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.

Fusarium wilt of banana (Musa spp.), a typical vascular wilt disease caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense race 4 (Foc4), seriously threatens banana production worldwide. Pathogens, including vascular wilt fungi, secrete small cysteine-rich proteins during colonization. Some of these proteins are required for pathogenicity. In this study, 106 small secretory proteins that contain a classic N-terminal signal peptide were identified using bioinformatic methods in Foc4. Among them, 11 proteins were selected to show transient expressions in tobacco. Interestingly, transient expression of FoSsp1 in tobacco, an uncharacterized protein (of 145 aa), induced necrotic cell death reactive oxygen burst, and callous deposition. Furthermore, the expression of FoSSP1 in Foc4 wild type (WT) was up-regulated during the stage of banana roots colonization. A split-marker approach was used to knock out FoSSP1 in the Foc4 WT strain. Compared with the WT, the deletion mutant Fossp1 was normal in growth rate but increased in conidiation and virulence. RT-qPCR analysis showed that the expression of four conidiation regulator genes in the Fossp1 deletion mutant was significantly decreased compared to the WT strain. In addition, the expression of four pathogenesis-related genes of bananas infected with Fossp1 deletion mutant was down-regulated in comparison with that of the WT. In summary, these results suggested that FoSSP1 is a putative elicitor that negatively regulates conidiation and pathogenicity in Foc4.

Powdery mildew severely affects several important crops and cash plants. Disruption of mildew resistance locus O ( MLO) genes elevates resistance against powdery mildew in several plants. However, whether rubber tree ( Heveae brasiliensis) MLO proteins are linked to susceptibility remains unknown, owing to technical limitations in the genetic manipulation of this woody plant. A previous study showed that the H. brasiliensis MLO-like protein HbMLO12 demonstrates high amino acid sequence similarity with the known Arabidopsis MLO protein AtMLO12. In this study, we investigated whether HbMLO12 regulates susceptibility to powdery mildew. H. brasiliensis leaves take up exogenously synthesized double-stranded RNAs (dsRNAs), and foliar application of dsRNA homologous to HbMLO12 gene specifically induces HbMLO12 silencing in H. brasiliensis leaf tissues. Notably, HbMLO12 silencing inhibited fungal infection and elevated the immune response during interaction with the rubber tree powdery mildew fungus. Furthermore, the heterologous expression of HbMLO12 suppressed bacterial flg22– and fungal chitin–induced immune responses and enhanced bacterial infection in Arabidopsis. Our study provides evidence that HbMLO12 contributes to susceptibility to powdery mildew. [ ] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus from P. striiformis strain GS-1, which was designated as “Puccinia striiformis mitovirus 2” (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 multiplied highly during Pst infection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 in Pst by barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity of Pst. These results indicate PsMV2 promotes host pathogenicity in Pst. Interestingly, PsMV2 was detected among a wide range of field isolates of Pst and may have coevolved with Pst in earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution in Pst which may offer new strategies for disease control.

Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt of banana (Musa spp.), a notorious soil-borne vascular fungal disease threatening the global banana industry. Phytopathogens secrete effectors to suppress plant immunity. However, little is known about the effectors of Foc race 4 (Foc4). In this study, we built a streamlined screening system (candidate effector prediction, RNA-seq-based expression level analysis, and cell death manipulative activity assessment based on transient expression in Nicotiana benthamiana) to identify candidate virulence-related effectors. In total, 80 candidate effector genes (CEGs) differentially expressed during plant colonization were predicted; 12 out of 15 characterized CEGs, including FoSSP17, could suppress BAX-triggered programmed cell death (PCD) in N. benthamiana and were induced during the infection of plants. FoSSP17 encodes a novel protein conserved in the Fusarium genus. FoSSP17 gene deletion mutants were not affected in vegetative growth and conidiation but showed reduced virulence. Furthermore, the deletion mutants triggered higher expression levels of host defense-related genes including PR1, PR3, PR5, and PR10. Signal peptide activity assay and subcellular localization assay suggested that FoSSP17 is a conventional secretory protein that exerts cell-death-suppressive activity inside plant cells. In addition, FoSSP17 suppressed pattern-triggered immunity in plants by inhibiting reactive oxygen species (ROS) accumulation, reducing callose deposition, and suppressing the expression of NbLOX and NbERF1 genes related to jasmonic acid (JA)-pathway and ethylene (ET)-pathway, respectively. Overall, a systemic screening of Foc4 candidate effectors reveals that FoSSP17 contributes to the virulence of Foc4 and suppresses pattern-triggered immunity in plants.