CN112592831A - Method for quickly separating single conidia of panax notoginseng teres - Google Patents
Method for quickly separating single conidia of panax notoginseng teres Download PDFInfo
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Abstract
The invention discloses a method for rapidly separating single conidia of panax notoginseng round spot germs, (1) air-drying panax notoginseng round spot leaves collected in the field at normal temperature; (2) observing the lesion site under a biological microscope, searching for a dense place of conidia, sucking 10 mu L of sterile water by using a pipette gun, beating the place, repeatedly sucking for 4-5 times, enabling the conidia on the lesion site to enter the sterile water to form a spore suspension, sucking the spore suspension onto an aqueous agar culture medium, and lightly coating the aqueous agar culture medium uniformly. (3) Immediately searching conidia on the surface of the water agar culture medium under a microscope, dipping sterile water by using a sterilization pick needle, and transferring to the position of the conidia to adsorb single conidia; (4) and (3) marking the picking needle adsorbed with the single spore on a PDA culture medium for multiple times, sealing the marking with a sealing film, and culturing in a constant-temperature incubator. According to the invention, single conidium is obtained by directly utilizing the adhesion effect of sterile water on the picking needle, so that the separation effect is more accurate and the pollution is not easy to occur.
Description
Technical Field
The invention relates to the technical field of spore separation, in particular to a method for quickly separating single conidium of panax notoginseng circinelloides.
Background
The pathogenic bacteria of the round spot disease of the panax notoginseng is Acer nikosporum (Mycocentrospora acerina (Hartig) Deighton), which belongs to the fungus of the Acer nikosporum, which is firstly discovered in Wenshan state in Yunnan province in 1993 and is a very serious disease which damages the overground part of the panax notoginseng. Acer nikosporum prefers low temperature and high humidity, and is rapidly propagated, the disease incidence can reach more than 80% in rainy season every year in pseudo-ginseng growing areas, and the pseudo-ginseng circular spot can cause the top-off of pseudo-ginseng in extreme conditions, thereby causing serious economic loss to Qinong. Acer nikosporum was first reported to infect leaf of Acer nikosporum in 1880 in Germany, which is mainly distributed in Europe and North America, has a wide host range, parasitizes in 16 plants such as Aceraceae, Cruciferae and Solanaceae, and causes diseases of various vegetables and ornamental plants such as carrot, lettuce and primula under low-temperature and high-humidity conditions (Fourier et al, 1995). At present, Acer nikosporum in China is reported to cause two plant diseases, one is asarum leaf blight, and the other is panax notoginseng round spot, which can cause destructive damage to hosts. (handsome et al, 1995; Cheng et al, 1997).
Acer nikosporum overwintering in soil or plant disease residues by virtue of chlamydospores is a main primary infection source of panax notoginseng round spot in the next year, the Acer nikosporum usually attacks and becomes popular in rainy seasons every year, the leaf spots in the early stage of attack are small and are circular or nearly circular water stain-shaped, brown infection dots can be seen in the centers of the spots, then the spots gradually expand, are brown or grey brown, and a ring-striated white, gray or pink white spore meristem pile can be observed on the surfaces of the spots. The conidia can be spread by wind power or rainwater splash, and the large-area occurrence of the panax notoginseng round spot is caused. Short and thick conidiophores of Acer nigeri conidia, full-wall buds combined with axial type spore production and obvious spore marks. Conidium has a length of (60-) 150-; sometimes there is a downward stab-like appendage filament on the side below the basal cell.
