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 AerialPlant Parts:
Almost the methoddescribed for isolating pathogens from leaves can also be used toisolate these pathogens from superficial infections ofstems, fruits, seeds, and other aerial plant parts. Entireseeds can be plated. In addition to these methods,however, pathogens can often be isolated easily frominfected stems and fruits in which the pathogen haspenetrated fairly deeply. This is accomplished by splittingthe stem or breaking the fruit from the healthy side firstand then tearing it apart toward and past the infectedmargin, thus exposing tissues not previously exposed tocontaminants and not touched by hand or knife andtherefore not contaminated. Small sections of tissue canbe cut from the freshly exposed area of the advancingmargin of the infection with a flamed scalpel and can beplated directly on the culture medium.
b- From Roots, Tubers, Fleshy Roots, and VegetableFruits in Contact with Soil
Isolating pathogens from any diseased plant tissue incontact with soil presents the additional problems ofnumerous saprophytic organisms invading the planttissue after it has been killed by the pathogen. For this
reason, the first step in isolating the pathogen is repeatedthorough washing of such diseased tissues to remove allsoil and most of the loose, decayed plant tissue in whichmost of the saprophytes are present. If the diseased rootis small, once it is washed thoroughly, pathogens can be
isolated from it by following one of the methodsdescribed for isolating pathogens from leaves. If isolationis attempted from fleshy roots or other fleshy tissuespenetrated only slightly by the pathogen and showing
only surface lesions, the tissue is washed free fromadhering soil, and several bits of tissue from the marginof the lesions are placed in Clorox solution. The tissuesections are picked from the solution one by one, blottedor washed in sterile water, and placed on agar in petriplates. If the pathogen has penetrated deeply into thefleshy tissue, the method described earlier for stems andfruit can be used most effectively, namely breaking thespecimens from the healthy side first and then tearingtoward the infected area and plating bits taken from thepreviously unexposed margin of the rot.