CN113652466B - Method for measuring conidium quality of corynespora polymorpha - Google Patents
Method for measuring conidium quality of corynespora polymorpha Download PDFInfo
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- CN113652466B CN113652466B CN202111002122.0A CN202111002122A CN113652466B CN 113652466 B CN113652466 B CN 113652466B CN 202111002122 A CN202111002122 A CN 202111002122A CN 113652466 B CN113652466 B CN 113652466B
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- 241000609458 Corynespora Species 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000000725 suspension Substances 0.000 claims abstract description 33
- 239000000843 powder Substances 0.000 claims abstract description 29
- 239000000463 material Substances 0.000 claims abstract description 22
- 239000000853 adhesive Substances 0.000 claims abstract description 14
- 230000001070 adhesive effect Effects 0.000 claims abstract description 14
- 238000004820 blood count Methods 0.000 claims abstract description 13
- 230000003287 optical effect Effects 0.000 claims abstract description 10
- 230000003213 activating effect Effects 0.000 claims abstract description 4
- 230000001902 propagating effect Effects 0.000 claims abstract description 4
- 238000005303 weighing Methods 0.000 claims description 22
- 239000011324 bead Substances 0.000 claims description 13
- 241000186216 Corynebacterium Species 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 11
- 230000010355 oscillation Effects 0.000 claims description 6
- 241000862992 Chondromyces Species 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 239000001965 potato dextrose agar Substances 0.000 claims description 3
- 241000132152 Polymyxa Species 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims 1
- 241000233866 Fungi Species 0.000 abstract description 16
- 230000008569 process Effects 0.000 abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 238000011160 research Methods 0.000 description 10
- 238000000691 measurement method Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 241001248590 Isaria Species 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 241000186031 Corynebacteriaceae Species 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
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- 238000005063 solubilization Methods 0.000 description 2
- 230000007928 solubilization Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 208000035742 Air-borne transmission Diseases 0.000 description 1
- 241000609455 Corynespora cassiicola Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000125496 Crepis vesicaria subsp. taraxacifolia Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000010726 Vigna sinensis Nutrition 0.000 description 1
- 244000042314 Vigna unguiculata Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000005557 airborne transmission Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000003749 cleanliness Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 244000053095 fungal pathogen Species 0.000 description 1
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Classifications
-
- 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
- C12Q1/06—Quantitative determination
Abstract
The invention discloses a method for measuring the conidium quality of corynespora polymorpha. The determination method comprises the following steps: activating and propagating the tested multi-main corynespora strain, sticking multi-main corynespora conidium by using an adhesive material, and obtaining the quality of multi-main corynespora conidium powder by using a difference method; preparing a suspension of conidium of corynespora polymorpha; the suspension was aspirated and dripped onto the surface of a hemocytometer, and the number of conidia of the corynespora polymorpha in the counting zone was counted under an optical microscope, and the mass of individual spores was obtained according to formula 1. The invention uses the analytical balance to weigh the mass of the spore powder, has simple operation, does not need special instruments and equipment, and is suitable for most fungus species; the spore number observed in the counting area is quantified by utilizing the blood cell counting plate under the optical microscope, so that the problem that the fungus spore individuals are tiny and cannot be directly counted by naked eyes can be effectively solved, and only the spore number in the counting area is counted, so that the time-consuming and labor-consuming process of counting all the sample numbers is avoided.
Description
Technical Field
The invention relates to a method for measuring the conidium quality of corynespora polymorpha, and belongs to the technical field of microorganisms.
Background
Corynespora polymorpha Corynespora cassiicola is a worldwide fungus and is commonly found in 57 countries, such as china, the united states, the netherlands, japan, and the like. The strain has a very wide host range, can infect 349 crops in 252 of 90 families, mainly comprises vegetables, fruits, flowers, grains, woods and the like, and has the most serious harm to commercial crops such as rubber, cotton, soybean and the like and vegetables such as cucumber, tomato, cowpea and the like. In 2005, the annual occurrence area of the leaf spot disease of the vegetable corynespora in China exceeds 1000 ten thousand mu, and the economic loss exceeds 50 hundred million yuan. Especially, the leaf spot disease of cucumber corynespora is spread over 19 urban areas such as Beijing, shandong, liaoning and the like until now, the incidence rate in the field is generally 10% -25%, and 60% -70% even 100% in severe cases, which forms a serious threat to the industrial development.
