阅读说明：本技术 一种玉米真菌性病原菌的分离方法 (Separation method of corn fungal pathogenic bacteria ) 是由 杨秀娟 甘林 代玉立 兰成忠 滕振勇 刘晓菲 卢学松 于 2021-09-24 设计创作，主要内容包括：本发明涉及植物病害病原菌分离技术领域,特别是涉及一种玉米真菌性病原菌的分离方法。本发明提供了一种玉米真菌性病原菌的分离方法,包括以下步骤：(1)在玉米病株发病部分的病健交界处截取发病组织,无菌水清洗后对所得发病组织进行第一培养,得培养物；(2)将培养物转移到玉米幼苗茎杆的切口处进行第二培养,得染菌茎杆；(3)将染菌茎杆上的病原菌菌丝转置PDA培养基进行第三培养,得病原真菌纯培养物。本发明所述的分离方法无需对病组织进行消毒,且具有分离病菌成功率高、种类多的优势,同时对发病后期含菌量少或受细菌感染的病样中真菌性病原菌的识别和分离具有较好的适用性。(The invention relates to the technical field of separation of plant disease pathogenic bacteria, in particular to a separation method of corn fungal pathogenic bacteria. The invention provides a separation method of corn fungal pathogenic bacteria, which comprises the following steps: (1) intercepting pathogenic tissues at the diseased part of a corn plant at a diseased key junction, and performing first culture on the obtained pathogenic tissues after cleaning with sterile water to obtain a culture; (2) transferring the culture to the incision of the corn seedling stem for second culture to obtain a fungus-infected stem; (3) and (3) transferring the pathogenic bacteria hyphae on the infected stems into a PDA culture medium for third culture to obtain a pure pathogenic fungus culture. The separation method provided by the invention does not need to disinfect the diseased tissue, has the advantages of high success rate and multiple types of separation of pathogenic bacteria, and has better applicability to identification and separation of fungal pathogenic bacteria in a diseased sample with low bacterial content or bacterial infection in the later stage of disease attack.)

1. A method for separating corn fungal pathogens is characterized by comprising the following steps:

(1) intercepting pathogenic tissues at the diseased part of a corn plant at a diseased key junction, and performing first culture on the obtained pathogenic tissues after cleaning with sterile water to obtain a culture;

(2) transferring the culture to the incision of the corn seedling stem for second culture to obtain a fungus-infected stem; the disease symptoms of the infected stems comprise discoloration of the infected stems, water-soaking symptoms of the infected stems, and velvet, flocculent or thin hyphae at the cut parts of the infected stems and adjacent tissues;

(3) and (3) transferring the pathogenic bacteria hyphae on the infected stems into a PDA culture medium for third culture to obtain a pure pathogenic fungus culture.

2. The isolation method according to claim 1, wherein the fungal pathogens include one or more of sheath rot pathogen, stem rot pathogen, sheath blight pathogen, and root rot pathogen.

3. The isolation method according to claim 1, wherein the maize seedlings are 2-4 leaf-old seedlings.

4. The separation method according to claim 1, wherein the temperature of the first culture in the step (1) is 20-25 ℃, the time is 1-3 d, and the humidity is 85-100 wt.%; the first culture mode is dark culture.

5. The separation method according to claim 1, wherein the temperature of the second culture in the step (2) is 20-25 ℃, the time is 1-2 d, and the humidity is 85-100 wt.%; the light intensity of the second culture is 1000Lx, and the illumination time is 10-14 h.

6. The method of claim 1, wherein the third culturing of step (3) comprises primary culturing and purification culturing; the temperature of the primary culture and the purification culture is 25-28 ℃, the time is 2-3 d, and the culture mode is dark culture.

7. The isolation method as claimed in claim 1, wherein the corn seedling stems are surface-sterilized before the transfer of the culture in step (2).

8. The method according to claim 1, wherein the diseased tissue in the step (1) has a length of 1 to 3 cm.

9. The isolation method according to claim 1, wherein the culture of step (1) comprises hyphae and/or spores.

10. The separation method of claim 1, wherein the incision is a sterile contaminated incision.

Technical Field

The invention relates to the technical field of separation of plant disease pathogenic bacteria, in particular to a separation method of corn fungal pathogenic bacteria.

Background

During the growth process of plants, the plants are easily infected by pathogenic bacteria and cause diseases. The successful separation and acquisition of target pathogenic bacteria from pathogenic tissues is a key link for the phytopathology research and the disease control.

Corn sheath rot, stem rot, sheath blight, root rot and the like are important stem and root diseases in world corn production, the pathogeny is complex, and the fungal pathogeny comprises various fusarium, pythium and rhizoctonia, the pathogeny singly or compositely infects tissues of corn roots, stems and stalks (leaf sheath, spikelet and vascular bundle) to cause the corn root and stem diseases, and the diseases are expressed as root rot, leaf sheath discoloration, stem, spikelet necrosis, leaf yellowing, plant withering or breaking and lodging.

However, the diseased tissue of the diseased plant is easily infected by saprophytic bacteria or bacteria in the later period along with the development of the disease or untimely collection and separation of the specimen, so that the separation and purification difficulty of pathogenic bacteria of the disease specimen is increased. At present, a common disinfectant is a tissue conventional disinfection method which is mostly used for separating the disease germs, and the common disinfectant comprises 75% of ethanol by volume percentage, 0.1% of mercuric chloride by mass percentage, 1% of sodium hypochlorite by mass percentage and the like, however, target germs are sensitive to disinfectant, the disease germs with small bacterium content are easy to kill in the single use or combined use treatment process of the disinfectant, and the mixed germs can grow rapidly on a culture medium, so that the separation effect and the success rate of the pathogenic germs are greatly influenced.

