CN114451163A - Walnut black spot pathogen infection test method - Google Patents
Walnut black spot pathogen infection test method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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Abstract
The invention discloses a walnut black spot pathogen infection test method, which belongs to the technical field of walnut disease resistance research, and comprises the steps of taking walnut tissue culture seedlings, cutting off calluses, placing cut stems into bacterial suspension, soaking for 15-25s, and immediately transferring to a new culture medium for culture; the OD600 of the bacterial suspension is 0.01-0.1. The method disclosed by the invention is simple to operate, stable in test conditions, accurate and reliable in result, capable of meeting the test requirements for the disease attack time, beneficial to research on phenotypic change of the inoculated walnut tissue culture seedling, physiological and biochemical change in the plant body and related immune response mechanisms, and significant for early screening of resistant germplasm.
Description
Technical Field
The invention belongs to the technical field of walnut disease resistance research, and particularly relates to a walnut black spot pathogen infection test method.
Background
The walnut black spot is a main disease of walnuts, and pathogenic bacteria of the disease mostly infect tender stems, tender leaves, fruits and the like of the walnuts and influence the vegetative growth and reproductive growth of the walnuts. At present, most of researches on the walnut black spot disease adopt field leaves for inoculation, but the in vitro leaf inoculation can only be used for preliminary screening of concentration and resistance and cannot be used for determining indexes, and field living body inoculation operation is labor-consuming, is easily limited by external environment, causes large inoculation test errors, and is not suitable for early screening of resistant germplasm.
Therefore, how to provide a walnut black spot pathogen infection test method which is convenient to inoculate, accurate in test result and beneficial to early screening of resistant germplasm is an urgent problem to be solved in the field.
Disclosure of Invention
The invention discloses a walnut black spot pathogen infection test method, which can effectively reduce test errors by inoculating pathogenic bacteria to a tissue culture seedling stem cut, thereby facilitating early screening of resistant germplasm.
In order to achieve the purpose, the invention adopts the following technical scheme:
a walnut black spot pathogen infection test method comprises the following steps: taking walnut tissue culture seedlings, cutting off and healing the seedlings, placing the cut stems into the bacterial suspension, soaking for 15-25s, and immediately transferring the cut stems to a new culture medium for culture;
bacterial suspension OD600＝0.01-0.1。
By carrying out bacterial suspension soaking treatment on the cut of the stem of the walnut tissue culture seedling, the pollution of pathogenic bacteria to a tissue culture medium can be reduced to the minimum extent, so that the secondary infection of the tissue culture seedling caused by mass propagation of the pathogenic bacteria on the culture medium is avoided, and the accuracy of a test result is influenced; the tissue culture seedlings subjected to the pathogen infection treatment of the invention have good vitality while having black spot symptoms at the stem and the leaf, can maintain growth for a long time without death, and further provide enough time for implementing related tests after pathogen inoculation; in addition, the tissue culture seedling has stable growth environment under the tissue culture condition, thereby reducing the test error caused by the environment difference and ensuring that the test result is more accurate; furthermore, the walnut tissue culture seedling has short propagation period, can maintain excellent genetic characteristics through asexual propagation, rarely generates genetic variation, and has higher credibility when being used for early screening of resistant germplasm.
Preferably, the culture conditions are 23 ℃ and the photoperiod is 16h light and 8h dark.
Preferably, a control group is set:
cutting off the callus of the walnut tissue culture seedlings with the same tender degree and growth vigor as the bacteria suspension group, placing the stem cut in sterile water, soaking for 15-25s, and immediately transferring to a new culture medium for culture.
Preferably, when the bacterial suspension group and the control group are inoculated in the same container, the control group is inoculated first.
Preferably, photographing recording and sampling tests are carried out on the tissue culture seedlings in the culture process.
Preferably, the tissue culture seedling container for photographing is used for fixing the tissue culture seedling in the photographing recording process:
the tissue culture seedling container for photographing comprises a dish cover, a dish bottom, a shielding piece and a clamping piece;
firstly, standing the bottom of the dish on the side, and clamping by a clamping piece to shield the bottom of the opening of the dish bottom by a shielding piece, wherein the shielding area does not exceed 1/2 of the area of the dish bottom;
and pouring the culture medium into the laterally erected dish bottom, removing the shielding piece and the clamping piece after the culture medium is solidified, and buckling the dish cover with the dish bottom for transferring the tissue culture seedlings.
