Isolated culture of plant pathogenic fungi

I.
Experimental Principle
Plant Disease
Fungi
mycelium within the
tissue, if given appropriate environmental conditions, except for individual species, can generally resume growth and reproduction. The separation of plant pathogens refers to the separation of pathogenic fungi from other germs from infected plant tissue through artificial
culture
, and the isolation of the isolated pathogens from the host plant, and then purifying the isolated pathogens in a suitable environment, a process known as the separation and culture of plant germs.
the separation of plant pathogenic fungi is generally the use of tissue separation method, that is, the cutting of small pieces of diseased tissue, after surface disinfection and sterilization of water wash, moved to artificial
flisting base
culture.
II,
Experimental Purpose
The separation and culture of plant pathogens is one of the most basic operational techniques of plant
pathology
experiment, which is a frequently used research method in the identification of primary diseases, observation of pathogen morphology, and cultivation of plant pathogenic inoculations. Through this experiment, the general principles and methods of plant pathogen isolation and culture are required.
, the
of experimental materials and preparation
1. Separation materials: new disease leaves of Alternaria kikuchiana, Pestnlotia sp and glomerella cingolata; Cytospora chrysosperma with new disease spots; and oil pine seeds.
2． Separation utensils (per group unit)
alcohol lamp 4, surgical shear 4, ophthalmology 4, PDA culture base 3 bottles, petri dish (9cm) 24 sets, small
berrott
(5 ml) 4, big berries 1, Slanted culture base 12 tubes, sterilized water 4 bottles, 75% alcohol bottle 1 (inside degreased cotton ball) 0.1% liter mercury bottle 1, 5% lactic acid bottle (60ml) 1, match 1 box, wet, dry gauze 4 sheets each.
4th,
methods and steps of the
(a) pre-separation preparations
1. Cleaning and disinfection of the working environment
isolation culture is generally carried out in sterile rooms, sterile boxes or sterile work stations (ultra-clean work stations), sterile rooms and sterile boxes are sprayed and dusted, and disinfected with drugs or ultraviolet radiation (commonly used disinfection drugs for 70% alcohol, 2% kerosene soap, 5% charcoal acid and other sprays. 20-30 minutes if exposed to UV light).
in the absence of the above equipment conditions, in the cleaning room closed doors and windows, to avoid air flow, after spray removal of air and ground dust operation, but also to obtain better results. Wipe the table before work, preferably with wet gauze. Place the items you need on the workbench in order to avoid moving around at work, and it is best for staff to wear sterilized work clothes, bring
masks
, wash their hands with soap, and wipe their hands with 70% alcohol or 0.1% neo-Ger.
2． Disinfectation of separation equipment
All utensils in contact with the separation material (knife, shear, tweezers, needles, etc.) should be sterile at any time (at least when used), these utensils are immersed in 70% alcohol, when used in the lamp flame sterilization burn alcohol, so 2-3 times (knife, scissors, tweezers, etc. should not burn too long on the lamp flame, to prevent fire). Sterilization must be repeated when used again. Petri dishes, tests, etc. should be dried and hot to sterilise. The culture base and distilled water used for washing or dilution need to be sterilized with high-pressure vapor in advance.
3． The choice of
can reduce the contamination of rotting bacteria by using newly ill plants, organs or tissues as separation materials. The internal or surface of any plant necrosis part may have the iniquity of decaying
microbial
, so the general speckled disease should be obtained from the adjacent sound tissue, that is, from the junction of disease and healthy tissue.