Sunflowers are much more than bright symbols of summer. They play a crucial role in global agriculture as the world’s fourth-largest oil crop. These plants not only provide cooking oil but also serve as a source of livestock feed, medicinal compounds, and ornamental beauty.
However, their significance is threatened by fungal pathogens such as Diaporthe helianthi. This pathogen causes Phomopsis stem canker, a disease responsible for devastating yield losses.
In a groundbreaking study published in Phytopathology Research (2025), researchers introduced a rapid, portable, and ultrasensitive diagnostic tool that combines Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a.
The Impact of Diaporthe helianthi
Diaporthe helianthi is a fungal pathogen that attacks sunflower stems, leaves, and seeds. First identified in Yugoslavia in 1980, the fungus has spread to regions including France, the United States, Italy, and Australia.
It causes gray-brown lesions at the stem-petiole junction, which soon encircle the entire stem. This damage leads to wilting, necrosis, and in severe cases, crop losses that can reach up to 40%, as was observed in France in 1984.
The spread is further exacerbated by infected seeds that act as carriers, enabling the fungus to cross international borders. In many countries, including China, D. helianthi is classified as a regulated quarantine pest—a term that refers to organisms absent or limited in a region but capable of causing significant harm if introduced.
Traditional Detection Methods and Their Limitations
Historically, detecting Diaporthe helianthi has involved methods such as culturing fungi, morphological analysis, and various PCR techniques. Each of these traditional approaches has significant drawbacks.
For example, real-time PCR requires expensive thermocyclers and several hours to deliver results. Morphological identification, on the other hand, depends on the expertise of skilled technicians who must distinguish fungal structures under a microscope.
Although multiplex PCR assays—using techniques like Dual Priming Oligonucleotides—improve specificity, they still depend on well-equipped laboratories. This reliance on laboratory infrastructure not only delays diagnosis but also increases the cost and complexity of disease detection.
The Breakthrough: RPA/CRISPR-Cas12a System
To overcome these challenges, the research team developed a diagnostic tool that merges Recombinase Polymerase Amplification (RPA) with CRISPR-Cas12a technology. The breakthrough lies in combining these two powerful techniques into a single system that offers speed, sensitivity, and portability.
The tool is designed to provide rapid and accurate results without the need for bulky and expensive laboratory equipment.
The integration of RPA and CRISPR-Cas12a ensures that even minute amounts of fungal DNA can be detected, making it possible to diagnose infections early and efficiently.
The simplicity and portability of this system mean that it can be used on-site in the field, offering a practical solution for farmers and agricultural inspectors alike.
How the System Works: RPA and CRISPR-Cas12a Explained
Targeting the Calmodulin (Cal) Gene
The first step in the system’s process involves selecting an appropriate target gene. The team chose the calmodulin gene (or Cal gene) from D. helianthi. Out of three potential targets—ITS, Cal, and EF1-α—the Cal gene proved most sensitive due to its high copy number and conserved sequence.
Specific primers and CRISPR RNA (crRNA) molecules were designed to bind accurately to this gene, ensuring that the diagnostic system could reliably detect the pathogen. This initial step is critical because the accuracy of any diagnostic method hinges on the specificity of the chosen target.
Recombinase Polymerase Amplification (RPA)
Once the target gene is selected, the system employs Recombinase Polymerase Amplification (RPA) to amplify the DNA. Unlike traditional PCR, which requires a series of temperature changes, RPA is an isothermal process that works at a constant 37°C.
In this process, short DNA sequences called primers attach to the target Cal gene. Recombinase enzymes help unwind the DNA to allow the primers to bind effectively, while a polymerase enzyme extends these primers to replicate the DNA.
This entire reaction takes only 25 minutes, yet it is powerful enough to amplify even trace amounts of fungal DNA. The rapid nature of RPA is particularly valuable in field settings where time and resources are limited.
CRISPR-Cas12a Detection
After the DNA is amplified, the system uses CRISPR-Cas12a for detection. CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, was originally discovered as a bacterial defense mechanism. In this diagnostic tool, CRISPR is repurposed to identify the amplified DNA.
The Cas12a enzyme, guided by crRNA, binds specifically to the amplified Cal gene. Upon binding, Cas12a exhibits collateral cleavage activity, meaning it starts cutting nearby single-stranded DNA (ssDNA) reporters. These reporters are tagged with markers such as fluorescent dyes (FAM) or biotin.
Two methods of detection are employed: one based on fluorescence, where the cleavage of FAM-BHQ1 reporters results in a visible signal, and another based on lateral flow strips, where cleaved FAM-biotin reporters produce clear lines on a test strip.
Both methods offer fast and unambiguous results, making the diagnostic process both efficient and user-friendly.
