The antimitotic properties of the benzimidazole fungicide carbendazim and a mechanism of resistance to this compound in Aspergillus nidulans

cum laude graduation (with distinction)

Systemic benzimidazole fungicides are well-known for their pronounced ability to control a large number of fungal plant diseases. On the other hand development of resistance in fungi to these compounds is as well widely known.

Biochemical aspects of both fungitoxic action and resistance in fungi are the subject of this thesis. It contains four articles describing a) the mechanism of action of carbendazim or methyl benzimidazol-2-yl carbamate, b) a mechanism of resistance to this compound and c) its metabolic conversion. Most of the work was done with Aspergillus nidulans, because this fungus is genetically well defined and new mutants can be readily characterized. Three strains were used, one strain with wild type sensitivity, one with an increased sensitivity and one with a decreased sensitivity to MBC. The behaviour of both mutant strains was due to a mutation in the benA locus.

In the first paper it has been shown that MBC is an effective inhibitor of mitosis in Aspergillus nidulans. Synthesis of DNA and RNA appeared also to be affected but this could be ascribed to the disturbance of the cell cycle, due to inhibition of mitosis. Hence, mitosis can be considered the process first affected by this compound.

In mammalian cells potent inhibitors of mitosis like colchicine, podophyllotoxin and vinblastine sulphate exert their action via interference with functioning and assembly of microtubules, the elements of the spindle. These compounds bind to tubulin, the dimeric subunit of microtubules, preventing in this way microtubule formation.

On the assumption that the antimitotic activity of MBC might be based on a similar binding, the MBC-binding properties of mycelial extracts were investigated. The results of these studies are presented in the second and third paper. An MBC-binding protein was found to be present in mycelial extracts which showed characteristic properties of tubulin. MBC binding was competitively inhibited by colchicine and oncodazole, a benzimidazole compound which binds to mammalian tubulin at the colchicine binding site. Partial purification of the binding activity resulted in a protein preparation in which the two tubulin monomers predominated. These results indicate that MBC is bound to fungal tubulin. Binding probably prevents microtubule formation or functioning which leads to disturbance of mitosis.

Study of the action of MBC at the molecular level also led to the elucidation of a mechanism of resistance of fungi to this compound. Results of binding experiments with a number of MBC-sensitive and MBC-resistant fungal species and strains suggested that the affinity of the binding protein for MBC determined the fungal response to the action of MBC. Evidence in favour of this hypothesis was obtained from detailed binding experiments with three A. nidulans strains. Results clearly indicated the relation between degree of sensitivity for MBC and magnitude of the binding constant for MBC and tubulin.

In the fourth paper the conversion of MBC to a non-toxic metabolite has been described. Conversion was found to occur in three strains of A. nidulans examined and in non of these cases it had any bearing on the mechanism of resistance to this compound. Wheather conversion of MBC plays any role in the mechanism of resistance in other fungi remains to be elucidated.

The identification of MBC as an antitubulin, a compound which binds to tubulin, indicates that antitubulins can be successful fungicides. However, a wide spread and frequent use of such compounds does not seem advisable for a number of reasons. Their action based on the specific interference with one target site is possibly subjected to any change in this site. A mutation leading to a decreased affinity can finally result in the development of a resistant fungal population which cannot be controlled effectively.

The use of these compounds which are selective with respect to plant pathogenic fungi may also have ecological implications. A shift in the dynamic equilibrium between the various components of the microflora in the soil and on the surface of the plant is conceivable. Naturally resistant, as well as resistant strains of normally sensitive plant pathogens might be favoured in their development, giving rise to increased disease incidence.

The potential hazard of large scale use of compounds with this type of action should also be kept in mind. Mitosis in all eukaryotes follows a basic pattern, and in nearly all eukaryotes microtubules play an essential role in the separation of the chromosomes. The structure of microtubules seems to be highly conserved during evolution and, therefore, agents interfering with microtubules might be active in a wide range of organisms. Although MBC is relatively non-toxic to mammals, which is probably due to a rapid metabolic conversion and excretion, as well as to low affinity of mammalian tubulin for this compound, its ability to induce abnormalities in nuclear division in some cells of these organisms has been demonstrated. Such an effect in reproductive cells may have severe consequences. The use of compounds which act on mitosis through interference with microtubules, therefore, implies a potential genetic risk for men.

Whatever may be the future of practical use of benzimidazole compounds in agriculture or medicine, they will undoubtedly continue to be valuable tools in the study of the structure and functioning of microtubules.