ISOLATION AND IDENTIFICATION OF PATHOGENS Once plant and soil samples have been collected, isolation and identification of the pathogen is the next task facing the investigator. To isolate a soilborne fungus from host tissue, the investigator must first decide upon the appropriateness of surface-disinfestation of the tissue. For large pieces of woody tissue (i.e., large tap roots), surface-disinfestation is often necessary to prevent the growth of surface-resident saprophytic 2*^1 fungi which might prevent the growth of the pathogen. However, for fine feeder roots, surface-disinfestation may kill pathogens such as Pythium spp. inside the roots g The choice of a surface-disinfestation treatment and the duration of the treatment will vary with the different groups of fungi. The next step in the isolation of the fungus from host tissue is the selection of an appropriate culture medium. Appendix A lists a number of general and highly specific media for this purpose. The isolation of soilbome pathogens from soil can employ soil-dilution plate techniques, selective (or semi-selective) . V-*, media, or baiting. In general, the soil-dilution plate technique (. (Tuite, 1979) involves drying the soil sample, followed by ' crushing it through a 2-mm sieve. A 10-g subsample is added .to 200 ml of 0.2% water agar (WA) (or 1% sodium carboxymethyl cellulose) in a screw-capped bottle. The ! contents are mixed thoroughly for 20jnin, then a 1-ml aliquot is withdrawn and added to 9 ml of 0.2% WA (or other diluent) and shaken for 4 min. A 1-ml aliquot is removed and added to 49; ml of 0.2% WA and shaken for 2 min. Using a wide-mouth pipette, transfer 1 ml of the final dilution to each of five sterile petri dishes and add 17 ml of the desired culture medium which has been cooled to 45C. Disperse soil particles by circular agitation of the petri dishes. The petri dishes are incubated under conditions appropriate for the fungus of interest. This description is offered only as a general illustration of the technique, and the actual amount of dilution of the soil sample will depend upon the number and type of fungal propagules present. If many are present (i.e., 10s CFU/g) then a higher dilution will be required than where the fungal population is less than 10 CFU/g.
A commonly used variant of the soil-dilution plate technique is the Warcup plate method (Tuite, 1969; Warcup, 1950) wherein a small soil sample (0.005 - 0.15 g) is transferred to a sterile petri dish and crushed in a drop of water. To the petri dish, add 8-10 ml of the desired growth medium. Gently swirl the plate to distribute the soil particles in the medium and incubate as appropriate for the particular fungus. Tuite (1969) recommends Czapek's agar (Appendix A) amended with 0.5% yeast extract and acidified to pH 4.0 with phosphoric acid. Many selective media have been devised for the recovery of soilborne fungi from soil samples. In principle, they all involve the addition of various antimicrobial chemicals to suppress the growth of other organisms which might interfere with the target organism. As a cautionary note in using selective media, the investigator should be aware that each selective medium was devised for use with one (or a limited number) of soil types. Thus, a medium which works perfectly well in Texas may perform poorly when used in Missouri. As an alternative to selective media, several investigators have used plant tissue as "bait" to isolate plant-pathogenic fungi (i.e., Pythium and Phytophthora spp.) from soil. The bait tissue is selected because it will selectively permit the growth of the desired fungus. Once the putative pathogen has been recovered in pure culture, the investigator is faced with the task of identifying it. The prerequisite for fungal identification is the production of all taxonomically useful structures for microscopic (or macroscopic) examination. For the Deuteromycotina, it is possible to induce many of these fungi to produce reproductive structures on microscope slides using the Riddell slide culture technique (Riddell, 1950). A 1-cnf square of potato dextrose agar (PDA, Appendix A), or other suitable culture medium, is aseptically placed in the center of a sterile glass microscope slide. A 2-mm2 inoculum-block of the fungus is aseptically placed at the center of each of the four edges of the agar square. A sterile cover slip is then applied to the agar square and the entire slide culture is incubated in a moist chamber under conditions appropriate for the particular fungus. When fungal growth has extended over several mm from the original inoculation sites, the cover slip is lifted off vertically, a drop of lactophenol (Appendix A) or other mounting medium (or stain) is added to the culture, then the cover slip is inverted and gently placed on a clean microscope slide. This first culture is now ready for microscopic examination. The 1-mm2 agar block is gently removed from the original microscope slide culture. Suitable mounting media or stains are applied, followed by gentle addition of a cover slip to complete the preparation of the second culture. Ultimately, identification of a particular fungus will require consultation with an appropriate monograph (or with an appropriate expert). However, there are several general mycological texts which may aid in identification. These include Hawksworth 1974, Hawksworth et al, 1983, and Domsch et al, 1980.
Isolation and identification of pathogens 
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