Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/μL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method’s user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/μL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.

Colletotrichum spp. are the causative agents of anthracnose of rubber trees, one of the most destructive diseases, resulting in substantial economic losses. To investigate the evolutionary characteristics of these pathogenic species, we first assembled the complete mitogenomes of four dominant pathogens, i.e., C. siamense, C. fructicola, C. wanningense and C. bannaense. Comparative analyses revealed that variations in their mitogenome size were primarily driven by intron expansion and expansion/contraction within the cox1, cob and nad genes. Moreover, we observed the strong conservation of gene content, mitochondrial DNA copy number, gene order and intron features within species complexes, but a clear divergence between them. Notably, further studies indicated that patterns such as genomic organization, selective pressures and codon usage were consistent across the genus, suggesting that Colletotrichum species complexes had followed distinct evolutionary trajectories, particularly in the arrangement of protein-coding genes. Therefore, this study systematically characterized the mitogenomes of the four major Colletotrichum species associated with rubber tree anthracnose and provided novel insights into the broad evolutionary mechanisms shaping Colletotrichum species complexes.

Powdery mildew caused by Erysiphe quercicola is a serious threat to natural rubber (Hevea brasiliensis) production, but the molecular basis of host immunity against this pathogen remains poorly understood. Here, we identify EqBPIE1 (E. quercicola Broad-spectrum Plant Immunity Elicitor 1), a fungal effector expressed during the mid-to-late colonization stage, that localizes to the cytosol, chloroplasts, and nucleus of host cells. EqBPIE1 enhances chloroplast-derived reactive oxygen species production, promotes perinuclear chloroplast clustering and stromule (stroma-filled tubule) formation, and thereby facilitates the relay of immune signals to the nucleus. In the nucleus, EqBPIE1 interacts with the NLR protein HbRG1 (H. brasiliensis Resistance Gene 1) to trigger hypersensitive cell death. Silencing EqBPIE1 increased susceptibility of H. brasiliensis to E. quercicola, whereas heterologous expression and foliar application conferred broad-spectrum resistance against fungal, bacterial, and oomycete pathogens in multiple plant species. Our findings establish an effector-mediated chloroplast-to-nucleus immune signaling pathway and highlight EqBPIE1 as a promising molecular tool for engineering durable, broad-spectrum disease resistance across crops.

Hevea brasiliensis (H. brasiliensis) is the principal source of natural rubber, but its productivity is severely threatened by powdery mildew caused by the biotrophic fungus Erysiphe quercicola (E. quercicola). Although circular RNAs (circRNAs) are emerging as key regulators in plant stress responses, their functions in H. brasiliensis immunity remain largely unexplored. Here, we aimed to systematically characterize circRNAs involved in the early immune response of H. brasiliensis to E. quercicola. Transcriptome sequencing and bioinformatic analyses identified 52 and 177 differentially expressed circRNAs (HbcircRNAs) at 1 and 3 days post-inoculation, respectively. Twelve HbcircRNAs with significant expression changes were validated, and nine were confirmed as true circRNAs. Functional assays using spray-induced gene silencing (SIGS) in H. brasiliensis leaves and heterologous overexpression in the Arabidopsis thaliana eds1 mutant, which is susceptible to E. quercicola, revealed that HbcircARF3, HbcircSCSA1, and HbcircARFGAP8, together with their homologous linear counterparts (HbLinearRNAs), exert distinct regulatory effects on disease resistance. Silencing of these HbcircRNAs enhanced host immunity, whereas overexpression increased susceptibility. Pathway analyses suggested their involvement in auxin signaling, mitochondrial energy metabolism and vesicle trafficking. Collectively, our findings uncover the differential regulatory roles of circular and linear RNAs in the H. brasiliensis–E. quercicola interaction, providing mechanistic insights and potential molecular targets for breeding disease-resistant H. brasiliensis.