The acer mono-spore strain is obtained by directly separating from field panax notoginseng round spot disease leaf disease spots because the acer mono-spore is difficult to generate conidia on an artificial culture medium, and a reliable technical means for enabling the acer mono-spore to stably generate the conidia on the artificial culture medium does not exist at present. CN102140432A discloses a method for separating individual spores of fungi, which mainly comprises the steps of inserting a glass capillary into a Pasteur pipette from the tip, picking the fungi material to be separated by using a dissecting needle, enabling the center of the orifice of the capillary to be opposite to and close to the spores, transferring the spores to the upper part of a culture medium, and completing the separation of the euspores. The key steps are that a part of fungus colonies or lesion tissues to be separated are picked by using a dissecting needle, and as for the lesion tissues, conidia are not necessarily arranged on the lesion tissues, and the lesion tissues are directly taken to be used as spore suspension, so that more impurities are generated, and the spores are not easy to disperse. In addition, the used tools comprise a Pasteur pipette and a capillary tube, a single spore is sucked into the capillary tube, the glue head is kept still, the technical and experience requirements of operators are high, and the method is not suitable for large-batch single-spore separation of the fungus. CN 108587929A discloses a method for rapidly separating Pyricularia oryzae monospore on a field Pyricularia oryzae spot, which mainly comprises the steps of coating rice leaves containing typical Pyricularia oryzae monospore on the surface of a water agar culture medium, culturing a water agar plate with Pyricularia oryzae conidia, searching single and germinated Pyricularia oryzae conidia under a stereoscopic microscope, and separating the Pyricularia oryzae monospore to obtain an agar block containing the Pyricularia oryzae monospore. The method directly coats the surface of the water agar culture medium with the scab, is inaccurate in positioning of conidia, has more other impurities, needs to cut an agar block containing the single spore, and has more operation steps and is relatively troublesome.
Therefore, in the research on the biological characteristics and pathogenic mechanism of the panax notoginseng round spot disease, the development of a rapid, stable and efficient monospore separation method suitable for separating a large batch of samples is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for rapidly separating single conidia of panax notoginseng teres punctatus comprises the following steps:
(1) collecting pseudo-ginseng leaves with typical pseudo-ginseng round spot disease spots in a field, wherein the spots are required to have obvious white mildew layers which can be seen by naked eyes, then putting the pseudo-ginseng leaves into paper sampling envelopes, paying attention to the fact that each leaf is independently placed into one envelope and cannot be overlapped, returning the pseudo-ginseng leaves to a laboratory, putting the pseudo-ginseng leaves into envelope paper bags, and naturally drying the pseudo-ginseng leaves in a ventilated and dry place;
(2) shearing the disease spot part of the air-dried disease spot leaf by using scissors, placing the cut disease spot part on a glass slide, observing the region of a conidium pile on the disease spot by using a 10 x 10 times microscope, locking the region, sucking sterile water by using a 10 mu L range pipette gun to hit the region and repeatedly sucking for 4-5 times, allowing conidia to enter the sterile water, finally sucking the spore liquid onto a water agar culture medium, and slightly and uniformly coating by using a sterilized coater;
(3) placing the water agar culture dish coated with conidia under a microscope for observation, dipping a pre-red pick needle in sterile water for several seconds and then moving the pick needle to the visual field of the microscope when a single conidia is observed, then slowly moving the pick needle to the vicinity of the conidia, slightly touching the conidia by one side of the pick needle to adhere the conidia to the pick needle, and then quickly slightly scratching a plurality of lines on the PDA culture dish by the tip of the pick needle;
(4) and (4) putting the marked PDA culture dish into a constant-temperature incubator for culture until the single conidia grow into colonies.
Further, the preparation method of the water agar medium in the step (2) comprises the following steps: weighing 20g of agar powder in 1L of distilled water or deionized water, heating and boiling until the agar powder is completely dissolved, subpackaging, sterilizing at 121 ℃ for 20 minutes under high pressure, and pouring the mixture into a flat plate for later use.
Further, the step (3) needs to be performed in a clean bench, and before the microscope is placed in the clean bench, the microscope body needs to be wiped by 75% alcohol, and the microscope can be used after being irradiated by an ultraviolet lamp for 30 minutes.
Further, in the step (3), microscopic observation of single conidia on the surface of the water agar is carried out under a 10 × 10 microscope.
Further, the picking needle in the step (3) is made by installing an inoculating filament on an inoculating stick, and when the inoculating needle is used, the inoculating needle needs to be burnt on an alcohol lamp in advance, and then the tip part of the inoculating needle is dipped in sterile water for a plurality of seconds.
Further, in the step (3), after a single conidium is observed under a microscope, the conidium is moved to the center of the visual field, the tip part of the picking needle is moved to the center of the visual field, then the tip part of the slow moving needle is close to the conidium, the conidium is adsorbed by the tip part, when the conidium is adsorbed on the picking needle under the observation of an ocular lens, the picking needle is rapidly transferred to slightly cut a plurality of lines on the PDA culture medium.
