Since the introduction of the systemic fungicides, fungicide resistance has become a serious problem in plant disease control. This study was carried out in order to contribute to the knowledge about the genetics of fungal resistance to fungicides both from a practical and a fundamental point of view.
The potential of a fungus to acquire fungicide resistance, measured as mutation frequency and degree of resistance, was investigated in various fungi. The fungicides employed were: the benzimidazole fungicides benomyl and thiabendazole; carboxin, an oxathiin compound; chloroneb, an aromatic hydrocarbon; imazalil, an imidazole derivative; and the two antibiotics cycloheximide and pimaricin, a glutarimide and a polyene antibiotic, respectively (chapter 3). Induced resistance occurred in all cases, in which it was searched for. The mutation frequencies varied from 10 -7
for pimaricin and benomyl to 2.10 -4
for chloroneb resistance (Table 6). Both mutation frequency and degree of resistance were independent on the fungal species. However, considerable differences between the fungicides were found in the highest level of resistance observed among the resistant strains. From the results it was concluded that imazalil and pimaricin, for which the level of resistance in relation to the wild type increased at most by a factor 10 and 4, respectively, might be used in agriculture without a considerable chance of interference with control. On the other hand the development of resistance to the other fungicides of 50-5000 times, makes their practical use rather questionable.
Pathogenicity and in vivo fungitoxicity tests were carried out with benomylresistant strains of five plant pathogens, viz. Cladosporium cucumerinum
, Cercosporella herpotrichoides
, Fusarium nivale
, Penicillium expansum
and Phialophora cinerescens
(chapter 4). Generally, a close correlation between resistance in vitro and in vivo could be established. With respect to pathogenicity some strains were as pathogenic as the wild type, others were less pathogenic and a few had lost their pathogenicity. However, the results did not allow the conclusion that loss of pathogenicity was due to the mutation to resistance.
Characterization of the resistant mutants in vitro was performed especially in Aspergillus nidulans
(chapter 5). Dosage response relationships of fungicide resistance in agar growth tests were presented. With the four fungi examined, viz. A. nidulans, Aspergillus niger
, P. expansum
and Ustilago maydis
crossresistance of the chloroneb-resistant strains was observed to pentachloronitrobenzene (PCNB), a related aromatic hydrocarbon fungicide, and to 3-phenylindole. Usually, resistance to either benomyl or thiabendazole involved resistance to the other compound but in rare cases resistance to benomyl only or more frequently to thiabendazole only occurred, the latter involving increased sensitivity to benomyl. This phenomenon, known as negative crossresistance was found in all five fungi examined, viz. A. nidulans
, A. niger
, P. expansum
, Rhodotorula rubra
and U. maydis.
number of pleiotropic effects in the imazalil-resistant mutants of A. nidulans
and A. niger
was found, viz. resistance and hypersensitivity to various inhibitors. This suggested that the action of imazalil might be sought in changes in cell membrane permeability.
It is known that for Saccharomyces cerevisiae
carboxin is ten times more toxic on a medium with acetate as sole carbon source than with glucose. This effect was verified in the wild-type strain of A. nidulans.
However, in the case of the carboxin-resistant strains this difference was the smaller the more resistant the strains were. This strengthened the observations that reactions in the tricarboxylic acid cycle are involved in the mechanism of action of carboxin, since carboxin has been shown to inhibit mitochondrial succinate oxidation.
Using genetically well-defined strains of A.nidulans,
it was possible to establish the genetic basis of characters such as patterns of cross-resistance. Genetic analysis with the non-pathogenic A.nidulans
was carried out by means of the sexual and parasexual cycles (chapter 6). A single nuclear gene relationship of all mutations to resistance was found. The number of loci involved in the resistance to a fungicide was determined by recombination analysis of different mutants. It appeared that with some fungicides such as chloroneb and oligomycin only one locus was responsible for all the mutations to resistance; on the other hand in the case of resistance to pimaricin and to carboxin two and three genes, respectively, were identified, whereas in the case of cycloheximide and imazalil resistance a multigenic: system was involved. High-level resistance to benomyl and/or thiabendazole appeared to be based on mutations at one locus. Twenty- three genes conferring resistance were assigned to seven different linkage groups (chromosomes), sixteen of which were mapped. Using heterozygous diploids the dominance behaviour of the mutations to resistance was studied. A range of degrees of dominance was found from a practically recessive condition of mutations to benomyl, chloroneb and pimaricin resistance to an almost complete dominance of some genes conferring carboxin and cycloheximide resistance. In the case of imazalil resistance a positive interaction of genes resulting in a high degree of resistance in recombinant strains was observed.
In order to verify the results obtained with A.nidulans
in plant-pathogenic fungi, genetic analysis was also performed with U.maydis,
using the sexual cycle, and with A.niger
by means of the parasexual cycle (chapter 7). As a result of this approach it was demonstrated that in A. niger
and U. maydis
benomyl and thiabendazole resistance including negative crossresistance was based, as in A.nidulans,
on one single gene in which all resistant strains carry a different mutation. Analysis through the parasexual cycle, which involves heterokaryosis, diploid formation and recovery of haploid and diploid segregants, gave similar results in A.niger
as known from the literature. In C.cucumerinum
for which a parasexual cycle was not known, diploid strains were isolated, from which recombinant haploids were also recovered, although, surprisingly, without observing a heterokaryotic state. The parasexual cycle in C.cucumerinum
provided information on the genetic basis of pathogenicity, because loss of pathogenicity of auxotrophic mutants was fully complemented in the heterozygous diploid strains, which appeared to be as pathogenic as the haploid prototrophic wild-type strain.