Tobacco Research Institute, Scafati 84018, Italy

Background and objectives
Thielaviopsis basicola (Chalara elegans) is the agent of black root rot, a widespread disease of tobacco with an uneven incidence in the field. On account of the broad host range of the fungus, attempts have been made in the past to evaluate if isolates within the species have any degree of specialization toward different host plants, but no conclusive results have been obtained [1]. Variation in host range and virulence of plant pathogenic fungi has been often reported in relation to the production of toxic metabolites. So far the ability of pathogenic isolates of T. basicola to release toxins involved as disease determinants has not been evaluated. Results of preliminary investigations on the subject are reported in the present paper.

Materials and methods
Production of toxic metabolites was evaluated in liquid cultures of three virulent isolates of the fungus recovered from diseased tobacco plants. Liquid cultures were prepared in 500-ml erlenmayer flasks containing 150 ml Oku & Nakanishi’s medium (ON) [2]. After 21 days cultures were filtered at 0.45 µm, and the culture filtrate was lyophilized until reduction to 1/25 of the starting volume. Biological activity of the culture filtrate was tested on the root system of tobacco plantlets grown on Hoagland & Knop's substrate in cylindrical transparent plastic containers (5x11 cm, four seedlings each). 1 ml concentrated culture filtrate was sterilized by filtration at 0.2 µm and inoculated on the substrate. Tobacco genotypes, both susceptible and resistant to black root rot, were tested, together with the main source of resistance to the disease in the Nicotiana genus (N. debneyi). Two sets of control containers were arranged, which received either 1 ml concentrated ON substrate or 1 ml chlamydospore suspension (5x105 ml) harvested on PDA cultures of the isolates under investigation. Concentrated culture filtrates were also fractionated by means of gel filtration through Sephadex G-25 in a 30-cm chromatographic column (eluant: distilled water). The eluted liquid was collected in 2 ml fractions; under the above-mentioned conditions, total elution of 6 ml concentrated culture filtrate entailed the collection of 80 fractions, which were tested on tobacco plantlets as described. A further assay was carried out by inoculating concentrated culture filtrate dialysed through a 500-Da cut-off cellulose ester membrane (Spectra/Por).

Results and conclusions
Inoculation of 1 ml concentrated culture filtrate of all isolates tested induced symptoms of root necrosis on tobacco plantlets within 3 days. The necrotic process was restricted to root tips and hindered the normal development of the root system, even if absorption of nutrients from the medium was not inhibited and growth of tobacco plants continued at a lesser extent. A gradual recovery from the initial damage was observed later in relation to the lack of continuous toxin release. Symptoms were similar to those observed soon after inoculation of the chlamydospore suspension, before pathogen colonization determined diffused root rot. They were evident on susceptible genotypes only, while both resistant genotypes and N. debneyi were unaffected. As fractions obtained by gel filtration were tested, the same symptoms were observed in containers where fractions had been inoculated. A comparison with data on elution of chemicals of known molecular weight in the same conditions allowed a rough estimate of about 400-500 Da molecular weight for compounds collected in the toxic fractions, which was confirmed by the observation that loss of toxicity occurred after dialysis of culture filtrates. The latter observation indicated that the biological activity of culture filtrates is due to toxic metabolites rather than pectic enzymes, which also have been reported to play a main role in damage to plants by the pathogen. The results obtained suggest that toxic metabolites can be regarded as virulence factors in T. basicola, as well as that resistance in tobacco may be related to the response to their action.

1. Corbaz R, 1985. Phytopathologische Zeitschrift 113, 289-299.
2. Oku H, Nakanishi T, 1963. Phytopathology 53, 1321-1325.