Untersuchungen über den Pflanzenparasiten Verticillium albo-atrum Reinke et Berth.

1. Morphology The Verticillium strains pathogenic on phanerogams are described chiefly in the literature under the names V. albo-atrum Reinke et Berth, and V. dahliae Klebahn. Flic difference between the two species is based mainly on the occurrence of a resting stage consisting of bundled dark hyphae in the species V. albo-atrum and of microsclerotia in the species V. dahliae. In later years Verticillium strains, which formed loose dark hyphae in vitro, were also brought into the species V. albo-atrum. In these investigations a large number of Verticillium strains were subjected to a further research, in order to find out if there really were two species. Attention was paid not only to the morphology, but also to some physiological aspects e.g. toxin formation and pathogenicity. From 70 Verticillium strains, isolated from different hosts and many places in the Netherlands, we found in two strains only the bundled dark hyphae, as described by Reinke and Berthold (1879) for the species V. albo-atrum. These bundled hyphae fell to pieces after some time and the individual cells then started budding, similarly to those of young microslerotia, with the result that the clumps of cells could no longer be distinguished from microsclerotia. These strains would then be considered as being in the species V. dahliae, so that they may be regarded as having moved from one “species” into the other. Attention was next paid to the Verticillium strains with loose dark hyphae the d.m.-type, these being compared with strains containing microsclerotia, the mscl-type. The measurements of the spores of both types provided no real basis for the separation of two species. There were indeed significant differences in the average length and width of the spores of some Verticillium strains, but these differences could not be correlated with the occurrence of microsclerotia or loose dark hyphae. Neither type could be distinguished on the basis of spore form. These findings are in agreement with those of Presley (1941), who obtained from one mono-spore culture saltants of both types. On morphological grounds the two species cannot be maintained and because the name V. albo-atrum __ Reinke and Berthold has priority, we must place all the strains investigated by the author in this species. 2. Physiological Properties a. The influence of temperature The reactions of the two types to high temperatures were different. In vitro the d.m.-type died sooner than the mscl-type at temperatures of 35° C and higher. The optimum and maximum temperatures for mycelial growth were also higher for the mscl-type than for the d.m.-type. A strain of the mscl-type was able to produce symptoms on and even to kill Impatiens balsamina plants, when these were incubated for 20 days after inoculation at a temperature of 35° G. A strain of the d.m.-type did not produce any symptom after the same treatment. This latter Verticillium strain had died in the plants during the incubation period at 35° C. This proves that the mscl-type is also better able to resist high temperatures in the plant. b. Toxin formation All Verticillium strains here investigated formed toxic substances in vitro. Leaves of tomato shoots, placed in the filtered culture liquid, wilted, the margins of the leaves curled, the intercostal areas became discoloured and the stems flaccid. In addition leafy shoots of Impatiens balsamina, Anthirrhinum majus and Mentha piperita were damaged by toxic substances present in the culture liquid. I he severity of the damage and the rapidity of the appearance of the symptoms were used as a standard for the phytotoxicity of the toxins formed. These proved to be specific to individual Verticillium strains; but the toxicity did not correlate with the pathogenicity of the strains investigated nor with the morphological type. c. Pathogenicity The penetration of the fungus into the plant takes place through the root-hairs or directly through the cortex of the root. The formation of “lignitubers” could not be found in the roots either of Lupinus luleus or of Impatiens balsamina. The spread of the fungus in the plant was traced in Impatiens balsamina and Fraxinus excelsior. It was found that spread of the fungus through the vessels was possible both by mycelium growth and also by the transport of spores, formed in the vessels, with the transpiration stream. Some strains which produced toxic substances, were scarcely pathogenic. Probably they are unable to penetrate or find difficulty in penetrating into the plant or else they are not able to spread within it. Other strains, which form toxic substances in vitro, could penetrate and spread in Solarium nigrum and Chrysanthemum leucanthemum; but these weeds showed no symptoms at all. This toleration could be explained by a far lower sensitivity of the tissues of these plants to the toxic substances formed by the fungus. The great diversity in character of the Verticillium strains in morphological as well as in physiological respects, makes it impossible to maintain the two species. On physiological