Frogeye leaf spot (FLS), caused by Cercospora sojina, is a significant disease affecting soybean crops economically. Seed inspection plays a vital role in reducing the incidence of this disease. However, limited research has been conducted on the fast and accurate identification of C. sojina in soybean seeds. In this study, we developed a rapid diagnostic method to detect C. sojina in soybean seeds using loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was utilized to identify specific sequences belonging to C. sojina. The LAMP primer set designed based on the selected specific sequence proved to be highly specific, distinguishing C. sojina from other common soybean pathogens and two maize pathogens of the Cercospora genus. The primer set exhibited remarkable sensitivity, detecting sixteen copies per reaction, which is approximately 1,000 times more sensitive than conventional PCR. The LAMP assays were applied for direct inspection of soybean seeds, revealing that twenty of the thirty tested soybean varieties were contaminated with C. sojina, with contamination rates ranging from 10% to 60%. The seeds of TongNong 943 and JiNong 38 had the highest contamination rate at 60%. Seeds coated with fungicide showed a reduced disease incidence, as demonstrated on PDA medium and in soil pots. In summary, our results indicate that rapid seed diagnosis using LAMP assays can effectively screen out seeds carrying C. sojina, providing a preventive strategy in managing FLS disease.

Northern corn leaf blight (NCLB), caused by Exserohilum turcicum, is one of the most devastating foliar diseases of maize. Rapid and accurate diagnosis for this disease is urgently needed but still limited. Here, we establish a field-deployable diagnostic method to detect E. turcicum based on loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was used to search for the specific sequences belonging to E. turcicum by comparing the whole genome sequence between E. turcicum and other known maize pathogens. Five LAMP primer sets were designed based on specific and single-copy fragments of E. turcicum. Post-LAMP analyses indicated that only the primer set, Et9468_set1, was the most suitable, producing a ladder-like amplification pattern in the agarose gel electrophoresis and a strong fluorescence signal in the presence of SYBR Green I. The LAMP assay using Et9468_set1 primers demonstrated a high level of specificity in distinguishing E. turcicum from six other common fungal pathogens of maize, as well as 12 more fungal and oomycete strains including the epiphytic fungi from maize leaves and other crop pathogens. Moreover, it exhibited remarkable sensitivity by detecting five copies per reaction, which was approximately 10⁴ times more sensitive compared with conventional PCR. The LAMP assay successfully detected E. turcicum in field maize leaves without DNA extraction, demonstrating its suitability for rapid on-spot detection of NCLB. Our study provides a direct LAMP diagnostic method to detect E. turcicum, which enables on-site pathogen detection in the field and the development of preventive strategies for NCLB management.

Gray leaf spot (GLS) is a major foliar disease of maize caused mainly by Cercospora zeae-maydis and Cercospora zeina. Although both species infect maize, the comparative transcriptional programs between isolates of different species during maize colonization remain largely unknown. We conducted time-course pathogen-focused RNA-seq of one Jilin isolate assigned to each species during colonization of maize B73 under a controlled inoculation system. RNA-seq reads were mapped to the public C. zeae-maydis SCOH1-5 and C. zeina CMW25467 v3 reference genomes for transcript quantification and putative functional annotation. The C. zeae-maydis isolate developed visible symptoms earlier and showed greater pathogen-derived transcript representation at 6 dpi. The C. zeae-maydis isolate expressed slightly more reference-annotated cell wall-degrading enzyme (CWDE)-associated loci than the C. zeina isolate, whereas the numbers of expressed candidate effector-associated loci were similar. The C. zeae-maydis isolate also showed earlier or stronger host-associated induction of several CWDE- and candidate effector-encoding genes. Both isolates showed post-inoculation induction of candidate transcripts related to lipid metabolism, protease-related functions, and cercosporin biosynthesis, while a subset differed between isolates in the timing or magnitude of expression. RT-qPCR broadly supported the temporal trends of selected candidates. Together, these results define shared and contrasting transcriptional programs of two isolates of different Cercospora species during maize colonization.