Benzimidazole antidote

Table 2. Results of Screening Extracts of Higher Plants for Benzimidazole Antidotes...

A preliminary screening test (TLC bioassay) for benzimidazole antidotes was carried out using 22 plant species, representing 18 plant families. As shown in Table 2 (A), out of the 22 plants tested, benzimidazole antidote activity was initially found only in Polygonum thunbergii (Polygonaceae). An aliquot, equivalent to 125 mg of fresh plant material, of the EtOAc soluble constituents from methanol extracts of P. thunbergii was sufficient to clearly show the antidote activity on a thin-layer plate [Table 2 (B)]. 

Three further benzimidazole antidotes have also been identified, two in P. lapathifolium subsp. nodosum, and one in P. thunbergii. Isolation and purification procedures for the two active compounds in P. lapathifolium are shown in Scheme 1 [13]. 

To further understand the properties of benzimidazole antidotes, the following experiments were carried out using the anthraquinone emodin (18) as a representative antidote found in the Polygonaceae. 

The charcoal method [50] was applied to determine free [2-,4C]-MBC in the binding test solution which consisted of crude fungal tubulin and [2-14C]-MBC. This method was successfully used to examine the ability of emodin to compete with [2-14C]-MBC bound to the colchicine site of 13-tubulin. As shown in Fig. 10A, the benzimidazole fungicide, nocodazole (16) can release [2-14C]-MBC bound to P-tubulin because of the property of 16 to compete with MBC. Under the same conditions, however, the antidote emodin (18) could not release the P-tubulin bound [2-l4C]-MBC (Fig. 10B). These results clearly suggest that the benzimidazole antidote emodin does not compete directly with the binding site for MBC in P-tubulin [44]. 

Fig. (11). Structures of the non-benzimidazole fungicides, diethofencarb (15) and zarilamide (65a) which inhibit tubulin assembly, 5-hydroxy-7-methoxychromone (64), an antagonist of benzimidazole antidotes, and centaureidin (65), a tubulin-interactive flavone.

We thank Professors R. Yokosawa, F. Tomita, and M. Fujimura for kindly supplying the fungal strains, Aphanomyces cochlioides, Cladosporium herbarum, and Neurospora crassa, respectively. Much of the research on which this review is based, has been carried out by the following students, Masters Y. Matsukura, N. Toda, and H. Katsuta (benzimidazole antidotes), and by Dr. T. Horio Masters T. Takayama, H. Kikuchi, K. Ohkawa, and M. Mizutani (zoospore attractants). 

Bioassays for Detection of Antidotes Against Benzimidazole Fungicides... 

Antidotes Against Benzimidazole Fungicides in Species of Polygonaceae... 

Fig. (5). Antidotes against benzimidazole fungicides found in the Polygonaceae.

Finally, a fifth antidote to benzimidazole fungicides was isolated from 13 kg of the aerial parts of P. thunbergii in very poor yield (ca 1 mg). However, comparison of its physicochemical and chromatographic properties with those of authentic 2,6-dimethoxy-p-benzoquinone (21) [13] confirmed that the compounds were identical. This benzoquinone has... 

The structures of compounds found in the Polygonaceae with antidote effects against benzimidazole fungicides, are shown in Fig. 5. Although,... 

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. 

It is now well known that the antimitotic benzimidazole fungicides do not prevent spore germination, but act by inhibiting the mycelial growth of many fungi. We have found that antidotes against benzimidazole... 

Finally, some herbicide antidotes are now commercially available [54], and the naturally-occurring isoflavone genistein (8) has been found to act as an antidote against the herbicides, haloxyflop and alloxydim [55]. However, as far as we are aware, no antidotes to benzimidazole-related compounds, or to other types of fungicides, have previously been reported. 

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