grounds it should be possible to distinguish within the species V. albo-atrum Reinke and Berthold a mycelial form and a microsclerotial form. 3. Specialization It was proved by cross inoculations with Verticillium strains of different origins on a great number of host plants, that the investigated strains certainly showed differences in pathogenicity, but specialized strains with a narrow host range (Nelson, 1950; Isaac, 1957) could not be found. Only the Verticillium strains isolated from tomato always produced more distinct symptoms after inoculation into tomato than those strains isolated from other plant species. These latter Verticillium strains proved by reisolation to possess a very poor power of spread in the tomato plant, while the Verticillium strains originally isolated from tomato could be re-isolated much more widely. In other experiments too, the tomato occupied a particular place as host. A Verticillium strain, originally isolated from Antirrhinum, proved to be more pathogenic towards tomato after three passages through this plant, while its pathogenicity to Antirrhinum did not change. A Verticillium strain isolated from tomato, however, kept its pathogenic character with respect to this plant after several passages through Antirrhinum. The pathogenicity with respect to Antirrhinum did not increase. It seems that the tomato affects pathogenicity in a peculiar and inexplicable manner. 4. The Behaviour of Verticillium in the Soil Verticillium spreads in sterile soil only by means of the growth of hyphae. In originally steamed soil, containing Impatiens plants, which were subsequently inoculated with a Verticillium spore suspension, it could be shown that spread was only by means of infected dead root particles. In non-sterilized soil the same result was achieved with plants of Anthirrhinum majus. The fungus could apparently maintain itself in competition with the microflora of the soil only on organic remains. Also Verticillium could not maintain itself, if inoculum was brought directly into originally steamed soil, without any other organic material. It died out, probably under the influence of other micro-organisms, which developed later. The fungus could spread in one season through non-steamed soil from the roots of Antirrhinum plants inoculated with both a strain of the d.m.-type and the mscltype, to neighbouring plants. In the following year Antirrhinum plants were again put out in the places occupied by the previous year’s plants. From the number which developed the disease it could be concluded, that the fungus had spread further than the previous year. In the following two years the experiment was continued. The number of Antirrhinum plants, that became attacked, then diminished markedly. The strain of the d.m.-type was no longer able to attack Antirrhinum plants four years after the beginning of the experiments; the strain of the mscl-type still attacked them, but only slightly, after the same period. It here appears that Verticillium has more the character of a soil-invader than of a soil-inhabitant ('Garrett, 1944). 5. The Germination of Microsclerotia New colonies easily developed from microsclerotia, which were brought on to an agar-medium. Probably the attached hyphae or the spores laying between the dark, thick-walled cells grew out. It was possible to kill these hyphae and spores by desinfection, drying or exposure to high temperature; no development of new colonies then took place. In no instance did the cells of the microsclerotia themselves germinate. Nor could root secretions stimulate the thick-walled cells to further development. Most probably the microsclerotia are organs with a stock of food, from which the old hyphae may profit and grow afresh. 6. The Control of Verticillium Infected dead root and stem remains can form a focus of contamination for the surrounding healthy roots. Contaminated plant material must therefore be destroyed or the destruction of it must be aided by soil cultivation. The degree of contamination of the soil can be decreased by the activity of soil micro-organisms. A good soil structure and an definite crop rotation will exercise a favourable influence on the state of health of the plants. The degree of contamination increases as a result of repeated cultivation of the same susceptible crop until the second year after infection. It is therefore necessary to cultivate a crop that is not susceptible to Verticillium the year after an infection, so that a building up of inoculum can be prevented. Some weeds may contain the fungus without any symptoms, or occasionally the plants are merely smaller than non-infected ones. From this point of view it is also of importance to pay attention to weed control. Control by chemical treatment still leaves much to be desired. The cultivation of resistant varieties will probably give some temporary improvement, because not all Verticillium strains have the same pathogenicity for a certain hostplant. We must, however, take into account that a variety, resistant in one place, need not be so in another because there may be other Verticillium strains present in the soil there.