Single spore isolation method of phytophthora capsici zoospores
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A technology of zoospores and Phytophthora capsici, which is applied to methods using spores, methods based on microorganisms, biochemical equipment and methods, and can solve problems such as long time-consuming, heavy workload, and complicated processes
Inactive Publication Date: 2016-08-24
NORTHEAST AGRICULTURAL UNIVERSITY
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[0003] At present, the method for the isolation of Phytophthora capsici zoospore monospore is basically with reference to Yuan Junhai and Yao Yuqi (2002) to the isolation method of potato infestans zoospore monospore: prepare zoosporangium or zoospore suspension, After zoospores germinate and produce germ tubes, carry out under the microscope, sterilize the coverslip on the stage and place it on a sterilized slide, take 10-15 μL of spore suspension, drop a drop on the coverslip, and then examine under the microscope , confirm that there is only one zoosporangium or zoospore in this drop of suspension, which is a monospore strain. To isolate zoospores of Phytophthora infestans, the m
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Embodiment 1
[0024] Embodiment 1, the mensuration of the mating type of Phytophthora capsici strain
[0025] The mating type of the Phytophthora capsici control strain A1 used in this example (Fu Yingkun, Tian Xiaoli, Li Yi, Li Li. Analysis and distribution characteristics of the mating type of Phytophthora capsici in Qinghai Province. Northern Horticulture, 2014 (18):
[0026] The experimental method is as follows:
[0027] (1) Isolation of Phytophthora capsici
[0028] Collect Phytophthora capsicum strains from the plots where peppers are planted, take several pieces of tissues at the junction of disease and health, treat them with 70% alcohol for 1-2s, then place them in 0.1% mercuric chloride for 3 minutes, change and wash them with sterile water 3 times, After drying, put it on the V8 juice agar medium plate with penicillin (50 μg / mL), culture in the dark at 26°C for 2-3 days, wait for colonies to grow, and purify for later use.
[0029] (2) zoospore single spore isolation
[0030]...
Embodiment 2
[0036] Embodiment 2, capsicum variety are to the mensuration of capsicum phytophthora disease resistance
[0037] (1) Cucumber planting
[0038] 20 cucumber varieties for testing, 20 seeds for each variety, 1 seed for each culture pot (8cm in diameter), 5 of which are blank controls, and the culture medium is black loam, placed in the greenhouse (13-33 ° C), every day Water once, and inoculate after 15 days.
[0039] (2) Expansion of test strains
[0040] Select 6 monospore strains of zoospores of Phytophthora capsici (see "Specific operation for the isolation of single spores of Phytophthora capsici zoospores" for the acquisition of single spores) and inoculate them into V8 (100% vegetable) juice medium plates to inoculate peppers respectively Phytophthora, sealed with parafilm, cultured in the dark at 25°C for 5 days to promote the growth of mycelia, then transferred to light (5 000lx) to remove the parafilm and cultivated at 23°C for 24h to promote the production of zoospor...
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Abstract
The invention relates to a single spore isolation method of phytophthora capsici zoospores. The single spore isolation method comprises the steps that phytophthora capsici is inoculated to a V8 culture medium, sealing is performed with a sealing membrane, culture at the constant temperature of 25 DEG C is performed for 5 days, then the sealing membrane is removed, irradiation is performed for 24 hours, sterile water of 4 DEG C is added to perform induction and constant-temperature release respectively for 25 minutes so as to obtain a zoospore suspension, the suspension is diluted to reach the standard that 1-2 spores exist in each (10*) view, 1 mL of the suspension is taken and added to a V8 culture medium flat plate containing antibiotics in an evenly coated mode, constant-temperature standing is performed for 6 hours, a single germinated zoospore is found through an inverted microscope and is cut by using a scalpel to form a 0.5 cm square mark, it is confirmed that single spores are transferred to a flat antibiotic plate, and purified spores are single spore plants. The single spore isolation method is simple and convenient to operate, high in accuracy and good in rapid separating effect, adopts simple devices and can separate single spores of a large number of phytophthora capsici zoospores within a short time.
Description
technical field [0001] The invention relates to the technical field of agricultural plant protection, in particular to a single spore separation method of zoospores of Phytophthora capsici. Background technique [0002] Pepper blight (Phytophthora blight) is a devastating disease caused by Phytophthora capsici L., which not only harms the roots, stems, branches, leaves and fruits of peppers, but also harms tomatoes, eggplants, cucumbers, melons, etc. Crops often cause a large area of dead plants. The rate of dead plants in a general field is 20%-30%, and it can reach more than 90% in severe cases. Pepper blight has a great impact on yield and quality. In severe cases, the yield can be reduced by more than 50%, and even devastating hazards. Pepper disease-resistant breeding is the most direct and effective way to prevent and control pepper blight. Large-scale screening of Phytophthora-resistant pepper germplasm resources is the premise of disease-resistant breeding, and t...
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