Airborne transmission is the root cause of broad host and broad geographical distribution of pathogenic fungi, and is an important way to cause short-time and large-area outbreaks of diseases. After the plants are infected by the corynespora polymorpha, symptoms (conidiophores and conidiophores) are generated on the surfaces of the attack parts, and the spores are released after maturation and rapidly spread along with air flow. The whole process can be completed in the field from the occurrence of central disease plants to the onset of disease in the whole shed (field) generally in 5 days and 2 days at maximum. The development of fungus air-borne research is a key for restraining disease spreading and spreading, and has important guiding significance for green, safe and high-quality production of crops.
Conidium is a asexual spore of fungi, has the capability of resisting adverse environments such as high temperature, low temperature, dryness, nutrition deficiency and the like, and is also an important propagation medium of fungal diseases. As a carrier of pathogens, spore mass is directly related to its buoyancy and sedimentation velocity, determines the distance and height of transmission, and obviously varies with species, which is an important factor affecting disease prevalence.
The spores of most fungi are micron-sized, the individuals are extremely tiny, and the accurate weighing of the mass of single conidium can not be directly realized by the existing instrument and equipment. To date, there is no suitable method for weighing fungal spores. The definite spore quality is the basis for developing the fungus air transmission research work, and is a scientific problem which is needed to be solved in the pathogenic epidemiology research for a long time, and has a non-negligible meaning in theory and practice. Therefore, there is an urgent need to find a new method for rapid and simple weighing of fungal spore mass.
Disclosure of Invention
The invention aims to provide a method for measuring the quality of conidium of corynespora polymorpha, which is simple to operate, simple and convenient in steps and high in applicability, and can be used for weighing the quality of conidium of fungi, so that the urgent requirement of research on propagation and popularity of fungi is met. The method can realize the weighing of most fungus spores, is helpful for the dermatologist to simulate and predict the germ transmission track, and promotes the further development of fungus epidemiology.
According to the invention, the mass of a large amount of spore powder is weighed by an analytical balance, so that the unrealizable individual research is converted into the operable group research, and the problem of availability of no instrument can be effectively solved; the quantitative observation of the number of spores by utilizing a blood cell counting plate under an optical microscope can effectively solve the problem that the individual fungus spores are tiny and cannot be directly identified and counted by naked eyes; in addition, only 0.1mm of the blood cell counting plate is needed 3 The spore number in the counting area is counted, so that the time-consuming and labor-consuming process of counting the number of all samples is avoided.
The invention realizes the determination of the conidium mass of the corynespora polymorpha by the following method: collecting and weighing the powder of the multi-main corynespora spores, preparing multi-main corynespora suspension, counting the number of the multi-main corynespora conidia in a counting area of a blood cell counting plate, and calculating the mass of single conidia of the multi-main corynespora by a formula.
Specifically, the method for measuring the conidium mass of the corynespora polymorpha provided by the invention comprises the following steps:
s1, activating and propagating a tested corynebacterium polymorpha strain, adhering the corynebacterium polymorpha conidium by using an adhesive material, and obtaining the quality of the corynebacterium polymorpha conidium powder by using a difference method;
s2, preparing a conidium suspension of the corynespora polymorpha;
s3, sucking the suspension of the conidium polycephalum corynespora and dripping the suspension onto the surface of a blood cell counting plate, counting the number of the conidium polycephalum corynespora in a counting area under an optical microscope, and obtaining the mass of single spores according to the following formula (1):
in the above measurement method, in step S1, activation is performed under the following conditions:
on potato dextrose agar medium, culturing in darkness at 28deg.C for 5 days;
propagation was performed under the following conditions:
the culture was carried out at 28℃under ultraviolet light of 265nm in wavelength and 50cm in height for 10 days.