Preferably, the tissue culture seedling container for photographing is directly used for inoculating the tissue culture seedling after being soaked in bacterial suspension or sterile water, or is used for fixing when photographing records need to be carried out in the tissue culture seedling culture process. This shoot and make the record of tissue culture seedling phenotype change more convenient with tissue culture seedling container, avoid tissue culture bottle reflection of light, take out the tissue culture seedling many times and easily infect, take a picture inconvenient problem in the superclean bench, and pour the culture medium in the culture dish of inclining upright and not only can realize the fixed of tissue culture seedling, can also provide sufficient nutrition for the growth of tissue culture seedling, also make the sample detection of tissue culture seedling more convenient.
Preferably, the shielding piece is of an L-shaped structure and comprises a baffle plate and a supporting plate which are perpendicular to each other;
the baffle is blocked at the bottom of the opening of the dish bottom by the clamping of the clamping piece.
Preferably, the clamping member comprises an elastic member and a pressing plate;
the clamp plate is fixed in the one end of layer board through the elastic component, and the elastic component gives the clamp plate towards the power of baffle effect, with the centre gripping at the bottom of the ware between clamp plate and baffle.
Preferably, the baffle is a transparent plate, so that the solidification condition of the culture medium can be observed conveniently.
In conclusion, the walnut black spot pathogen infection test method is simple to operate, stable in test conditions, accurate and reliable in result, and capable of meeting test requirements for disease attack time, facilitating research on phenotype change of inoculated walnut tissue culture seedlings, physiological and biochemical change in plants and relevant immune response mechanisms, and has important significance for early screening of resistant germplasm.
Drawings
FIG. 1 shows the results of the in vitro leaf soaking inoculation and needle-prick inoculation tests of the tissue culture seedlings;
FIG. 2 shows the results of the spray inoculation test (OD) for tissue culture seedlings600＝0.05，20dpi)；
FIG. 3 shows the results of the test of the stem cut soaking inoculation (OD) of the tissue culture seedlings600＝0.05)；
FIG. 4 is a DAB staining diagram of 25d leaf after stem cut soaking inoculation of tissue culture seedlings;
FIG. 5 shows the results of the test of the stem cut soaking inoculation (OD) of the tissue culture seedlings600＝0.1)；
The left is a comparison graph before inoculation, and the right is a comparison graph when a bacterial suspension group (T group) tends to die;
FIG. 6 shows the bottom and lid of a container for tissue culture seedlings for photography;
FIG. 7 shows the position of the shield of the tissue culture container for photographing;
FIG. 8 is a view showing a combination of the constitution of pouring the culture medium into the container for photo-taking tissue culture seedlings;
FIG. 9 shows the state of a tissue culture seedling cultured using a tissue culture seedling container for photography;
FIG. 10 is a view showing a combination configuration when a tissue culture seedling is cultured using a tissue culture seedling container for photography;
FIG. 11 is a first view showing the placement of the L-shaped shield;
FIG. 12 is a second view of the L-shaped shield;
fig. 13 is a schematic structural view showing the dish bottom clamped between the pressing plate and the baffle.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Taking Pantoea agglomerans (Pantoea agglomerans) as an example, taking fragrant walnuts as materials, carrying out a walnut black spot pathogen infection test:
separating Pantoea agglomerans from walnut black spot disease plants, activating, centrifuging to obtain precipitate, and resuspending to OD with sterile water600Bacterial suspension was prepared for use at 0.05 and 0.1.
Taking 'fragrant' walnut branches as explants, culturing in a walnut DKW culture medium (DKW/Juglans Base Salts, Beijing Kudiebo science and technology Limited, product number PM1390-50L) for about 14 days under the conditions of 23 ℃, 16h of illumination and 8h of darkness, transferring into a new culture medium, and subculturing under the conditions as follows: DKW medium +0.75 mg/L6-BA +0.01mg/L IBA +30g/L sucrose +2.5g/L Gelzan, pH 5.5-5.6.
Selecting walnut tissue culture seedlings with consistent tender degree and growth vigor after subculture for about 10-14 days, and taking walnut tissue culture seedling leaves to respectively perform needle-punching inoculation and soaking inoculation; selecting walnut tissue culture seedlings with about 10-14d after subculture, consistent tender degree and growth vigor, and respectively carrying out spray inoculation and stem cut soaking inoculation:
(1) carrying out needle-prick inoculation on in vitro leaves of the tissue culture seedlings:
taking off the leaves of the walnut tissue culture seedlings, temporarily preserving moisture in sterile water, placing the moisture-preserving leaves on sterile filter paper after the leaves are completely collected, sucking off excessive moisture, then paving the leaves on different culture media, carrying out acupuncture inoculation, taking water as a reference, and repeating for many times. After inoculation is finished, the culture medium is placed in an illumination incubator with the temperature of 28 ℃ and the illumination for 16h and the darkness for 8h for observation.