Disclosure of Invention

In order to solve the problems, the invention provides a method for separating corn fungal pathogens. The separation method provided by the invention does not need to disinfect the pathogenic tissues, has the advantage of high success rate of separating pathogenic bacteria, and has better applicability to the identification and separation of fungal pathogenic bacteria in a disease sample with low bacterial content or bacterial infection at the later stage of the disease.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a separation method of corn fungal pathogenic bacteria, which comprises the following steps:

(1) intercepting pathogenic tissues at the diseased part of a corn plant at a diseased key junction, and performing first culture on the obtained pathogenic tissues after cleaning with sterile water to obtain a culture;

(2) transferring the culture to the incision of the corn seedling stem for second culture to obtain a fungus-infected stem; the disease symptoms of the infected stems comprise discoloration of the infected stems, water-soaking symptoms of the infected stems, and velvet, flocculent or thin hyphae at the cut parts of the infected stems and adjacent tissues;

(3) and (3) transferring the pathogenic bacteria hyphae on the infected stems into a PDA culture medium for third culture to obtain a pure pathogenic fungus culture.

Preferably, the fungal pathogens include one or more of sheath rot pathogen, stem rot pathogen, sheath blight pathogen and root rot pathogen.

Preferably, the corn seedlings are 2-4 leaf-old seedlings.

Preferably, the temperature of the first culture in the step (1) is 20-25 ℃, the time is 1-3 d, and the humidity is 85-100 wt.%; the first culture mode is dark culture.

Preferably, the temperature of the second culture in the step (2) is 20-25 ℃, the time is 1-2 d, and the humidity is 85-100 wt.%; the light intensity of the second culture is 1000Lx, and the illumination time is 10-14 h.

Preferably, the third culture of step (3) comprises a primary culture and a purification culture; the temperature of the primary culture and the purification culture is 25-28 ℃, the time is 2-3 d, and the culture mode is dark culture.

Preferably, the corn seedling stems are surface sterilized prior to transferring the culture in step (2).

Preferably, the length of the diseased tissue in the step (1) is 1-3 cm.

Preferably, the culture of step (1) comprises hyphae and/or spores.

Preferably, the incision is a sterile and contamination-free incision.

Has the advantages that:

the invention provides a separation method of corn fungal pathogenic bacteria, which comprises the following steps: taking a diseased tissue at a diseased part of a diseased plant, cleaning the diseased tissue with sterile water, and performing first culture to obtain a culture; transferring the culture to the incision of the corn seedling stem for second culture to obtain a fungus-infected stem; the disease symptoms of the infected stems comprise discoloration of the infected stems, water-soaking symptoms of the infected stems, and velvet, flocculent or thin hyphae at the cut parts of the infected stems and adjacent tissues; and (3) transferring the pathogenic bacteria hyphae on the infected stems into a PDA culture medium for third culture to obtain a pure pathogenic fungus culture. The method does not disinfect the diseased part of the diseased plant, can avoid the influence of a disinfectant on the viability of a small amount of pathogenic bacteria at the diseased part, infects the corn seedling stems through the culture of diseased tissues of the diseased plant, ensures that cuts of the corn seedling stems are directly contacted with target pathogenic bacteria, can lead the target pathogenic bacteria to be rapidly propagated and expanded, reduces the interference of mixed bacteria on the separation process of the specimen target pathogenic bacteria, finally realizes the rapid identification, selection and trapping of the target pathogenic bacteria, reduces the blindness of the separation and storage of the pathogenic bacteria in the diseased sample, has the advantages of simplicity, convenience, high efficiency and the like, can more comprehensively separate and obtain a plurality of pathogenic bacteria strains from the diseased plant, has the advantages of simplicity, high efficiency, high success rate of separating the pathogenic bacteria and multiple types, is particularly suitable for the separation of pathogenic fungi in the diseased sample which has low bacteria content or is infected by the mixed bacteria at the later stage of disease, the application of the method has important significance for developing the research on the variety diversity of the pathogenic bacteria of the stem diseases of the corn and the scientific treatment of the diseases.

Moreover, when the cut of the stem of the corn seedling is contacted with target pathogenic bacteria, the stem with the bacteria is discolored and has water stain symptoms, and villous, flocculent or thin hypha grows out; if the cut of the stem is contacted with other miscellaneous bacteria or bacteria suspected to be pathogenic fungi, the stem shows that aseptic silks appear, or some compact hyphae or overgrown rhizopus nigricans hyphae appear, the target pathogenic bacteria can be quickly identified, selected and trapped, and the technical effect of simplicity, convenience and high efficiency is further ensured to be realized.

Furthermore, the corn seedling stems with the leaf age of 2-4 have the advantages of being easy to obtain and disinfect, a small amount of disinfectant still remains on the surfaces and the incisions of the corn seedling stems after the corn seedling stems are disinfected, when the incisions are instantly contacted with the surface layers of hyphae on diseased tissues, the disinfectant can disinfect the diseased tissues and a small amount of mixed bacteria on the surface layers of the hyphae, meanwhile, the disinfectant can also play a certain biological filtering role on bacteria, and target pathogenic bacteria can be quickly identified and trapped.

Drawings

FIG. 1 is a diagram showing the state of a diseased tissue (diseased tissue) being subjected to a wet culture in a moisture-retaining cassette;

FIG. 2 is a view of corn seedlings and corn seedling stems for identifying and trapping pathogens, wherein A is corn seedling and B is corn seedling stem;

FIG. 3 shows suspected pathogenic bacteria hyphae and mixed fungi hyphae appearing in the moisture-retaining treated diseased tissue, wherein the suspected pathogenic bacteria hyphae are on the left side of the culture dish, and the mixed fungi hyphae are on the right side;

FIG. 4 shows the growth of hyphae in the moist culture of diseased tissue with high and low bacterial contents for 3 days, where A is the diseased tissue with high bacterial content and B is the diseased tissue with low bacterial content;

FIG. 5 is a picture of the stem of a maize seedling, which is obtained by dipping a cut into a moist-keeping tissue to pick up suspected pathogenic bacteria hyphae, and then culturing for 2 days, wherein the left picture is a picture of the stem infected with sheath rot pathogen, and the right picture is a picture of the stem infected with root rot pathogen, sheath blight pathogen and saprolegnia pathogen from left to right;

FIG. 6 shows the isolation of Fusarium, a target strain, from infected shoot stalks, without the concomitant presence of bacteria, wherein A is a pathogen cultured for 3 days and B is a pathogen cultured for 2 days;

