阅读说明：本技术 植物细菌性维管束病害的人工接种方法 (Artificial inoculation method for plant bacterial vascular bundle diseases ) 是由 欧阳秋飞 李曦 钟丽华 韦兰洁 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种植物细菌性维管束病害的人工接种方法,包括以下步骤：步骤1)、菌液的制备：将病原菌接种于NA培养基上,30℃培养24h后再转接一次,用灭菌的0.2～0.3mol/L的PB缓冲液将平板上的菌苔洗下,并制备成菌体浓度为0.5×10～(8)～1.5×10～(8)cfu/mL的菌悬液；步骤2)、离体枝条的处理：从木本植物上剪取顶部带有4～5片展开叶片的新生的枝条,放入75％的乙醇溶液中消毒1～3min后,用灭菌水冲洗多次；步骤3)、接种：将枝条插入装有1/3～1/2体积菌悬液的三角瓶或玻璃杯中,并用棉花或海绵包住枝条封口固定,再置于人工气候箱中培养。本发明能够对木本植株进行植物细菌性维管束病害的人工接种,具有操作简单,接种效果高效且稳定的有益效果。(The invention discloses an artificial inoculation method for plant bacterial vascular bundle diseases, which comprises the following steps: step 1), preparation of bacterial liquid: inoculating the pathogenic bacteria to NA culture medium, culturing at 30 deg.C for 24 hr, inoculating, washing off thallus Porphyrae with sterilized PB buffer solution of 0.2-0.3 mol/L, and making into thallus concentration of 0.5 × 10 8 ～1.5×10 8 cfu/mL of bacterial suspension; step 2), treatment of isolated branches: shearing a new branch with 4-5 unfolded leaves at the top from a woody plant, placing the new branch into a 75% ethanol solution for disinfection for 1-3 min, and washing with sterilized water for multiple times; step 3), inoculation: inserting the branches into a triangular flask or a glass cup filled with bacterial suspension with the volume of 1/3-1/2, wrapping the branches with cotton or sponge, sealing and fixing, and then placing the branches in a climatic chamber for culturing. The invention can carry out artificial inoculation on the plant bacterial vascular bundle diseases of woody plants, and has the advantages of simple operation, high-efficiency and stable inoculation effectHas the beneficial effects.)

1. The artificial inoculation method for the plant bacterial vascular bundle disease is characterized by comprising the following steps:

step 1), preparation of bacterial liquid: inoculating the pathogenic bacteria to NA culture medium, culturing at 30 deg.C for 24 hr, inoculating, washing off thallus Porphyrae with sterilized PB buffer solution of 0.2-0.3 mol/L, and making into thallus concentration of 0.5 × 10⁸～1.5×10⁸cfu/mL of bacterial suspension;

step 2), treatment of isolated branches: shearing a new branch with 4-5 unfolded leaves at the top from a woody plant, placing the new branch into a 75% ethanol solution for disinfection for 1-3 min, and washing with sterilized water for multiple times;

step 3), inoculation: inserting the branches into a triangular flask or a glass cup filled with bacterial suspension with the volume of 1/3-1/2, wrapping the branches with cotton or sponge, sealing and fixing, and then placing the branches in a climatic chamber for culturing.

2. The method for artificially inoculating bacterial vascular bundle disease in plants according to claim 1, wherein in step 2), the shoots are cut from woody plants, immediately inserted into sterile water, and left in the dark for 24 hours, and then the shoots with consistent growth and health are selected for sterilization.

3. A method of artificially inoculating a plant bacterial vascular bundle disease as claimed in claim 1 wherein the concentration of PB buffer in step 1) is 0.25 mol/L.

4. The method for artificially inoculating bacterial vascular bundle disease of plants according to claim 1, wherein the bacterial concentration of the bacterial suspension prepared in the step 1) is 1X 10⁸cfu/mL。

5. A method of artificially inoculating bacterial vascular bundle disease in a plant as claimed in claim 1 wherein the woody plant is mulberry.

6. The artificial inoculation method of plant bacterial vascular bundle disease as claimed in claim 1, wherein in step 3), the temperature in the artificial climate box is 28-32 ℃, the humidity is 90-95%, the illumination is 24h dark on day 1, 6h illumination and 18h dark on day 2, and 12h illumination and 12h dark after day 3; the light intensity after day 2 was 5000 lx.

