CN112195217A - Method for determining pathogenicity of pathogenic bacteria of weedy rice and identification device - Google Patents
Method for determining pathogenicity of pathogenic bacteria of weedy rice and identification device Download PDFInfo
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Abstract
The invention belongs to the technical field of determination of pathogenic bacteria of weedy rice, and discloses a method and a device for determining the pathogenicity of the pathogenic bacteria of the weedy rice, wherein the method for determining the pathogenicity of the pathogenic bacteria of the weedy rice comprises the following steps: collecting weed rice disease specimens in a rice planting area; separating the fungi from the stem, the leaf and the seeds within 3 days after the collection of the fresh-keeping preserved strain, purifying and preserving the separated fungi, and separating pathogenic fungi infecting the weedy rice from the air-dried preserved specimen by using a tissue culture separation method; identifying pathogenic bacteria of the weed rice: culturing the strain at constant temperature, recording the morphology, color and growth speed of a bacterial colony, and analyzing sporulation condition and morphological characteristics; and (4) determining pathogenicity of pathogenic bacteria of the weed rice. The invention establishes a Ningxia weedy rice pathogenic fungi resource library, screens pathogenic fungi resources with strong pathogenicity to weedy rice, no pathogenicity to rice or little influence on rice, and provides a foundation for developing a fungi herbicide to prevent and control weedy rice.
Description
Technical Field
The invention belongs to the technical field of determination of pathogenic bacteria of weedy rice, and particularly relates to a determination method and an identification device for pathogenicity of pathogenic bacteria of weedy rice.
Background
At present, weedy rice is also called wild rice and miscellaneous rice, is a global weed, weedy rice associated with cultivated rice is widely distributed in many countries and regions in the world, the morphological characteristics of the weedy rice are between wild rice and cultivated indica-japonica rice, most weedy rice is very similar to wild rice, mature seeds are light, strong in dormancy and easy to fall, the tillering period and the flowering period are earlier than those of the varieties of the accompanying cultivated rice, compared with the cultivated rice, the weedy rice is in the dominant position of competing nutrients, moisture and illumination in a rice field, so that the yield of the rice is reduced, the quality of the rice is reduced, the yield reduction caused by the weeds in the rice production can reach 4.3% -8.4%, and the yield reduction is greatly reduced in severe cases.
In recent years, weedy rice begins to occur in a rice transplanting area in the north, the distribution range is gradually expanded, the harm to the yield and the quality of cultivated rice is more and more serious, the weedy rice and the cultivated rice are of the same species, but the weedy rice belongs to the wild type, the natural survival law is followed in the survival and propagation process, the characteristics of the weedy rice, such as phenotype, stress resistance and the like have certain difference with the cultivated rice, the pathogenic fungi of weeds are natural enemies of the weeds, a plurality of successful examples of preventing and controlling the weeds by pathogenic microorganisms exist, the biological herbicide developed by the fungi is called as the fungal herbicide, although the method for preventing and controlling the weedy rice by using the biotechnology is still in the theoretical and early exploration stage at present, the assumption for preventing and controlling the weeds by using the biological technology has wide prospect, therefore, the method for preventing and controlling the harm of the weedy rice by using the pathogenic fungi herbicide developed by using the pathogenic fungi is a, to date, no investigation and research on pathogenic fungi of weedy rice has been reported.
The patent application number is CN201911057540.2, and the patent name is an identification method and application of pathogenic bacteria of weedy rice, and discloses an identification method and application of pathogenic bacteria of weedy rice, and the specific method comprises the following steps: widely collecting weed rice disease specimens, and separating, purifying and storing pathogenic bacteria of weed rice: identification of pathogenic bacteria of the weed rice and determination of pathogenicity of the pathogenic bacteria of the weed rice. The method has the problems that the effect of preventing and controlling the weedy rice is poor, the investigation, identification and pathogenicity research on the pathogenic bacteria of the weedy rice are less, the main diseases and pathogenic bacteria types of the weedy rice cannot be ascertained, a pathogenic fungi resource library of the weedy rice cannot be established, and a foundation cannot be provided for developing a fungal herbicide to prevent and control the weedy rice.
Through the above analysis, the problems and defects of the prior art are as follows: the prior art has poor effect on weed rice control measures, has less investigation, identification and pathogenicity research on weed rice pathogenic bacteria, can not find main diseases and pathogenic bacteria species of weed rice, can not establish a weed rice pathogenic fungi resource library, and can not provide a basis for developing fungal herbicides to control weed rice.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for determining the pathogenicity of pathogenic bacteria of weedy rice and an identification device.