FIG. 7 shows the separation of target strains Pythium species and Rhizoctonia solani from the infected stems of seedlings without the concomitant presence of bacteria, wherein A is Pythium species and B is Rhizoctonia solani;

FIG. 8 shows that the strain isolated from the Changtai collected diseased strain shows a variety of plate forms, wherein A-F are all fusarium;

FIG. 9 shows the results of the separation of the diseased tissue of the specimen 3 by a conventional separation method (75% ethanol for 30-45 s), wherein the first row shows the results from left to right which are obvious for the colony-infecting bacteria separated from the diseased tissue, and the second row shows the results from left to right which are not obvious for the colony-infecting bacteria separated from the diseased tissue;

FIG. 10 is a graph of colonies isolated from the stem of a contaminated maize seedling and colonies streaked on a PDA plate by an inoculating needle picking hyphae, where the left side is a graph of colonies isolated from the stem of a maize seedling, and the middle and right sides are streaked colonies;

FIG. 11 shows the case of bacteria isolated from the hyphal-grown moisturized and cultured diseased tissue by a conventional isolation method (75% ethanol 3045s), wherein the first row and the second row, from left to right, are both obvious results of bacterial colonies isolated from the diseased tissue infecting miscellaneous bacteria;

FIG. 12 shows the growth of hyphae 3 days after dipping the hyphae with the sheath of leaves in adult stage of corn, the stem of rice seedling and the stem of corn seedling, from left to right in turn;

FIG. 13 shows the growth of hyphae 3 days after the hyphae are picked up with stems and leaf sheaths of maize in the adult stage;

FIG. 14 shows the separation of pathogenic bacteria after the stem of rice seedling is dipped into hyphae;

FIG. 15 shows the separation of pathogenic bacteria after the stem and leaf sheath of maize in adult stage are dipped to take hyphae, wherein the stem and leaf sheath are on the left side and the leaf sheath is on the right side of the stem and leaf sheath;

FIG. 16 shows that no hyphae grow out or hyphae overgrow grow out after the cut of the stem of the corn seedling is treated by rhizopus for 3 days;

FIG. 17 shows that no hyphae grow out of cut stem of corn seedling treated with Trichoderma, unknown fungi, and bacteria for 3 days;

FIG. 18 shows the re-separation and purification effect of the stem of the maize seedling infected with the pathogenic bacteria and the pathogenic bacteria after the plate is dipped to culture the pathogenic bacteria infected with the bacteria, wherein the left picture is the colony of the pathogenic bacteria infected with the bacteria, the middle picture is the stem of the maize seedling infected with the bacteria, the right picture is the colony of the pathogenic bacteria successfully separated from the stem of the maize seedling infected with the bacteria, and the left picture and the right picture are the colonies of the pathogenic bacteria without bacterial infection.

Detailed Description

The invention provides a separation method of corn fungal pathogenic bacteria, which comprises the following steps:

(1) intercepting pathogenic tissues at the diseased part of a corn plant at a diseased key junction, and performing first culture on the obtained pathogenic tissues after cleaning with sterile water to obtain a culture;

(2) transferring the culture to the incision of the corn seedling stem for second culture to obtain a fungus-infected stem; the disease symptoms of the infected stems comprise discoloration of the infected stems, water-soaking symptoms of the infected stems, and velvet, flocculent or thin hyphae at the cut parts of the infected stems and adjacent tissues;

(3) and (3) transferring the pathogenic bacteria hyphae on the infected stems into a PDA culture medium for third culture to obtain a pure pathogenic fungus culture.

The method comprises the steps of intercepting pathogenic tissues at the disease-key junction of the diseased part of a corn diseased plant, cleaning the pathogenic tissues with sterile water, and carrying out first culture on the obtained pathogenic tissues to obtain a culture. The invention directly cultures the pathogenic tissue without sterilizing the pathogenic tissue, thereby ensuring that target pathogenic bacteria are trapped from the specimen pathogenic tissue with low bacteria content.

In the present invention, the number of washing is preferably 2 to 3. In the invention, the length of the pathogenic tissue is preferably 1-3 cm, more preferably 1.5-2.0 cm; the pathogenic tissues with specific lengths are intercepted at the disease-health junction, so that a small amount of pathogenic bacteria can be propagated under the condition that the pathogenic tissues are moisturized, spores and hyphae are generated, the tissues at the disease-health junction are usually effective parts for separating the pathogenic bacteria, the pathogenic bacteria are more active and less in infectious microbes, and the pathogenic bacteria intercepted at the disease-health junction on the pathogenic tissues can grow and propagate by utilizing the residual nutrition of the pathogenic tissues. The intercepting mode is not particularly limited, and the mode known by the technicians in the field is adopted; in a specific embodiment of the present invention, the cutting is preferably performed by cutting diseased tissue with sterile scissors.

In the invention, the corn disease plants comprise corn sheath rot disease plants, corn stem rot disease plants, corn root rot disease plants, corn sheath blight disease plants and corn composite disease plants; the complex disease comprises sheath rot, stem rot, sheath rot and sheath blight; the fungal pathogenic bacteria preferably comprise one or more of sheath rot pathogenic bacteria, stem rot pathogenic bacteria, sheath blight pathogenic bacteria and root rot pathogenic bacteria.

In the present invention, the diseased portion preferably includes a leaf sheath, a stem or a rhizome portion; the growth period of the corn diseased plant is not particularly limited, namely the corn diseased plant can be separated in the whole growth period.

In the invention, when the fungal pathogenic bacteria are sheath rot pathogenic bacteria, the pathogenic part is preferably a leaf sheath, and the growth period of a diseased plant carrying the sheath rot pathogenic bacteria is preferably 8-9 leaf period, androgenesis period, kernel establishment period and milk maturation period; when the fungal pathogenic bacteria are stem rot pathogenic bacteria, the diseased part is preferably leaf sheath and stem, and the growth period of the diseased plant carrying the stem rot pathogenic bacteria is preferably the seed establishment period, the milk maturity period and the androgenesis period; when the fungal pathogenic bacteria are sheath blight pathogenic bacteria, the diseased part is preferably a leaf sheath, and the growth period of the diseased plant carrying the sheath blight pathogenic bacteria is preferably an androgenesis period and a kernel establishment period; when the fungal pathogenic bacteria are root rot pathogenic bacteria, the diseased part is preferably one or more of roots, roots and stems, and the growth period of the diseased plant carrying the root rot pathogenic bacteria is preferably 3-4 leaf seedling period and jointing period.

