CN105875200A - Method for inoculating Fungi - Google Patents

Abstract

The invention discloses a method for inoculating fungi, as well as an inoculation device and a culture medium used in the method. The fungal inoculation method of the present invention is to sterilize the metal inoculation tool with a pointed end and wooden or bamboo parts on it, and then transfer the aforementioned inoculation tool to the target fungus in an aseptic environment. The culture medium is cultivated after the culture medium, and then the aforementioned inoculation tool is transferred to the medium with the target fungus in it under a sterile environment, and the culture medium is cultivated, so that the inoculation tool made of wooden or bamboo parts Infect the body with bacteria, then take out the inoculation tool and insert its tip into the inoculation site of the host, so that the woody or bamboo inoculum fungus that has been infected on the inoculation tool can infect the host and realize the inoculation operation. The invention has the characteristics of accuracy, convenience, time saving and labor saving, etc., and can inoculate relatively hard parts such as forest trees.

Description

A kind of fungal inoculation method

technical field

The present invention relates to a fungal inoculation method, and the inoculation utensil and culture medium used in the method. Specifically, it is an inoculation method in which the target fungus is contacted with the inoculated part of the host plant by using the inoculation tool, and the fungus invades the host plant. Inoculation equipment and culture medium.

Background technique

The process of artificially contacting pathogenic fungi with the susceptible parts of the host plant to create conditions for the pathogen to invade and induce the disease of the host is called inoculation. Inoculation is an important step in the process of syndrome and disease. Inoculation is required when studying the pathogenesis of parasitic phenomena, measuring the disease resistance of varieties, and evaluating the effect of pesticides on disease prevention. Therefore, the inoculation method directly affects the test effect.

The method of artificial inoculation of plant diseases is designed according to the infection mode and infection route of the disease. There are many types of plant diseases, and their infection modes and infection routes are different. There are two types of fungal inoculation methods: methods without inoculation tools and methods with inoculation tools. Among them, the methods with inoculation tools are:

1) Spray method, this method is to spray the prepared spore suspension on the surface of the host, and the pathogenic bacteria can invade from the stomata, wound or epidermis.

2) The smearing method, that is, the pathogenic bacteria are spread on the surface of the plant with a brush or brush, so that the spores can germinate and invade.

3) Injection method, the spore suspension is injected into the growing point or tender part with a syringe.

4) Acupuncture method, after artificially manufacturing mechanical damage, paste the bacteria cake on the wound to induce disease.

The existing inoculation methods can be found in Fang Zhongda, 1998, "Plant Disease Research Methods". However, traditional inoculation methods often have disadvantages such as low inoculation efficiency, slow symptom onset, poor inoculation consistency, and methods 1 and 2 in the aforementioned methods also need to be kept moist for 48 hours after inoculation, while methods 3 and 4 are for the seedling stage. Smaller individuals, as well as tissue structures such as detached leaves, are difficult to quantitatively implement infection.

Bacterial inoculation methods have improved a lot in recent years, while fungal inoculation methods have changed little.

Chinese invention patent 201410604178.7 discloses a method for making edible fungus wooden swab strains, which belongs to the field of edible fungus production and planting. The method comprises the steps of: fixing gauze or kraft paper to one end of a wooden stick, putting it into lime water and soaking until there is no white center inside the wooden stick, taking out the wooden stick and drying the surface water; mixing flour, corn flour, white sugar, gypsum, phosphoric acid Potassium dihydrogen, magnesium sulfate, _gufengsu , and vitamin B1 are added to water, and heated to a paste to obtain an adhesive; mix the wooden stick with the adhesive and stir until the adhesive is evenly attached to the surface of the wooden stick, and then add bran Stir the skin or rice bran until it evenly adheres to 70-80% of the surface of the wooden stick, put the wooden stick into a polypropylene bag or a strain bottle, seal it and sterilize it; Cultivate at 26°C for 10-20 days, and the hyphae will grow. But this wooden stick strain is not suitable for fungal inoculation, and its preparation method is too complicated.

Chinese invention patent application 201310720075.2 discloses a method for inoculating wooden nails. The wooden nails used in this patent are made of branches, waste materials, defective food sticks, fried dough sticks, and apricot sticks with a thickness of 0.5cm. Rod-shaped wooden nails are soaked in normal temperature white sugar water with 5% concentration for 24 hours, pulled out and then mixed with 10% nutrient material of the bacterial nail volume and can be bagged for inoculation. But this dowel will soften when immersed in the substratum, and then use this dowel on the harder host of texture, such as on trees, it may not be able to puncture the host during inoculation, so as to realize the inoculation operation.

