Isolation and culture of plant pathogenic fungi is a frequently used research tool for (1) identification of the original pest (2) observation of pathogen morphology (3) cultivation of plant disease inoculum.
Operation method
Isolation and culture of plant pathogenic fungi
Principle
Fungal mycelia in diseased plant tissues can generally resume growth and reproduction if given suitable environmental conditions, except for a few species. The isolation of plant pathogenic fungi is to separate the pathogenic fungi from the diseased plant tissues from other stray fungi through artificial culture, and separate them from the host plant, and then purify the isolated pathogenic fungi in suitable environment, this process is always called the isolation culture of plant pathogenic fungi. The isolation of plant pathogenic fungi generally adopts the tissue isolation method, that is, small pieces of diseased tissues are cut, washed with surface disinfection and sterilized water, and then transferred to artificial culture medium for cultivation.
Materials and Instruments
Plant material Move (i) Preparation before separation 1. Cleaning and sterilization of the working environment Separation culture is generally carried out in the aseptic room, aseptic box or aseptic bench (ultra-clean bench), the aseptic room and aseptic box should be sprayed to remove dust and disinfected with drugs or ultraviolet irradiation (commonly used disinfectant drugs such as 70% alcohol, 2% coal phenol soap solution, 5% carbolic acid solution spray. If the ultraviolet light is used, it takes 20-30 minutes to irradiate). In the absence of the above equipment conditions, in a clean room, close the doors and windows to avoid air flow, after the spray to remove air and ground dust after the operation, you can also get better results. Wipe the table clean before working, preferably with a damp gauze. Will need to use the items in order on the worktable, to avoid moving around when working, the staff is best to wear sterilized overalls, put on a mask, and wash their hands with soap, with 70% alcohol or 0.1% Neosporin handrub. 2. Sterilization of separation equipment Where the separation of material contact with the utensils (knife, scissors, forceps, needles, etc.) should be kept sterile at any time (at least in the use of), these utensils will be immersed in 70% alcohol, the use of the lamp flame sterilized and burned alcohol, so 2-3 times (knife, scissors, forceps, etc. should not be burned on the lamp flame for too long to prevent annealing). Sterilization must be repeated when using again. Petri dishes, tests, etc. should be sterilized by dry heat. Culture medium and distilled water for washing or dilution need to be autoclaved beforehand. 3. Selection of isolation materials Using newly diseased plants, organs or tissues as isolation materials can reduce the contamination of saprophytic bacteria. Inside or on the surface of any necrotic part of the plant, there may be saprophytic microorganisms saprophytic, so the general spot disease should be obtained from the neighboring sound tissues, i.e., from the junction of diseased and healthy tissues to isolate the material. (ii) Isolation of plant pathogenic bacteria 1. Isolation of pathogenic bacteria from leaf spots and branch and rod spots (non-vascular infestation) First, select fresh diseased leaves with typical symptoms as isolation materials, and follow the steps below: ① Preparation of culture medium plate: Heat the PDA culture medium in microwave oven to melt the test, and pour the melted culture medium into the sterilized culture medium with aseptic operation method, 12 ml per dish, which can form a 2-3 mm thick plate. (To prevent bacterial contamination, pour the dish before adding 6% lactic acid 4-6 drops to each triangular bottle). ② Diseased leaves or diseased branches rinsed by tap water, cut from the spot turnover 1-2 mm at the healthy tissue parts (in the case of branches, the cortex with spots peeled off, but). ③ Take 10 ml small beaker sterilized by wine belt, put in the separated material, pour 0.1% liter of mercury solution appropriate amount for surface disinfection for one minute, or 1% bleach disinfection can be used. ④After disinfection, pour out the disinfectant solution and rinse with sterile water for 2--3, and do not pour out the sterile water for the last time. ⑤ With sterilized forceps, cut the isolated material into 2--3 mm sized squares in sterile water, and each piece of tissue should be diseased and healthy tissue. Use sterilized tweezers to clip the cut material, put it into the culture medium plate, and gently press it. 4--5 pieces per dish, discharged evenly. (6) Use crayon to indicate the isolation code, date and name on the petri dish, and turn the petri dish over and place it at 23-25℃. ⑦ 3 - 4 days later selected by the separation of the material on the growth of typical and no stray colonies, in the edge of the colony with mycelium with the transfer needle picked a small piece of medium, transferred to the center of the test tube slant medium, at 25 ℃ in the incubator culture. 2. Separation of pathogens from seeds The whole seed or a part of the seed is rinsed, then surface disinfection (mercuric chloride or bleach), and finally washed with sterilized water and transferred to the artificial medium plate culture (or directly placed in the dish moisturized culture at 25 ℃ in a warm box). 3. Isolation of pathogens jeopardizing the transmission tissues (represented by wilt) Firstly, the isolated stems are surface sterilized, then the epidermis is peeled off with a sterilized knife, and small pieces of discolored vascular tissues are cut, and then bleach is used for performance disinfection, and then moved to the medium plane for cultivation. 4. Purification of isolates The aforementioned methods to obtain isolates, must be purified, in order to become cultured, the purification method has a single spore separation method and the side of the continuous dilution culture method, the latter is simple, in order to general research commonly used. Continuous dilution method: for fungal material, it is from the edge of a typical colony cut a small piece of medium containing mycelium, transplanted on another medium plate, to be formed after the colony, and then transplanted in accordance with this method. So several times, until the colony morphology is typical, no stray bacteria can be moved into the slant test tube culture preservation. Caveat 1. Isolation cultures are usually performed in a sterile room, a sterile box or on a sterile bench (ultra-clean bench). 2. Close doors and windows in clean rooms to avoid air movement. 3. all utensils (knives, scissors, forceps, needles, etc.) that come into contact with the isolated material should be kept sterile at all times (at least while in use). Common Problems Preparation of solutions 1. Preparation of 0.1% ascending mercury solution: 1 g of ascending mercury, 2.5 ml of concentrated hydrochloric acid; add 1000 ml of water. Note that the ascending mercury should be dissolved in hydrochloric acid first, and then diluted with water. 2、P.D.A culture medium composition; potato 200 grams, glucose 10-20 grams, 17-20 grams of watercress, water 1000 milliliters. 3、Mizuna culture medium composition, 17-20 grams of watercress, water 1000 milliliters. For more product details, please visit Aladdin Scientific website.
70% alcohol Coal phenol soap PDA medium Lactic acid Slant medium 75% alcohol
Alcohol lamp Surgical scissors Ophthalmic forceps Petri dishes Small beakers Large beakers Thermostat