阅读说明：本技术 一种多主棒孢菌分生孢子质量的测定方法 (Method for measuring quality of conidia of corynebacterium polystachyum ) 是由 柴阿丽 李宝聚 赵倩 石延霞 谢学文 李磊 范腾飞 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种多主棒孢菌分生孢子质量的测定方法。所述测定方法包括如下步骤：将供试多主棒孢菌菌株进行活化和扩繁,利用粘附材料粘取多主棒孢菌分生孢子,利用差值法得到多主棒孢菌孢子粉的质量；配制多主棒孢菌分生孢子悬浮液；吸取悬浮液并滴加至血球计数板的表面,在光学显微镜下统计计数区多主棒孢菌分生孢子的数量,按照式1得到单个孢子的质量。本发明利用分析天平对孢子粉的质量进行称量,操作简单,无需特殊仪器设备,适用于绝大多数真菌物种；在光学显微镜下利用血球计数板定量计数区观察到的孢子个数,不仅能有效解决真菌孢子个体微小,肉眼无法直接计数的问题,而且仅对计数区的孢子个数进行统计,避免了对全部样品个数进行统计耗时、费力的过程。(The invention discloses a method for measuring the quality of conidia of a polyspora clavuligera. The determination method comprises the following steps: activating and propagating the tested corynespora polybotrya strain, picking up the conidia of the corynespora polybotrya by using an adhesive material, and obtaining the quality of the spore powder of the corynespora polybotrya by using a difference method; preparing a conidium suspension of the corynebacterium polystachyum; and (3) sucking the suspension, dropwise adding the suspension onto the surface of a blood counting chamber, counting the number of conidia of the corynebacterium polybotrys in the counting area under an optical microscope, and obtaining the quality of a single spore according to the formula 1. The method utilizes the analytical balance to weigh the mass of the spore powder, is simple to operate, does not need special instruments and equipment, and is suitable for most fungal species; the spore number observed in the quantitative counting area of the blood counting chamber is utilized under an optical microscope, so that the problems that fungal spore individuals are small and naked eyes cannot count directly can be effectively solved, the spore number in the counting area is only counted, and the time-consuming and labor-consuming process of counting all the sample numbers is avoided.)

1. A method for measuring the quality of conidia of a polyspora, which comprises the following steps:

s1, activating and expanding the test corynebacterium polygamum strains, picking up the conidia of the corynebacterium polygamum by using an adhesive material, and obtaining the quality of the spore powder of the corynebacterium polygamum by using a difference method;

s2, preparing a conidium suspension of the corynebacterium polygamum;

s3, sucking the conidia suspension of the corynebacterium polybotrys and dripping the conidia suspension onto the surface of a blood counting chamber, counting the number of the conidia of the corynebacterium polybotrys in a counting area under an optical microscope, and obtaining the quality of a single spore according to the following formula (1):

2. the method for measuring according to claim 1, wherein: in step S1, activation is performed under the following conditions:

culturing on potato glucose agar medium at 28 deg.C in dark for 5 days;

propagation was carried out under the following conditions:

culturing at 28 deg.C under ultraviolet light with wavelength of 265nm and height of 50cm for 10 days.

3. The assay method according to claim 1 or 2, characterized in that: in step S1, the adhesive material is weighing paper;

the weighing paper is 3-3.5 cm multiplied by 1-1.5 cm.

4. The assay method according to any one of claims 1 to 3, wherein: in step S1, the quality of the polyspora spore powder is obtained according to the following method:

placing the adhesive material adhered with the conidia of the multi-main corynebacterium in a sterile centrifuge tube, and then centrifuging; and after the centrifugation is finished, weighing the total mass of the centrifuge tube and the multi-principle corynebacterium sp spore powder, and further obtaining the mass of the multi-principle corynebacterium sp spore powder.

5. The method for measuring according to claim 4, wherein: the centrifugation conditions were:

the rotation speed is 8000-12000 rpm, and the time is 1-2 min.

6. The assay method according to any one of claims 1 to 5, wherein: in step S2, an ethanol aqueous solution with a volume content of 70% is used to prepare the corynebacterium polygamum conidia suspension.

7. The assay method according to any one of claims 1 to 6, wherein: in the step S2, adding grinding beads to carry out vortex oscillation in the process of preparing the polyspora conidia suspension;

the diameter of the grinding beads is 1-3 mm, 6-10 grinding beads are added into each centrifugal tube, and the vortex oscillation time is 3-5 s.

