Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/CRISPR-Cas12a method for potential on-site disease diagnosis.

BACKGROUND: Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora. RESULTS: The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12 days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60 min. CONCLUSION: The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic beads or rapid DNA extraction, isothermal nucleic acid amplification at 39 °C, CRISPR-Cas12a cleavage reaction at 37 °C, and lateral flow assay or endpoint fluorescence visualization at room temperature. The RPA and CRISPR-Cas12a reagents can be preloaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR-Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, and has promising applications for rapid on-site disease screening, port inspection, or controlling fungal pathogen transmission in crop. © 2023 Society of Chemical Industry.

Nanopore/Illumina Hybrid Whole-Genome Sequence Resource of Plenodomus lindquistii Strain US01 Infecting Sunflower.

Plenodomus lindquistii (basionym: Leptosphaeria lindquistii) is the most prevalent fungus pathogen causing the black stem disease in sunflower. To date, only one whole-genome sequence is reported in NCBI, but no complete gene annotation has been submitted. Herein, we sequenced a Leptosphaeria lindquistii isolate from sunflower seeds imported from the United States using Oxford Nanopore long-read sequencing and Illumina short-read sequencing. We generated a 38.6-Mb genome assembly consisting of 29 contigs with an N50 of 1.689 Mb and a maximum length of 5.316 Mb. In the genome assembly, we identified 13,481 protein-coding genes, of which 12,531 were annotated. The high-quality assembly and annotation of the Leptosphaeria lindquistii genome will allow us to conduct further studies for the specific identification of pathogens causing sunflower black stem disease and provide a better understanding of the infection mechanisms of Leptosphaeria lindquistii.

A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops.

Establishing a portable diagnostic method for identifying plant pathogens is essential to prevent the spread of plant disease, especially in field and customs inspections. Leptosphaeria maculans (L. maculans) is an aggressive fungus, which causes severe phoma stem canker of Brassica napus, responsible for major yield losses of oilseed rape worldwide. In this study, CRISPR/Cas12a-based detection system and recombinase polymerase amplification (RPA) technique were employed to develop a rapid and sensitive detection method for identifying L. maculans. The involved RPA pre-amplification and CRISPR/Cas12a cleavage confer considerable sensitivity and selectivity, which can be finished within 45 min with a LOD of 4.7 genomic DNA copies. This detection system was further developed to two portable platforms, i.e., one-pot lateral flow detection and all-in-one chip lateral flow assay (AOCLFA), which integrates the lyophilized recombinase polymerase amplification (RPA) reagents and lyophilized Cas12a cleavage reagents in one tube or chip. The developed portable platforms have flexible portability and simple operation for the detection of L. maculans from plant tissues in the field. The proposed portable suitcase containing the minimum equipment, regents, and AOCLFA meets the practical needs of rapid on-site disease screening of plant fungi, port quarantine, or pathogen spreading control.

RPA/CRISPR/Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment.

Toxoplasma gondii is an opportunistic pathogen widely distributed within the world, poses a huge threat to human health, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection of T. gondii by combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. A glass microfiber filter device used for the first step can efficiently extract T. gondii from low-concentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and both reactions can be performed on a low-cost thermal controller (∼37 °C), avoiding the drawbacks of the step-by-step addition of components. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon of T. gondii over other parasites with a limit of detection of 3.3 copies/µL. The visual signal readout can be easily realized by a fluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas.

Rapid detection of the pathogenic fungi causing blackleg of Brassica napus using a portable real-time fluorescence detector.

The fungus Leptosphaeria maculans leading to Phoma stem canker (blackleg) of Brassica napus (oilseed rape, canola) produces the phytotoxin sirodesmin PL, which is responsible for major yield losses of oilseed rape worldwide. Polymerase chain reaction (PCR) remains the gold standard diagnostic tool for L. maculans, but the required expensive equipment and long time make it inappropriate for fast field test. Herein, a portable system for rapid assaying L. maculans and L. biglobosa is designed around recombinase polymerase amplification (RPA) with fluorescent probe as the signal indicator, which allowed the real-time assay of amplification performed on a portable device between 37 and 42 °C. The time needed to observe the positive reaction results is controlled within 30 min. The proposed assay system is a good choice for on-site disease screening of oilseed rape plant where rapid detection is valuable, including port quarantine, agriculture quality testing, and pathogen spreading control.