In the invention, the temperature of the first culture is preferably 20-25 ℃, and more preferably 21-24 ℃; the first culture time is preferably 1-3 d, and more preferably 1.5-2.5 d; the humidity of the first culture is preferably 85-100 wt.%, and more preferably 90-95 wt.%; the first culturing mode is preferably dark culturing. In an embodiment of the invention, the first culture is preferably performed in a petri dish; the culture dish is preferably a sterile culture dish; the culture dish is preferably placed in a plastic moisture preservation box; the colour of the plastic is preferably transparent. In the present invention, the culture preferably includes hyphae and/or spores, more preferably hyphae.

After the culture is obtained, the culture is transferred to the incision of the corn seedling stem for second culture to obtain a fungus-infected stem; the disease symptoms of the infected stems comprise discoloration of the infected stems, water-soaking symptoms of the infected stems, and the appearance of velvet, flocculent or thin hyphae at the incision parts of the infected stems and adjacent tissues. The incision is preferably an incision without mixed bacteria pollution, and is more preferably a middle incision; the intermediate incision is preferably an incision cut from the middle of a sterilized stem; the cut obtained by cutting the stem in the middle of the stem can ensure no pollution, and the cut or the cut caused by cutting or shearing by a tool in the process of obtaining the stem from the seedling cultured by the seedling culture plate can be infected by factors such as a shear, mixed bacteria on the surface of the plant, mixed bacteria in the ambient air and the like. In the invention, the corn seedlings are preferably 2-4-leaf-old seedlings. In the invention, the temperature of the second culture is preferably 20-25 ℃, and more preferably 21-24 ℃; the second culture time is preferably 1-2 d; the humidity of the second culture is preferably 85-100 wt.%, and more preferably 90-95 wt.%; the light intensity of the second culture is preferably 1000 Lx; the illumination for the second culture is preferably 10-14 h, and more preferably 10-12 h. In the present invention, the second culture is preferably performed in a petri dish; the culture dish is preferably a sterile culture dish; the culture dish is preferably placed in a plastic moisture preservation box; the colour of the plastic is preferably transparent. In the present invention, the culture preferably includes hyphae and/or spores, more preferably hyphae.

In the present invention, the culture is transferred in a manner not limited in any way, and the method is well known to those skilled in the art; in a particular embodiment of the invention, the means of transfer of the culture is preferably dipping.

In the invention, the corn seedlings with the leaf age of 2-4 are preferably sourced from indoor nutrient soil for 10-20 days, the indoor cultured corn seedlings are usually weak, and the stems of the corn seedlings are easy to trap target pathogenic bacteria and can enable the pathogenic bacteria to grow rapidly. The source of the nutrient soil is not particularly limited, and the nutrient soil can be obtained by conventional purchase by a person skilled in the art; in a specific embodiment of the invention, the nutrient soil is preferably topiramate. The method for cultivating the maize seedlings is not particularly limited, and a mode which is well known by a person skilled in the art can be adopted; in the specific embodiment of the invention, the method for cultivating the maize seedlings is preferably cultivation by utilizing indoor nutrient soil; the cultivation temperature is preferably 20-25 ℃; the culture mode is preferably light and dark alternation, and more preferably the light irradiation time: dark time 12 h: 12 h; the light intensity is preferably 1000 Lx.

In the invention, the length of the stem of the corn seedling is preferably 4-6 cm; the corn seedling stem is preferably a stem obtained after leaves and roots are removed; the invention does not limit the way of removing the blade and the root at all, and the way known by the technicians in the field can be adopted; in a specific embodiment of the present invention, the manner of removing the leaves and roots is preferably by using sterile scissors.

The present invention preferably surface disinfects the corn seedling stems before transferring the culture. In the present invention, the disinfectant used for the disinfection is preferably ethanol; the volume percentage content of the ethanol is preferably 75%; the time for disinfection is preferably 30-45 s; the sterilization is preferably performed on a clean bench. In the present invention, after the sterilization, the sterilized stems obtained by the sterilization are preferably placed in a petri dish; the culture dish is preferably a sterile culture dish.

After the infected stems are obtained, the pathogenic bacteria hyphae on the infected stems are transferred to a PDA culture medium for third culture, and the pure culture of the pathogenic fungi is obtained. In the present invention, the culture medium used for the third culture is preferably PDA culture medium; the PDA culture medium preferably comprises the following components in parts by mass: 200g of potato, 20g of glucose and 1000ml of water; the third culture preferably comprises a primary culture and a purification culture; the temperature of the primary culture is preferably 25 ℃; the time for primary culture is preferably 2-3 d; the primary culture mode is preferably dark culture; the temperature of the purification culture is preferably 25 ℃; the time for the purification culture is preferably 2-3 d.

In order to further illustrate the present invention, the following detailed description of the isolation method of corn fungal pathogens is provided with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Test specimen book:

(1) the corn sheath rot disease strain (collected from Changtai, the maturity stage of the growing period), the corn stalk rot disease strain (collected from Jianyang, the androgenesis period, the growing period, the male stage), the corn root rot disease strain (collected from Fuzhou, 4-5 leaf seedling stage), and the corn sheath blight disease strain (collected from Min, the androgenesis period, the male stage).

The test method comprises the following steps:

collecting typical corn sheath rot disease plants, typical corn stalk rot disease plants, typical corn root rot disease plants and typical corn sheath blight disease plants from a diseased field, removing root soil and leaves of the plants, and separately wrapping with sterilized newspaper and bringing back to a laboratory.