The culture medium used comprises:
culture medium 1: DKW medium +0.75 mg/L6-BA +0.01mg/L I-BA +30g/L sucrose +2.5g/LGelzan, pH 5.5-5.6.
Culture medium 2: DKW medium +30g/L sucrose +2.5g/L Gelzan, pH 5.5-5.6.
(2) Soaking and inoculating the cultured seedling in vitro leaves:
after the leaves of the walnut tissue culture seedlings are taken down, temporarily preserving moisture in sterile water, after the leaves are taken, placing the moisture-preserving leaves on sterile filter paper to absorb excess moisture, respectively soaking the leaves in the sterile water and bacterial suspension for 20s, then drying excess liquid on the surfaces of the leaves on the sterile filter paper, then paving the leaves on a culture medium (which is inoculated with acupuncture), placing the leaves in an illumination incubator with the temperature of 28 ℃, the illumination for 16h and the darkness for 8h, and repeating the steps for many times.
(3) Spray inoculation of tissue culture seedlings:
sterilizing PP material with a spray can in a super clean bench under ultraviolet for 30min, soaking in 75% alcohol for 30min, and rinsing with sterile water for three times; adding the bacterial suspension into a sprinkling can, opening a bottle cap of a tissue culture bottle, sprinkling the bacterial suspension on the surface of a leaf of a tissue culture seedling, spraying the bacterial suspension to moisten the surface of the leaf, covering the bottle cap of the tissue culture bottle, moving the bottle cap of the tissue culture bottle to an illumination incubator with 23 ℃, 16h illumination and 8h darkness, setting a control group for spraying sterile water, and repeating the steps for many times.
(4) Soaking and inoculating the stem cut of the tissue culture seedling:
cutting off the callus at the lower part of the tissue culture seedling in a super clean bench, and reserving stem and leaf parts with consistent tender degree and approximately equal stem length at the upper part; using gun-shaped forceps to clamp the tissue culture seedling, only enabling the cut of the stem part of the tissue culture seedling to be soaked in a 1.5mL sterile centrifuge tube containing 800 mu L of bacterial suspension for 20s, and enabling the rest parts not to contact the bacterial suspension, and setting a control group soaked in sterile water; after the incision soaking of each group of tissue culture seedlings is finished, the tissue culture seedlings are respectively immediately transferred to a tissue culture bottle filled with a new culture medium (DKW culture medium +0.75 mg/L6-BA +0.01mg/L IBA +30g/L sucrose +2.5g/L Gelzan, pH is 5.5-5.6), cultured in an illumination incubator with 16h illumination and 8h darkness at 23 ℃, and the operation is repeated for many times.
In addition, a test that the tissue culture seedlings of the bacterial suspension group and the tissue culture seedlings of the control group are cultured in the same tissue culture bottle is set, the tissue culture seedlings of the control group are soaked in sterile water in the test process, and are immediately transferred to a culture medium after being soaked, and then the tissue culture seedlings of the bacterial suspension group are soaked in bacterial suspension and are immediately transferred to the culture medium after being soaked. Culturing at 23 deg.C in 16h light and 8h dark light incubator, and repeating the steps.
In the culture process, photographing, recording and sampling tests are carried out on the in vitro leaves and the tissue culture seedlings.
The experimental results of the needle-prick inoculation and the immersion inoculation of the isolated leaves of the tissue culture seedlings show that the resistance of a single isolated leaf to pathogenic bacteria is weak, and 3d leaves obviously change color after inoculation (figure 1); however, the in vitro leaf inoculation method has larger error, and partial control group leaves also have the phenomenon of blackening and dying; in addition, the leaves in vitro can not keep vitality for a long time, and the leaves die greatly 1 month after inoculation.
The results of the spray inoculation test of the tissue culture seedlings show that the bacterial suspension group can keep vitality for a long time (figure 2), and the stems and leaves can have black spot symptoms, but the spray inoculation range is not easy to control, so that the bacterial suspension falls on a culture medium, the lower callus and the upper leaves are dually infected, the infection conditions of the tissue culture seedlings are large in individual difference, and the tissue culture seedlings cannot be used for subsequent experimental research.