Contents of the invention

The invention provides a fungal inoculation method which can overcome the deficiencies of the prior art, and provides the culture medium and inoculation utensils used in the method at the same time.

The fungal inoculation method of the present invention also uses an inoculation tool to contact the target fungus with the inoculation site of the host, so that the fungus can invade the host. The inoculation tool of the component is sterilized, and then the aforementioned inoculation tool is transferred to the medium containing the target fungus in a sterile environment, and then the culture medium is cultured, and then the aforementioned inoculation tool is transferred into it in a sterile environment After the culture medium of the target fungus is available, the medium is cultivated, and the inoculum composed of wooden or bamboo parts on the inoculation tool is infected with bacteria, and then the inoculation tool is taken out and its tip is inserted into the inoculation site of the host, so that the inoculation tool has been infected. The woody or bamboo inoculum fungus infects the host to realize the inoculation operation.

The medium used in the fungal inoculation method of the present invention is to add 20 grams of glucose in 1 L of potato decoction.

The inoculum used in the inoculation method of the present invention is at least made of a rod-shaped metal piece with a tip and wood or bamboo embedded on the rod-shaped metal piece or other materials that can allow bacteria and fungi to adhere to form an inoculum, wherein the inoculation At least a part of the body is exposed on the outer surface of the rod-shaped metal piece.

The inoculum used in the inoculation method of the present invention can also be made of wood or bamboo with a cone at one end, an inoculum that is set as a cylinder after the cone, and a metal tip that is fixed on the head of the cone.

Preferably, the metal tip of the inoculation tool of the present invention is embedded and fixed with the wooden or bamboo cone, or fixed with threads.

The inoculation tool of the present invention is characterized in that the metal used for the inoculation tool is stainless steel.

The advantages of the present invention are:

1. Because the present invention adopts a metal rod-shaped structure with a tip, especially the stainless steel needle tip is convenient for tissue stabbing, and is especially suitable for inoculation of hard tree trunks, and there is no shortage of inoculation dowel softening in the prior art. On the other hand, the inoculation set of the present invention can be reused as long as it is washed and sterilized after use, and the inoculum can also be replaced under special circumstances, so the use of the inoculation set of the present invention can save costs.

2. The woody or bamboo inoculum provided on the inoculation tool of the present invention can be infected with bacteria to infect the host, and at the same time it is more convenient to accurately observe the invasion point of pathogenic bacteria, and accurately observe and count at fixed points.

3. Save time. The method of the present invention is the same as other methods, needs the pure culture of pathogenic bacteria, but in the past all is cultured on solid medium, waits for pathogenic bacteria to produce spores. Some such as Alternaria sporulation are faster, but many pathogenic bacteria have a long sporulation period and a small sporulation amount. It is necessary to prepare a spore suspension with a concentration of 1×10 ⁶ /ml, which often requires a large number of pathogenic bacteria to multiply. 20-60 days range. In the method of the present invention, the inoculum is cultivated in the liquid base, so that the pathogenic bacteria can grow rapidly and fully infect the bacteria, and this process only needs 10-15 days.

4. The error is small. In the past, when preparing spore suspension or spore smear, in order to ensure a higher concentration of pathogenic bacteria, it was necessary to prepare the spore suspension with a hemocytometer. However, the statistical data of the hemocytometer is only a statistical estimate of multiple conversion, which often has a large error with the actual situation (for example, the spore concentration of the hemocytometer is 1×10 ⁶ /ml, but the actual may only be 1×10 ⁵ /ml, the difference can be up to 10 times). The method of the invention does not need to prepare the spore suspension, and can ensure that the amount of bacteria carried by each inoculum is uniform on the shaker, avoiding errors.

5. Good uniformity. Traditional spore suspension inoculation, smear inoculation and other methods only inoculate plants with a certain concentration of spores, but the uniformity is difficult to ensure, especially for small individuals such as seeds and seedlings, and the phenomenon of missed inoculation may occur (such as spray inoculation Method: Some seed surfaces may be in the spray, exposed to 10 spores, while others may not have 1). In the method of the present invention, in the preparation process of the inoculation tool, the uniform speed shaker makes the woody or bamboo inoculum on the inoculation tool fully mixed with the pathogenic bacteria, which ensures the consistency of the amount of bacteria carried by each inoculation tool from the maximum probability. Plants, to ensure the uniformity of the number of pathogens during the inoculation process, consistent with the treatment.

