CN106399451B - Culture medium for detecting rice seed carrying fungi - Google Patents
Culture medium for detecting rice seed carrying fungi Download PDFInfo
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- CN106399451B CN106399451B CN201610814181.0A CN201610814181A CN106399451B CN 106399451 B CN106399451 B CN 106399451B CN 201610814181 A CN201610814181 A CN 201610814181A CN 106399451 B CN106399451 B CN 106399451B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a culture medium for detecting fungi carried by rice seeds. The culture medium contains 5g of peptone, 10g of glucose, 1g of monopotassium phosphate, 0.25g of MgSO4 & 7H2O 0.25, 15g of agar, 0.033g of Bengal, 0.1g of chloramphenicol, 0.05g of copper sulfate, 0.029g of zinc nitrate and 0.0001g of cadaverine per liter of sterilized water. The culture medium is an improvement of a Bengal culture medium, rice seeds carrying fungi can be detected more by the culture medium, the operation is simple and convenient, the fungi are not easily polluted by bacteria, and the diameter of the fungus colony is smaller than that of the fungus colony cultured by using a PDA culture medium in the prior art, so that the separation and statistics of the fungi are facilitated.
Description
Technical Field
The invention belongs to the technical field of seed quality detection, and particularly relates to a method for detecting fungi carried by rice seeds by improving a Bengal culture medium.
Background
At present, the detection of the health degree of seeds becomes a routine measure in the process of seed quality assessment and plant quarantine in many countries, becomes an indispensable component in a seed quality control system, and special seed health determination laboratories are established in some countries. The seed health measurement is to determine whether the seeds carry pathogenic bacteria (such as fungi, bacteria and viruses), harmful animals (such as nematodes and pests) and other health conditions, namely to detect the types and the number of the pests and diseases carried by the seeds, wherein the detection of the fungi carried by the seeds is an important content in the detection of the health degree of the seeds. The systematic study of seed health began, as international rice institute established the department of seed health (SEED HEALTH UNIT) in 1983 and the seed health laboratory established in 1987.
The traditional methods for detecting fungi carried by seeds include a washing method, an agar dish method and a filter paper moisturizing method.
The washing method comprises the following steps: putting the seeds into a sterilized 250mL triangular flask, then adding 25mL of sterilized water, sealing the triangular flask, then placing the triangular flask into a shaking table with the rotation speed of 150rpm and the temperature of 25 ℃ for fully shaking for 1h, collecting the suspension into a sterilized 10mL centrifugal tube, centrifuging for 10min with the rotation speed of 3000rpm, removing the supernatant, and then adding 200 mu L of sterile water for suspension precipitation. The suspension was diluted with sterile water in a 10-fold series of concentration gradients and then plated on PDA plates for testing. The washing method can only separate part of fungi carried on the surface of the seeds, the fungi which are infected into the seeds cannot be separated, and the types and the quantity of the fungi carried on the surface of the seeds can be completely counted by a plurality of tests only by diluting the washing liquid.
Moisturizing filter paper method: the method comprises the steps of firstly disinfecting the surface of a test seed by 5% sodium hypochlorite (NaClO), then cleaning for 3 times by using sterile water, absorbing surface moisture of the disinfected seed by using filter paper, placing the disinfected seed in 9cm culture dishes padded with the sterile filter paper, overlapping two layers of sterile filter paper in each culture dish, wetting the sterile filter paper by using the sterile water, placing the seed in the wetted sterile filter paper, culturing for 12 hours under the condition of alternate illumination (6 hours)/darkness (6 hours) in a 25 ℃ incubator, then placing the seed in a-20 ℃ refrigerator for 12 hours to kill the swollen seed so as to inhibit the seed from sprouting, and then placing the seed in the 25 ℃ incubator for 5 days after illumination (12 hours)/darkness (12 hours) for culture. The moisture-retaining filter paper method can avoid the interference of bacteria on the separation of fungi, but because the nutrient conditions for the growth of the fungi are only supplied for the seeds, when a plurality of fungi grow on the same seeds, the fungi are difficult to separate.
