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.