Botanic Gardens Trust, Sydney, Australia

Pathogenicity Tests

The pathogenicity of a fungus should be assessed using a technique which enables the reproduction of typical symptoms of the disease over the time-scale relevant to the commercial glasshouse or field situation. The following guidelines are recommended for the design of meaningful tests of pathogenicity.

Typical wild-type cultures should be used for the preparation of inoculum. Ideally these cultures should have been recently isolated from diseased plant material and maintained on a low nutrient medium such as CLA. Alternatively lyophilised cultures with a similar history can be used. Isolates that have degenerated (mutated), or that have been subcultured repeatedly on rich media should be avoided as they are likely to be avirulent.
The nature and amount of inoculum used in pathogenicity tests should conform to the inoculum causing the disease under field conditions. It may be necessary to make assumptions in this respect as pathogenicity tests are usually initiated prior to detailed studies on the mode of persistence and dispersal of the species in question.

The cultivars used in the pathogenicity test should be identical to those on which the disease has been observed in the field. These cultivars should be grown under environmental conditions similar to those which prevail in the growing season in areas where the disease occurs. Plants should be grown in containers which permit normal growth and development during the period of the test.

Some pathogens only cause severe disease in plants which have been subjected to stress. Thus the technique selected for the pathogenicity test must enable the application of stress if it is thought to be a key factor predisposing the plant to the disease. Stress, for example, may be caused by inadequate soil moisture, extremes of temperature or herbicides.

The design of pathogenicity tests of suspected soilborne pathogens is difficult because the nature and amount of inoculum, and the soil have a significant effect on infection and disease development. Certain structures produced in culture which have the potential to act as soilborne inoculum may not persist in soil and are thus unsuitable for pathogenicity studies. Conidia of many Fusarium species, for example, are rapidly lysed in natural soil at higher temperatures. In contrast, chlamydospores may persist for long periods in soil and are the main mode of survival for a number of soilborne pathogens. Chlamydospores should normally be used as the inoculum of F. oxysporum and F. solani. The diseases caused by these two fungi can be reproduced using relatively low levels of propagules, if the isolates are virulent. Inoculum densities as low as 50-100 propagules/g soil can be quite satisfactory for reproducing typical symptoms of disease. Some pathogens mainly persist in soil as hyphae in host residues. Suitable inoculum of these species can be prepared from cultures grown on sterile natural substrates such as wheat chaff or corn stalks. The infested tissue is dried and milled before addition to soil. Wheat, barley or oat chaff (with or without grain) is an appropriate substrate for F. pseudograminearum, F. culmorum and F. avenaceum. Corn stalks are an appropriate substrate for F. moniliforme. This type of inoculum can be mixed evenly through the soil (1-2% w/w) or added as a fine horizontal layer of inoculum within the soil profile.

The soil (sterile, pasteurised or untreated) has a critical influence on the severity of disease caused by Fusarium, and other soilborne pathogens. Thus the soil selected for a pathogenicity test should be similar to that associated with the disease under investigation in respect of physical and chemical properties. A sterile soil should be used if the disease is normally restricted to sterile soils. Crown and root rot of tomato caused by F. oxysporum f. sp. radicis-lycopersici, for example, is mainly a problem in fumigated soil. Conversely an untreated field soil should be used in respect of field crop diseases such as crown and root rots of wheat and stalk rots of corn and sorghum.

The untreated field soil should be free from soilborne fungal and other pathogens of the relevant host, and from residual herbicides. Such soil can be obtained from a field that has been planted to other crops, or that has been long-fallowed.

The microflora in untreated field soil inhibits (suppresses) the activity of a soilborne pathogen. The removal of this inhibitory effect by sterilisation allows many soilborne fungal pathogens to proliferate vegetatively following the addition of inoculum to the sterile soil. The pathogen consequently causes greater disease in sterile than in untreated soil. This occurs because the level of inoculum is higher following the vegetative growth, and there is no microbial inhibition of the pre-penetration phase of infection.
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