In the above measurement method, in step S1, the adhesive material is weighing paper;
the size of the weighing paper is 3-3.5 cm multiplied by 1-1.5 cm.
In the above measurement method, in step S1, the mass of the corynespora polymorpha spore powder is obtained according to the following method:
placing the adhesive material adhered with the multi-main coryneform conidium into a sterile centrifuge tube, and centrifuging; after the centrifugation is finished, weighing the total mass of the centrifuge tube and the multi-main corynespora spore powder, and further obtaining the mass of the multi-main corynespora spore powder;
during centrifugation, the side of the adhesive material is pressed by a tube cover, so that the adhesive material is prevented from being compressed at the bottom of the centrifuge tube during centrifugation.
In the above measurement method, the centrifugation conditions are as follows:
the rotating speed is 8000-12000 rpm, and the time is 1-2 min.
In the above measurement method, in step S2, the suspension of the conidium polycephalum coryneform bacteria is prepared by using an ethanol aqueous solution with a volume content of 70%.
In the above measurement method, in step S2, during the process of preparing the above-mentioned multi-main corynespora conidium suspension, grinding beads are added to perform vortex oscillation;
the diameter of the grinding beads is 1-3 mm, 6-10 grinding beads are added into each centrifuge tube, and the vortex oscillation time is 3-5 s.
In the above measurement method, in step S3, the magnification of the optical microscope is 100 times;
the number of conidia of the corynespora polymorpha in the counting zone of the hemocytometer is the average of 10 samples taken at random.
In the above measurement method, in step S3, when the density of the suspension of the conidia of the corynebacterium polymorpha is 1mg/mL, the volume of the counting zone is 0.1mm 3 When the mass of individual spores is obtained according to formula (2):
the invention uses the analytical balance to weigh the mass of the spore powder, has simple operation, does not need special instruments and equipment, can be carried out in a general microbiological laboratory, and is suitable for most fungus species. The number of spores observed in the quantitative counting area by utilizing the blood cell counting plate under an optical microscope can not only effectively solve the problem that the individual fungal spores are tiny and cannot be directly counted by naked eyes; and only the spore number in the counting area is counted, so that the time-consuming and labor-consuming process of counting the number of all samples is avoided. The invention greatly improves the speed and feasibility of weighing the fungus spores, and is convenient for the subsequent research of other fungus workers.
Drawings
FIG. 1 is a schematic illustration of different types of spore adhesion materials collected for C.polymorpha; wherein, fig. 1A1, 1A2 and 1A3 are respectively weighing paper, tinfoil paper and common printing paper for spore collection; 1B1, 1B2 and 1B3 are centrifugal tubes for weighing paper, tinfoil paper and common printing paper after centrifugation; fig. 1C1, 1C2, and 1C3 are respectively a weighing sheet, a tinfoil sheet, and a plain printing sheet after centrifugation.
FIG. 2 is a schematic representation of the configuration of a suspension of Isaria polymorpha and its microscopic view; wherein, fig. 2A and fig. 2B are respectively spore suspensions prepared by sterile water and 70% ethanol solution in a centrifuge tube; fig. 2C and 2D are microscopic views of the spore suspension without and after shaking with the addition of grinding beads, respectively, in a centrifuge tube.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The invention provides a method for measuring the conidium quality of corynespora multocida, which comprises the steps of collecting and weighing the conidium powder of the corynespora multocida, preparing spore suspension, counting the number of spores in a counting area of a blood cell counting plate and the like, and specifically comprises the following steps:
1) Activated test corynespora polymorpha strains were propagated under ultraviolet light (cultured at 28 ℃ for 10 days) to induce mass production of spores. When the colony grows up to be full of the culture medium, adhering spore powder by using weighing paper, centrifuging for 2min at 12000rpm in a 1.5mL sterile centrifuge tube, collecting the spore powder to the bottom of the centrifuge tube, and calculating by a mass difference method to obtain the mass of the spore powder.