The agar plate method is a method for separating and checking fungi carried by seeds, which is specified in national standard GB/T3543.7-1995 crop seeds-other project inspection. At present, the agar plate method in the national standard GB/T3543.7-1995 is basically adopted for the detection of fungi carried by seeds in China.
The agar plate method specified in the national Standard GB/T3543.7-1995: taking a proper amount of seeds to soak in a 5% sodium hypochlorite solution for 8min for surface disinfection, then washing with sterile water for 3 times, uniformly placing on a PDA (personal digital assistant) plate with the diameter of 9cm after drying, placing 10 seeds in each dish, repeating each treatment for 4 times, and using a blank PDA plate as an environment control under the same operation (the PDA plate needs to be added with a proper amount of streptomycin to inhibit the growth of bacteria during manufacturing). Culturing in 25 deg.C incubator for 6d under 12h light/dark alternation, checking, and recording the species and amount of fungi carried by seeds and different anatomical parts. Although the agar plate method can better separate fungi carried by seeds, the fungi can not be separated smoothly due to the interference of bacteria during separation, and part of the fungi grow faster, such as Penicillium (Penicillium), Aspergillus (Aspergillus), Pythium (Pythium) and Rhizopus (Rhizopus), after the fungi are cultured for 6 days, the diameter of a single colony is 4 cm-5 cm, and even the single colony grows all over the plate, so that the separation of other fungi is interfered.
As the fungus can generate conidium which is fast in transmission speed along with air flow, water flow and the like, the fungus carried by the seeds has huge harm to agricultural production, once the fungus is attacked, the fungus is difficult to control, for example, rice blast can be carried by the seeds, and destructive disasters can be caused to the production after the fungus is attacked, so that a method for separating and detecting the fungus carried by the seeds is developed, and the method has important significance for detecting the situation of the fungus carried by the seeds and preventing destructive diseases.
The formula of the culture medium of the Bengal red is 5g of peptone, 10g of glucose, 1g of monopotassium phosphate, 0.5g of magnesium sulfate, 15g of agar, 0.033g of Bengal red and 0.1g of chloramphenicol. Bengal red medium is mainly used for the counting, isolation and cultivation of Molds (Molds) and yeasts (Yeast).
Disclosure of Invention
The invention aims to solve the technical problems that the separated fungi are easily interfered by bacteria in the prior art of seed health detection, so that the number of the separated fungi is small, the fungi carried by seeds cannot be completely reflected, and during separation, part of the fungi grow at a high speed, the diameter of bacterial colonies is large, the separation of other fungi is interfered, and the fungi are difficult to separate independently.
In order to solve the technical problems, the invention provides a culture medium for detecting fungi carried by rice seeds, which is improved on the basis of a Bengal culture medium.
The invention provides a culture medium for detecting rice seed carried fungi, which comprises the following components in part by weight: 5g of peptone, 10g of glucose, 1g of monopotassium phosphate, 0.25g of MgSO4 & 7H2O 0.25, 15g of agar, 0.033g of Bengal, 0.1g of chloramphenicol, 0.05g of copper sulfate, 0.029g of zinc nitrate and 0.0001g of cadaverine are added to each liter of sterilized water.
The preparation method of the culture medium for detecting the fungi carried by the rice seeds comprises the following steps: placing the components in a triangular flask according to the formula of the culture medium for detecting the fungi carried by the rice seeds, adding 1L of water, performing moist heat sterilization at 121 ℃ and 0.1-0.15 MPa for 20min according to a moist heat sterilization method, and then placing the triangular flask in a medicine cabinet for storage or subpackaging the triangular flask into culture dishes with the diameter of 9cm for later use.
Compared with the prior art, the invention has the beneficial effects that:
1. The culture medium of the invention is added with 0.05g of copper sulfate (CuSO4), 0.029g of zinc nitrate (Zn (NO3)2) and 0.0001g of cadaverine (1, 5-Pentanedium) on the basis of the original Monascus culture medium, the usage amount of magnesium sulfate (MgSO4) is reduced, the separated fungus species are increased, only Penicillium (Penicillium), Aspergillus (Aspergillus), Rhizopus (Rhizopus), Pythium (Pyroluum) and Fusarium (Fusarium) fungi can be separated by using the Monascus culture medium under the condition of the same seed lot in most cases by using the traditional flat-plate method, and the fungi of the genus Helminthosporium (Bipolaris), Curvularia (Curvularia) and Nodulisporium (Nodulisporium) can be separated in addition to the fungi of the genus Helminthosporium after the culture medium formula is improved.
