Isolation of pathogen from Plant materials:
a- Aerial parts:
Leaves
The most common method however for isolating pathogens from
infected plant parts involve:
1- cutting several small sections 5 to 10 mm square from the margin
of the infected lesion to contain both diseased and healthy–looking
tissue (see fig. 4-6 ).
2- These are placed in one of the surface disinfectant solution (3.5%
sodium hypochlorite) for 1 min. this may be diluted, in which case
the material must be immersed for a longer time. making sure that
the surface do get wet.
3- It is then washed three times in sterilized distilled water. Use three
beakers and transfer the material with forceps. It can be dried on
clean tissue-paper and then plated on nutrient medium, usually
three to five per dish for 7 days.
FIGURE
From Stems, Fruits, Seeds, and Other Aerial Plant Parts:
Almost the method described for isolating pathogens from leaves
can also be used to isolate these pathogens from superficial infections of
stems, fruits, seeds, and other aerial plant parts. Entire seeds can be
plated. In addition to these methods, however, pathogens can often be
isolated easily from infected stems and fruits in which the pathogen has
penetrated fairly deeply. This is accomplished by splitting the stem or
breaking the fruit from the healthy side first and then tearing it apart
toward and past the infected margin, thus exposing tissues not previously
exposed to contaminants and not touched by hand or knife and therefore
not contaminated. Small sections of tissue can be cut from the freshly
exposed area of the advancing margin of the infection with a flamed
scalpel and can be plated directly on the culture medium.
b- From Roots, Tubers, Fleshy Roots, and Vegetable Fruits in
Contact with Soil
Isolating pathogens from any diseased plant tissue in contact with
soil presents the additional problems of numerous saprophytic organisms
invading the plant tissue after it has been killed by the pathogen. For this
reason, the first step in isolating the pathogen is repeated thorough
washing of such diseased tissues to remove all soil and most of the loose,
decayed plant tissue in which most of the saprophytes are present. If the
diseased root is small, once it is washed thoroughly, pathogens can be
isolated from it by following one of the methods described for isolating
pathogens from leaves. If isolation is attempted from fleshy roots or other
fleshy tissues penetrated only slightly by the pathogen and showing
only surface lesions, the tissue is washed free from adhering soil, and
several bits of tissue from the margin of the lesions are placed in Clorox
solution. The tissue sections are picked from the solution one by one,
blotted or washed in sterile water, and placed on agar in petri plates. If the
pathogen has penetrated deeply into the fleshy tissue, the method
described earlier for stems and fruit can be used most effectively, namely
breaking the specimens from the healthy side first and then tearing
toward the infected area and plating bits taken from the previously
unexposed margin of the rot.