The invention is realized in such a way that a method for determining the pathogenicity of pathogenic bacteria of weedy rice comprises the following steps:
step one, collecting a weed rice disease specimen in a rice planting area;
the method for collecting the weed rice disease specimen in the rice planting area comprises the following steps:
collecting weed rice specimens with diseases from a rice planting area at different periods; collecting samples which are easy to separate and can produce conidium strains according to the classification of stems, leaves and seeds, placing the samples in a fresh-keeping bag, and preserving the samples at 4 ℃ and preserving the samples in an aseptic air-drying way;
step two, separation, purification and preservation of pathogenic bacteria of the weed rice: separating the fungi from the stem, the leaf and the seeds within 3 days after the collection of the fresh-keeping preserved strain, purifying and preserving the separated fungi, and separating pathogenic fungi infecting the weedy rice from the air-dried preserved specimen by using a tissue culture separation method; through the processes of purification culture, monospore separation, monospore culture, colony propagation expansion and strain preservation, storing in a PC tube filled with sterile water, a weed rice pathogenic bacteria resource living body storage library is constructed, which comprises the following steps:
selecting diseased plant, determining stem, leaf and seed part of the plant, cleaning the boundary of diseased key with clear water, washing with sterile water several times in clean bench, and cutting into 0.25-0.5cm with sterilized scissors2Cutting into blocks;
soaking the cut blocks with 75% (v/v) alcohol for 10-12s to remove surface bubbles, soaking with 5% (m/v) sodium hypochlorite for 5min, immediately washing with sterile water for 3 times after soaking, placing on PDA solid culture medium, placing 5 tissue blocks in each dish, and culturing in an incubator at 26 deg.C;
after 5 days of constant temperature culture at 26 ℃, cutting hyphae from the edge of the separated pathogenic fungi colony of the weedy rice to a new PDA culture medium, and repeating the two times of purification culture;
culturing the purified fungi for 7 days, shaking the cultured spores in new culture medium, separating single spores in a super clean bench under microscope after 10-16h, selecting single spores to culture in PDA culture medium for 5 days, selecting mycelia to culture in WA culture medium for 5 days, and cutting the colonies into 0.5cm2The cutting blocks are put into a sterilized PC tube filled with sterile water for storage;
step three, identifying pathogenic bacteria of the weed rice: culturing the strain at constant temperature, recording the morphology, color and growth speed of a bacterial colony, and analyzing sporulation condition and morphological characteristics;
the identification of pathogenic bacteria of the weed rice comprises the following steps:
inoculating the purified strain on a PDA culture medium, culturing at a constant temperature of 26 ℃ for 5-10 days, recording the morphology, color and growth speed of a bacterial colony, and preparing a slide specimen to microscopically observe the sporulation condition and morphological characteristics of the slide specimen, including hypha morphology, spore length and spore width;
if the strain is not easy to produce spores on the PDA culture medium, promoting the spores;
according to the colony morphology, the size, the length-width ratio, the number of diaphragms and the shape of hyphae under a microscope, preliminarily identifying the bacterial strains as 6 genera which are fusarium, eudiplodiella, alternaria, nigrospora and pyricularia respectively;
step four, determining pathogenicity of pathogenic bacteria of the weed rice: and (3) performing pathogenic bacteria activation propagation, spore suspension preparation, weedy rice test plant cultivation, spray inoculation and pathogenicity analysis, and further screening a strong pathogenic strain.
Further, in the second step, 82 strains which produce conidia are preserved together in the living body storage library of the pathogenic bacteria resource of the weedy rice.
Further, in step three, the spore promotion comprises:
(1) putting a bacterium block with the diameter of 2cm in a culture dish paved with two layers of wet filter paper;
(2) inoculating the strain in water agar medium (TWA + W) containing wheat straw;
(3) after 10 days of culture, slide specimens were prepared, and the spore morphology and the spore size were measured.
Further, in the fourth step, the pathogenic bacteria pathogenicity of the weedy rice is determined, and the pathogenic bacteria pathogenicity determination method comprises the following steps:
step A, separating a pure culture of the inoculated and diseased plant;
step B, preparing spore suspension;
step C, cultivating a weedy rice test plant;
d, carrying out weed rice inoculation;
and step E, investigating the incidence of the inoculated strain.
Further, in step A, said isolated pure culture from a seed-diseased plant comprises:
separating and purifying in vitro or artificial culture medium according to the Koehz's rule with the presence of a microorganism on diseased plants; inoculating the pure culture to healthy plants of the same variety to generate diseases with the same symptoms; then separating the pure culture from the inoculated and diseased plant, wherein the character of the pure culture is the same as that of the inoculum; and (3) detecting the pathogenicity of the pathogenic fungi of the weedy rice.
Further, in step B, the preparing of the spore suspension comprises:
1) selecting a strain with strong spore production capacity, and inoculating the strain on a rice flour culture medium plate;
2) placing the culture medium in a constant-temperature incubator at 26 ℃ for 12h, culturing in the dark at intervals for 7-10 days, and observing sporulation conditions;
3) when the spore is produced more, adding sterilized water to brush the bacterial colony with a brush pen gently, filtering with two layers of sterilized lens paper, collecting spore filtrate, adding tween 80, and preparing into 30ml spore suspension; a suspension of 30 spores per field of view under 100-fold microscopy was obtained.
