Site of Action and Mechanism(s) of Resistance
Site/Mode of action
Many cellular processes in eukaryotic organisms depend on proper functioning of the cytoskeleton. Cell division, maintenance of cell shape, intracellular movement of organelles, all of these processes depend on the cytoskeleton. In filamentous fungi, the cytoskeleton also plays a key role in signal transduction, adhesion, hyphal tip growth, septation, motility when flagella are involved as well as in pathogenesis. Microtubules, one type of cytoskeleton filament, regulate organelle position and movement within the cell. Microtubules are long, hollow cylinders of repeating dimers of α- and ß-tubulin. Although highly conserved, the amino acid sequences in tubulin genes may vary significantly without destroying gene function. As a consequence, sensitivity to inhibitors also is highly variable. In fact sufficient diversity is present to allow selection of a wide range of inhibitors in many diverse target organisms, as well as host selectivity necessary for successful therapeutic use. Over the past 50 years, microtubule formation and functioning has been a fruitful target not only for fungicides but for other pesticides, including insecticides, miticides, nematicides and herbicides.
Benzimidazoles are potent inhibitors of ß-tubulin polymerization in many species of fungi and have been used for plant disease control since the late 1960’s. Although direct interactions of inhibitors with ß-tubulin seem to be the predominant inhibitory processes for this chemical class, interactions with other forms of tubulin as well as differential interactions with tubulin in the free and polymerized states also have been reported.
Depending on their concentration, time of incubation and type of media, benzimidazoles appear to induce abnormalities in spore germination, germ tube elongation, cellular multiplication, and mycelial growth of sensitive fungi. The initial stages of spore germination often are not affected by benzimidazoles. Benzimidazoles prevent cell division,however, that is required for continued germ tube elongation and infection.
Benzimidazole fungicides control a remarkably broad spectrum of plant pathogenic fungi but they do not control Oomycetes like Pythium, Phytophthora and organisms that are responsible for downy mildew diseases of many crops. Interestingly, the benzamide fungicide zoxamide introduced more recently has been reported to affect tubulin and microtubule interactions in Oomycetes as well as other fungi.
Mechanism of resistance
Studies have demonstrated that fungicidal activity and resistance are determined by affinity of specific inhibitors for target sites on the ß-tubulin protein. Alterations in the α-tubulin gene affecting sensitivity to benzimidazole fungicides usually are of little practical significance for resistance management since they result in increased sensitivity to inhibitors. Resistance to benzimidazole fungicides is related primarily to specific alterations in the binding sites on the ß-tubulin protein. Many mutations conferring resistance to benzimidazoles have been identified in the ß-tubulin gene in laboratory studies with a wide range of different fungi. Loss of field performance, however, has been associated with 6 target site mutations, at codons 6, 50, 167, 198, 200, 240. The most common and significant mutations occur at codons 198 and 200. At codon 198, mutants vary from moderately to very highly resistant depending on the specific amino acid replacing glutamic acid present in the sensitive wild type. At codon 200, resistant isolates are characterized by a single substitution of phenylalanine by tyrosine (F200Y) that generally confers moderate to high levels of benzimidazole resistance.
Other possible resistance mechanisms have also been reported. In some fungi like Colletotrichum acutatum inherently less sensitive to benzimidazoles, overexpression of the β-tubulin protein may be a factor contributing to increased tolerance. In some cases, low levels of resistance have been associated with multidrug resistance. Other mechanisms like increased metabolism or reduced uptake of inhibitors do not appear to be important factors for this area of chemistry.
Cross-resistance with other fungicides
There is generally no cross-resistance between benzimidazoles and commercial fungicides from other chemical classes.Therefore combinations of benzimidazoles with companion fungicides with different target sites are an effective means of reducing resistance risk.
The phenomenon of negative cross resistance has also been reported between benzimidazole fungicides and a few other chemical classes, including N-phenylcarbamates and more recently benzamides. For example, fungal isolates sensitive to benzimidazoles are resistant to N-phenylcarbamates like diethofencarb. Isolates that are resistant to benzimidazoles, however, are sensitive to N-phenylcarbamates. This phenomenon allowed mixtures of these two classes to be used commercially in European vineyards for resistance management in Botrytis cinerea until isolates with the F200Y mutation appeared with resistance to both classes.. Although isolates resistant to both classes have been detected, the low frequencies of double resistant isolates results in this mixture remaining effective in some areas.
For N-phenylcarbarmates and benzamides, negative cross-resistance is dependent on the specific mutations present at codon 198 of the ß-tubulin gene. Wild-type isolates that are sensitive to benzimidazoles are insensitive to diethofencarb and zoxamide. In the presence of a glutamic acid to alanine mutation (E198A), isolates lose sensitivity to benzimidazoles but become sensitive to diethofencarb and zoxamide. A glutamic acid to lysine mutation (E198K), however, results in loss of sensitivity to all 3 classes.
There have been a few reports of negative cross resistance within the benzimidazole class of chemistry. In Aspergillus nidulans, for example, a mutation at codon 165 conferred resistance to thiabendazole but increased sensitivity to carbendazim. A mutation at codon 167 in Saccharomyces cerevisiae resulted in resistance to carbendazim but increased sensitivity to benomyl. For practical applications, however, fungal isolates resistant to one benzimidazole fungicide usually are resistant to other members of this chemical class. Therefore combinations of various benzimidazole fungicides cannot be used as a resistance management tool.
Fitness and persistence of resistant isolates
Some mutations in the β-tubulin gene caused both increased and decreased stability of microtubules after assembly, both effects reducing fitness of the affected organisms. Several mutations also resulted in increased sensitivity to temperature extremes. Reduced fitness associated with benzimidazole resistance suggested that resistance may not be stable and could decline if selection pressure provided by repeated fungicide applications was reduced or eliminated. Practical experience however, demonstrated that benzimidazole resistance remained stable in some areas 10 years or more after benzimidazole use was stopped. There have been a few exceptions. Benomyl was successfully used for over 10 years in some parts of the United States for cucurbit powdery mildew control even though resistant isolates were first detected before first commercial use of the fungicide. Monitoring demonstrated that the proportion of resistant isolates dropped after reduction of fungicide use and field efficacy was regained. With repeated use, however, the proportion of benomyl-resistant isolates increased and field efficacy decreased to the point where benomyl was no longer highly effective. A similar situation was reported for benzimidazoles used for the control of banana Sigatoka in Latin America. Fitness penalties associated with mutations therefore cannot be relied on to reduce resistance risk.