The Influence of Antagonistic Fungi on Thielaviopsis basicola (Berk. et Br.) Ferraris

That the damage which Thielaviopsis basicola (Berk, et Br.) Ferraris may cause to a definite host plant, is not always equally severe, has been known already for a long time. This variability has usually been ascribed to physical factors operating in the soil, but another circumstance might also be of importance, viz. the presence of micro-organisms acting as antagonists. It is rather striking that the influence which such organisms might exercise on Thielaviopsis basicola, has received so far little or no attention, and for this reason a study of this problem in vitro as well as in the soil seemed appropriate. Thielaviopsis basicola was isolated from the roots of Primula obconica as well as from those of Nicotiana glutinosa, and by using the isolation method of Yarwood (1946) it was found that at Baarn (Netherlands) this root parasite is very common in garden soil. In order to obtain antagonists, samples of various kinds of soil were plated out, and the fungi that developed on the plates, were isolated and tested as to their power to inhibit the growth of Thielaviopsis. On cherry agar about 38 of them showed an antagonistic effect. In further experiments the antagonistic activity of these fungi was estimated by means of filtrates obtained from cultures in cherry juice. In this way about 20 % of the 38 fungi were found to cause in Thielaviopsis a notable growth inhibition. The strongest antagonistic activity was found in the culture filtrates of ~ ~ – – – . Aspergillus fumigatus Fres., Penicillium expansum (Link) Thom, Penicillium spinulosum (Link) Thom, Penicillium spiculisporum Lehman and Penicillium roqueforti Thom. The two last-mentioned species were so far unknown as producers of antibiotic substances. The composition of the culture medium in which the fungi were grown, appeared to exercise a marked influence on the antibiotic activity of the culture filtrates as observed in cultures of Thielaviopsis. From a Czapek-Dox medium, in most instances, a more active filtrate was obtained than from a cherry-juice medium or from a culture in potato extract. In the Czapek medium, moreover, the carbon source proved to be of importance; in the case of Penicillium roqueforti saccharose gave the most active filtrate, whereas with Penicillium spiculisporum glucose and maltose proved to be more suitable. It was found, moreover, that in the case of Penicillium roqueforti the activity of the filtrates increased when the concentration of the saccharose in the Czapek medium was raised from 1 % to 5 %. The antibiotic activity of the filtrate of this fungus appeared to decrease when corn steep liquor was present. The growth-inhibiting substances in the culture filtrates, with the exception of those present in the filtrate of Aspergillus fumigatus, proved to be able to withstand a temperature of 103 G for 10 minutes; at room temperature they retained their activity for at least 45 days. Penicllium roqueforti proved to produce its growth-inhibiting substance(s) mainly in the period of active growth, i.e. during the first 15 days. When the fungus was kept for more than 30 days on the same medium, the antibiotic activity of the filtrate decreased. Different strains of Penicillium roqueforti were found to differ as to the inhibiting effect of their culture filtrates, whereas different strains of Thielaviopsis basicola reacted more or less in the same way on the culture filtrate of this antagonist. The relation between the natural microflora of different soils and the development of" Thielaviopsis in the latter were also studied. To this end the soils were periodically inoculated with a suspension obtained from a Thielaviopsis * – ,— culture. Two different soils were tested, viz. a “diseased” soil, i.e. a soil in which diseased olants of Primula obconica had grown, and a “healthy” soil, i.e. a soil in which the Primulas had remained healthy. In the “diseased” soil, at the beginning of the experiment, the presence of Thielaviopsis could not be demonstrated by means of the plate method; that it nevertheless was present, follows from the fact that Primulas which were subsequently planted in this soil also became infected. Suspensions containing the mycelium and the spores of Thielaviopsis were added every other day, from the 4th March to the 22nd March, and every time before a new dose was given, a soil sample was taken in which the composition of the microflora was quantitatively determined. It appeared that the frequency of the sod fungi was not influenced by the periodic inoculations with Thielaviopsis. Contrary to the expectation, the frequency of the antagonists too remained the same. During the first 6 days of the experiment the number of Thielaviopsis • – – i colonies remained high, but then a sharp decrease was noted. It was largest in the soil m which the microflora was best developed, i.e. in the “diseased” soil. During the later part of the experiment the lower level was maintained. Shortly after the additions of the Thielaviopsis suspension were stopped, the number of Thielaviopsis colonies in the soil samples decreased with 50 to 60 %. When the experiment was ended, the soil was planted with Primula obconica, and then it appeared that the infection of the latter was heaviest in the “healthy” soil that had periodically been inoculated with Thielaviopsis. This is in agreement witn me unding mat the number of ' ' ' Thielaviopsis colonies that could be isolated from the diseased sod underwent a stronger decrease than the number that could be isolated irom the healthy” soil. The experiment with the Primulas is also of interest because it shows that the presence of a parasite can sometimes be demonstrated by means of a susceptible host where attempts to isolate it by means of the plate method remain unsuccessful. In the experiments on the influence exercised by various antagonists on the pathogenicity oi Phielaviopsis, Nicotiana glutinosa r . , w was used as a test plant. It is very susceptible to infection by * * Thielaviopsis. First the influence exercised by various external conditions on the growth of Nicotiana glutinosa and on its infection by Thielaviopsis was investigated in order to find a suitable combination of conditions for the cultivation of "this plant, i.e. a combination that docs not cause a syndrome by which the symptoms of the disease might be obscured. It appeared that in direct sunlight the plants grew slowly and remained stunted; the leaves showed a yellow discoloration, and flowering started at an early stage. These symptoms may easily be mistaken for those caused by an infection with Thielaviopsis. A temperature of 25° C proved to be slightly better than a temperature of 19° G, but the humidity of the atmosphere had hardly any effect. i he degree to which the roots were infected, showed little or no correlation with the external conditions, but this did not apply to the plant as a whole, for the infected plants that had developed in direct sunlight were at the end of the experiment at the verge of death, whereas the infected plants that were grown in the shade, though they had lost a good deal of their vigour, were still in a fairly good condition. The “condition-index” calculated for the two groups shows, on the other hand, that the decrease in vigour shown by the shaded plants was proportionally larger than that of the sunlight plants (Fig. 1). The amount of inoculum that is required for a severe infection of Nicotiana glulinosa, proved to be 3000-3500 infection units (chlamydospores and conidia) per cc soil. A dose of this strength was used for the assessment of the pathogenicity of this root parasite in sterilized soils to which an antagonist had been added, and also in an unsterilized soil, i.e. in a soil in which the natural microflora still was present. The conditions under which this experiment was carried out, were shade, a temperature of 25° C and a high relative humidity, because this are the circumstances which proved to be not only favourable for the development of the plants, but which also allowed an accurate assessment of the severity of the disease symptoms caused by Thielaviopsis basicola in the presence or absence of antagonists. In sterilized soil the infection of Nicotiana glutinosa by Thielaviopsis proved to be inhibited to some extent when the soil was at the same time infested with Penicillium expansum, Penicillium spiculisporum or Penicillium spinulosum. Aspergillus fumigatus had no effect. The experiment with unsterilized soil showed that the inhibition exercised by the microflora which is normally present in the soil, is far more effective than that which could be obtained in a sterilized soil by inoculation with one of the above-mentioned antagonists (Fig. 2). Of all the antagonists tested, the three Penicillia proved to be most effective against Thielaviopsis basicola in the soil as well as in vitro.