Qin Peng,Xinchang Hao,Chunyue Liu,Xiuhuan Li,Xingxing Lu,Xili Liu
Florylpicoxamid, a QiI fungicide, demonstrates broad-spectrum activity against a wide range of phytopathogenic organisms belonging to the phyla Basidiomycota and Ascomycota. Nevertheless, the potential for resistance and the underlying resistance mechanisms of Corynespora cassiicola against florylpicoxamid are still not fully understood. We determined the baseline sensitivity levels of 101 C. cassiicola isolates to florylpicoxamid. The EC 50 values varied from 0.01 to 1.18 μg/mL with an average of 0.50 μg/mL. Laboratory-induced fungicide adaptation of nine wild-type isolates generated seven C. cassiicola mutants exhibiting high level of resistance to florylpicoxamid, all originating from a single parental isolate. The mutants maintained their resistance even after undergoing ten successive cultivations on a medium devoid of fungicides. No cross-resistance was detected between florylpicoxamid and pyraclostrobin, fluopyram, prochloraz, or propineb. Five of the resistant mutants showed an improved compound fitness index (CFI) relative to their parental isolate, whereas the remainder displayed either a reduced or comparable CFI. All seven of the resistant mutants displayed an A37V substitution within the CcCytb protein, which was responsible for the resistance to florylpicoxamid, as validated through molecular docking analysis. Furthermore, an allele-specific PCR (AS-PCR) method for detecting the CcCytb A37V mutation was successfully established. In summarize, the findings of this study indicate a moderate risk of C. cassiicola developing resistance to florylpicoxamid, with the A37V substitution in CcCytb playing a key role in this resistance, detectable through the use of specific AS-PCR primers.
Florylpicoxamid (FLCD) is a novel broad-spectrum fungicide with considerable application potential. However, its residues in food may pose health risks, and a rapid detection method is currently lacking. Although immunoassays are well-established for detecting small-molecule contaminants, the development of corresponding antibodies for FLCD has been hampered by the complex and cyanide-dependent synthesis of its traditional haptens. To overcome this limitation, this study introduces a sub-similarity principle for rational hapten design. Guided by computational chemistry, this approach involves designing haptens that retain the feature structure essential for eliciting specific immune responses, while substituting the distal molecular regions with simpler, lower-cost, and more synthetically accessible analogues. Subsequently, three FLCD haptens featuring distinct spacer arms were designed, and the first monoclonal antibody (mAb) specific to FLCD was successfully developed, with an IC 50 of 0.6 μg/L and negligible cross-reactivities below 0.1 % to other 5 picolinamide pesticides. Investigation of the mAb's recognition mechanism further confirmed the reliability of the sub-similarity principle. Utilizing this mAb, an indirect competitive enzyme-linked immunosorbent assay was developed for the rapid screening of FLCD residues in tomatoes, grapes, milk, and chicken, achieving limits of detection (LODs) ranging from 0.52 to 1.46 μg/L. Furthermore, a lateral flow immunoassay was constructed for on-site detection, yielding visual LODs of 1.0–3.0 μg/L and cut-off values of 1.2–4.0 μg/L. This study not only presents a novel strategy for generating antibodies against small molecules, but also addresses a critical gap in the rapid detection of FLCD.
危险品化学品经营许可证（带存储）