purification adopts observation of colony morphology and microscopic examination to remove the mixed bacteria and suspected strains.
8. The method of claim 1, wherein: the strain of ustilaginoidea virens in the step S4 is stored for a short period, and the ustilaginoidea virens is transferred to a sterile PSA slant culture medium to be cultured for 7-10 days, and then is stored at 4-10 ℃ and transferred for 1 time in at least 3 months.
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