Further, the PDA flat plate marked with the lines in the step (4) is placed into an illumination incubator for culture, the temperature is set to be 20 ℃, the full illumination culture is carried out, and obvious monospore bacterial colonies can be seen after the culture is carried out for 5-7 days.
Compared with the prior art, the invention has the advantages that: by adopting the technical scheme, the invention has the following advantages:
(1) the invention provides a new monospore separation method aiming at panax notoginseng round spot pathogenic bacteria, and fills the blank that no method for separating the monospores of the pathogenic fungi exists in the prior art. In addition, the method has the advantages of simple operation process, short period and high success rate, and is suitable for processing large-batch samples.
(2) According to the invention, conidium suspension liquid is not required to be prepared independently, and the conidium is directly obtained at the lesion site without dilution, so that the method is convenient and fast, saves time and improves efficiency.
(3) The invention does not need to make agar slides and cut agar blocks, but directly utilizes the adhesion effect of the sterile water on the picking needle to obtain single conidia, the separation effect is more accurate, and the pollution is not easy to occur.
(4) Tools used by the invention, such as a microscope, a paper envelope, an inoculation rod, an inoculation wire, a liquid transfer gun and the like are laboratory stock materials, special equipment does not need to be purchased, and the operation can be met in a common laboratory. In addition, the invention has simple operation, can be mastered by general experimenters after a plurality of times of training, and is simple and easy to learn.
Drawings
FIG. 1 is a schematic diagram of the operation of a pipette;
FIG. 2 shows molecular spores of the air-dried diseased leaf and spot of round spot of Panax notoginseng;
FIG. 3 is a view showing repeated aspiration of the conidium mass of the lesion with a pipette;
FIG. 4 shows picking single conidia on the surface of water agar medium;
FIG. 5 is a single conidia of the surface of a microscopic agar medium;
FIG. 6 is a PDA culture dish after streaking;
FIG. 7 is the morphology of a single conidiophore after 5 days of culture in an incubator.
Wherein, 1, a pipette, 2, spore liquid absorbed by the pipette head, 3, panax notoginseng round spot disease spots, 4 and panax notoginseng leaves.
Detailed Description
The method for rapidly separating single conidia of panax notoginseng teres variegatus of the present invention is further described in detail with reference to the accompanying drawings.
The present invention will be described in detail with reference to the accompanying drawings.
A method for rapidly separating single conidia of panax notoginseng teres punctatus comprises the following steps:
(1) collecting pseudo-ginseng leaves 4 with typical pseudo-ginseng round spot disease spots 3 in a field, wherein obvious white mildew layers which can be seen by naked eyes are required to be arranged at the spots, then putting the spots into paper sampling envelopes, paying attention to that each leaf is independently placed into one envelope and cannot be superposed, conveying the leaves back to a laboratory, putting the diseased leaves into envelope paper bags, and naturally drying the leaves in a ventilated and dry place;
(2) shearing the disease spot part of the air-dried disease spot leaf by using scissors, placing the cut disease spot part on a glass slide, observing the region of the conidium pile on the disease spot by using a 10 x 10 times microscope, locking the region, sucking sterile water by using a 10 mu L range pipette 1 to beat the region, repeatedly sucking for 4-5 times, allowing conidia to enter the sterile water, sucking the spore liquid 2 to a water agar culture medium, and lightly and uniformly coating by using a sterilized coating device;
(3) placing the water agar culture dish coated with conidia under a microscope for observation, dipping a pre-red pick needle in sterile water for several seconds and then moving the pick needle to the visual field of the microscope when a single conidia is observed, then slowly moving the pick needle to the vicinity of the conidia, slightly touching the conidia by one side of the pick needle to adhere the conidia to the pick needle, and then quickly slightly scratching a plurality of lines on the PDA culture dish by the tip of the pick needle;
(4) and (4) putting the marked PDA culture dish into a constant-temperature incubator for culture until the single conidia grow into colonies.