6. Rapid onset. Spray inoculation, smear inoculation and other methods need to wait for pathogenic bacteria to germinate, germ tubes grow, and then form epispores to invade plants. They will be affected by the waxy layer of plant epidermis and cuticle, and it usually takes about 10 days for symptoms to appear. The method of the present invention is an improved acupuncture inoculation, directly causes wounds, can quickly cause pathogens to invade plants, and symptoms can be observed in about 3-4 days, to investigate the damage caused by pathogenic bacteria to plants, and to measure the growth of pathogenic bacteria in plants , Expansion speed.

7. Strong fixed point. In the past inoculation methods, it was not easy to determine which spores germinated and invaded plants from a macro perspective, and the specific observation was easily affected by the mutual influence of multiple invasion points. However, in the method of the present invention, the invasion point is fixed, that is, the infection starts from the acupuncture point, and it is easy to observe the process and degree of the pathogenic bacteria invading the plant, and measure the expansion speed of the pathogenic bacteria on the plant at fixed points.

8. The inoculation tool of the present invention is more practical and can make the operation more convenient and simple. After the traditional acupuncture method, spore suspension or fungus cake is also used for inoculation. Spore suspensions still have the above-mentioned disadvantages. However, the bacteria cake inoculation method has the following problems: 1. The bacteria cakes produced in different areas of the colony have different bacterial loads and pathogenic bacteria activities, so the consistency between different bacteria cake treatments is poor; 2. The bacteria cake inoculation process requires other tools to assist in making wounds. Also closed moisturizing, easy to pollute; wooden inoculation tools are prone to softening and are difficult to pierce into harder hosts. The front end of the inoculation tool of the present invention is a metal tip, which itself is a tool for making wounds; in addition, it is made of metal, and due to the material texture, it has considerable rigidity and is easy to penetrate into the hard wooden host. When the inoculum interacts with the culture medium, it will not affect the rigidity of its metal tip, and will not cause the existing wooden nail front end to soften; the embedded wood or bamboo parts in the inoculum itself form an inoculum, which It can be co-cultured with pathogenic bacteria and can carry a large number of pathogenic bacteria, making the inoculation operation simple and convenient. Therefore, the method and the inoculation tool of the present invention integrate multiple functions and make the operation easier.

9. The cost of the medium used is lower. The medium used in the present invention is potato decoction with glucose added. And the currently used fungal culture medium is glucose agar medium (PDA), and although the method of the present invention is still a kind of PDA medium, there is no need to add agar, so the cost of its preparation is lower than that of the prior art.

Description of drawings

Accompanying drawing 1 is the schematic view of the inoculation tool of the present invention. Among the figure: 1 is the rod-shaped metal part of the inoculation tool, 2 is the wooden or bamboo part embedded on the rod-shaped metal part, and 3 is the tip part of the rod-shaped metal part.

Accompanying drawing 2 is A-A sectional schematic diagram of accompanying drawing 1.

Accompanying drawing 3 is the schematic diagram of another embodiment of the inoculation device of the present invention.

Accompanying drawing 4 is the photo of the present invention after inoculation.

Accompanying drawing 5 is the comparison photograph adopting the method of the present invention to inoculate and not inoculate.

detailed description

The following are examples of the present invention.

An embodiment of the inoculation tool of the present invention, as shown in Figure 1, is made of a rod-shaped metal piece 1 and a wooden or bamboo inoculum 2 embedded in the rod-shaped metal piece, and the front end of the rod-shaped metal piece 1 is a tip portion. Can offer a through hole on the bar-shaped metal part 1 as shown in accompanying drawing 1 in actual preparation, then inlay a piece of inoculum 2 by wooden or bamboo material in the through hole, the two ends of inoculum 2 will be able to like this exposed to contact with culture bacteria. The inoculation tool can also be made to provide a blind hole on the rod-shaped metal part 1, and inlay a piece of inoculum by wood or bamboo material in the blind hole. In this structure, the inoculum will only have one exposed outside. The top of the hole can be provided with a top wire, and the rotation of the top wire can push the inoculum inlaid into the blind hole out of the blind hole for easy replacement.

The third embodiment of the inoculation tool of the present invention is shown in Fig. 3, and it is made of wooden or bamboo one end band cone, the cone is set as a cylinder behind the cone, and the metal tip embedded in the cone head constitutes. In the inoculation device of the present invention, the metal tip is inlaid and fixed with the wooden or bamboo cone, or fixed with threads, that is, the metal tip and the wooden or bamboo cone and the metal tip are matched respectively. Sets the female thread of the mating male thread.

The used metal of the inoculation tool of the present invention is preferably stainless steel.

The dimensions of a practical article of the inoculum according to the invention are as follows: the cylindrical part is 10 mm long, the diameter is 2 mm, and the tip part is 5 mm long.

