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To determine with certainty that a particular microorganism is the cause of a disease rather than some incidental contaminant, it is necessary to critically examine its relationship with the host. This dilemma was first recognized in studies of pathogens of humans and other animals. In 1876, Robert Koch provided the first experimental proof of disease causation by applying a set of rules which have since come to be known as Koch's postulates. Koch considered that these rules must be satisfied before any microorganism can be regarded as a pathogen. The rules involve five steps outlined below.

1 The suspected pathogen must be consistently associated with the same symptoms.

2 The organism should be isolated into culture, away from the host. This precludes the possibility that the disease may be due to malignant tissues or other disorders of the host itself.

3 The organism should then be reinoculated into a healthy host.

4 Symptoms should then develop which are identical to those observed in the original outbreak of disease.

5 The causal agent should be reisolated from the test host into pure culture and be shown to be identical to the microorganism initially isolated.

An actual example of the use of Koch's rules is shown in Figure 2.5. An apparently new disease of orange trees, with symptoms of chlorosis, stunting, and dieback of branches, was reported in South America. Leaves from affected trees were surface sterilized and plated onto a nutrient medium. Colonies of a small, gram‐negative bacterium were obtained. Suspensions of the bacterium were then injected into healthy citrus saplings, and after a period of incubation, some of these artificially inoculated trees developed symptoms very similar to those seen in the original infected tree. The same small bacterium was reisolated from these trees.

This procedure completed Koch's postulates and showed that the new disease, named citrus variegated chlorosis, was due to a bacterium. In reality, a lot more work, including light and electron microscopy and the use of specific antisera, was required to actually identify the agent as a new strain of the xylem‐inhabiting pathogen Xylella fastidiosa. A few years later, in 2000, X. fastidiosa became the first cellular plant pathogen to have its complete genome sequenced.

Procedures for the detection and diagnosis of specific pathogens are described in more detail in Chapter 4.

This example shows that even today, Koch's rules are still relevant, although they cannot be rigidly applied in their original form to all pathogens. The most important exceptions in plant pathology are when the pathogen cannot be grown in artificial culture, for example the viruses and some biotrophic fungi. The problem of isolating viruses from their host plants is generally overcome by using indicator plants. These are alternative hosts which develop symptoms which are specific for a particular virus. Healthy specimens of the original host may then be reinoculated. In addition, electron microscopy of plant sap or of purified crystalline samples of the virus, coupled with serological techniques, may be employed to investigate the type(s) of virus present at each step of the procedure. There are also a number of new and powerful methods for detecting nucleic acid sequences specific to particular pathogens.

Figure 2.5 Use of Koch’s postulates to establish the etiology of a new disease of citrus caused by the bacterium Xylella fastidiosa.

Source: Based on Hartung et al. (1994).

The application of Koch's rules to nonculturable fungal pathogens presents fewer problems because these agents produce spores. Such propagules can be removed from the host and then used in reinoculation experiments. In many instances, spore morphology is also a valuable aid to identification of the inoculated and reisolated pathogens.

Further difficulties in satisfying these postulates may be experienced in cases where symptoms result from mixed infections or when dealing with previously undescribed disease agents. For instance, few pathologists would have predicted the existence of the viroids, which scarcely conform to our preconceptions of a successful parasite (see Chapter 3).