8. The assay method according to any one of claims 1 to 7, wherein: in step S3, the magnification of the optical microscope is 100 times;

the counting area of the blood counting plate is the average value of 10 random sampling times of the number of the conidia of the corynebacterium polystachyum.

9. The assay method according to any one of claims 1 to 8, wherein: in step S3, when the density of the corynebacterium polyspora conidia suspension is 1mg/mL, the volume of the counting area is 0.1mm³The mass of individual spores was then obtained according to formula (2):

Technical Field

The invention relates to a method for measuring the quality of conidia of polyspora, belonging to the technical field of microorganisms.

Background

The corynebacterium polymorpha corynebacterium cassiicola is a worldwide fungus and commonly occurs in 57 countries such as china, the united states, the netherlands, japan, and the like. The host range of the strain is very wide, and the strain can infect 349 crops of 252 genus of 90 family, mainly comprising vegetables, fruits, flowers, grains, woods and the like, wherein the strain has the most serious harm to economic crops such as rubber, cotton, soybeans and the like and vegetables such as cucumbers, tomatoes, cowpeas and the like. Since 2005, the vegetable leaf blotch in China has occurred in an area of over 1000 million acres per year, and the economic loss is over 50 million yuan. Especially cucumber corynespora leaf spot disease, has spread to 19 provinces such as Beijing, Shandong, Liaoning, etc., the field incidence is generally 10% -25%, and can reach 60% -70% or even 100% when serious, thus forming a serious threat to the industry development.

Air transmission is the root cause of the broad host and wide regional distribution of pathogenic fungi, and is an important way for causing the outbreak of diseases in short time and large area. After the corynebacterium polygamum infects plants, symptoms (conidiophores and conidia) are generated on the surfaces of diseased parts, and the spores are released after being mature and are rapidly diffused along with airflow. The disease is generally required to be 5 days from the occurrence of central diseased plants to the occurrence of diseases in the whole shed (field), and the whole process can be completed in 2 days at the fastest speed. The development of the fungus air transmission research is the key point for restraining the spread of the disease, and has important guiding significance for green, safe and high-quality production of crops.

Conidia are asexual spores of fungi, have the capability of resisting adverse environments such as high temperature, low temperature, dryness and nutritional deficiency, and are important transmission media of fungal diseases. As a pathogen transmitter, spore mass is directly related to buoyancy and sedimentation velocity thereof, determines transmission distance and height, obviously varies with species, and is an important factor influencing disease prevalence.

Spores of most fungi are in micron-sized and are extremely tiny, and the existing instrument and equipment cannot directly realize accurate weighing of the mass of a single conidium. There is no suitable method for weighing the mass of fungal spores to date. The clear spore quality is the basis for developing the work of fungus airborne research, and is a scientific problem to be solved urgently by pathogenic epidemiological 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 rapidly and easily weighing the fungal spore mass.

Disclosure of Invention

The invention aims to provide a method for measuring the conidium quality of corynebacterium polystachyum, which is simple to operate, simple and convenient in steps and strong in applicability, can be used for weighing the conidium quality of fungi, and solves the urgent need of the propagation and prevalence research of the fungi. The method can realize the weighing of the mass of most fungal spores, is helpful for a mycologist to simulate and predict the germ propagation track, and promotes the further development of the fungal epidemiology.

The invention adopts the analytical balance to weigh the mass of a large amount of spore powder, converts the unachievable individual research into the operable group research, and can effectively solve the problem of availability of equipment without instruments; the number of spores is quantitatively observed by using a blood counting chamber under an optical microscope, so that the problem that the individual fungal spores are tiny and cannot be directly identified and counted by naked eyes can be effectively solved; in addition, only 0.1mm of blood counting plate is needed³The counting of the number of spores in the counting area avoids the time-consuming and labor-consuming process of counting the number of all samples.

The invention realizes the determination of the quality of the conidia of the corynebacterium polystachyum by the following method: collecting and weighing the powder of the spores of the polyspora, preparing suspension of the polyspora, counting the number of the conidia of the polyspora in a counting area of a blood counting chamber, and calculating the mass of the single conidia of the polyspora through a formula.