2. Is not easy to be polluted by bacteria. In the prior art, after antibiotic (usually streptomycin sulfate) is added into PDA culture medium regulated by agar plate law in national standard GB/T3543.7-1995 crop seed inspection regulation-inspection of other projects, interference of bacteria cannot be completely avoided, once the bacteria are nourished, the surface of the whole culture medium can be quickly filled, growth of fungi is influenced, even if the fungi are reluctantly separated out, because the bacteria are mixed, purification is required for many times, workload and pollution probability are increased, pure culture of target fungi is difficult to obtain, and under the condition that all measures are operated according to the national standard, the general bacterial pollution rate is 5% -10%. The bacterial contamination rate is below 4% by using the culture medium of the invention.
3. Facilitating the separation of fungi. In the prior art, the national standard GB/T3543.7-1995 crop seed inspection regulation-PDA culture medium used in other project inspection regulations has excess nutrition, so that part of fungi such as penicillium, aspergillus, fusarium, pythium and the like have high growth speed and excessively large colony diameter, and after 6 days of culture, the colony diameter is 4-5 cm, and some of the fungi even grow in the whole dish, so that a single colony is difficult to effectively pick for identification and analysis. And the growth of other fungi is inhibited, so that the detection result is inaccurate, after the culture medium is used, because the nutrient components of the culture medium only meet the basic requirement of the growth of the fungi, the diameter of a fungus colony is 2 cm-3 cm after the culture is carried out for 6 days, and is smaller than the diameter (4 cm-5 cm) of the fungus colony cultured by using a PDA culture medium in the prior art, so that the effective and accurate separation can be realized.
4. The statistics of the carrying rate of the seed fungi is convenient. The statistics of the carrying rate of the seed fungi is to count the bacterial colonies growing on each seed according to the result of plate culture, and to respectively pick and identify the bacterial colonies, and to count the number of the seeds with fungi, the number of different types of fungi and the total number of the bacterial colonies in all the repeated tests. And then calculating the bacteria carrying rate of the batch of seeds and the occurrence frequency of different types of fungi, if pathogenic bacteria are detected and the occurrence frequency is high, treating the seeds to avoid causing diseases after planting. The culture medium has small colony diameter for fungus growth, so that when a plurality of fungi are grown on one seed, the mutual interference can be effectively avoided, and the statistics of the carrying rate of the fungi in the seed is facilitated. The culture medium used in the traditional plate method has excessive nutrition, so that the diameter of partial fungus colonies growing faster is too large, and the culture medium strives for living space of other fungi to cause the fungi to grow difficultly, so that detection omission occurs, and the risk of field planting is increased.
Drawings
FIG. 1: example 1 comparison of the effect of the inventive culture medium and PDA culture medium on the detection of rice seed bacteria-carrying condition is shown. FIG. 1A is a diagram showing the examination of the presence of bacteria in rice seeds cultured in the medium of the present invention, and FIG. 1B is a diagram showing the examination of the presence of bacteria in rice seeds cultured in the PDA medium prescribed by the agar plate method in the national Standard GB/T3543.7-1995 agricultural crop seed test protocol-other project test. In FIG. 1A, the notation: 1 is a helminthosporium (Bipolaris) fungus, 2 is a Fusarium (Fusarium) fungus, 3 is a Curvularia (Curvularia) fungus, and 4 is a nodularia (Nodulisporium) fungus; labels in FIG. 1B: 5 is a Fusarium (Fusarium) fungus and 6 is a helminthosporium (Bipolaris) fungus.
