Xanthomonas campestris pv. campestris (Xcc), causes the disease known as black rot in brassicas. The disease is found globally and is one of the most serious bacterial diseases affecting brassica crops. The disease is spread through infected seeds and can be dispersed by water between plants. Although copper-based treatments can prevent disease, there is no cure for infected plants. The use of disease-free seed and early eradication of sick plants is the best method to suppress impacts. This, however, requires rapid and accurate diagnostics.
Several molecular diagnostic methods are available, however, current PCR based assays do not have the sensitivity or specificity required for diagnostic use. While LAMP assays are more sensitive, they are complex to design. A CRISPR-Cas diagnostic assay could be the solution, as they are more sensitive than PCR and less complex compared to LAMP assays. CRISPR-Cas based assays have been used to detect plant pathogenic bacteria, viruses and insect pests. Various CRISPR-Cas based assays are available for pests in brassicas, however, to this date no assay is available for Xcc.
This study aims to develop a CRISPR-Cas based assay for diagnosing Xcc in Brassica and compare the sensitivity and specificity against existing diagnostic methods. An assay was developed, targeting a gene region unique to Xcc, which is amplified using an RPA reaction. This was then followed by CRISPR-Cas12a based cleavage of the amplicon that enables fluorescence or lateral flow read out. Initial testing shows the assay works. Further testing will reveal its suitability as a diagnostic method and its potential as an in-field test.