Identifying a Pathogenic Fungus

William W. Diehl.

From the beginning to the end of its life the health of every seed plant, wild or cultivated, is affected by fungi.

Even though a seed within a fruit or capsule may be sterile, it comes into contact with fungal spores and hyphae as soon as it is exposed to the air or is in contact with the ground. Spores are microscopic, seedlike, reproductive bodies, and hyphae are the microscopic vegative growths of fungi.

The air is literally charged with spores, and the soils of the whole earth are full of living spores and hyphae of different kinds of fungi. Most of the fungi are innocuous. Many are beneficial. But some thousands of recognizably different kinds of fungi are now known to be pathogens, or agents of disease, in plants.

Practical measures for the prevention and control of plant diseases depends in large part upon scientific knowledge of each pathogen and its role in nature. Since there are more than 100,000 recorded names of supposedly different kinds or species of fungi, the specific identification of a single specimen or culture of a fungus involves the exclusion of some 99,999 names. That is a technical problem akin in complexity and difficulty to the isolation and identification of any one out of 100,000 chemical compounds.

But the problem is not insuperable. There is a general procedure that leads the way out of the apparent chaos of more than 100,000 names.

First of all the specimen must be subjected to critical examination in order to determine any and all features that characterize it. Spores and fructifications, or spore-bearing structures, are the most significant features for diagnostic purposes.

To see the features to best advantage under the different powers of the compound microscope requires special preparation for each kind of material. The form as well as the texture of a fungal fructification, whether moldlike and fluffy or a solid structure, will determine the best method of treatment.

Most fructifications of microfungi are best viewed at first in place by reflected light with a hand lens or, better yet, a stereoscopic microscope, followed by examination under the different powers of the compound microscope of a very minute fragment mounted in water or in a staining medium.

Molds are more or less easily mounted in water or special mounting media, although they frequently require preliminary treatment with a fixing fluid to prevent a loss of spore heads, chains, or other delicately attached structures. Nevertheless, if immature stages are placed directly in the mountant, those structures that are too readily detached when mature often tend to remain in place so that their genesis is more readily observed.

If a fructification is large and opaque its anatomy can be discerned only in sections. Microtome sections made from materials imbedded in paraffin or nitrocellulose are the acme in elegance and are essential if good photo-micrographic records are desired. Under ordinary circumstances, however, their preparation is too time-consuming to be justified, since for most practical purposes satisfactory sections are quickly made free-hand or by means of the freezing microtome. With moderate practice, excellent freehand sections can be made using elder pith, carrot, or other convenient plant material as a clamp to hold a fragment firmly while slicing a number of sections among which only the best need be selected for study. If the material is too scanty to permit wastage or if the operator has not mastered the more rapid technique of free-hand sectioning, recourse may be had to the freezing microtome. Although a second-rate instrument as microtomes go, it has its advantages. Fungal structures that are too hard for easy sectioning or, after sectioning, are too impenetrable to transmitted light may usually be softened or cleared by soaking for a suitable period in some softener or clearing agent, such as a solution of potassium hydroxide or chloral hydrate. Clearing agents effectively remove fats and oils. Often after their use, structural details not otherwise evident are rendered more distinguishable, especially if they are stained. Certain mounting media, which clear and stain at one operation, are distinctly advantageous although the unstained aqueous mounts are usually satisfactory, especially so for water molds if the microscope illuminant is properly adjusted. A phase microscope is of decided advantage for living materials.

If the living specimen or culture possesses well-marked, matured spore-bearing structures, it is usually adequate for study. But if it bears no fructifications or only immature ones, they may often be produced or forced into recognizable maturity by such expedients as immersing them in water or keeping them in a moist atmosphere for a convenient period. Moist chambers are easily improvised by placing wet blotting paper under a bell jar or in a closed mason jar. It is sometimes preferable to keep specimens moist by having them wrapped in a wet towel. If there is any likelihood that the fungus requires an especially low or high temperature for maturation, that condition should be met.

Diagnostic features of many fungi are best developed through pure culture on selected artificial media in petri dishes. Standard media are particular combinations of nutrients and agar gel, but there is a wide choice of formulas. The growth reactions of some species are characteristic on certain media and not on others. In pure culture on artificial media a species may, furthermore, present a different appearance from that in nature. Hence it may be needful to grow it upon the natural substrate to obtain the development of normal fructifications.