Cutting 1-3 cm of diseased tissues at diseased tissue healthy junctions of the 4 diseased plants by using sterile scissors (wherein the number of the diseased tissues randomly cut by the specimens 1, 2, 3 and 4 is respectively 30, 10 and 10, the diseased tissues of the corn sheath rot disease plant are cut at the diseased tissue healthy junctions of the leaf sheath part, the diseased tissues of the corn stalk rot disease plant are cut at the diseased tissue healthy junctions of the leaf sheath part and the stalk part, the diseased tissues of the corn root rot disease plant are cut at the diseased tissue healthy junctions of the root part and the rhizome part, the diseased tissues of the corn sheath blight disease plant are cut at the diseased tissue healthy junctions of the leaf sheath part), cleaning the diseased tissues of the corn sheath disease plant for 2-3 times by using sterile water, placing the mycelia in a sterile culture dish, placing the culture dish in a transparent plastic moisture preservation box (4 layers of moisture paper extraction are padded in the culture dish), and performing first culture (moisture preservation culture) for 1-3 days at the temperature of 20-25 ℃ and the humidity of 85-100 wt.% to obtain four types of diseased tissue mycelia (the recorded photos of the culture process are shown in figure 1).

The culture process finds that hyphae appear if the diseased tissue with high bacterial content is subjected to moisture preservation culture for 1 day, and thin hyphae can be seen only if the diseased tissue with low bacterial content needs 3 days, so that the quantity of the bacterial content of the diseased tissue can be preliminarily judged by virtue of the high and low hyphae newly grown after the moisture preservation treatment. Wherein, the result after 2 days of culture is shown in figure 3, as can be seen from figure 3, pathogenic tissues are treated in a moisture preservation box for 2 days, hypha of pathogenic bacteria grow out, but mixed bacteria also appear, so that obvious mixed bacteria appear in some diseased tissues, and the target pathogenic bacteria can be screened once on the basis, if hypha with unknown suspicious symptoms are not needed; as shown in FIG. 4, the results of 3 days of culture show that hyphae can grow in the diseased tissue with high and low bacterial contents by the isolation method of the present invention after the tissue is moisturized and cultured for 3 days, as can be seen from FIG. 4.

2-leaf 1-core corn seedlings are cultivated for 10 days in nutrient soil (topira pusilla) of a seedling tray at the indoor temperature of 20-25 ℃ under the condition of light-dark alternation (12h light/12 h dark, namely the illumination time: the dark time is 12 h: 12h, and the light intensity is 1000Lx), leaves and roots are removed by sterile scissors, and the stems of the corn seedlings are kept to be 4-6 cm long (figure 2).

Soaking and disinfecting the stems of the corn seedlings on a superclean workbench for 30-45 s by using 75% ethanol by volume, putting the stems into a sterile culture dish, and cutting the stems into 2 sections from the middle by using a sterile knife. Clamping sterilized maize seedling stems by using sterile tweezers, quickly dipping one end of a middle notch to obtain target hyphae on moisturizing culture diseased tissues to obtain infected stems, wherein the number of tested maize seedling stems is 60, and 1 maize seedling stem is dipped by the hyphae on each diseased tissue.

And respectively placing the stems of the corn seedlings in an aseptic culture dish, placing the culture dish in the transparent plastic moisture preservation box, and carrying out second culture (moisture preservation treatment) for 1-2 days under the conditions of 20-25 ℃, 85-100 wt.% of humidity and 1000Lx of light intensity to obtain the stems with the infected bacteria, wherein the samples 1, 2, 3 and 4 respectively have 25, 8 and 6 stems with the infected bacteria successfully, namely the cut of 47 stems with the infected bacteria has fungus hyphae growing out, and 13 stems have no infected hyphae and are possibly infected with bacteria or germs. The fungus-infected stem is used for treating the disease that the fungus-infected stem is discolored, the fungus-infected stem is in a water stain disease state, and the cut of the fungus-infected stem and the adjacent tissues thereof have villous, flocculent or thin hypha. The results after successful transfection are shown in fig. 5, and it can be seen that, after the corn seedling stem is infected with sheath rot and stem rot germs, the hyphae are down-like and flocculent, after the corn seedling stem is infected with sheath blight germs, the hyphae are thin but do not overgrow and have no cysts, and after the corn seedling stem is infected with root rot germs, the hyphae are down-like and flocculent, and some are thin;

the results of 2 days and 3 days of fusarium-dipped stem culture are shown in fig. 6. The results of the isolation of the seedling stalk isolate of Pythium species or Rhizoctonia solani are shown in FIG. 7. As can be seen from FIGS. 6 and 7, the stems of the infected seedlings obtained by the method of the present invention showed colonies of the target strain, and no bacterial association was observed.

Cutting the part with the hyphae on the infected stalk into 2-3 mm diseased tissues by using a sterile knife, transferring the diseased tissues into a PDA (potato) culture medium (200 g, 20g of glucose and 1000ml of water) for primary culture, wherein the primary culture is dark culture, the temperature of the primary culture is 25-28 ℃, the time is 2-3 d, cutting the separated hyphae at the edges of the bacterial colony of the pathogenic fungus together with the culture medium, and placing the cut hyphae on a new PDA culture medium for purification culture, wherein the temperature of the purification culture is 25-28 ℃, and the time is 2-3 d, so as to obtain the pure culture of the pathogenic fungus. The obtained culture was observed, and the identification was carried out by utilizing the hypha form of Fusarium in a villiform state, the hypha form of Pythium species in a flocculent state, and the hypha form of Rhizoctonia solani in a thin state.

The test results are as follows: the strains obtained by separating the 1 st test sample are all fusarium, the strains obtained by separating the 2 nd test sample are all pythium, the strains obtained by separating the 3 rd test sample are fusarium, and the strains obtained by separating the 4 th test sample are rhizoctonia solani (4 test samples are respectively taken from a strain with typical corn sheath rot disease, a strain with typical disease corn stalk rot disease, a strain with typical corn root rot disease and a strain with typical corn sheath rot disease). The strain transfected from the Changtai disease sample shows obvious morphological diversity, as can be seen from figure 8, fusarium species are rich, and various fusarium can be separated by adopting the method of the invention. Therefore, the separation method has the advantages of high efficiency, high success rate and multiple types of separation of pathogenic bacteria, has better applicability to the identification and separation of pathogenic fungi in disease strains with low bacteria content or infected by bacteria in the later period of disease onset, can successfully separate the pathogenic bacteria for multiple times, and has good repeatability.

