Seedling-stage injection inoculation method for zucchini cucumber mosaic virus
A cucumber mosaic virus, injection inoculation technology, applied in biochemical equipment and methods, microbe measurement/inspection, etc., can solve the problems of late inoculation time, unfavorable identification, and inability to clarify pathogens, etc., to achieve fast growth and shorten Time, good effect of resistance
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Embodiment 1
[0027] Example 1 The incidence of CMV in zucchini seedlings by friction inoculation and injection inoculation
[0028] The inoculation method used was as follows:
[0029] (1) After the zucchini seeds were sterilized in 2% sodium hypochlorite for 10 minutes, rinse them with sterile water for 3-4 times;
[0030] (2) Zucchini seeds were sterilized and germinated in a Petri dish at 30°C in the dark for 36 hours. After the seeds germinate, they are sown in a 7×7 nutrient pot for growth. During the growth period, the temperature is 25°C day / night 20°C, and the seedlings can be inoculated when 2 cotyledons are fully unfolded. Specific forms such as figure 1 ;
[0031] (3) Take the purified cucumber mosaic virus disease bacterial strain, artificially inject and inoculate it on cotyledon flattening stage zucchini seedlings, and harvest the diseased leaves three weeks after the inoculation; have both;
[0032] (4) Add 2.5 ml of 0.01 mol / L phosphate buffer solution with a pH value ...
Embodiment 2
[0037] Example 2 RT-PCR detection of field-onset zucchini virus disease infestation species
[0038] 1. Take tender leaves from the zucchini leaves of each plant obtained in Example 1, and store them at -80°C after rapid liquid nitrogen freezing;
[0039] 2. Extraction of total RNA
[0040]Weigh 0.5g of each sample, put them into a -80°C deep-frozen mortar and add liquid nitrogen to fully grind, quickly transfer to a sterile 1.5mL Eppendorf tube pre-cooled by liquid nitrogen, and add 1mL Trizol pre-cooled at 4°C for extraction Mix well and let stand for 5 minutes, then centrifuge at 12,000g for 5 minutes at 4°C. The supernatant was extracted once with 200 μL chloroform, and centrifuged at 12,000 g for 15 min at 4°C. Add an equal volume of isopropanol to the supernatant, invert and mix well, let stand for 10 min, and centrifuge at 12,000 g for 15 min at 4°C. Discard the supernatant, wash the pellet once with 70% pre-cooled ethanol, and dissolve it in 30 μL DEPC-treated ddH ...
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