Example 1:
a. a penman collects a panax notoginseng round spot disease sample in 7 months in 2019 in Shanshan Zhongbei county of Yunnan province, selects panax notoginseng leaves with typical characteristics of panax notoginseng round spot disease and white or brown mildew layers on the disease spots, puts the panax notoginseng leaves into paper envelopes after being cut off by scissors, divides one envelope into a plurality of diseased leaves, and then brings the envelopes back to a laboratory to be put in a ventilated and dry place for natural air drying.
b. Media required during the isolation of spores: the preparation method of the water agar culture medium comprises the following steps: weighing 20g of agar powder in 1L of distilled water or deionized water, heating and boiling until the agar powder is completely dissolved, subpackaging, sterilizing at 121 ℃ for 20 minutes under high pressure, and pouring the mixture into a flat plate for later use; the preparation method of the PDA culture medium comprises the following steps: 200g of cleaned and peeled potatoes are weighed, cut into small pieces and put into a pot, 800mL of water is added, the mixture is boiled for about 20 minutes, and then the mixture is filtered on a beaker by using a plurality of layers of gauze. Adding the filtrate into a pot, adding 20g agar powder and 20g glucose, heating with slow fire, pouring into a beaker after agar is completely dissolved, adding water to constant volume of 1L, autoclaving at 121 deg.C for 20 min, and pouring into a flat plate for use
c. The air-dried leaf blade is cut with scissors to get the lesion site, and put on a slide glass to observe the area of the conidium heap on the lesion site under a microscope at 10 × 10 times, after locking the area of the conidium heap (figure 2), 10 μ L of sterile water is sucked by a 10 μ L pipette 1 and hit on the area and repeatedly sucked 4-5 times (figure 3), the conidium will enter into the sterile water, finally, the sterile water containing the conidium is hit on the water agar medium and is lightly coated with a sterilized spreader.
d. The surface of a microscope is wiped completely by 75% alcohol and then the microscope is placed in an ultra-clean workbench, a single conidium on the surface of water agar is observed under 10 to 10 times after the ultraviolet lamp irradiates for 30 minutes, after the single conidium is observed (figure 4), the single conidium is moved to the center of the visual field of the microscope, then a picking needle is held by the right hand, the tip of the picking needle is burnt red by the alcohol lamp and then is placed in sterile water to dip in the picking needle for a plurality of seconds, the tip of the picking needle is moved to the center of the visual field, the picking needle is slowly moved to the vicinity of the conidium, and the single conidium is lightly dipped (figure 5).
e. After the inoculating needle is successfully dipped into a single conidium, the inoculating needle is quickly removed, the conidium is inoculated on a culture medium by a method of streaking on a PDA culture medium (figure 6), then the culture medium is placed in a constant-temperature illumination incubator, the temperature is constant at 20 ℃, the culture medium is subjected to full illumination culture, and a colony formed by the single conidium can be observed after 5 days (figure 7).
Example 2:
a. the pencil writer collects the panax notoginseng round spot disease leaves in a panax notoginseng base under the forest of Pu' er city billcang county in 2019 in 9 months, selects panax notoginseng leaves with the typical characteristics of panax notoginseng round spot and white or brown mildew layers on the disease spots, puts the panax notoginseng leaves into paper envelopes after being cut by scissors, divides one envelope into a plurality of diseased leaves, and then brings the envelopes back to a laboratory to be put in a ventilated and dry place for natural air drying.
b. Cutting the air-dried leaf blade with scissors to obtain the scab part, placing the scab part on a glass slide, observing the area of the conidium pile on the scab part under a 10 x 10 times microscope by using a microscope, locking the area of the conidium pile, marking the position by using a marking pen, then taking down the leaf blade and the glass slide, sucking 10 mu L of sterile water on a laboratory desktop by using a 10 mu L pipette 1, repeatedly sucking 4-5 times at the marked position, allowing the conidium to enter the sterile water, finally spraying the spore liquid on a water agar culture medium, and lightly and uniformly coating by using a sterilized coating device.
c. The surface of a microscope is wiped completely by 75% alcohol and then the microscope is placed in an ultra-clean workbench, a single conidium on the surface of water agar is observed under 10 to 10 times after the ultraviolet lamp irradiates for 30 minutes, the single conidium is moved to the center of the visual field of the microscope after the single conidium is observed, then a picking needle is held by the right hand, the tip of the picking needle is burnt red by the alcohol lamp and then is placed in sterile water to dip in the picking needle for several seconds, the tip of the picking needle is moved to the center of the visual field, the picking needle is slowly moved to the vicinity of the conidium, and the single conidium is lightly dipped.