Example 2

Test specimen book:

specimen 1: the leaf sheath tissue of the corn sheath rot disease plant has hypha (collected from Changtai in the growth period and the establishment period of grains), the specimen is fresh, and the separation time and the sampling time are different by 1 day. The leaf sheath disease tissues of the germs to be separated are equally divided into 2 parts.

Specimen 2: the corn sheath rot and stem rot are mixed to infect a diseased plant, hypha is not seen in the tissue of the diseased plant, the diseased plant is infected with bacteria (collected from a building, at the early heading stage of the growth period), the specimen is not fresh, and the separation time and the sampling time are different by 3 days. The diseased tissues of the leaf sheath and the stem base of the pathogen to be separated are equally divided into 3 parts.

Specimen 3: the hypha is not seen in the tissues of a diseased corn sheath rot plant, the diseased corn sheath rot plant is accompanied by bacterial infection (collected from Minjian, in the growth period of 8-9 leaves), the specimen is not fresh and wilted due to improper treatment in the mailing process, and the separation time is 4 days different from the sampling time. The leaf sheath disease tissues of the germs to be separated are equally divided into 3 parts.

The experimental method comprises the following steps:

the test method of example 1 was used, the only difference being: hyphae obtained from each diseased tissue culture were dipped into 1 cut of the stem of maize seedling and 10 diseased tissues were provided per specimen, i.e. repeated 10 times.

Comparative example 1

Test specimen book:

the same as in example 2.

Experimental procedure (conventional separation method 1):

shearing the tissues at the disease-health junction of the diseased tissues into 0.5-0.8 cm diseased tissues, and obtaining 10 diseased tissues in total, wherein the diseased tissues and the diseased tissues in the embodiment 2 are from the same plant, washing the diseased tissues for 2-3 times by using sterile water, then disinfecting the diseased tissues for 30-45 s by using 75% ethanol with volume percentage, washing the diseased tissues for 2-3 times by using the sterile water, absorbing excess water by using sterile filter paper, transferring the excess water into a PDA culture medium (the components of each liter of PDA culture medium are 200g of potato, 20g of glucose, 15-17 g of agar and 1000ml of distilled water) containing 60ppm of rifampicin, and observing the separation result of pathogenic bacteria after 3-4 days.

Comparative example 2

Test specimen book:

same as example 2, but without separation of specimen 1.

Experimental procedure (conventional separation method 2):

cutting the tissues at the disease-health junction of the diseased tissues into 0.5-0.8 cm diseased tissues, and taking 10 diseased tissues in total, wherein the diseased tissues and the diseased tissues in the embodiment 2 are from the same plant, cleaning the diseased tissues for 2-3 times by using sterile water, soaking and sterilizing the diseased tissues for 30-45 s by using 75% ethanol in percentage by volume, then carrying out 0.1% mercuric rise for 3min, flushing the diseased tissues for 2-3 times by using the sterile water, then carrying out sterile filtration on the sterilized tissues, transferring the sterilized tissues into a PDA culture medium containing 60ppm of rifampicin, and observing the separation result of pathogenic bacteria after 3-4 days.

The results of the pathogen investigation of example 2, comparative example 1 and comparative example 2 are shown in table 1.

TABLE 1 comparison of pathogen results obtained by different methods for separating samples from different sources

The results are shown in table 1, where specimen 1: by adopting the separation method, 10 pathological tissues of suspected pathogenic bacteria hyphae (preliminarily similar to target hyphae according to hypha forms) are grown out after moisture preservation in 10 tested pathological tissues, 8 corn seedling stems grown by fungal hyphae are cut, and 2 corn seedling stems not grown by fungal hyphae are cut, so that pathogenic fungi strains are separated from 8 corn seedling stems grown by fungal hyphae in the cut, the pathogenic fungi strains are not associated, the separation success rate of the pathogenic fungi strains of the stems is 100%, and finally 8 fusarium strains are obtained by separation; adopting the separation method of comparative example 1, 3 diseased tissues infected with bacteria, 5 non-hyphal diseased tissues and 2 hyphal pathogenic tissues in 10 diseased tissues to be tested without accompanying bacteria, and finally separating to obtain 2 fusarium strains;

specimen 2: by adopting the separation method, in 10 disease tissues to be tested, 8 suspected disease fungus hypha blocks grow out after moisture preservation, 6 stems growing out from the cut fungi hypha are separated out, 6 pathogenic fungus strains are separated out, no bacterium is associated, and finally 6 fusarium strains are obtained by separation; adopting the separation method of comparative example 1, 6 diseased tissues infected with bacteria, 2 tissues not growing hypha and 2 pathogenic tissues growing hypha in 10 diseased tissues tested without accompanying bacteria, and finally separating to obtain 2 fusarium strains; adopting the separation method of the comparative example 2, 0 diseased tissue infected with bacteria and 10 non-growing hyphal diseased tissues in 10 tested diseased tissues are separated to obtain 0 fungus strain finally;

specimen 3: by adopting the separation method, 7 suspected pathogenic bacteria hypha blocks grow out after moisture preservation in 10 tested diseased tissues, 6 corn seedling stems grow out by the cut fungi hypha, and finally 6 fusarium strains are obtained by separation; adopting the separation method of comparative example 1, 2 diseased tissues infected with bacteria, 8 diseased tissues with hyphae and 5 accompanying bacteria are obtained from 10 diseased tissues tested, and 3 germs are finally obtained by separation, wherein the 5 separated accompanying bacteria tissues are shown in figure 9, wherein the bacterial colony infected with the diseased tissue in the first row is obvious from left to right, and the bacterial colony infected with the diseased tissue in the second row is not obvious from left to right; by adopting the separation method of comparative example 2, 3 diseased tissues infected with bacteria and 7 non-growing hyphal diseased tissues in 10 diseased tissues to be tested are finally separated to obtain 0 strain of fungi.