Further, in step C, the cultivation of the weedy rice test plant comprises:
step I, soaking weed rice seeds to be tested in 5% (m/v) sodium hypochlorite for 5-8min for surface sterilization;
step II, filling the nutrient soil and the matrix for planting into an aluminum basin for moist heat sterilization, and filling into a flowerpot after sterilization;
step III, sowing 20 seeds of the weedy rice to be tested with the surface sterilized in each pot, and sowing 6 pots;
and IV, cultivating in a greenhouse at the temperature of 20-30 ℃, watering on time, observing the growth condition, placing the plant in an inoculation box when the plant grows to the 3-5 leaf stage, and keeping the plant with consistent growth for later use.
Further, in step D, said performing weedy rice inoculation comprises:
the inoculation adopts an in vitro culture method, the spore suspension is dripped on the in vitro new leaves of the 3-5-leaf weedy rice for inoculation, and the obtained product is put into a constant temperature incubator at 26 ℃ for moisture-preserving aseptic culture for ten days; measuring the disease spot area ten days later, and determining the pathogenicity of the inoculated strain to the weedy rice according to the area size;
sterilizing culture dish and filter paper under high pressure, taking off new leaves with consistent growth size, sterilizing with alcohol, cutting into 5cm long, mixing well spore suspension, uniformly dripping three drops on the leaves, inoculating new weed rice leaves with each strain in 3 dishes, and taking 3 drops of sterile water as control; and (3) placing the inoculated leaves in a prepared special sterilization constant-temperature incubator for moisture preservation culture, illuminating for 12h every day, and keeping the humidity to ensure the condition of the leaves for inoculation and disease attack.
Further, in step E, the investigation of the onset of the inoculated strain comprises:
investigating the disease condition of the weed rice leaves ten days after inoculation; analyzing and recording the disease symptoms of the weeds-inoculated rice fungi on the leaves; analyzing the shape and the size of the disease spot, and photographing the diseased leaves; ten days later, the area of the lesion is measured, and the pathogenicity is determined.
Another object of the present invention is to provide a weedy rice pathogenic bacteria pathogenicity measuring apparatus for carrying out the method for measuring weedy rice pathogenic bacteria pathogenicity.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention collects weed rice disease specimens, stores the specimens in a classified manner, and separates and picks single spores of the specimens and stores effective strains.
The invention identifies and classifies pathogenic bacteria of weedy rice. And carrying out data statistics and arrangement.
Aiming at the problems that the harm of the weedy rice to the rice is aggravated year by year and the effect of the existing prevention and control measures is poor, the invention develops the investigation, identification and pathogenicity research of the pathogenic bacteria of the weedy rice on the basis of the biological prevention and control of the weedy rice, so as to find out the main diseases and the pathogenic bacteria types of the weedy rice, establish the Ningxia weedy rice pathogenic fungi resource library, screen the pathogenic bacteria resources which have strong pathogenicity to the weedy rice and have no pathogenicity or little influence on the rice, and provide a basis for developing a fungi herbicide to prevent and control the wee.
132 parts of a weed rice disease specimen is collected, and a weed rice pathogenic fungus resource library is preliminarily constructed; preserving 82 strains of the weedy rice fungal disease strain; perfects the technology of separating, purifying and preserving the fungi on the weedy rice; the species to which 82 strains belong was morphologically identified; 30 parts of each type of weedy rice are collected.
The invention widely collects weed rice disease specimens; the separation and preservation work of the strain is accelerated, and morphological and molecular biological identification is deeply carried out; the weed rice pathogenicity identification technology is integrated and perfected; the pathogenicity of the strain with strong pathogenicity to the weedy rice is identified, and the strain with strong pathogenicity to the weedy rice and weak pathogenicity to the rice is screened out.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flowchart of a method for determining pathogenicity of pathogenic bacteria of weedy rice according to an embodiment of the present invention.
FIG. 2 is a flow chart of spore promotion provided by the embodiment of the invention.
FIG. 3 is a flowchart of pathogenic bacteria pathogenicity of weedy rice provided by an embodiment of the present invention.
FIG. 4 is a flow chart for performing the preparation of a spore suspension provided by an embodiment of the present invention.
FIG. 5 is a flow chart of the cultivation of a weedy rice test plant according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for determining the pathogenicity of pathogenic bacteria of weedy rice and an identification device, and the invention is described in detail with reference to the attached drawings.
The method for determining the pathogenicity of pathogenic bacteria of the weedy rice comprises the following steps:
s101, collecting weed rice disease specimens in a rice planting area.
