Benzimidazole-resistant strain

Leroux and Gredt reported that benzimidazole-resistant strains of Botrytis cinerea and Penicillium expansum exhibited negatively correlated cross resistance to herbicidal N-phenylcarbamates such as barban, chlorpropham, and chlorbufam (10, 11, 12). Based on their observation, Sumitomo scientists evaluated many examples of N-phenylcarbamates to search for compounds with potent fungitoxicity and no phytotoxicity. This effort was eventually successful, leading to new fungicides such as MDPC ( , 21 ) and diethofencarb (14, 23, 24) (Figure 2). 

Some years ago, two groups of compounds, i.e. If-phenylcarbamates and N-phenylformamidoximes were synthesized in Japan and their specific antifungal activity against benzimidazole-resistant strains was reported ( 2, 3J. In France, where benzimidazole-resistant strains of the gray mold Botrvtis cinerea predominate in some areas, a N-phenylcarbamate compound diethofencarb is already in use as a mixture with a benzimidazole fungicide carbendazim. 

Diethofencarb can be used against benzimidazole-resistant strains of Botrytis spp. It also inhibits mitosis. It is quickly degraded in soil and in animals through oxidation of the 4-ethoxy group. 

Recently, the carbamate fungicide diethofencarb (15) which takes advantage of a negatively-correlated cross-resistance phenomenon, has been developed as a potent fungicide against benzimidazole-resistant fungi [45, 46]. Studies on the mode of action of 15 have revealed that P-tubulin in the benzimidazole-resistant strain F914 of N. crassa has an affinity for diethofencarb (15) rather than for benzimidazole derivatives, whilst in a wild-type strain of N. crassa there was an inverse affinity [46, 47]. 

According to the results of Fujimura et al. [45], the minimum inhibitory concentrations (MICs) of MBC (12) and diethofencarb (15) for wild-type N. crassa were 0.2 ppm and >100 ppm, whereas for N. crassa F914 they were >100 ppm and 0.1 ppm, respectively. Therefore, an experiment using two combination systems, (1) benzimidazole-resistant strain + diethofencarb + emodin, and (2) benzimidazole-susceptible strain + MBC + emodin was carried out in order to obtain data on whether emodin could exhibit antidote activity in both systems. In this experiment, only emodin was used as an antidote because of the relatively weak activity of other antidotes in the interaction of MBC with wild-type N. crassa [44]. The results are summarized in Table 6, and indicate that emodin (18) can also act as an antidote against diethofencarb (15) in a benzimidazole-resistant strain of N. crassa. These and other results (Tables 4 and 6) are indicative of the versatility of emodin (18) as an antidote to the action of certain types of fungicide which possess an affinity for fungal P-tubulin, and may suggest that the antidote effect of emodin is closely linked to the function of P-tubulin. 

Although it was proposed that inhibition of HCMV DNA maturation by the benzimidazole ribonucleoside BDCRB is mediated through the UL89 gene product, and resistance to TCRB maps to the two ORFs UL89 and UL56, there was no cross-resistance of an HCMV AD 169 sulphonamide-resistant strain to BDCRB (Reefschlaeger et al. 1999). 

Thus, they prevent an arrangement of the spindle apparatus. The tubuline-benzimidazole-interactions have been studied in detail (.6). It is known that carbendazim, for example, after entering the nucleus, specifically binds to the 3 -subunit of tubuline and by this inhibits the dimerization of the a and 3 -subunits to a functional tubuline unit. Resistant strains possess altered 0 -subunits with a decreased affinity for benzimidazoles (7). The modes of action of other inhibitors of mitosis, like dicarboximides, aromatic hydrocarbons, or dithiocarbamates, have not yet been precisely described on a molecular basis. 

Mechanism of Action of jV-Phenylcarbamates in Benzimidazole-Resistant Neurospora Strains... 

However difference aspect was observed in resistance to MBC between N. crassa isolates in laboratory and B. cinerea isolates from the field. Laboratory-generated mutants of N. crassa showed various levels of resistance to MBC, and a specific one showed negatively correlated cross resistance to diethofencarb. Others showed double resistance to these chemicals. On the other hand, most of the benzimidazole resistants isolated from the field, especially in in the case of B. cinerea, were supersensitive to diethofencarb. This difference between laboratory and field strains may derive from a difference in fitness among the resistant strains of the plant pathogen. 

Bennet, E. M. and Bryant, C. (1984) Energy metabolism of adult Haemonchus contortus in vitro a comparison of benzimidazole-sensitive and resistant strains. Mol. Biochem. Parasitol. 10 335-346. 

Recent studies have shown that a strain of Neurospora crassa resistant to benzimidazole carbamates but sensitive to the phenylcarbamates, MDPC and dlethofencarb, resulted from a mutational change in the -tubulin gene (1 0). The change of a single amino acid from glutamic acid to glycine in -tubulin apparently accounted for benzimidazole resistance in this mutant strain. Revertants of this mutant were resistant to dlethofencarb but exhibited the same benzimidazole sensitivity as the wild type. 

When we introduce new fungicides, it is necessaiy to evaluate how quickly resistant strains can appear and increase with the use of fungicides. It is generally useful to elucidate the mode of action of newly developed products when predicting the inherent risk for resistance development, as most fungicides that have encountered resistance so far were site-specific inhibitors such as benzimidazoles, Qols and MBI-Ds. 

L.G. Davidse and W. Flach, Differential Binding of Benzimidazol-2-yl Carbamate to Fungal Tubulin as a Mechanism of Resistance to this Antimitotic Agent in Mutant Strains of Aspergillus nidulans J. Cell. Biol., 1977, 72, 174-193. 

We used N. crassa as a model fungus and attempted to elucidate the mechanism of negatively correlated cross resistance between benzimidazoles and N-phenylcarbamates. MBC resistant mutants were isolated from the wild type strain of N. crassa by UV treatment. Selection of the MBC resistant mutants was carried out on medium containing 50ppm MBC. MBC (carbendazim), the degradation product of benomyl and thiophanate-methyl, appears to be the active form in fungi (1). 

Considerable interest is given nowadays to the possibility of using natural products as parasiticides in agriculture to replace or enhance phytochemical products, because of the serious environmental problems caused by the often indiscriminate use of pesticides. Massive use of synthetic fungicides against parasites has led to the appearance of strains which are resistant to both the dicarboximide derivatives (iprodione, procymidone, vinclozolin [64-65] and the benzimidazoles (benomyl, carbendazim, thio-phanates, etc.) [66-67] making it much more difficult to control the pathogens. 

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