FIG. 2: example 2 comparison of the effect of the inventive culture medium and PDA culture medium on the detection of rice seed bacteria-carrying condition is shown. FIG. 2A is a graph showing the effect of bacteria-carrying detection of rice seeds cultured in the red medium of the present invention, and FIG. 2B is a graph showing the effect of bacteria-carrying detection of rice seeds cultured in the PDA medium prescribed by the agar plate method in the national Standard GB/T3543.7-1995 agricultural crop seed test protocol-other project test. Labeled in FIG. 2A: 1 is a Penicillium (Penicillium) fungus, 2 is a Pythium (Pythium) fungus, marked in fig. 2B: 3 is a Pythium (Pythium) fungus and 4 is a Penicillium (Penicillium) fungus.
FIG. 3: FIG. 1 is a microscopic image of isolated fungal spores carrying bacteria from rice seeds, labeled in FIG. 3: microscopic images of isolated fungal spores were obtained from culture medium of the present invention, wherein 1 is Helminthosporium (Bipolaris), 2 is Fusarium (Fusarium), 3 is Curvularia (Curvularia), and 4 is Nodulisporium. In FIG. 3, the microscopic images of fungal spores isolated from the PDA medium specified by agar plate method in national Standard GB/T3543.7-1995 crop seed test protocol-other project test-are shown in Fusarium (Fusarium) at 5 and Helminthosporium (Bipolaris) at 6.
FIG. 4: FIG. 2 is a microscopic image of isolated fungal spores carrying bacteria from rice seeds, marked in FIG. 4: the microscopic pictures of the isolated fungal spores of the culture medium of the invention are 1 of Penicillium (Penicillium) and 2 of Pythium (Pythium). In FIG. 4, the microscopic pictures of the isolated fungal spores are taken for 3 Pythium (Pythium) and 4 Penicillium (Penicillium) in the PDA medium specified by agar plate method in the national standard GB/T3543.7-1995 crop seed test protocol-other project test.
Detailed Description
the reagents referred to in the following examples are all commercially available. Each example is not specifically illustrated as a conventional method.
Example 1
The specific steps of detecting rice seeds by using the culture medium are as follows:
(1) the preparation of the culture medium comprises the steps of weighing the components according to the formula of the culture medium, putting the components into a triangular flask, adding 1L of water, boiling for 15min, performing moist heat sterilization for 20min at 121 ℃ and 0.15MPa according to a moist heat sterilization method, and subpackaging the components into a culture dish with the diameter of 9cm for later use (if the components are not used temporarily, the components can be left in the triangular flask, and then subpackaging the components after the next sterilization); the formula of the culture medium is as follows: per liter of sterilized water were added Peptone (Peptone)5g, Glucose (Glucose)10g, monopotassium phosphate (KH2PO4)1g, MgSO4 & 7H2O 0.25g, agar (agar powder)15g, Bengal red (Tetrachlorofluoscein) 0.033g, Chloramphenicol (Chloramphenicol)0.1g, copper sulfate (CuSO4)0.5g, zinc nitrate (Zn (NO3)20.029g, cadaverine (1,5-Pentanediamine)0.0001 g.
(2) After the culture medium in the culture dish is solidified, performing surface disinfection on seeds for 8min by using a sodium hypochlorite solution with the mass fraction of 5%, then uniformly placing the seeds on the culture medium in the culture dish, then placing the culture dish in an illumination incubator at the temperature of 25 ℃, the illumination intensity of 9000Lx, and the illumination (12 h)/darkness (12h) for 1d, after the seeds are sucked and swelled, placing the culture dish in a-20 ℃ refrigerator for 12h, then taking out the culture dish, keeping the temperature at 28 ℃, the illumination intensity of 9000Lx, and the light period of 12h light period/12 h dark period, culturing for 6d, then picking the grown fungi, observing morphological characteristics under a microscope, and checking the types of the fungi carried by the seeds.
in this example, a total of 4 replicates of the same variety of seeds of red rice, Yuanyang (moon valley) were performed, with 10 seeds placed in each dish. The Yuanyang red rice (moon valley) rice seeds are Yuanyang local cultivars, and are collected from local farmers and can be purchased from the market.