Inoculation method of the present invention is as follows:

1. Preparation of culture medium

Cut 200g of potatoes into small pieces, add water and boil until rotten (boil for 20-30 minutes, if it can be pierced by a glass rod, filter it with gauze and put it into the pot, add 1000ml of water, add 20g of glucose to it, and then autoclave at 121°C Bacteria for 30min, then subpackaged in 250ml Erlenmeyer flasks and stored for later use.

2. Culture of pathogenic fungi

Make the purified target pathogenic bacteria into a 5mm diameter bacterial cake, add it to the liquid medium in the aforementioned Erlenmeyer flask, place it on a constant temperature shaker (adjust the temperature according to different pathogenic bacteria), rotate at a speed of 150r/min, and cultivate for 10-15 days. Pathogens continue to grow.

3. Preparation of inoculation set

Sterilize the inoculation tool at 121°C for 30 minutes under high pressure for 2 times, immerse it in the potato dextrose liquid medium for the third time to sterilize, then transfer the inoculation tool to the triangular flask for liquid culture of pathogenic bacteria in a sterile environment, and add a few Ten inoculation kits. Then continue to put it on a constant temperature shaker (adjust the temperature according to different pathogenic bacteria) to carry out pathogenic bacteria culture, the shaker speed is 150r/min, and the culture time is about 72h.

4. Vaccination

Take out the infected inoculum from the culture medium, and use the tip of its front to pierce the seedling rhizomes, leaves or tree stems determined to be the host under a sterile environment, causing artificial trauma, so that the inoculum part is separated from the host. The contact of the host inoculation site can promote the invasion of pathogenic bacteria from the wound, and it can be used for experiments such as in vitro leaf infection and disease system infection. See attached drawing 4.

Accompanying drawing 5 is the comparison photograph adopting the method of the present invention to inoculate and not inoculate. It can be seen from accompanying drawing 5 that the host located at the lower part of the photo has been inoculated and infected, and the inoculation site has shown infection, although the plant at the top of the photo has also been stabbed, but does not show infection.

In order to test the reliability of the inoculum, put 10 inoculation devices in a sterilized petri dish (90mm), add 5ml of sterile water, stir and shake well, and wash off the spores on the surface of the inoculum to form a suspension. Count the number of spores in 20 fields of view under a common microscope (100×), and calculate the number of spores on each inoculum according to the diameter of the field of view of the microscope and the inner diameter of the petri dish. A total of 10 repetitions. Measured with the eyepiece micrometer, the diameter of each field of view under the microscope 100× is 2mm, the inner diameter of the bottom cover of the 90mm culture dish is 85mm, and the bottom cover of the culture dish has a total of 1805.25 fields of view, and the number of spores carried by each inoculum is:

. The number of spores carried by each inoculum ranged from 48 to 62, with an average of 56. After SPSS analysis of variance, there was no significant difference in the number of spores carried by each inoculum.

It can be seen from the above that the inoculation method of the present invention has the characteristics of accuracy and convenience. It can be inoculated on hard parts such as forest trees, and the inoculum can be directly fixed into the plant, saving time and effort.

Claims (7)

1. A fungal inoculation method, using an inoculation tool to make the target fungus contact with the inoculation site of the host, so that the fungus invades the host, it is characterized in that: at first its one end made of metal is a tip with wooden or bamboo parts on it The inoculation tool is sterilized, and then the aforementioned inoculation tool is transferred to the culture medium with the target fungus in the aseptic environment, and the medium is cultivated, then the inoculation tool is taken out and the tip of the inoculation tool is inserted into the host inoculation site. 2. the substratum that a kind of fungal inoculation method described in claim 1 uses is characterized in that used substratum is to be added with 20 grams of glucose in 1L potato decoction. 3. Its one end that a kind of fungus inoculation method described in claim 1 uses has the inoculum with tip, it is characterized in that described inoculation tool is at least made of rod-shaped metal piece with tip and wood inlaid on the rod-shaped metal piece Or bamboo or other materials that can allow bacteria and fungi to adhere to form the inoculum, wherein at least a part of the inoculum is exposed on the outer surface of the rod-shaped metal piece. 4. the end that a kind of fungus inoculation method described in claim 1 uses has the inoculum with tip, it is characterized in that by wooden or bamboo its one end band cone, the inoculum that is set to cylinder after the cone and Consists of a metal tip fixed to the head of a cone. 5. The inoculation device according to claim 4, characterized in that the metal tip is inlaid and fixed with the wooden or bamboo cone. 6. The inoculation device according to claim 4, characterized in that the metal tip and the wooden or bamboo cone are fixed with threads. 7. The inoculation tool according to claim 3, 5 or 6, characterized in that the used metal of the inoculation tool is stainless steel.