S102, separation, purification and preservation of pathogenic bacteria of the weed rice: the strain preserved in a fresh-keeping way is used for separating fungi from stems, leaves and seeds within 3 days after collection, and purifying and preserving the separated fungi.
S103, identifying pathogenic bacteria of the weed rice: the strain is cultured at constant temperature, the morphology, color and growth speed of the colony are recorded, and the sporulation condition and morphological characteristics are analyzed.
S104, determining pathogenicity of pathogenic bacteria of the weed rice: and (3) performing pathogenic bacteria activation propagation, spore suspension preparation, weedy rice test plant cultivation, spray inoculation and pathogenicity analysis, and further screening a strong pathogenic strain.
In step S102, 82 strains producing conidia are co-stored in the weedy rice pathogenic bacterium resource living body storage library provided in the embodiment of the present invention.
As shown in fig. 2, in step S103, the spore promotion provided in the embodiment of the present invention includes:
s201, putting a fungus block with the diameter of 2cm into a culture dish paved with two layers of wet filter paper;
s202, inoculating the strain into a water agar culture medium (TWA + W) containing wheat straws;
s203, culturing for 10 days, preparing a slide specimen, recording the shape of the spore and measuring the size of the spore.
As shown in fig. 3, in step S104, the pathogenic determination of pathogenic bacteria of weedy rice provided by the embodiment of the present invention includes:
s301, separating a pure culture of the inoculated and diseased plant;
s302, preparing spore suspension;
s303, cultivating a weedy rice test plant;
s304, carrying out weed rice inoculation;
s305, the onset of the inoculated strain is investigated.
In step S301, the isolation of a pure culture from a plant with a seed disease provided by the embodiments of the present invention comprises:
separating and purifying in vitro or artificial culture medium according to the Koehz's rule with the presence of a microorganism on diseased plants; inoculating the pure culture to healthy plants of the same variety to generate diseases with the same symptoms; then separating the pure culture from the inoculated and diseased plant, wherein the character of the pure culture is the same as that of the inoculum; and (3) detecting the pathogenicity of the pathogenic fungi of the weedy rice.
As shown in fig. 4, in step S302, the spore suspension preparation provided by the embodiment of the present invention includes:
s401, selecting a strain with strong spore production capacity, and inoculating the strain on a rice flour culture medium plate;
s402, placing the culture medium in a constant-temperature incubator at 26 ℃ for 12h, culturing in the dark at intervals for 7-10 days, and observing sporulation conditions;
s403, when the spore yield is high, adding sterile water to brush the bacterial colony down by using a writing brush, filtering the bacterial colony by using two layers of sterile lens paper, collecting spore filtrate, adding Tween 80 into the spore filtrate, and preparing 30ml of spore suspension; a suspension of 30 spores per field of view under 100-fold microscopy was obtained.
As shown in fig. 5, in step S303, the cultivation of the weedy rice test plant according to the embodiment of the present invention includes:
s501, soaking the weed rice seeds to be tested in 5% (m/v) sodium hypochlorite for 5-8min for surface sterilization;
s502, filling nutrient soil and a matrix for planting into an aluminum basin for damp-heat sterilization, and filling into a flowerpot after sterilization;
s503, sowing 20 seeds of the weedy rice to be tested with the surface sterilized in each pot, and sowing 6 pots;
s504, cultivating in a greenhouse at the temperature of 20-30 ℃, watering on time, observing the growth condition, placing the plant in an inoculation box when the plant grows to the 3-5 leaf stage, and keeping the plant with consistent growth for later use.
In step S304, the weedy rice inoculation provided by the embodiment of the present invention includes:
the inoculation adopts an in vitro culture method, the spore suspension is dripped on the in vitro new leaves of the 3-5-leaf weedy rice for inoculation, and the obtained product is put into a constant temperature incubator at 26 ℃ for moisture-preserving aseptic culture for ten days; measuring the disease spot area ten days later, and determining the pathogenicity of the inoculated strain to the weedy rice according to the area size;
sterilizing culture dish and filter paper under high pressure, taking off new leaves with consistent growth size, sterilizing with alcohol, cutting into 5cm long, mixing well spore suspension, uniformly dripping three drops on the leaves, inoculating new weed rice leaves with each strain in 3 dishes, and taking 3 drops of sterile water as control; and (3) placing the inoculated leaves in a prepared special sterilization constant-temperature incubator for moisture preservation culture, illuminating for 12h every day, and keeping the humidity to ensure the condition of the leaves for inoculation and disease attack.
In step S305, the investigation of the onset of the inoculated strain according to the embodiment of the present invention includes:
investigating the disease condition of the weed rice leaves ten days after inoculation; analyzing and recording the disease symptoms of the weeds-inoculated rice fungi on the leaves; analyzing the shape and the size of the disease spot, and photographing the diseased leaves; ten days later, the area of the lesion is measured, and the pathogenicity is determined.
The following specific tests further describe the invention.