The test result of the stem cut soaking inoculation shows that the bacterial suspension group can survive 30 days after the tissue culture seedling is inoculated, and the bacterial suspension group presents obvious black spot disease symptoms, and the growth vigor is obviously different from that of a control group (figures 3 and 4). When the tissue culture seedlings of the bacterial suspension group and the tissue culture seedlings of the control group are cultured in the same tissue culture bottle, the tissue culture seedlings of the control group are not influenced (figure 5).
Example 2
On the basis of example 1, the tissue culture seedling container for photographing was used for fixing the tissue culture seedlings in the photographing recording process:
as shown in fig. 6-8, the tissue culture seedling container for photographing comprises a dish cover 1, a dish bottom 2, a shielding piece 3 and a clamping piece 4; wherein, the depth of the dish bottom 2 is 5cm, the diameter is 12cm, and the dish cover 1 is matched with the dish bottom 2; the shielding piece 3 is a transparent plate, and the clamping piece 4 is a clamp.
Firstly, the dish bottom 1 is erected laterally and is clamped by the clamping piece 4, so that the shielding piece 3 shields the bottom of the opening of the dish bottom 1, and the shielding area does not exceed 1/2 of the area of the dish bottom 1;
pouring a culture medium into the laterally standing dish bottom 1, removing the shielding part 3 and the clamping part 4 after the culture medium is solidified, and buckling the dish cover 2 and the dish bottom 1 for transferring tissue culture seedlings.
The tissue culture seedling container for photographing can be directly used for inoculating a tissue culture seedling after being soaked in a bacterial suspension or sterile water, or used for fixing when photographing records are required in the culture process of the tissue culture seedling after inoculation (figure 9). When tissue culture seedlings are cultured using this container, the barrier 3 can be held by the holder 4 and fixed to the end of the dish bottom 1 or the dish lid 2, and a fulcrum is provided for the vertical culture of the tissue culture seedlings (fig. 10).
In another preferred embodiment, as shown in fig. 11 and 12, the shielding member 3 is of an "L" shape, and includes a baffle 31 and a supporting plate 32 which are perpendicular to each other, and the baffle 31 is transparent; the baffle 31 is blocked at the bottom of the opening of the dish bottom 1 by clamping of the clamping piece 4, and the supporting plate 32 is supported at the bottom of the dish bottom 1 or arranged at the other side of the dish bottom 1 to provide support for the side standing of the dish bottom 1; pouring the culture medium, after tissue culture seedlings are transplanted, continuously fixing the shielding part 3 at the bottom of the dish 1 or one side of the dish cover 2 by using the clamping part 4, and further providing a fulcrum for the vertical culture of the tissue culture seedlings.
In another preferred embodiment, as shown in fig. 13, the clamping member includes an elastic member (not shown in the drawings) and a pressing plate 41; the pressing plate 41 is fixed at one end of the supporting plate 32 through an elastic piece, the elastic piece gives the pressing plate 41 a force acting towards the baffle plate 31, the dish bottom 1 is clamped between the pressing plate 41 and the baffle plate 31, and the culture medium is convenient to pour.
And after the culture medium is solidified, transplanting the tissue culture seedlings, taking down the dish bottom 1, covering the dish cover 2, and clamping the buckled dish between the pressing plate 41 and the baffle 31 to perform vertical culture of the tissue culture seedlings.
Example 3
In different areas and differentRespectively separating multiple Pantoea agglomerans (Pantoea agglomerans), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Xanthomonas Campestris and Pantoea ananatis (Pantoea ananatis) from walnut melasma plants, centrifuging the activated bacterial liquid, taking the precipitate, and suspending the precipitate with sterile water until OD is achieved600Bacterial suspension was prepared for use at 0.01, 0.05, 0.1.
Inoculating by using each bacterial suspension according to the method for soaking and inoculating the stem cut of the tissue culture seedling in the embodiment 1;
the results showed that the Pantoea agglomerans inoculated at OD600 of 0.01 showed the onset of disease symptoms about 7 days after inoculation, the leaves showed brown lesions or microspots at about 10dpi, and the leaves showed the remarkable inoculation phenomenon at 14dpi, and could sustain the vitality for about 35 days.