7. The artificial inoculation method for plant bacterial vascular bundle diseases according to claim 1, wherein the branches are treated before step 3), four cross wounds are firstly cut at the middle lower part of the branches by a sterilization scalpel, the four cross wounds are spirally arranged along the vertical direction, absorbent cotton soaked with a mixed solution of zeatin and gibberellin is wrapped on the four cross wounds, a preservative film is used for wrapping and moisturizing, and after 1h, the preservative film and the absorbent cotton are removed, so that the branches are treated; immediately inserting the treated branches into the bacterial suspension, wherein any cross wound is not inserted into the bacterial suspension; wherein the concentration of the mixed solution of the zeatin and the gibberellin is 100mg/kg, and the mass ratio of the zeatin to the gibberellin is 1: 3.

8. A method of artificially inoculating a plant bacterial vascular bundle disease according to claim 7 wherein any cross wound has a length of 1/3 to 1/2 of the circumference of a shoot and a depth of 3 to 5 mm.

Technical Field

The invention relates to the technical field of plant protection. More particularly, the invention relates to an artificial inoculation method for bacterial vascular bundle diseases of plants.

Background

The plant bacterial vascular bundle diseases are common plant disease types in agricultural production and are caused by pathogenic bacteria, common bacterial wilt and bacterial wilt can be caused, and the disease can cause failure in delivery when serious. Bacterial vascular bundle pathogenic bacteria generally invade through radicles or wounds, spread in plant vascular bundles, block ducts, secrete toxins, damage ducts, block moisture transportation and further wither the overground part.

The pathogeny of the disease needs to be accurately identified for effective prevention and treatment of the disease, and the artificial inoculation method is a common means for identifying the pathogeny. At present, the artificial inoculation method of plant bacterial vascular bundle diseases is generally an injection bacterial liquid method, a needle punching method, a bacterial liquid root irrigation method and the like, but for some woody plants, the bacterial liquid of the injection bacterial liquid method is not easy to be injected into the plant body; the amount of bacteria carried by the needle punching method is limited, and when the temperature is dry or the humidity is low, the inoculated bacteria volume is volatile and deactivated; the bacterial liquid root irrigation method often cannot cause the seedling to be ill. Therefore, in the study of bacterial vascular bundle diseases in plants, particularly woody plants, there is a need for a rapid, efficient and reproducible artificial inoculation method.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide an artificial inoculation method for plant bacterial vascular bundle diseases, which can be used for artificially inoculating the plant bacterial vascular bundle diseases to woody plants and has the advantages of simple operation, high efficiency and stability of the inoculation effect.

To achieve these objects and other advantages in accordance with the purpose of the present invention, there is provided a method for artificial inoculation of a bacterial vascular bundle disease of a plant, comprising the steps of:

step 1), preparation of bacterial liquid: inoculating the pathogenic bacteria to NA culture medium, culturing at 30 deg.C for 24 hr, inoculating, washing off thallus Porphyrae with sterilized PB buffer solution of 0.2-0.3 mol/L, and making into thallus concentration of 0.5 × 10⁸～1.5×10⁸cfu/mL of bacterial suspension;

step 2), treatment of isolated branches: shearing a new branch with 4-5 unfolded leaves at the top from a woody plant, placing the new branch into a 75% ethanol solution for disinfection for 1-3 min, and washing with sterilized water for multiple times;

step 3), inoculation: inserting the branches into a triangular flask or a glass cup filled with bacterial suspension with the volume of 1/3-1/2, wrapping the branches with cotton or sponge, sealing and fixing, and then placing the branches in a climatic chamber for culturing.

Preferably, in the step 2), after the branches are cut from the woody plants, the branches are immediately inserted into sterile water and placed in a dark place for 24 hours, and then the branches with consistent growth vigor and healthy growth are selected for disinfection.

Preferably, the concentration of PB buffer in step 1) is 0.25 mol/L.

Preferably, the bacterial concentration of the bacterial suspension prepared in the step 1) is 1X 10⁸cfu/mL。

Preferably, the woody plant is mulberry.

Preferably, in the step 3), the temperature in the artificial climate box is 28-32 ℃, the humidity is 90-95%, the illumination is 24h dark on day 1, 6h illumination and 18h dark on day 2, and 12h illumination and 12h dark after day 3; the light intensity after day 2 was 5000 lx.