1. Extensive collection of weed rice disease specimens:
weed rice specimens with diseases are collected from the whole rice planting area at different periods. Collecting samples which are easy to separate and can produce conidium strains according to the classification of stems, leaves and seeds, putting the samples in a fresh-keeping bag to be taken back, and preserving the samples at 4 ℃ and preserving the samples in a sterile air-drying way. 132 samples of each type are collected at 26 places in the 4 cities of the Yinhuang irrigation district. See table 1 for details.
Table 1: gathering a list of weed rice specimens:
2. separation, purification and preservation of pathogenic bacteria of weedy rice:
the fungus is separated from the strain which is preserved in a fresh-keeping way according to three parts of stems, leaves and seeds within 3 days after the collection. And (3) separating pathogenic fungi infecting the weedy rice from the air-dried and preserved specimen by using a tissue culture separation method. The weedy rice pathogenic bacteria resource living body storage library is constructed by the working processes of purification culture, monospore separation, monospore culture, colony propagation expansion and strain storage and storing in a PC tube filled with sterile water.
2.1, method:
selecting stem, leaf and seed parts of diseased plant, cleaning the boundary of diseased key with clear water, washing with sterile water in a superclean bench for several times, and cutting into 0.25-0.5cm with sterilized scissors2Soaking the small blocks in 75% (v/v) alcohol for 10s to eliminate surface air bubbles, soaking in 5% (m/v) sodium hypochlorite for 5min, immediately washing with sterile water for 3 times, placing on PDA solid culture medium, placing 5 blocks of tissue blocks on each dish, and culturing in an incubator at 26 deg.C. After 5 days of constant temperature culture at 26 ℃, hyphae are cut from the edge of the separated pathogenic fungi colony of the weed rice to a new PDA culture medium, and the purification culture is repeated twice. Culturing the purified fungi for 7 days, shaking the produced spores in new culture medium for more than ten hours, separating single spores in a super clean bench under microscope, culturing the single spores in PDA culture medium for 5 days, and picking mycelia in WA culture mediumAfter 5 days of culture, the colonies were cut into 0.5cm2The small pieces were stored in sterilized PC tubes filled with sterile water.
2.2 test results:
strain 82, which can produce conidia, was co-preserved.
3. Identification of pathogenic bacteria of weed rice:
3.1 method:
and (3) morphological identification, namely culturing the strain at constant temperature, recording the morphology, color and growth speed of a bacterial colony, and microscopically observing the sporulation condition and morphological characteristics of the bacterial colony, wherein the spore morphology mainly comprises hypha morphology, spore morphology and spore length and width.
Inoculating the purified strain on a PDA culture medium, culturing at a constant temperature of 26 ℃ for generally 5-10 days, recording the morphology, color and growth speed of a bacterial colony, and making a slide specimen to microscopically observe the sporulation condition and morphological characteristics of the slide specimen, including hypha morphology; spore morphology, spore length, and spore width. If the strain is not susceptible to sporulation on PDA medium, sporulation can be carried out as follows (Fangzhou, 1998): firstly, placing a bacterium block with the diameter of 2cm in a culture dish paved with two layers of wet filter paper; secondly, the bacterial strain is inoculated in a water agar culture medium (TWA + W) containing wheat straws. The slide specimens were prepared at 10 days, and the spore morphology was recorded and the size of the spores was measured by observing and recording the spore morphology using a photomicrograph system (Olympus, model BX 53F), as described in detail.
3.2 test results:
according to colony morphology, the size of spores under a microscope, aspect ratio, number of septa and shape of hyphae, the 82 bacterial strains are preliminarily identified as 6 genera which are fusarium, child's umbilicaria, alternaria, nigrospora and pyricularia respectively. See table 2 specifically:
table 2: weed rice isolation strain types and proportions:
3. pathogenic bacterium pathogenicity determination of weedy rice
The method mainly comprises the steps of pathogenic bacteria activation propagation, spore suspension preparation, weedy rice test plant cultivation, spray inoculation and pathogenicity investigation, and then the strong pathogenic strains are screened.
3.1 method:
3.1.1 pathogenicity of the weed rice isolate is determined according to the Koch's Rule, which is usually accompanied by the presence of a microorganism on the diseased plant, isolated and purified on an isolated or artificial medium to obtain a pure culture; inoculating the pure culture to healthy plants of the same variety to generate diseases with the same symptoms; then separating the pure culture from the inoculated and diseased plant, and the character is the same as that of the inoculum. So as to detect the pathogenicity of the pathogenic fungi of the weedy rice.
3.1.2 spore suspension preparation: selecting a strain with strong spore production capacity, inoculating the strain on a rice flour culture medium flat plate, culturing for 7-10 days in a constant temperature incubator at 26 ℃ for 12h in the dark at intervals, observing the spore production condition, adding sterile water to brush down bacterial colonies gently by using a writing brush when the spore production is more, filtering by using two layers of sterile lens paper, collecting spore filtrate, and adding tween 80 to prepare 30ml of spore suspension. A suspension of 30 spores per field of view under 100-fold microscopy was obtained.