Therefore, the corn stalk rot and sheath rot pathogenic bacteria are sensitive to 0.1% of mercuric chloride, cannot be separated out, are separated by using 75% ethanol, and have certain killing power on a small amount of survival pathogenic bacteria in diseased tissues. Meanwhile, the freshness of the sample can directly influence the separation effect of pathogenic bacteria, some samples can be infected by bacteria, and in the process of adopting the conventional separation method, a small amount of bacteria can easily and quickly grow on a PDA culture medium, so that the separation of target pathogenic bacteria is influenced, therefore, bacterial colonies symbiotic with fungi and bacteria appear, and other steps are usually required to be added for purification by adopting the conventional separation method. The number of the strains obtained by the method is obviously higher than that of the strains obtained by the conventional separation method, and the method has the advantages of multiple pathogenic strain separation and high separation efficiency.

Example 3

Test materials: the corn sheath rot disease strain has no hypha in the tissue of the disease strain, and the separation time and the sampling time are different by 3 days along with bacterial infection (collected from Ou establishment and the initial heading stage in the growth period).

The test method comprises the following steps:

the effect of nick hyphae growth and the effect of strain isolation were observed after 2 to 3 days, as in example 1.

Comparative example 3

Test specimen book:

the same as in example 3.

The test method comprises the following steps:

on the basis of example 1, hyphae are picked by an inoculating needle from the cut of 8 maize seedling stems with hyphae and directly streaked on a PDA plate (PDA culture medium is poured into the plate to obtain the PDA plate, wherein each liter of PDA culture medium comprises 200g of potatoes, 20g of glucose, 15-17 g of agar and 1000ml of distilled water) for 8 positions, and the growth effect of the cut hyphae and the strain separation effect are observed after 2-3 days.

Comparative example 4

Test specimen book:

the same as in example 3.

The test method comprises the following steps:

on the basis of the embodiment 1, 8 corn seedling stems with hyphae are sterilized by 75% ethanol for 30-45 s, washed by sterile water for 1-2 times, diseased tissues are cut into 2-3 mm and transferred into a PDA culture medium, and the growth effect of the hyphae at the cut and the separation effect of the strains are observed after 2-4 days.

Comparative example 5

Test specimen book:

the same as in example 3.

The test method comprises the following steps:

on the basis of the embodiment 1, 8 moisturizing culture diseased tissues with hyphae are disinfected by 75% ethanol for 30-45 s, washed by sterile water for 2-3 times, cut into 2-3 mm diseased tissues and transferred into a PDA culture medium, and the incision hyphae growth effect and the strain separation effect are achieved after 2-3 days.

Comparative example 6

Test specimen book:

the same as in example 3.

The test method comprises the following steps:

the same procedure as in example 1 was followed except that: and (3) replacing the rice seedling stems (the length is 2-3cm, the width is 0.3cm) after 18 days of sowing with the corn seedling stems for 8 treatments, and observing the growth effect of cut hypha and the strain separation effect after 2-5 days.

Comparative example 7

Test specimen book:

the same as in example 3.

The test method comprises the following steps:

on the basis of example 1, 8-10 leaf corn adult-stage stem leaf sheaths (with the length of 2-3cm and the width of 0.3cm for replacing corn seedling stems) planted in the field are taken for 12 treatments, and the growth effect of cut hyphae and the strain separation effect are observed after 2-5 days.

Comparative example 8

Test specimen book:

the same as in example 3.

The test method comprises the following steps:

the same procedure as in example 1 was followed except that: and (3) replacing the corn seedling stems with the adult corn stems (the length is 3-4 cm, the width is 3cm) stripped of the leaf sheaths for 8 treatments, observing the growth effect of cut hyphae and the strain separation effect after 2-5 days, and finding out the results shown in the figure 10-15.

The diseased tissue hyphae picked up by the incisions of examples 3 and comparative examples 3 to 8 were of the same origin, and the hyphae growth effect and the strain isolation effect of the incisions of examples 3 and comparative examples 3 to 8 were observed 2 to 3 days later.

By adopting the separation method, 7 stalks with cut and fungal hypha growing are found in 8 treated maize seedling stalks; the results of the moisture retention treatment for 2 days are shown in the right-most picture of FIG. 12, and hyphae were abundant in the cut of the stem of the corn seedling. Meanwhile, the success rate of separating the fungus strains at the stalk of the corn seedling with the fungus hypha is 100 percent. Therefore, the corn seedling stem cut can quickly trap the dominant pathogenic bacteria on the diseased tissue, the stem can preferentially select the pathogenic bacteria for 1 time, and the separation and purification of the target pathogenic bacteria are easy.

After 4 days of culture by the separation method of comparative example 3, 6 mycelia were not grown and 2 target bacteria were grown, and the result of streaking colonies on the PDA plate by the mycelia separated from the stem of the maize seedling infected with the inoculum needle is shown in FIG. 10 (the left side is a colony pattern separated from the stem of the maize seedling, and the middle and right sides are streaked colonies), it can be seen that the colonies grown by the mycelia are small and the mycelia are thin;

the separation method of comparative example 4 is adopted, and after 3 days of culture, 5 stems are found to separate target germs, and 3 stems do not grow hypha, which shows that 75% ethanol has influence on germs;

the results of 3 days of culture using the isolation method of comparative example 5 are shown in FIG. 11, in which although pathogenic bacteria were isolated, various miscellaneous bacteria and bacteria appeared, and this method was found to affect the effect of further isolation of the target bacteria.