Preferably, the branches are treated before the step 3), firstly, a sterilization scalpel is used for cutting four cross wounds at the middle lower part of the branches, the four cross wounds are spirally arranged along the vertical direction, then the four cross wounds are wrapped by the absorbent cotton soaked with the mixed solution of the zeatin and the gibberellin, the absorbent cotton is wrapped by a preservative film for moisturizing, and after the preservative film and the absorbent cotton are kept for 1 hour, the treatment on the branches is completed; immediately inserting the treated branches into the bacterial suspension, wherein any cross wound is not inserted into the bacterial suspension; wherein the concentration of the mixed solution of the zeatin and the gibberellin is 100mg/kg, and the mass ratio of the zeatin to the gibberellin is 1: 3.

Preferably, the length of any cross wound is 1/3-1/2 of the circumference of the branch, and the depth is 3-5 mm.

The invention at least comprises the following beneficial effects:

according to the invention, branches are selected as inoculation materials and placed in the bacterial suspension for inoculation, and PB buffer solution is used for preparing the bacterial suspension, so that the artificial inoculation of plant bacterial vascular bundle diseases can be carried out on woody plants, and the method has the beneficial effects of simple operation, short experimental period, high efficiency and stability in inoculation effect;

secondly, after the branches are cut from the woody plant, the branches are immediately inserted into sterile water and placed in a dark place for 24 hours, and then the branches with consistent growth vigor and health are selected for disinfection, so that the experimental error can be further reduced, and the stability and reliability of the inoculation effect can be improved;

thirdly, the branches are treated before inoculation, so that the stability and the high efficiency of the inoculation effect can be further improved.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a disease symptom chart of the mulberry twig inoculated by the method of example 2 for 3-7 days.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

< example 1>

An artificial inoculation method for plant bacterial vascular bundle diseases comprises the following steps:

step 1), preparation of bacterial liquid: inoculating purchased Enterobacter mori in NA culture medium, culturing at 30 deg.C for 24 hr, transferring again, washing off thallus Porphyrae on plate with sterilized 0.2mol/L PB buffer solution, and making into thallus concentration of 0.5 × 10⁸cfu/mL of bacterial suspension;

step 2), treatment of isolated branches: shearing a new branch with 4 unfolded leaves at the top from a mulberry, placing the new branch into a 75% ethanol solution for disinfection for 1min, and washing with sterilized water for 3 times;

step 3), inoculation: the branches are inserted into a triangular flask filled with 1/3-volume bacterial suspension, are wrapped by sterile cotton, are sealed and fixed, and are cultured in a climatic chamber.

< example 2>

An artificial inoculation method for plant bacterial vascular bundle diseases comprises the following steps:

step 1), preparation of bacterial liquid: inoculating Enterobacter mori to NA medium, culturing at 30 deg.C for 24 hr, transferring, washing off thallus Porphyrae with sterilized 0.25mol/L PB buffer solution, and making into thallus concentration of 1 × 10⁸cfu/mL of bacterial suspension;

step 2), treatment of isolated branches: shearing a new branch with 4 unfolded leaves at the top from a mulberry, placing the new branch into a 75% ethanol solution for disinfection for 2min, and washing with sterilized water for 3 times;

step 3), inoculation: the branches are inserted into a triangular flask filled with 1/3-volume bacterial suspension, are wrapped by sterile cotton, are sealed and fixed, and are cultured in a climatic chamber.

< example 3>

An artificial inoculation method for plant bacterial vascular bundle diseases comprises the following steps:

step 1), preparation of bacterial liquid: inoculating Enterobacter mori to NA medium, culturing at 30 deg.C for 24 hr, transferring, washing off thallus Porphyrae with sterilized 0.3mol/L PB buffer solution, and making into thallus concentration of 1.5 × 10⁸cfu/mL of bacterial suspension;

step 2), treatment of isolated branches: shearing a new branch with 4 unfolded leaves at the top from a mulberry, placing the new branch into a 75% ethanol solution for disinfection for 3min, and washing with sterilized water for 3 times;

step 3), inoculation: the branches are inserted into a triangular flask filled with 1/3-volume bacterial suspension, are wrapped by sterile cotton, are sealed and fixed, and are cultured in a climatic chamber.

< example 4>

Different from the embodiment 2, the method is that in the step 2), after the branches are cut from the mulberry, the branches are immediately inserted into sterile water and placed in a dark place for 24 hours, and then the branches with consistent growth vigor and healthy are selected for disinfection; the other steps and parameters were the same as in example 2.