3.1.3 cultivation of weedy rice test plants: the weedy rice seeds to be tested are soaked for 5-8min by 5% (m/v) of sodium hypochlorite for surface sterilization. The nutrient soil and the matrix for planting are filled into an aluminum pot for moist heat sterilization, the aluminum pot is filled into a flowerpot after sterilization, 20 seeds of the weedy rice to be tested after surface sterilization are planted in each pot, and 6 pots are planted for each kind of the weedy rice. Culturing in a greenhouse at 20-30 deg.C, watering on time, observing growth condition, placing in an inoculation box when the plant grows to 3-5 leaves, and keeping the plant with uniform growth for use.
3.1.4 weedy rice inoculation method: the inoculation adopts an in vitro culture method, the spore suspension is dripped on the in vitro new leaves of the 3-5-leaf weedy rice for inoculation, the obtained product is put into a constant temperature incubator at 26 ℃ for moisture-preserving sterile culture for ten days, the lesion area is measured after ten days, and the pathogenicity of the inoculated strain to the weedy rice is determined according to the area size. Firstly, sterilizing a culture dish and filter paper under high pressure, taking off new leaves with consistent growth and size, disinfecting the new leaves with alcohol, cutting the new leaves into 5cm long, uniformly mixing the prepared spore suspension liquid on the leaves, uniformly dripping three drops of the spore suspension liquid on the leaves, inoculating 3 dishes of new weed rice leaves on each strain, and taking 3 dishes of sterile water as a control. And (3) placing the inoculated leaves in a prepared special sterilization constant-temperature incubator for moisture preservation culture, illuminating for 12h every day, and keeping the humidity to ensure the condition of the leaves for inoculation and disease attack.
3.1.5 investigation of the onset of the inoculated strains: ten days after inoculation, the disease condition of the weed rice leaves is investigated. The observation and record of the disease symptoms of the weed rice inoculated fungi on the leaves. The shape and size of the disease spots are observed, and the diseased leaves are photographed and recorded. The area of the lesion is measured ten days later to determine pathogenicity.
3.2 test results:
by measuring the inoculated lesion area of 28 strains, the bipolaris and the pyricularia are preliminarily found to have stronger pathogenic effect on the weedy rice.
The invention is further described below in connection with the positive effects.
132 parts of a weed rice disease specimen is collected, and a weed rice pathogenic fungus resource library is preliminarily constructed; 82 strains of the weedy rice fungal disease strain were preserved.
Perfects the technology of separating, purifying and preserving the fungi on the weedy rice.
The species to which the 82 strains belong was morphologically identified.
30 parts of each type of weedy rice are collected.
A weed rice disease specimen is widely collected;
the separation and preservation work of the strain is accelerated, and morphological and molecular biological identification is deeply carried out;
the weed rice pathogenicity identification technology is integrated and perfected;
the pathogenicity of the strain with strong pathogenicity to the weedy rice is identified, and the strain with strong pathogenicity to the weedy rice and weak pathogenicity to the rice is screened out.
In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, should not be construed as limiting the invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for determining the pathogenicity of pathogenic bacteria of weedy rice is characterized by comprising the following steps:
step one, collecting a weed rice disease specimen in a rice planting area;
the method for collecting the weed rice disease specimen in the rice planting area comprises the following steps:
collecting weed rice specimens with diseases from a rice planting area at different periods; collecting samples which are easy to separate and can produce conidium strains according to the classification of stems, leaves and seeds, placing the samples in a fresh-keeping bag, and preserving the samples at 4 ℃ and preserving the samples in an aseptic air-drying way;
step two, separation, purification and preservation of pathogenic bacteria of the weed rice: separating the fungi from the stem, the leaf and the seeds within 3 days after the collection of the fresh-keeping preserved strain, purifying and preserving the separated fungi, and separating pathogenic fungi infecting the weedy rice from the air-dried preserved specimen by using a tissue culture separation method; through the processes of purification culture, monospore separation, monospore culture, colony propagation expansion and strain preservation, storing in a PC tube filled with sterile water, a weed rice pathogenic bacteria resource living body storage library is constructed, which comprises the following steps:
selecting diseased plant, determining stem, leaf and seed part of the plant, cleaning the boundary of diseased key with clear water, washing with sterile water several times in clean bench, and cutting into 0.25-0.5cm with sterilized scissors2Cutting into blocks;
soaking the cut blocks with 75% (v/v) alcohol for 10-12s to remove surface bubbles, soaking with 5% (m/v) sodium hypochlorite for 5min, immediately washing with sterile water for 3 times after soaking, placing on PDA solid culture medium, placing 5 tissue blocks in each dish, and culturing in an incubator at 26 deg.C;
