Production technology of Armillaria mellea and germinating bacteria of Gastrodia elata

Artificial cultivation of Gastrodia elata needs two kinds of symbiotic bacteria: germinating bacteria and Armillaria mellea in the process of sexual reproduction. In the production practice, the author found out the culture medium and production technology which can grow both of the two kinds of symbiotic bacteria. The specific practices are as follows.

1 preparation of culture medium

1.1liquid medium potato (sprouting, peeling, slicing) 100g, hard sawdust, wheat bran, corn flour 50g each, tap water or natural water 1300mL, boiled in a double pot for 30 minutes, then filtered with double gauze. Add 20g glucose, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 0.5% peptone and 10mLVB1 to 1000mL. After all dissolving and shaking, they are separately packed in triangular bottles or canned bottles of 500mL, about 150mL in each bottle, or in a test tube, with a volume of 1 inch 4 of the test tube volume. The cotton stopper is used for the triangular bottle and the test tube, and the can bottle is sealed with 2 layers of Kraft paper or 4 layers of newspaper covered with plastic sheeting, then fasten the mouth with a leather band. Features: high technical content, fast reproduction, good growth can be used, but can not be preserved for a long time.

The filtrate was prepared according to the operation procedure of 1.1.The filtrate was prepared by adding 18g Agar to the filtrate, then dissolved and watered to 1000mL, 20g glucose, 2g potassium dihydrogen phosphate, 1g magnesium sulfate, 5g peptone and 10mgVB1 were added while hot, then dissolved and shaken evenly, then packed in the test tube, loaded with 110g of the test tube capacity, and then plugged with cotton.

1.3 sawdust medium hard sawdust 50%, corncob (crushed into small granules) 25%, wheat bran 15%, cornmeal 10%, plus glucose 0.5%, sucrose 0.5%, gypsum 1%, lime 0.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, 2%VB1. Mix the raw materials and dry materials such as gypsum and lime, then dissolve all the above-mentioned soluble excipients in water and mix them into the raw materials, so that the water content is 70%, mix them fully, put them into the test tube, and compact them properly. The loading volume is 1 stroke 2 of the test tube volume, and then plug the mouth with cotton. Features: resistant to storage, easy to mail.

2 sterilization

Portable high pressure sterilizer can also be used instead of civil pressure cooker.

2.1When liquid and slant solid medium are sterilized, keep steady for 30 minutes when the steam pressure is 1.4kg/ square centimeters, and leave the pot when the temperature in the pot drops to 50-70 ℃. After the liquid medium came out of the pot, it was directly put into the inoculation box and inoculated when the temperature dropped below 25 ℃. The bevel solid medium should be inclined to an inclined plane when the liquid state (the length of the bevel was about 3 × 5 of the length of the test tube), and moved into the inoculation box after the medium was naturally solidified.

2.2 during the sterilization of sawdust culture medium, the steam pressure was kept stable for 45 minutes at 1.4kg/ square centimeters for 60 minutes. After natural cooling, the pot was put directly into the inoculation box.

Note: the sterilization pot should add enough water in advance. After boiling, you must wait for the internal cold air to drain and then press the valve. After reaching the pressure, adjust the heat source to maintain the pressure. After sterilization, the blood pressure should not be reduced too quickly to avoid flushing off the cotton stopper.

3 inoculation

It must be carried out strictly in accordance with the requirements of aseptic operation under aseptic conditions.

3.1 inoculating liquid medium is only necessary to pick 1 or 2 pieces of peanut seed (seed block with mycelium) from the test tube of the mother seed into the flask. When connected to the test tube liquid medium, the seed block can be smaller.

3.2 inoculating Armillaria mellea in inclined medium should be inoculated when there is condensed water on the slope, which is beneficial to the germination of Armillaria mellea. 1-3 mother seeds were connected to the slope of each test tube, and the germinating bacteria should be inoculated when there was no condensed water on the surface of the culture medium. One or two mother seeds are connected to the slope of each test tube.

3.3 sawdust medium cannot be stored after sterilization and should be inoculated as soon as possible. During inoculation, the inoculation hook should be used to press the seed block appropriately to make it in close contact with the culture medium to facilitate the germination and growth of the strain.

4 cultivation

After inoculation, put it in the incubator or incubator as soon as possible and culture it at a constant temperature of 22 to 24 ℃.

4.1 in the process of liquid culture, the shaking table culture should be started from the second day; when manual operation, it is necessary to shake it 6-8 times a day. If it is installed in test tube, it should be put diagonally after shaking, and the culture can be terminated in about 7 days (the culture time of germinating bacteria is 2-3 days longer than that of Armillaria mellea). It should be used in production as soon as possible. The mature culture medium should be clear, light brown or khaki. If turbidity, liquid surface film or a large number of foam is miscellaneous bacteria pollution, should be abandoned.

4.2 the bevel species should be laid flat during culture, and the mycelium germinated and gradually radiated to the surrounding area could be seen in about 48 hours. It can be used in production when the mycelium runs through the inclined medium for 10-15 days (the culture time of germinating bacteria needs to be longer). The Armillaria mellea can be preserved for 40 days and the germinating bacteria can be preserved for 60 days in the refrigerator of 04,4 ℃.

4.3 sawdust seeds should be placed upright after inoculation to prevent seed block vibration. After 15-20 days of culture, Armillaria mellea hyphae or germinating hyphae can be used in production when they reach the bottom of the bottle. Keep it in the refrigerator of 04℃ and keep the quality unchanged for 6 months. When the sawdust bacteria stored in the refrigerator for a long time are used in production, in order to prevent the infection of miscellaneous bacteria during inoculation, the seeds can be inoculated from the bottom of the test tube.