Specifically, the method for measuring the conidium quality of the corynebacterium polystachyum provided by the invention comprises the following steps:

s1, activating and expanding the test corynebacterium polygamum strains, picking up the conidia of the corynebacterium polygamum by using an adhesive material, and obtaining the quality of the spore powder of the corynebacterium polygamum by using a difference method;

s2, preparing a conidium suspension of the corynebacterium polygamum;

s3, sucking the conidia suspension of the corynebacterium polybotrys and dripping the conidia suspension onto the surface of a blood counting chamber, counting the number of the conidia of the corynebacterium polybotrys in a counting area under an optical microscope, and obtaining the quality of a single spore according to the following formula (1):

in the above measurement method, in step S1, activation is performed under the following conditions:

culturing on potato glucose agar medium at 28 deg.C in dark for 5 days;

propagation was carried out under the following conditions:

culturing at 28 deg.C under ultraviolet light with wavelength of 265nm and height of 50cm for 10 days.

In the above-described measuring method, in step S1, the adhesive material is weighing paper;

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 spore powder of polyspora is obtained as follows:

placing the adhesive material adhered with the conidia of the multi-main corynebacterium in a sterile centrifuge tube, and then centrifuging; after the centrifugation is finished, weighing the total mass of the centrifuge tube and the multi-principle corynebacterium sp spore powder, and further obtaining the mass of the multi-principle corynebacterium sp spore powder;

during centrifugation, the side of the adhesive material is pressed by a tube cover to prevent the adhesive material from being compressed at the bottom of the centrifugal tube during centrifugation.

In the above measurement method, the centrifugation conditions are as follows:

the rotation speed is 8000-12000 rpm, and the time is 1-2 min.

In the above determination method, in step S2, the conidia suspension of corynebacterium polystachyum is prepared using an ethanol aqueous solution with a volume content of 70%.

In the above-described measurement method, in step S2, grinding beads are added to the suspension of the conidia of corynebacterium polygamum to perform vortex oscillation;

the diameter of the grinding beads is 1-3 mm, 6-10 grinding beads are added into each centrifugal 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 counting area of the blood counting plate is the average value of 10 random sampling times of the number of the conidia of the corynebacterium polystachyum.

In the above-mentioned measuring method, in step S3, when the density of the conidia suspension of the Cladosporium irregulare is 1mg/mL, the volume of the counting region is 0.1mm³The mass of individual spores was then obtained according to formula (2):

the invention utilizes the analytical balance to weigh the spore powder, has simple operation, does not need special instruments and equipment, can be carried out in a common microorganism laboratory, and is suitable for most fungus species. The number of spores observed in a quantitative counting area of a blood counting chamber under an optical microscope can effectively solve the problem that the number of fungal spore individuals is tiny and cannot be directly counted by naked eyes; and the counting is only carried out on the number of spores in the counting area, 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 the feasibility of weighing the fungal spore quality, and is convenient for the follow-up research of other fungus workers.

Drawings

FIG. 1 is a schematic diagram showing the collection of different types of spore adhesion materials on a plurality of Excellent spores; wherein, fig. 1a1, fig. 1a2 and fig. 1A3 are weighing paper, tinfoil paper and common printing paper for spore collection, respectively; FIG. 1B1, FIG. 1B2, FIG. 1B3 are centrifuge tubes for weighing paper, tinfoil paper, and common printing paper after centrifugation, respectively; fig. 1C1, 1C2, and 1C3 show centrifuged weighing paper, foil paper, and plain printing paper, respectively.

FIG. 2 shows the configuration of a suspension of Cladosporium polyspora and its microscopic field of view; wherein, fig. 2A and fig. 2B are spore suspensions prepared in a centrifuge tube by using sterile water and a 70% ethanol solution, respectively; FIGS. 2C and 2D are microscopic views of the spore suspension in the centrifuge tube without and with shaking of the grinding beads, respectively.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention provides a method for measuring the conidium quality of a polyspora, which comprises the steps of collecting and weighing sporophore spore powder of the polyspora, configuring spore suspension, counting the number of spores in a counting area of a blood counting plate and the like, and specifically comprises the following steps:

1) the activated test strains of Bacteroides polygama were propagated under UV light (incubation for 10 days at 28 ℃) to induce mass production of spores. And when the bacterial colony grows over the culture medium, adhering spore powder to weighing paper, centrifuging at 12000rpm in a 1.5mL sterile centrifuge tube for 2min, 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) A spore suspension with the density of 1mg/mL is prepared by using an ethanol solution with the volume fraction of 70 percent, and the spore is thoroughly dispersed and the spore suspension is uniform and stable by adding grinding beads and carrying out vortex oscillation in the process.