after 5 days of constant temperature culture at 26 ℃, cutting hyphae from the edge of the separated pathogenic fungi colony of the weedy rice to a new PDA culture medium, and repeating the two times of purification culture;
culturing the purified fungi for 7 days, shaking the cultured spores in new culture medium, separating single spores in a super clean bench under microscope after 10-16h, selecting single spores to culture in PDA culture medium for 5 days, selecting mycelia to culture in WA culture medium for 5 days, and cutting the colonies into 0.5cm2The cutting blocks are put into a sterilized PC tube filled with sterile water for storage;
step three, identifying pathogenic bacteria of the weed rice: culturing the strain at constant temperature, recording the morphology, color and growth speed of a bacterial colony, and analyzing sporulation condition and morphological characteristics;
the identification of pathogenic bacteria of the weed rice comprises the following steps:
inoculating the purified strain on a PDA culture medium, culturing at a constant temperature of 26 ℃ for 5-10 days, recording the morphology, color and growth speed of a bacterial colony, and preparing a slide specimen to microscopically observe the sporulation condition and morphological characteristics of the slide specimen, including hypha morphology, spore length and spore width;
if the strain is not easy to produce spores on the PDA culture medium, promoting the spores;
according to the colony morphology, the size, the length-width ratio, the number of diaphragms and the shape of hyphae under a microscope, preliminarily identifying the bacterial strains as 6 genera which are fusarium, eudiplodiella, alternaria, nigrospora and pyricularia respectively;
step four, determining pathogenicity of pathogenic bacteria of the weed rice: and (3) performing pathogenic bacteria activation propagation, spore suspension preparation, weedy rice test plant cultivation, spray inoculation and pathogenicity analysis, and further screening a strong pathogenic strain.
2. The method for determining pathogenicity of pathogenic bacteria of weedy rice according to claim 1, wherein in the second step, 82 strains producing conidia are co-preserved in the living-body stock of pathogenic bacteria of weedy rice.
3. The method for determining pathogenicity of pathogenic bacteria of weedy rice according to claim 1, wherein the sporulation is carried out in step three and comprises:
(1) putting a bacterium block with the diameter of 2cm in a culture dish paved with two layers of wet filter paper;
(2) inoculating the strain in water agar medium (TWA + W) containing wheat straw;
(3) after 10 days of culture, slide specimens were prepared, and the spore morphology and the spore size were measured.
4. The method for determining the pathogenicity of pathogenic bacteria of weedy rice according to claim 1, wherein the determination of the pathogenicity of pathogenic bacteria of weedy rice in the fourth step comprises:
step A, separating a pure culture of the inoculated and diseased plant;
step B, preparing spore suspension;
step C, cultivating a weedy rice test plant;
d, carrying out weed rice inoculation;
and step E, investigating the incidence of the inoculated strain.
5. The method for determining pathogenicity of pathogenic bacteria of weedy rice as claimed in claim 4, wherein in step A, said isolation of a pure culture thereof from a plant with an inoculated disease comprises:
separating and purifying in vitro or artificial culture medium according to the Koehz's rule with the presence of a microorganism on diseased plants; inoculating the pure culture to healthy plants of the same variety to generate diseases with the same symptoms; then separating the pure culture from the inoculated and diseased plant, wherein the character of the pure culture is the same as that of the inoculum; and (3) detecting the pathogenicity of the pathogenic fungi of the weedy rice.
6. The method for determining pathogenicity of pathogenic bacteria of weedy rice according to claim 4, wherein in step B, the preparation of the spore suspension is carried out and comprises:
1) selecting a strain with strong spore production capacity, and inoculating the strain on a rice flour culture medium plate;
2) placing the culture medium in a constant-temperature incubator at 26 ℃ for 12h, culturing in the dark at intervals for 7-10 days, and observing sporulation conditions;
3) when the spore is produced more, adding sterilized water to brush the bacterial colony with a brush pen gently, filtering with two layers of sterilized lens paper, collecting spore filtrate, adding tween 80, and preparing into 30ml spore suspension; a suspension of 30 spores per field of view under 100-fold microscopy was obtained.
7. The method for determining pathogenicity of pathogenic bacteria of weedy rice as claimed in claim 4, wherein in step C, the cultivation of the weedy rice test plant comprises:
step I, soaking weed rice seeds to be tested in 5% (m/v) sodium hypochlorite for 5-8min for surface sterilization;
step II, filling the nutrient soil and the matrix for planting into an aluminum basin for moist heat sterilization, and filling into a flowerpot after sterilization;
step III, sowing 20 seeds of the weedy rice to be tested with the surface sterilized in each pot, and sowing 6 pots;
and IV, cultivating in a greenhouse at the temperature of 20-30 ℃, watering on time, observing the growth condition, placing the plant in an inoculation box when the plant grows to the 3-5 leaf stage, and keeping the plant with consistent growth for later use.