< example 5>

The difference from the embodiment 2 lies in that the branch is treated before the step 3), four cross wounds are firstly cut at the middle lower part of the branch by a sterilization scalpel and are spirally arranged along the vertical direction, then the four cross wounds are wrapped by sterile absorbent cotton soaked with mixed solution of zeatin and gibberellin, the four cross wounds are wrapped by a preservative film for moisturizing, and after the preservative film and the absorbent cotton are kept for 1 hour, the preservative film and the absorbent cotton are removed, so that the treatment of the branch is finished; immediately inserting the treated branches into the bacterial suspension, wherein any cross wound is not inserted into the bacterial suspension; wherein the concentration of the mixed solution of the zeatin and the gibberellin is 100mg/kg, the mass ratio of the zeatin to the gibberellin is 1:3, the length of any cross wound is 1/3 of the circumference of a branch, and the depth is 4 mm; the other steps and parameters were the same as in example 2.

< example 6>

Different from the embodiment 2, the method is that in the step 2), after the branches are cut from the mulberry, the branches are immediately inserted into sterile water and placed in a dark place for 24 hours, and then the branches with consistent growth vigor and healthy are selected for disinfection; treating the branches before step 3), firstly cutting four cross wounds at the middle lower part of the branches by using a sterilization scalpel, spirally arranging the four cross wounds along the vertical direction, then wrapping the four cross wounds by using sterile absorbent cotton soaked with a mixed solution of zeatin and gibberellin, wrapping the four cross wounds by using a preservative film for moisturizing, and removing the preservative film and the absorbent cotton after keeping for 1h, thus finishing the treatment of the branches; immediately inserting the treated branches into the bacterial suspension, wherein any cross wound is not inserted into the bacterial suspension; wherein the concentration of the mixed solution of the zeatin and the gibberellin is 100mg/kg, the mass ratio of the zeatin to the gibberellin is 1:3, the length of any cross wound is 1/3 of the circumference of a branch, and the depth is 4 mm; the other steps and parameters were the same as in example 2.

< comparative example 1>

Inoculation is performed by a bacterial solution injection method, which is different from < example 2> in the following steps of 3): inserting the branches into a triangular flask filled with 1/3 volume 0.25mol/L PB buffer solution, wrapping the branches with sterile cotton, sealing and fixing, extracting bacterial suspension with a sterilization syringe, and injecting about 0.02ml of bacterial suspension at the axilla of the 2 nd to 4 th leaf; the other steps and parameters were the same as in example 2.

< comparative example 2>

The inoculation was carried out by needle punching, which is different from < example 2> in step 1), preparation of lawn: scribing or coating the purchased enterobacter mori on an NA culture medium, culturing for 24 hours at 30 ℃, and then transferring once again until bacterial lawn grows for later use;

step 3), inoculation: inserting the branches into a triangular flask filled with 1/3 volume 0.25mol/L PB buffer solution, wrapping the branches with sterile cotton, sealing and fixing, dipping bacterial lawn with sterilized toothpicks, piercing the axilla of 2-4 leaves to a depth of about 3mm, and pulling out the toothpicks after piercing; the other steps and parameters were the same as in example 2.

< comparative example 3>

Inoculating by adopting a bacterial liquid root irrigation method, which is different from the embodiment 2), and culturing mulberry seedlings by the steps of: accelerating germination of the disinfected mulberry seeds in a constant temperature box at 28 ℃, transplanting the seedlings into culture bags filled with sterilized seedling raising soil, and transplanting 1 seedling in 1 culture bag until 5-6 true leaves grow out of the mulberry seedlings;

step 3), inoculation: using a sterile knife to form a root wound at a position 1cm away from the root base on one side of the mulberry seedling, then pouring about 50ml of bacterial suspension into the wound position, pouring 1 time every 1 day, and pouring 3 times together; the other steps and parameters were the same as in example 2.

< comparative example 4>

And<example 2>Except that, in the step 1), the bacterial lawn on the plate was washed off with sterilized 0.25mol/L PBS buffer and prepared to have a bacterial density of 1X 10⁸cfu/mL of bacterial suspension; the other steps and parameters were the same as in example 2.

< comparative example 5>

And<example 2>Except that, in the step 1), the bacterial lawn on the plate was washed with sterilized water and prepared to have a bacterial density of 1X 10⁸cfu/mL of bacterial suspension; the other steps and parameters were the same as in example 2.