After the moisture preservation treatment for 24 hours by the separation method of example 6, 4 hyphae are grown in the cut of the stem of 8 treated rice seedlings, but the hyphae are extremely thin, the separation condition of the pathogenic bacteria is shown in fig. 14, 2 pathogenic bacteria are separated from the 4 hyphae-grown rice seedling stems, no bacteria are associated, and 2 mixed bacteria (rhizopus and unknown fungi) are separated; the processing result of the continuous moisture retention treatment for 2 days is shown in the middle picture of fig. 12, hyphae growing from the cut of the stem of the original rice seedling are degraded, and the cut is dried and shrunk;

after the moisture retention treatment for 24 hours by the separation method of example 7, hyphae did not grow in 12 adult stage corn leaf sheaths treated, and the result of continuing the moisture retention treatment for 2 days was shown in the rightmost picture of fig. 12 and fig. 13, most of the hyphae did not successfully contaminate the 12 adult stage corn stalk leaf sheaths, 2 hyphae grew out of the 12 adult stage corn stalk leaf sheaths, and 2 pathogenic bacteria and associated bacteria were separated out of the 2 hyphae-grown leaf sheaths. Pathogenic bacteria are not separated from 2 stalks of the corns with the hyphae in the adult plant period, and are all mixed bacteria;

after the separation method of the embodiment 8 is adopted for moisture retention treatment for 24 hours, 8 treated adult-stage corn stalks with leaf sheaths removed do not grow hyphae; the results of the moisture retention treatment for 2 days are shown in fig. 13, most of the stems are not successfully infected with bacteria, 2 thin hyphae grow out from 8 stalk cuts of the corn at the adult plant stage with the leaf sheath removed, pathogenic bacteria are not separated from 2 stalk cuts of the corn at the adult plant stage with the hyphae and are all mixed bacteria, and as shown in fig. 15, the diseased tissues on the flat plate have obvious mixed bacteria growth, so that the stalk cuts of the corn at the adult plant stage are not easily disinfected by 75% ethanol completely.

According to the method, although pathogenic bacteria can be trapped by adopting the stems of the rice seedlings and the leaf sheaths of 8-10 leaf corns planted in the field in the adult plant period, the effect of trapping the pathogenic bacteria and the propagation quantity and separation effect of pathogenic bacteria hyphae are not equal to those of the stems of the corn seedlings.

Example 4

Test strains:

rhizopus, non-pathogenic bacteria, trichoderma, unknown fungi (dark colonies on PDA plates), all from corn stalk diseased tissue, suspected saprophytic bacteria.

The test method comprises the following steps:

the procedure of example 1 was followed except that the sterilized maize seedling stem was directly dipped into hyphae, spores or lawn of the test strain (microorganism) plated on PDA, and the procedure was repeated 6 times.

Placing the corn seedling stems in an aseptic culture dish, placing the culture dish in the transparent plastic box, carrying out moisture preservation treatment for 2-3 days under the moisture preservation condition, and observing the cut and stem tissue diseases of the corn seedling stems.

The incision and stalk tissue disorders of the examples were investigated and the results are shown in figures 16 and 17:

example 4 the results of 3 days of stalk cuts of corn seedlings treated with rhizopus are shown in fig. 16, some of which have sterile silks and some of which have white hyphae but show overgrowth, while the results of 3 days of stalk cuts of corn seedlings treated with trichoderma, unknown fungi and bacteria are shown in fig. 17, and none of which have white hyphae.

Therefore, whether hyphae on diseased tissues are pathogenic bacteria or not can be preliminarily identified through the hyphae growth and the hyphae shape of the cut of the stem of the corn seedling.

Example 5

And (3) disinfecting 45s by using 75% ethanol, dipping one end of the corn seedling stem cut with the length of 4cm on a flat plate, culturing fusarium oxysporum f.sp.vaginalis infected with bacteria, and performing moisture-preserving culture for 2-3 days to find that the cut of the treated corn seedling stem has hypha.

And (3) taking diseased tissues of 2-3 mm of diseased tissue stems of the diseased tissues at a position 0.5mm away from the contact end of the hyphae, transferring the diseased tissues into a PDA culture medium for culture, wherein the result of the culture for 3 days is shown in figure 18, and after the diseased tissues are transferred into the PDA culture medium for culture, pathogenic bacteria grow out and no bacterial infection exists.

The pathogenic fungi are difficult to purify after infecting the bacteria, and the bacteria are still accompanied after the conventional method repeatedly purifies for a plurality of generations. The corn seedling stem cut provided by the invention has a good purification effect on pathogenic bacteria of plate culture infected bacteria, and the treated corn seedling stem has a certain disinfection effect and a certain biological filtration effect on non-pathogenic bacteria.

As is clear from the above-mentioned examples, the method of the present invention does not require sterilization of the diseased site of the diseased plant, can avoid the effect of a disinfectant on the viability of a small amount of germs in the diseased tissue, and the incision of the stem of the corn seedling is contacted with target pathogenic bacteria, so that the pathogenic bacteria can be rapidly propagated and expanded, the interference of mixed bacteria to the separation process of the specimen target fungi is reduced, the pathogenic bacteria are finally rapidly identified, selected and trapped purposefully, the blindness of separation and storage of the pathogenic bacteria in a disease sample is reduced, the method has the advantages of simplicity, convenience, high efficiency and the like, various pathogenic bacteria strains can be more comprehensively separated from the disease strain, and the method has the advantages of simplicity, convenience and high efficiency. Moreover, if the cut of the stem of the corn seedling is contacted with target pathogenic bacteria, the color of the stem with the bacteria is changed, the stem is in a water stain disease state, and villiform, flocculent or thin hypha grows out, if the cut of the stem is contacted with other mixed bacteria or bacteria of suspected pathogenic fungi, the stem shows that aseptic silks appear, or some compact or overgrown rhizopus nigricans hypha appear, the pathogenic bacteria can be quickly identified, selected and trapped, and the simple, convenient and efficient technical effect is further ensured to be realized. In addition, the corn seedling stems with the leaf age of 2-4 have the advantages of being easy to obtain and disinfect, a small amount of disinfectant still remains on the surfaces and the incisions of the corn seedling stems after the corn seedling stems are disinfected, when the incisions are instantly contacted with the surface layers of hyphae on the diseased tissues, the disinfectant can disinfect the diseased tissues and a small amount of mixed bacteria on the surface layers of the hyphae, meanwhile, the disinfectant can also play a certain biological filtering role on the bacteria, and pathogenic bacteria can be quickly identified and trapped.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.