8. The method for determining pathogenic pathogenicity of weedy rice pathogens according to claim 4, wherein in step D, the weedy rice inoculation comprises:
the inoculation adopts an in vitro culture method, the spore suspension is dripped on the in vitro new leaves of the 3-5-leaf weedy rice for inoculation, and the obtained product is put into a constant temperature incubator at 26 ℃ for moisture-preserving aseptic culture for ten days; measuring the disease spot area ten days later, and determining the pathogenicity of the inoculated strain to the weedy rice according to the area size;
sterilizing culture dish and filter paper under high pressure, taking off new leaves with consistent growth size, sterilizing with alcohol, cutting into 5cm long, mixing well spore suspension, uniformly dripping three drops on the leaves, inoculating new weed rice leaves with each strain in 3 dishes, and taking 3 drops of sterile water as control; and (3) placing the inoculated leaves in a prepared special sterilization constant-temperature incubator for moisture preservation culture, illuminating for 12h every day, and keeping the humidity to ensure the condition of the leaves for inoculation and disease attack.
9. The method for determining pathogenicity of pathogenic bacteria of weedy rice according to claim 4, wherein the investigation of the onset of the inoculated strain in step E comprises:
investigating the disease condition of the weed rice leaves ten days after inoculation; analyzing and recording the disease symptoms of the weeds-inoculated rice fungi on the leaves; analyzing the shape and the size of the disease spot, and photographing the diseased leaves; ten days later, the area of the lesion is measured, and the pathogenicity is determined.
10. A weedy rice pathogenic bacterium pathogenicity measuring apparatus for carrying out the method for measuring weedy rice pathogenic bacterium pathogenicity according to any one of claims 1 to 9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607346A (en) * | 2019-11-01 | 2019-12-24 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Identification method and application of pathogenic bacteria of weedy rice |
| CN115216409A (en) * | 2022-08-10 | 2022-10-21 | 武汉轻工大学 | Biological prevention and control method for radix seu folium Tetrastigmatis Hypoglauci |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020057463A (en) * | 2001-01-05 | 2002-07-11 | 이병길 | Differential Screening Methods for Antagonistic Microorganism against Plant Pathogenic fungi and Antagonists selected therefrom |
| CN103667080A (en) * | 2013-12-04 | 2014-03-26 | 中国农业科学院油料作物研究所 | Method for isolating plant pathogenic fungus |
| CN105803040A (en) * | 2014-12-31 | 2016-07-27 | 姜汉军 | Method for identifying pathogenic bacteria of potato early blight |
| CN106222097A (en) * | 2016-08-27 | 2016-12-14 | 四川省农业科学院土壤肥料研究所 | A kind of isolation and identification method of sorghum hybrid sudangrass Leaf blotch pathogeny |
| CN107488610A (en) * | 2017-07-31 | 2017-12-19 | 中国水稻研究所 | A kind of isolation and identification method of paddy bacterial fringe rot pathogen |
| CN109493920A (en) * | 2018-12-19 | 2019-03-19 | 江苏省农业科学院 | A kind of method and application of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny |
| CN110607346A (en) * | 2019-11-01 | 2019-12-24 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Identification method and application of pathogenic bacteria of weedy rice |
-
2020
- 2020-10-30 CN CN202011185477.3A patent/CN112195217A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020057463A (en) * | 2001-01-05 | 2002-07-11 | 이병길 | Differential Screening Methods for Antagonistic Microorganism against Plant Pathogenic fungi and Antagonists selected therefrom |
| CN103667080A (en) * | 2013-12-04 | 2014-03-26 | 中国农业科学院油料作物研究所 | Method for isolating plant pathogenic fungus |
| CN105803040A (en) * | 2014-12-31 | 2016-07-27 | 姜汉军 | Method for identifying pathogenic bacteria of potato early blight |
| CN106222097A (en) * | 2016-08-27 | 2016-12-14 | 四川省农业科学院土壤肥料研究所 | A kind of isolation and identification method of sorghum hybrid sudangrass Leaf blotch pathogeny |
| CN107488610A (en) * | 2017-07-31 | 2017-12-19 | 中国水稻研究所 | A kind of isolation and identification method of paddy bacterial fringe rot pathogen |
| CN109493920A (en) * | 2018-12-19 | 2019-03-19 | 江苏省农业科学院 | A kind of method and application of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny |
| CN110607346A (en) * | 2019-11-01 | 2019-12-24 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Identification method and application of pathogenic bacteria of weedy rice |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607346A (en) * | 2019-11-01 | 2019-12-24 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Identification method and application of pathogenic bacteria of weedy rice |
| CN115216409A (en) * | 2022-08-10 | 2022-10-21 | 武汉轻工大学 | Biological prevention and control method for radix seu folium Tetrastigmatis Hypoglauci |
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