Control 1(CK 1):
Comparative example 1A PDA medium (potato dextrose medium) for seed germ detection was prepared from the seeds of the Red Rice of Yuanyang of example 1 by the procedure of national Standard GB/T3543.7-1995, which was defined by the agar plate method in the examination of other items, using 200g of potato dextrose (20 g), 15g of agar powder, 1L of tap water, adjusting pH to 7, boiling for 15min, placing in a 1L Erlenmeyer flask, moist heat sterilizing at 121 deg.C, 0.1 MPa-0.15 MPa for 20min, and packaging in a 9cm diameter Petri dish, the excess medium being left in the Erlenmeyer flask, and then sterilizing again for packaging in a moist heat sterilization method, 4 repeated experiments were carried out, each dish being provided with 10 seeds.
The experimental effects are detailed in fig. 1 and 3:
In FIG. 1, 4 fungi (FIG. 1A, 1 in FIG. 1A is a helminthosporium umbilicifolium (Bipolaris) fungus, 2 is a Fusarium (Fusarium) fungus, 3 is a Curvularia (Curvularia) fungus, and 4 is a nodularium (Nodulisporium) fungus) were detected in the culture medium of the present invention, and the spore image of the isolated fungus is shown in FIG. 3, and the diameter of the fungus colony is 2-3 cm, which is convenient for picking and observation. 2 kinds of fungi (shown in figure 1B, 5 is Fusarium (Fusarium) fungi, 6 is Bipolaris (Bipolaris) fungi, the spore picture of the separated fungi is shown in figure 3), the diameter of the fungal colony is 4 cm-5 cm, the bacterial colony grows all over the dish, the bacterial colonies are overlapped and grow, and the separation is difficult, and the picking, observation and counting are difficult.
Example 2
Example 2 the experimental results are shown in fig. 2 and 4, except that the rice seeds of red rice (moon valley) of Yuanyang are taken, and the experimental results are the same as in example 1.
In FIG. 2, 2 fungi were detected in the medium of the present invention, (FIG. 2A shows that 1 in FIG. 2A is Penicillium (Penicillium) fungus and 2 is Pythium (Pythium) fungus, and the spore of the isolated fungus is shown in FIG. 4), and the diameter of the fungus colony is 2 cm-3 cm, which is convenient for picking and observing. 2 kinds of fungi (shown in figure 2B, 3 in figure 2B is Pythium fungus, 4 is Penicillium fungus, and the picture of spore of the separated fungi is shown in figure 4) are detected by the PDA culture medium, fungal colonies almost grow over a culture dish, the two kinds of fungi are overlapped in growth and difficult to separate, the number of bacteria carried by a single seed is difficult to count, and the germination of the seed influences the separation effect of the fungi carried by the seed. Since the detection of the fungi carried by the seeds is related to the types and the number of the fungi carried by the seeds, in the detection shown in FIG. 2, although only 2 kinds of fungi are detected by the culture medium disclosed by the invention and the traditional PDA culture medium, the fungus colonies separated by the culture medium disclosed by the invention are small and easy to pick and count, the fungus colonies separated by the traditional PDA culture medium grow too fast, the fungi carried by each seed are difficult to distinguish, and the colonies are overlapped and difficult to pick, observe and count.
Therefore, after using the medium of the present invention, more fungi of the genus Curvularia (Curvularia), the genus Nodulisporium (Nodulisporium) could be isolated than the conventional PDA medium of the national Standard GB/T3543.7-1995, and for the experiment for detecting the same fungi, as in example 2: the colony diameter of the Penicillium (Penicillium) and Pythium (Pythium) is smaller than that of the traditional PDA culture medium on the culture medium, so that the culture medium is easy to pick and separate, and the statistics of the carrying rate of the seed fungi is facilitated. The culture medium is simple and convenient to operate and is not easy to be polluted by bacteria, and the bacterial pollution rate is below 4%.
Claims (1)
1. A culture medium for detecting rice seeds carrying fungi is characterized in that: 5g of peptone, 10g of glucose, 1g of monopotassium phosphate, 0.25g of MgSO4 & 7H2O 0.25, 15g of agar, 0.033g of Bengal, 0.1g of chloramphenicol, 0.05g of copper sulfate, 0.029g of zinc nitrate and 0.0001g of cadaverine are added to each liter of sterilized water.
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