3) Taking 10 μ L of prepared spore suspension, dripping into counting area of blood counting plate, and counting volume under 100 times optical microscope to be 0.1mm³The number of the conidia of the corynebacterium polystachyum in the counting area is an average value of 10 random sampling times.

4) The mass of a single polyspora conidia is calculated by a simplified mass calculation formula (2)).

The invention takes the corynebacterium polystachyum strain GS1 (Wujun, the research on genetic diversity of the corynebacterium polystachyum in China, Chinese academy of agricultural sciences, 2019.) as a test material, the mass of spore powder is weighed three times each time, an average value is obtained, the number of spores in a counting area of a blood counting plate is an average value of 10 sampling statistics, and three times of repetition are arranged. The results show that: the mass of single conidia of the polyspora clavuligera is 5.69 +/-0.67 ng.

Example 1 screening of spore powder for optimum adhesion Material

And slightly sticking and pressing the clean adhesive material on a multi-main-clavispora flat plate full of culture medium to stick a layer of spore powder on the surface of the adhesive material. The spore powder-adhered material was then carefully placed into an empty 1.5mL sterile centrifuge tube using forceps, and the sides of the material were pressed with the tube lid at 12000rpm and centrifuged for 2 min.

Set up and weigh paper, tin foil paper, 3 kinds of materials altogether of ordinary printing paper, through observing the spore powder volume that centrifugation back centrifuging tube bottom was collected and the clean degree on adhesion material surface after the centrifugation, select best spore powder adhesion material.

As a result, as shown in FIG. 1, the materials from which spores are easily centrifuged are weighing paper and tinfoil paper, and a large amount of spores remain after centrifugation of general printing paper. After additional centrifugation, the amount of spore powder at the bottom of the centrifuge tube containing the weighing paper was significantly greater than that at the bottom of the centrifuge tube containing the tinfoil paper. Therefore, the most suitable material for spore adhesion is the weighing paper.

Example 2 screening of optimal preparation method of spore suspension

According to the mass of the spore powder collected at the bottom of the centrifuge tube, a proper amount of solution is added to prepare spore suspension with the density of 1 mg/mL. Sterile water and 70% ethanol 2 solutions were set up and the optimal solution for spore suspension was determined by observing the dissolution of the spore powder after vortexing for 5s and the wall build-up in the centrifuge tube. Subsequently, both treatments of no and addition of grinding beads in the centrifuge tube were set up and the optimal method for spore suspension preparation was determined by observing the degree of dispersion of spores in the spore suspension after vortexing for 5 s.

The results are shown in FIG. 2, and it can be seen that the spore powder was dissolved with 70% ethanol to a greater extent after vortexing than directly with sterile water, while the residual spore suspension on the centrifuge tube walls was significantly less water soluble. In addition, as the corynebacterium polygamum is streptozotocin, spores of the spore suspension prepared by the treatment without adding the grinding beads agglomerate and gather, and the dispersion degree of the spores of the spore suspension prepared by the treatment with adding the grinding beads is greatly improved, so that the subsequent counting by using a blood counting chamber 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 centrifugal tube and vortex oscillation is carried out.

Example 3 measurement of the quality of conidia of the Phycomyces polystachyus

Using a pipette to suck 10 mu L of the suspension of the polyspora spinosa with the density of 1mg/mL, dripping the suspension into a counting area of a blood counting chamber, and counting the volume of 0.1mm under a 100-time optical microscope³The number of conidia of the corynebacterium polystachyum in the zone was counted and 10 fields were recorded for each replicate observation. The mass of the individual conidia was then calculated according to equation (2).

TABLE 1 statistics of the quality calculation data of the conidia of the multi-dominant corynebacteria

As shown in Table 1, the mass of a single conidium of the Bacterocarpus polystachyus is 4.95-6.25 ng, and the average is 5.69 + -0.67 ng.

The method is suitable for measuring the mass of the conidia of the corynebacterium polystachyum, uses the analytical balance to weigh the mass of a large amount of spore powder, converts the unachievable individual research into the operable group research, and can effectively solve the problem of availability of equipment without instruments; the spore powder is prepared into spore suspension, and the number of spores observed in a counting area of the blood counting chamber is only counted, so that the problems that the fungal spore individuals are tiny and can not be identified by naked eyes are solved, and the time-consuming and labor-consuming process of counting the number of all samples to be detected is greatly simplified.

The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive activities should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.