When the pantoea agglomerans is inoculated according to the OD600 of 0.05, disease symptoms can be shown at 3dpi after inoculation, brown lesions or spots begin to appear at the edge of the leaf or in the leaf, and an obvious inoculation phenomenon can be shown at about 7-8dpi, so that the vitality can be maintained for about 30 d; the inoculation concentration has high morbidity speed, and the plant has long life-sustaining time, and can meet the research requirements of phenotype change of the inoculated walnut tissue culture seedling, physiological and biochemical change in the plant body and related immune response mechanisms.
When the pantoea agglomerans is inoculated according to OD600 being 0.1, disease symptoms can be shown at 1dpi after inoculation, differences can be shown among inoculation strains at about 3dpi, differences are shown among different resistant strains after tissue culture seedling inoculation at about 7-8dpi, obvious inoculation phenomena can be shown at about 7-8dpi, and the vitality can be maintained to be 20-25 d; the concentration is quick in onset, but the survival time of the plants is short, so that the method is suitable for quick screening of different resistant plants.
When pseudomonas aeruginosa, flavomonas aeruginosa and pantoea ananatis are inoculated according to OD600 of 0.05, the disease is fast to occur, and the survival time is longer.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A walnut black spot pathogen infection test method is characterized in that,
taking walnut tissue culture seedlings, cutting off and healing the seedlings, placing the cut stems into the bacterial suspension, soaking for 15-25s, and immediately transferring the cut stems to a new culture medium for culture;
the bacterial suspension OD600＝0.01-0.1。
2. The walnut black spot pathogen infection test method according to claim 1,
the culture conditions were 23 deg.C, the photoperiod was 16h light and 8h dark.
3. The walnut black spot pathogen infection test method according to claim 1,
also includes setting contrast group:
cutting off the callus of the walnut tissue culture seedlings with the same tender degree and growth vigor as the bacteria suspension group, placing the stem cut in sterile water, soaking for 15-25s, and immediately transferring to a new culture medium for culture.
4. The walnut black spot pathogen infection test method according to claim 3,
when the bacterial suspension group and the control group are inoculated in the same container, the control group is inoculated first.
5. The walnut black spot pathogen infection test method according to claim 3,
and carrying out photographing record and sampling test on the tissue culture seedlings in the culture process.
6. The walnut black spot pathogen infection test method according to claim 5,
the tissue culture seedling container for photographing is used for fixing the tissue culture seedling in the photographing recording process:
the tissue culture seedling container for photographing comprises a dish cover, a dish bottom, a shielding piece and a clamping piece;
firstly, standing the bottom of the dish on the side, and clamping by a clamping piece to shield the bottom of the opening of the dish bottom by a shielding piece, wherein the shielding area does not exceed 1/2 of the area of the dish bottom;
and pouring the culture medium into the laterally erected dish bottom, removing the shielding piece and the clamping piece after the culture medium is solidified, and buckling the dish cover with the dish bottom for transferring the tissue culture seedlings.
7. The walnut black spot pathogen infection test method according to claim 6,
the tissue culture seedling container for photographing is directly used for inoculating the tissue culture seedlings after being soaked in bacterial suspension or sterile water, or is used for fixing the tissue culture seedlings when photographing records need to be carried out in the culture process.
8. The walnut black spot pathogen infection test method according to claim 6,
the shielding piece is of an L-shaped structure and comprises a baffle plate and a supporting plate which are perpendicular to each other;
the baffle is blocked at the bottom of the opening of the dish bottom by the clamping of the clamping piece.
9. The walnut black spot pathogen infection test method according to claim 8,
the clamping piece comprises an elastic piece and a pressing plate;
the pressing plate is fixed at one end of the supporting plate through the elastic piece, and the elastic piece gives the pressing plate a force acting towards the baffle plate to clamp the dish bottom between the pressing plate and the baffle plate.
10. The walnut black spot pathogen infection test method according to claim 8,
the baffle is a transparent plate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105028204A (en) * | 2015-08-06 | 2015-11-11 | 新疆玉维鲜农业科技中心 | Screening method for virus-free potato test-tube plantlet early blight prevention |
| CN112111551A (en) * | 2020-09-24 | 2020-12-22 | 河南农业大学 | Method for identifying resistance of potato to black shank |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105028204A (en) * | 2015-08-06 | 2015-11-11 | 新疆玉维鲜农业科技中心 | Screening method for virus-free potato test-tube plantlet early blight prevention |
| CN112111551A (en) * | 2020-09-24 | 2020-12-22 | 河南农业大学 | Method for identifying resistance of potato to black shank |
Non-Patent Citations (1)
| Title |
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| 王艳丽等: "桉树抗青枯病的鉴定技术", 《林业科学》 * |
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