< evaluation of inoculation Effect >

Inoculating by adopting the methods of the embodiments 1-6 and the comparative examples 1-5, wherein the variety of the mulberry is Guisang you 12, each treatment is repeated by 20 times, the inoculated PB buffer solution of 0.25mol/L is taken as a control group 1, the inoculated PBS buffer solution of 0.25mol/L is taken as a control group 2, and the inoculated sterile water is taken as a control group 3; the temperature in the artificial climate box is 32 ℃, the humidity is 95%, the illumination is 24h darkness at day 1, 6h illumination and 18h darkness at day 2, and 12h illumination and 12h darkness after day 3; the light intensity after day 2 was 5000 lx;

observation of results and investigation of disease conditions: observing the disease condition every day after inoculation and culture, performing disease condition investigation 7 days after inoculation and culture, counting and calculating the disease rate and disease condition index, wherein the disease grading standard adopted by the disease condition investigation is as follows:

grade 0, no symptoms;

grade 1, 1 leaf wilting or withering and necrosis;

grade 2, 2 leaves wither or withered and necrotic;

grade 3, 3 leaves wither or withered and necrotic;

4-grade, 4 leaves wither or withered and necrotic;

incidence (%) × 100% (number of diseased plants/total number of inoculated plants);

the disease index is [ Σ (representative value at each disease level × number of disease plants at that level)/(total number of investigated plants × 4) ] × 100.

TABLE 1 comparison of pathogenic rates of pathogenic bacteria of Mulberry bacterial wilt using different artificial inoculation methods

Group of	Incidence (%)	Index of disease condition
			Example 1	80.31±2.32	80.32±2.13
Example 2	82.15±2.27	84.45±2.02
			Example 3	81.42±2.41	83.63±2.25
Example 4	83.01±0.98	84.78±0.74
			Example 5	92.52±1.94	91.51±1.77
Example 6	93.21±0.69	95.32±0.53
			Comparative example 1	45.74±2.52	55.19±2.42
Comparative example 2	35.46±2.43	58.91±2.18
			Comparative example 3	0	0
Comparative example 4	75.12±2.26	64.45±1.95
			Comparative example 5	73.32±2.15	62.25±1.87
Control group 1	0	0
			Control group 2	0	0
Control group 3	0	0

As can be seen from table 1, compared with comparative examples 1-3, the morbidity and disease index of example 2 are both significantly improved, and thus, the branches are selected as the inoculation material to be placed in the bacterial suspension for inoculation, so that the artificial inoculation of the plant bacterial vascular bundle diseases can be performed on woody plants, and the method has the beneficial effects of simple operation, short experimental period, and high and stable inoculation effect; moreover, compared with comparative examples 4-5, the morbidity and disease index of example 2 are both obviously improved, and the inoculation effect is better when the bacterial suspension is prepared by using the PB buffer solution;

compared with example 2, the standard deviation of the morbidity and the standard deviation of the disease index of example 4 are both obviously reduced, and compared with example 5, the standard deviation of the morbidity and the standard deviation of the disease index of example 6 are also obviously reduced, so that the branches are immediately inserted into sterile water after being cut from the woody plant, placed in a dark place for 24 hours, and then the branches with consistent growth and health are selected for disinfection, thereby further reducing experimental errors and improving the stability and reliability of the inoculation effect;

compared with the embodiment 2, the morbidity and disease index of the embodiment 5 are obviously improved, so that the treatment of the cross wound by the zeatin and gibberellin solution before the branch is inoculated is beneficial to promoting the transfer of organic substances in the branch and maintaining the growth activity of the branch, and the reliability, stability and high efficiency of the inoculation effect can be further improved;

in addition, the leaves of the branches of examples 1-4 and comparative examples 4-5 begin to show obvious wilting symptoms 3 days after inoculation, and the leaves are curled and scorched 7 days after inoculation (figure 1), the bark of the branches is peeled off, and the xylem of the branches turns brown; after the branches of the embodiments 5-6 are inoculated for 2 days, obvious wilting symptoms appear on leaves, and after 7 days of inoculation, the epidermis is peeled off, and the xylem of the branches is brown; 4 days after inoculation, leaf leaves of the branches in the comparative examples 1-2 begin to show wilting symptoms, 7 days after inoculation, the skin is peeled off, xylem of the branches turns brown, no obvious wilting symptom appears on the leaves in the comparative example 3 and the comparative examples 1-3, the skin is peeled off, and the xylem of the branches does not appear brown; therefore, the method has the beneficial effects of high efficiency and stability of the inoculation effect.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.