2) An ethanol solution with the volume fraction of 70% is used for preparing a spore suspension with the density of 1mg/mL, and the process ensures thorough spore dispersion and uniform and stable spore suspension by adding grinding beads and carrying out vortex oscillation.
3) Taking 10 μl of the prepared spore suspension, dripping into counting area of blood cell counting plate, and counting with 100 times optical microscope to obtain a volume of 0.1mm 3 The number of conidia of the corynespora polymorpha in the counting zone is the average of 10 samples taken randomly.
4) The mass of the single multi-main corynespora conidia is calculated by a simplified mass calculation formula (2)).
The invention takes the multi-main corynespora strain GS1 ('Wu Jun. Research on genetic diversity of multi-main corynespora in China. National academy of agricultural sciences, 2019.') as a test material, the mass of spore powder is weighed three times each time to obtain an average value, and the number of spores in a counting area of a blood cell counting plate is the average value of 10 sampling statistics, and three times of repetition are arranged. The results show that: the mass of the single conidium of the corynebacterium polymorpha is 5.69+/-0.67 ng.
Example 1 screening of spore powder for optimal adhesion Material
Lightly sticking the clean adhesive material on a multi-main corynespora plate full of culture medium to stick a layer of spore powder on the surface of the adhesive material. The material of the adhesive spore powder was then carefully placed into an empty 1.5mL sterile centrifuge tube using forceps, the side of the material was pressed with a tube cap, and centrifuged at 12000rpm for 2min.
The weighing paper, the tinfoil paper and the common printing paper are arranged for 3 materials, and the best spore powder adhesive material is screened out by observing the spore powder amount collected at the bottom of the centrifugal tube after centrifugation and the cleanliness of the surface of the adhesive material after centrifugation.
As a result, as shown in FIG. 1, materials which are easily centrifuged by the spore quilt are weighing paper and tinfoil paper, and a large amount of spores remain after the ordinary printing paper is centrifuged. In addition, after centrifugation, the spore powder amount at the bottom of the centrifuge tube with the weighing paper is obviously more than that at the bottom of the centrifuge tube with the tinfoil paper. Thus, the most suitable material for spore adhesion is weighing paper.
Example 2 screening of the optimal formulation of spore suspension
According to the mass of spore powder collected at the bottom of the centrifuge tube, adding a proper amount of solution to prepare spore suspension with the density of 1 mg/mL. Sterile water and 70% ethanol 2 solutions were set and the optimal solution for spore suspension was determined by observing the dissolution of the spore powder after 5s vortex shaking and the wall built-up in the centrifuge tube. Subsequently, the two treatments, no grinding beads and grinding beads added, were placed in the centrifuge tube and the optimal method for spore suspension configuration was determined by observing the degree of dispersion of spores in the spore suspension after vortexing for 5s.
As shown in FIG. 2, it can be seen that the use of 70% ethanol to solubilize the spore powder, the degree of solubilization of the spore suspension after vortexing was superior to direct solubilization using sterile water, while the residual spore suspension on the centrifuge tube wall was significantly less water soluble. In addition, as the multi-main coryneform bacteria are in chain growth, spore suspension spores in the treatment configuration without adding grinding beads are clustered, and the discrete degree of spore suspension spores in the treatment configuration with adding grinding beads is greatly improved, so that the subsequent counting by using a blood cell counting plate is facilitated. Therefore, the optimal preparation method of the spore suspension preferably adopts 70% ethanol as a solvent, and a proper amount of grinding beads are placed in a centrifuge tube to perform vortex oscillation.
Example 3 determination of conidium mass of Isaria polymorpha
mu.L of the suspension of Isaria polymorpha having a density of 1mg/mL was pipetted using a pipette, added dropwise to the counting area of a blood cell counting plate, and the statistical volume was 0.1mm under a 100-fold optical microscope 3 The number of conidia of the corynespora polymorpha in the counting zone was recorded as 10 fields per repeat observation. The mass of individual conidia is then calculated according to formula (2).
