Single spore isolation method of phytophthora capsici zoospores

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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Problems solved by technology

[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 main method is to dilute them to a certain concentration, apply them on the rye and oat basal medium, and check the single colony after a certain period of time to confirm the single spore strain. This method may also isolate the fungus The colony formed by silk, and once the colony is formed, it is not easy to determine whether it is a colony formed by monospores; the method for the isolation of Phytophthora capsici monosporangia basically adopts Chai Guixian et al. (2010), mainly for the single cyst of Phytophthora capsici zoosporangia separate

[0004] The methods reported above have problems such as heavy workload, insufficient accuracy, long time-consuming, complicated process, etc.

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