Stable and efficient single spore isolation method for plasmodiophora brassicae woronin serving as protist with strong parasitism

Abstract

The invention provides a stable and efficient single spore isolation method for plasmodiophora brassicae woronin serving as a protist with strong parasitism. The stable and efficient single spore isolation method comprises the following steps of: preparing a relatively pure spore suspension by using an improved sucrose gradient centrifugation method, calculating the concentration of the spore suspension by using a blood counting chamber, on the basis of the spore suspension with a certain concentration, obtaining single-dormancy spores by using an agar block planar capillary imprinting method,and then inoculating the single-dormancy spores to robust root hairs of the susceptible variety ECD-05 in an ECD system for accelerating germination for 2-3 days. Compared with previous related research methods, the method disclosed by the invention mainly overcomes the problems that: single spores are tiny and not easy to position under a microscope; the visual field of a liquid droplet method is layered too much; furthermore, the single spores are easy to dry and cannot be absorbed; the single spores cannot be accurately picked through micromanipulation and the like. The combination of theplanar capillary imprinting method can greatly reduce the visual field range needing to be inspected during microscopic examination; furthermore, the blocked agar is easy to grab; in addition, advanced automatic instruments and equipment are not needed; the operability is high; and the single spore isolation efficiency can be remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of microorganism single spore separation, and in particular relates to a separation technology of single dormant spores of the strong parasitic protozoan Plasmodium brassicae that cannot be cultivated in an artificial medium. Background technique [0002] Brassicaceae clubroot is a worldwide soil-borne disease caused by Plasmodiophora brassicae Woronin infection, and is an important root disease of Brassicaceae plants. P.brassicae is an obligate living parasite. In 1995, Ainsworth formally classified P. brassicae into the genus P.brassicae. Studies have shown that there is pathogenic differentiation of Plasmodium species due to differences in physiological races, and naturally occurring populations are often highly mixed. Mananares et al. found that the existing host identification method can also subdivide the pathogenic type within the physiological race, and proposed that only the test results using mono...

AI Technical Summary

Problems solved by technology

[0003] There are mainly four methods for the isolation and inoculation of single spores of pathogenic microorganisms: one is the automatic system, which can achieve a success rate of 40% to 50% under certain conditions for the isolation of single spores of Plasmodium; The use is restricted by the conditions of the laboratory; the second is the direct capillary method, but it is more suitable for microorganisms that can grow in artificial culture medium and grow hyphae; the third is the glass needle method, which requires high environmental conditions for production, and its application is limited by spores Size requirements (to be visible under an anatomical microscope), and the operation is relatively cumbersome; the fourth is the convenient and quick coating plate method, but it is not suitable for the separation of strongly parasitic Plasmodium

In addition, there are also reports on the isolation of single spores of Plasmodium, mainly including the capillary punching method, the imprinting method and the dilution drop method. These methods alone have certain theoretical support, but most of them are time-consuming and laborious, and the operability is not good. , it is difficult to separate

Images