| JS399-19,2-cyano-3-amino-3-phenylancryic acetate is a novel cyanoacrylate fungicide introduced by Jiangsu Branch of National Pesticide Research & Department South Center. This fungicide was demonstrated to have activity against Fusarium. spp, especially for Fusarium graminearum. In order to develop a sound recommendation for the use and exploitation of a new crop fungicide, sufficient biological mode of action and genetic analysis of its resistance should be studied.In vitro, the inhibitory activity of the fungicide against mycelial growth of both sensitive and resistant isolates of F. graminearum was measured and the influence of the fungicide on conidial germination of F. graminearum was determined. The results showed that JS399-19 could strongly inhibit the mycelial growth of a sensitive isolate of F. graminearum with the EC50 values 0.092-0.141μg/mL. JS399-19 decreased the speed of conidial germination of the sensitive isolate of F. graminearum, strongly inhibited the germtube growth of the conidia, and affected the conidia germination by causing the ratio of the tubes germinated from the basal and that from the middle parts of the conidia increased. Moreover, JS399-19 could cause abnormality of conidia and the tubes of the sensitive isolate, by inducing the conidia to swell, and inducing the tubes to swell and contort, respectively. However, this fungicide weakly affected the JS399-19-resistant isolates. JS399-19 could hardly inhibit the respiration of and pyruvic acid production of F. graminearum, suggesting that JS399-19 might not directly affect the respiration pathway of this fungus.Fifty nitrate nonutilizing mutants (nit) were obtained by transferring the 3 JS399-19-resistant mutants of F. graminearum on MMC media. The results showed that there were no significant differences in mycelial growth rate, cultural characters and pathogenicity between JS399-19-resistant nit mutants and their parental isolates. But the conidiophore production and perithecigerous capacity changed more or less in some mutants. Results also indicated that there were no cross-resistance toward chlorate and fungicide JS399-19 in F. graminearum, and the resistance to both chlorate and JS399-19 could be transferred by asexual reproduction steadily. Therefore, the nit can be used as a genetic marker for genetic studies of F. graminearum resistant to fungicide JS399-19.In vitro, a total of 86 JS399-19-resistant mutants were recovered from 5 wild-type strain by ultra-violet (UV) irradiating and fungicide training with the high frequency 1.25×10-7-2.29×10-7 for UV and 2.5-6.0% for fungicide training, respectively. The results also revealed that most of the resistant mutants belong to MR or HR. Although JS399-19-resistance mutation did not change the vegetative compatibility of F. graminearum, nevertheless, JS399-19-resistance could not be transferred by hyphal fusion or could be transferred with very low chance between two vegetatively compatible isolates. Therefore, hyphal fusion presumably took very little part in the development of JS399-19-resistant population in F. graminearum. Forty-three isolates sensitive to fungicide JS399-19 were collected from three commercial wheat fields of China. Forty-five isolates resistant to JS399-19 which had already been recovered from five sensitive isolates by selection for resistance to the fungicide JS399-19 were selected. Three sensitivity levels were identified:sensitive (S), moderately resistant (MR) and highly resistant (HR) to JS399-19 based on their sensitivity to JS399-19. All the conidia and ascospore progeny of three representative isolates (2043, Y2021B and YNT, representing S, MR and HR) exhibited unchanged resistance level to JS399-19, indicating resistance stability in asexual and self-crossed reproduction. Eight isolates representing three sensitivity level phenotypes were randomly selected for a study on the inheritance of JS399-19 resistance by analyzing the sensitivity of hybrid F1 progeny. The nitrate nonutilizing mutant (nit) was used as a genetic marker to confirm that individual perithecia were the result of out-crossing. Six crosses were assessed:S×S, S×HR, MR×HR, HR×HR, and MR×S. In crosses between the parents with different sensitivity levels, such as S×HR, MR×HR and MR×S, the progeny fit a 1:1 segregation ratio of the two parental phenotypes. No segregation was observed in the crosses of S×S and HR×HR. We concluded that the MR and HR phenotypes in F. graminearum were presumably conferred by different allelic mutations within the same locus. In these isolates, resistance to JS399-19 was not affected by modifying genes or cytoplasmic components.In order to study sexual recombination of F. graminearum, six selected isolates were adopted as parents and crossed in three designed pairs under laboratory conditions and under field conditions, respectively, by using nitrate non-utilizing (nit) mutants and carbendazim-resistance as genetic markers. Three sexual recombinants from each of the three combinations were randomly selected to compare the major biological properties with their parental isolates. The results showed that the nit gene and carbendazim-resistance gene could be recombined by sexual crosses. There were no significant differences in mycelial growth, traits of culture and pathogenicity between the sexual recombinants and their parental isolates. Sporulation and perithecigerous capacities, however, changed more or less in some sexual recombinants. The fitness of the sexual recombinants was comparable to the parents in general. Over 100 putative self-crossing or outcrossing perithecia for each cross were randomly sampled on the surface of the haulms of dead rice for each pair of the two parents and the results showed that about 5.7-20.9% outcrossing frequency occurred in the three crosses and confirmed sexual recombination under field conditions. Therefore, it is concluded that sexual recombination may play an important role in carbendazim resistance development and genetic evolution of F. graminearum populations.To evaluate the potential risk of resistance development in MBC-resistant F. graminearum isolates to the new fungicide JS399-19, five isolates (MBC-resistant or-sensitive) which were classified into three different sensitivity phenotypes, such as sensitive (S), moderately resistant (MR), and highly resistant (HR) to MBC, were selected to induce JS399-19-resistant mutants by selection for resistance on potato sucrose agar (PSA) plates amended with 10μg/mL JS399-19. Totally, twenty-four JS399-19-resistant mutants were obtained by selection for resistance to the new fungicide from the selected MBC-resistant or -sensitive isolates. All of the resistant mutants maintained their resistance to JS399-19 and/or MBC through 8 transfers on PSA plates for 40 days and when stored on PSA slants at 4℃for 60 days. It was hypothesized that MBC resistance and JS399-19 resistance could not be exchanged by mycelial fusion or there is a small chance to be exchanged between two compatible isolates, which might delay the development of JS399-19 resistance in field MBC-resistant F. graminearum isolates. The mycelial growth and conidial production capacity were decreased in some resistant mutants, indicating that a fitness cost was associated with JS399-19 resistant phenotypes of F. graminearum isolates. However, most of the mutants resistant to both MBC and JS399-19, exhibited high sexual reproduction capacity and pathogenicity as their parental isolates. Nevertheless, most of these mutants possessed fitness levels comparable to their parents. The results on the efficacy of the two fungicides for controlling FHB incited by the fungicide-resistant mutants were generally in consistence with that of in vitro sensitivity tests. JS399-19 was effective in controlling FHB caused by MBC-resistant isolates under field conditions, while it was not effective in controlling FHB caused by the JS399-19 resistant isolates and the special isolates resistant to both MBC and JS399-19. Moreover, the efficacy of MBC+JS399-19 was also significantly lower when controlling FHB caused by the special isolates resistant to both MBC and JS399-19 than when controlling disease caused by the sensitive isolates, the MBC-resistant isolates or JS399-19-resistant isolates. All these results indicated that JS399-19 possessed a high risk in the development of resistance in MBC-resistant and -sensitive F. graminearum isolates, and double resistance to the two fungicides could presumable emerge and become a practical problem when both of the fungicides were extensively used. Appropriate precautions against JS399-19 resistance development in natural populations should be taken into account to avoid unexpected control failures and to sustain the usefulness of MBC and the new product JS399-19. |
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