Method for separating single oospore of phytophthora infestans
A potato late blight and oospore technology, applied in the biological field, can solve the problems of difficult operation, time-consuming, low success rate, etc., and achieve the effect of reducing operation difficulty, saving operation time and high success rate
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Embodiment 1
[0019] (1) Activate the P. infestans strain 902-3 (collected from Yunnan in 2015), inoculate the strain in rye medium, and culture it in the dark at 19°C for 16 days to collect mycelia;
[0020] (2) Weigh 80 mg of mycelia into a 1.5 mL centrifuge tube, add 1 mL of sterile water and place in a refrigerator at 4°C for 3 hours at low temperature to stimulate sporangia to release zoospores;
[0021] (3) Then centrifuge at 1000r / min for 2min, remove the supernatant, and add 30mg / mL collapsing enzyme hydrolyzed hyphae, sporangia and zoospores, wherein the collapsing enzyme is prepared with PBS buffer solution with pH=7.0 , and filtered with a 0.22 μm bacterial filter;
[0022] (4) Centrifuge at 1000r / min for 1min, remove the supernatant, add 1mL of sterile distilled water; filter through nylon meshes with pore sizes of 76μm, 40μm and 20μm in sequence, rinse the 20μm nylon mesh in a small beaker with sterile water, collect oospore suspension;
[0023] (5) Use a hemocytometer to adj...
Embodiment 2
[0028] (1) Activate the P. infestans strain 902-3 (collected from Yunnan in 2015), inoculate the strain in rye medium, and culture it in the dark at 19°C for 15 days to collect mycelia;
[0029] (2) Weigh 100mg of mycelia into a 1.5mL centrifuge tube, add 1mL of sterile water and place in a refrigerator at 4°C for 2-3h at low temperature to stimulate sporangia to release zoospores;
[0030] (3) Then centrifuge at 1000r / min for 2min, remove the supernatant, and add 30mg / mL collapsing enzyme hydrolyzed hyphae, sporangia and zoospores, wherein the collapsing enzyme is prepared with PBS buffer solution with pH=7.0 , and filtered with a 0.22 μm bacterial filter;
[0031] (4) Centrifuge at 1000r / min for 1min, remove the supernatant, add 1mL of sterile distilled water; filter through nylon meshes with pore sizes of 76μm, 40μm and 20μm in sequence, rinse the 20μm nylon mesh in a small beaker with sterile water, collect oospore suspension;
[0032] (5) Use a hemocytometer to adjust t...
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