d. After the inoculating needle is successfully dipped with single conidium, the inoculating needle is quickly moved out, the conidium is inoculated on a culture medium by a method of streaking on a PDA culture medium, then the culture medium is placed in a constant-temperature illumination incubator, the temperature is constant at 20 ℃, the culture medium is fully illuminated, and colonies formed by the single conidium can be observed after 5 days.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A method for rapidly separating single conidia of panax notoginseng teres, which is characterized in that: the method comprises the following steps:
(1) collecting pseudo-ginseng leaves with typical pseudo-ginseng round spot disease spots in a field, wherein the spots are required to have obvious white mildew layers which can be seen by naked eyes, then putting the pseudo-ginseng leaves into paper sampling envelopes, paying attention to the fact that each leaf is independently placed into one envelope and cannot be overlapped, returning the pseudo-ginseng leaves to a laboratory, putting the pseudo-ginseng leaves into envelope paper bags, and naturally drying the pseudo-ginseng leaves in a ventilated and dry place;
(2) shearing the disease spot part of the air-dried disease spot leaf by using scissors, placing the cut disease spot part on a glass slide, observing the region of a conidium pile on the disease spot by using a 10 x 10 times microscope, locking the region, sucking sterile water by using a 10 mu L range pipette gun to hit the region and repeatedly sucking for 4-5 times, allowing conidia to enter the sterile water, finally sucking the spore liquid onto a water agar culture medium, and slightly and uniformly coating by using a sterilized coater;
(3) placing the water agar culture dish coated with conidia under a microscope for observation, dipping a pre-red pick needle in sterile water for several seconds and then moving the pick needle to the visual field of the microscope when a single conidia is observed, then slowly moving the pick needle to the vicinity of the conidia, slightly touching the conidia by one side of the pick needle to adhere the conidia to the pick needle, and then quickly slightly scratching a plurality of lines on the PDA culture dish by the tip of the pick needle;
(4) and (4) putting the marked PDA culture dish into a constant-temperature incubator for culture until the single conidia grow into colonies.
2. The method for rapidly separating single conidia of panax notoginseng round spot germ as claimed in claim 1, wherein: the preparation method of the water agar culture medium in the step (2) comprises the following steps: weighing 20g of agar powder in 1L of distilled water or deionized water, heating and boiling until the agar powder is completely dissolved, subpackaging, sterilizing at 121 ℃ for 20 minutes under high pressure, and pouring the mixture into a flat plate for later use.
3. The method for rapidly separating single conidia of panax notoginseng round spot germ as claimed in claim 1, wherein: the step (3) needs to be carried out in a super clean bench, before the microscope is placed on the super clean bench, the microscope body needs to be wiped by 75% alcohol, and the microscope can be used after being irradiated by an ultraviolet lamp for 30 minutes.
4. The method for rapidly separating single conidia of panax notoginseng round spot germ as claimed in claim 1, wherein: in the step (3), the single conidia on the surface of the water agar is observed under a 10-by-10 microscope.
5. The method for rapidly separating single conidia of panax notoginseng round spot germ as claimed in claim 1, wherein: the picking needle in the step (3) is made by installing an inoculation wire on an inoculation rod, and when the picking needle is used, the picking needle needs to be burnt on an alcohol lamp in advance, and then the tip part of the picking needle is dipped in sterile water for a plurality of seconds.
6. The method for rapidly separating single conidia of panax notoginseng round spot germ as claimed in claim 1, wherein: and (3) after single conidium is observed under a microscope, moving the conidium to the center of a visual field, simultaneously moving the tip part of the picking needle to the center of the visual field, then slowly moving the tip part of the picking needle to be close to the conidium, adsorbing the conidium by using the tip part of the picking needle, and rapidly transferring the picking needle to slightly draw a plurality of lines on a PDA culture medium when the conidium is observed to be adsorbed on the picking needle under an ocular lens.
7. The method for rapidly separating single conidia of panax notoginseng round spot germ as claimed in claim 1, wherein: and (4) putting the PDA flat plate marked with the lines in the step (4) into an illumination incubator for culture, setting the temperature to be 20 ℃, carrying out full illumination culture, and culturing for 5-7 days to obtain obvious single spore colonies.
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