TABLE 1 statistics of calculated mass of conidia of Isaria polymorpha
As shown in Table 1, the mass of single conidium of Corynebacterium polymyxa was 4.95-6.25 ng, and the average was 5.69.+ -. 0.67ng.
The method is suitable for measuring the mass of the conidium of the corynespora polymorpha, and the mass of a large amount of spore powder is weighed by using an analytical balance, so that the unrealizable individual research is converted into the operable group research, and the problem that no instrument is available can be effectively solved; by preparing spore powder into spore suspension, only counting the number of spores observed in a counting area of a blood cell counting plate, the problem that fungus spores are tiny and unrecognizable by naked eyes is solved, and the time-consuming and labor-consuming process of counting the number of all samples to be measured is greatly simplified.
The above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that do not undergo the inventive actions should be covered in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.
Claims (7)
1. A method for measuring the conidium mass of corynespora polymorpha comprises the following steps:
s1, activating and propagating a tested corynebacterium polymorpha strain, adhering the corynebacterium polymorpha conidium by using an adhesive material, and obtaining the quality of the corynebacterium polymorpha conidium powder by using a difference method;
activating and propagating the tested corynebacterium polymorpha strain on a potato dextrose agar culture medium;
the adhesive material is weighing paper;
in the step S1, the mass of the corynespora polymorpha spore powder is obtained according to the following method:
placing the adhesive material adhered with the multi-main coryneform conidium into a sterile centrifuge tube, and centrifuging; after the centrifugation is finished, weighing the total mass of the centrifuge tube and the multi-main corynespora spore powder, and further obtaining the mass of the multi-main corynespora spore powder;
s2, preparing a multi-main corynespora conidium suspension by adopting an ethanol water solution with the volume content of 70%;
in the step S2, during the preparation of the multi-main corynespora conidium suspension, grinding beads are added for vortex oscillation;
s3, sucking the suspension of the conidium polycephalum corynespora and dripping the suspension onto the surface of a blood cell counting plate, counting the number of the conidium polycephalum corynespora in a counting area under an optical microscope, and obtaining the mass of single spores according to the following formula (1):
2. the method according to claim 1, wherein: in step S1, the activation is performed under the following conditions:
on potato dextrose agar medium, culturing in darkness at 28deg.C for 5 days;
propagation was performed under the following conditions:
the culture was carried out at 28℃under ultraviolet light of 265nm in wavelength and 50cm in height for 10 days.
3. The assay method according to claim 1 or 2, wherein: in step S1, the size of the weighing paper is (3-3.5) cm× (1-1.5) cm.
4. The method according to claim 1, wherein: the centrifugation conditions are as follows: the rotating speed is 8000-12000 rpm, and the time is 1-2 min.
5. The assay method according to claim 1 or 2, wherein: in the step S2, the diameter of the grinding beads is 1-3 mm, 6-10 grinding beads are added into each centrifuge tube, and the vortex oscillation time is 3-5S.
6. The assay method according to claim 1 or 2, wherein: in step S3, the magnification of the optical microscope is 100 times;
the number of conidia of the corynespora polymorpha in the counting zone of the hemocytometer is the average of 10 samples taken at random.
7. The assay method according to claim 1 or 2, wherein: in step S3, when the density of the conidium suspension of the corynebacterium polymyxa is 1mg/mL, the volume of the counting area is 0.1mm 3 When the mass of individual spores is obtained according to formula (2):
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| CN110777082A (en) * | 2019-12-06 | 2020-02-11 | 海南大学 | Solid culture medium for improving spore yield of isaria clavuligerus and culture method |
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| Title |
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| 松栎柱锈菌春孢子显微计数方法的筛选;许佳瑶;赵博;瓮岳太;张月;杨波;薛煜;;东北林业大学学报(03);第110-113页 * |
| 橡胶树多主棒孢菌室内产孢条件的优化;时涛;蔡吉苗;李超萍;林春花;刘先宝;黄贵修;;热带作物学报(第01期);第98-105页 * |
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