Artificial culture, breeding and preservation method of plant root-knot nematode
A technique for artificially cultivating and root-knot nematodes, applied in the field of laboratory reproduction and preservation away from host plants, can solve the problems of investing a lot of time, and achieve the effect of shortening time, strong operability, and fast propagation method
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Embodiment 1
[0019] Embodiment 1: the preparation of the medium for cultivating Fusarium oxysporum f.sp.cubense
[0020] Preparation of test tube slant medium: weigh 10 grams of glucose, 5 grams of peptone, KH 2 PO 4 1 g, MgSO 4 ·7H 2 O 0.5 g, dissolved in 1000 ml of water, add 15-18 g of agar, heat and boil until the agar melts, make up the volume to 1000 ml, after cooling properly, put it into test tubes, the filling volume is 1 / 5-1 of the test tube height / 6. Plug the test tube mouth with a test tube stopper, autoclave at 121°C for 25 minutes, take it out while it is hot, place it on an inclined plane until it cools down, and the height of the inclined plane should not exceed 1 / 3 of the height of the test tube.
[0021] Preparation of liquid seed medium: weigh 10 grams of glucose, 5 grams of peptone, KH 2 PO 4 1 g, MgSO 4 ·7H 2 O 0.5 g, dissolved in 1000 ml of water, then sub-packaged, the liquid volume is 20-30ml / 100ml triangular flask, plugged with a cotton plug, autoclaved a...
Embodiment 2
[0023] Embodiment 2: the preparation of Fusarium oxysporum f.sp.cubense spore liquid
[0024] The Fusarium oxysporum f.sp.cubense described in the present invention is a bacterial species preserved by the inclined-plane cold storage method in our laboratory.
[0025] a) Activation of Fusarium oxysporum f.sp.cubense strain
[0026] The method of activating the strains preserved on a slant: pick a strain with a diameter of about 8 mm from the strains preserved on a slant and inoculate it into the slant culture medium of a test tube for activation, cultivate it in an environment of 28 ± 2°C for 3-5 days and then use it for later use.
[0027] b) Cultivation of seed solution
[0028] From the Fusarium bacterium activated in the test tube, pick three bacterium pieces and inoculate them respectively in three 100ml triangular flasks with 25ml liquid seed culture medium, place them on a shaker, the rotating speed of the shaker is 180r / min, at 28 Continue to cultivate in the environm...
Embodiment 3
[0032] Embodiment 3: the preparation of double culture medium
[0033] After the prepared solid medium is hot or melted by heating, pour it into a 9cm sterile petri dish in a sterilized ultra-clean workbench. , too little will limit the living space of nematodes, and too much will not be conducive to the separation of nematodes. After the medium is cooled and solidified, add 0.1-0.2ml of Fusarium oxysporumf.sp.cubense spore liquid to each plate, spread evenly, seal with a parafilm, and incubate upside down in a constant temperature incubator at 28°C for 3-5 days, white bacteria After the silk grows all over the plate, it is the double medium.
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