Method for rapid propagation and separation of pine wood nematodes

Abstract

The invention discloses a method for rapid propagation and separation of pine wood nematodes. The method comprises the following steps: inoculating Sphaeropsis sapinea onto a PDA culture medium, carrying out culturing in an incubator with a constant temperature of 26 DEG C for 5 days, and inoculating the pine wood nematodes into culture dishes when the culture dishes are almost full of hyphae; uniformly spraying a proper amount of sterile water onto the inner surfaces of culture dish covers, adding 5 to 10 ml of sterilized water after 4 days so as to flush the culture dish covers, collecting water obtained by flushing of the culture dish covers, and carrying out centrifuging so as to be able to obtain a great amount of pine wood nematodes; after completion of collection, continuing spraying a proper amount of sterile water onto the inner surface of the culture dishes uniformly, carrying out repeated culturing for 2 to 3 times, after completion of culturing, flushing the culture dish covers with sterile water, collecting water obtained by flushing of the culture dish covers, and carrying out centrifuging so as to be able to obtain the pine wood nematodes; and slicing the culture medium into small pieces at the same time, placing the small culture medium pieces into a Baermann funnel so as to separate the pine wood nematodes from the culture medium, placing the separated pine wood nematodes into a refrigerator with a temperature of 4 DEG C, and carrying out storage for subsequent usage. Compared with a traditional culturing method, the method provided by the invention can rapidly propagate a great amount of pine wood nematodes, has simple separation method and can greatly save time.

Description

technical field [0001] The invention belongs to the technical field of nematode preparation, and in particular relates to a rapid propagation and isolation method of pine xylophilus. Background technique [0002] Pine wilt disease (Pine wilt disease) is a dangerous disease caused by the pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, which causes rapid withering and death of pine trees. Due to its serious harm and difficult prevention and control, it has been highly valued by countries all over the world, and has been listed as an important dangerous forest disease, and has been enacted as a key quarantine object by more than 40 countries. The pine wood nematode originally originated in the United States, and was introduced to Japan, China, Korea, Mexico, Portugal and Spain through the timber trade, and pine xylophilus disease occurred successively. As of 2017, pine wood nematode disease in my country has spread to Taiwan, Liaoning, Jiangsu, Zheji...

AI Technical Summary

Problems solved by technology

The traditional B. xylophilus breeding and cultivation process needs to separate the nematodes through the Baemann funnel method. The steps are cumbersome, time-consuming and laborious, and the number of reproduction is limited, which limits the research on the control of B. xylophilus.

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