CRISPR/Cas12a Combined with RAA for On-Site Detection of ALS W574L Mutation in Three <i>Alopecurus</i> Species: A Visual Approach for Herbicide Resistance Monitoring

The genus <i>Alopecurus</i> encompasses several weed species, including <i>Alopecurus japonicus</i>, <i>Alopecurus aequalis</i>, and <i>Alopecurus myosuroides</i>, which represent significant threats to agricultural productivity, particularly in wheat and oilseed rape fields. ALS-inhibiting herbicides have been extensively used for controlling <i>Alopecurus</i> weeds. However, the widespread use of these herbicides has led to the rapid emergence of resistance in <i>Alopecurus</i> populations with the Trp-574-Leu (W574L) mutation in the ALS gene being one of the most common resistance mechanisms. This study aims to develop a novel molecular detection method combining recombinase-aided amplification (RAA) with CRISPR/Cas12a technology to detect the W574L mutation in <i>Alopecurus</i> species. The method was optimized for key parameters, balancing efficiency with experimental costs, and was evaluated for specificity, sensitivity, and field applicability. This approach offers a rapid, accurate, and visual tool for identifying W574L target-site resistance in <i>A. japonicus</i>, <i>A. aequalis</i>, and <i>A. myosuroides</i>, with significant potential for monitoring resistance and enhancing weed management strategies.