Key Findings: Sensitivity, Specificity, and Speed
One of the most remarkable features of the RPA/CRISPR-Cas12a system is its sensitivity.
In fluorescence detection mode the system is capable of detecting as little as 0.1 pg/μL of D. helianthi DNA, which is roughly equivalent to 1.4 DNA copies per microliter.
In lateral flow detection mode, the threshold is slightly higher at 1 pg/μL, or 14 copies per microliter. This level of sensitivity is nearly 100 times better than that of conventional real-time PCR, which typically requires around 10 pg/μL (or 140 copies per microliter) for detection.
Equally impressive is the system’s specificity. The diagnostic tool was rigorously tested against eight related pathogens, including species such as Leptosphaeria lindquistii, Sclerotinia sclerotiorum, and Verticillium dahliae.
In all cases, the tool was able to distinguish D. helianthi with high precision, showing no cross-reactivity even when tested with DNA from healthy sunflower leaves. This high specificity ensures that false positives are minimized, providing farmers and inspectors with reliable results that are critical for effective disease management.
Speed is another significant advantage of this new diagnostic system. From the start of the RPA reaction to the final CRISPR-Cas12a detection step, the entire process takes only 45 minutes. This rapid turnaround is a game changer for field diagnostics.
Instead of waiting several hours or even days for lab-based tests, users can obtain accurate results in less than an hour. The combination of high sensitivity, excellent specificity, and fast results makes the RPA/CRISPR-Cas12a system a truly revolutionary tool for detecting sunflower diseases.
Real-World Validation and Early Detection
The research team did not limit their study to controlled laboratory conditions; they also tested the system on actual sunflower tissues to simulate real-world conditions. In experiments designed to mimic natural infections, healthy sunflower leaves, stems, and seed coats were artificially spiked with D. helianthi mycelia.
The diagnostic tool successfully detected the fungus in all tissue types, although the results were clearest in the sunflower stems.
For instance, the fluorescence intensity observed in stems reached 84% of that seen with pure fungal DNA, demonstrating the system’s robustness even in complex biological samples.
In another set of experiments, sunflower stems were inoculated with D. helianthi spores and monitored over a period of 15 days. Remarkably, the system was able to detect the pathogen as early as five days post-inoculation using fluorescence detection.
In comparison, traditional methods such as lateral flow strips and real-time PCR only yielded positive results at 10 days post-inoculation. By the 15th day, all methods confirmed the presence of the fungus, but the rapid response provided by the RPA/CRISPR-Cas12a system allowed for early intervention.
This early detection is critical because it enables farmers to quickly identify and remove infected plants, thereby preventing the spread of the disease and minimizing crop losses.
Technical Optimization and System Fine-Tuning
A major part of the study involved refining the diagnostic system to ensure it was both fast and accurate. The research team experimented with various durations for the RPA reaction, testing times ranging from 10 to 40 minutes.
They found that a 25-minute reaction time offered the best balance between speed and yield. In other words, this duration allowed for sufficient DNA amplification without delaying the overall process.
The team also experimented with the concentration of reporters used in the CRISPR-Cas12a detection step. For the fluorescence-based detection method, a concentration of 0.2 µmol/L of FAM-BHQ1 reporters was determined to provide the best signal clarity while also being cost-effective.
Similarly, for the lateral flow detection method, a 0.2 µmol/L concentration of FAM-biotin reporters was optimal for generating clear and easily interpretable test lines on the strips. Additionally, the CRISPR-Cas12a incubation time was optimized at 20 minutes.
Shorter incubation periods, such as 5 to 10 minutes, sometimes resulted in faint lines that could lead to false-negative results. This careful fine-tuning of the system demonstrates the thorough approach taken by the researchers to ensure that the final diagnostic tool was both reliable and efficient.
Implications for Agriculture and Trade
The breakthrough achieved with the RPA/CRISPR-Cas12a system has significant implications for agriculture and international trade. One of the most immediate benefits is its potential use in quarantine inspections.
Ports and border control agencies can now quickly screen imported sunflower seeds for the presence of D. helianthi. This rapid screening helps prevent the cross-border spread of the pathogen, protecting local agriculture and maintaining the integrity of international trade.
In the field, the system offers an invaluable tool for farmers. Regular monitoring of crops becomes feasible, enabling the early detection of infections. Early diagnosis is essential because it allows for targeted treatment interventions that can save entire fields from devastation.
Moreover, the cost-effectiveness of this system is a major advantage. Traditional laboratory tests can be expensive and time-consuming, sometimes costing hundreds of dollars per test. By contrast, the RPA/CRISPR-Cas12a system reduces the cost of detection to just tens of dollars per test.
This affordability makes advanced diagnostic technology accessible to a wider range of users, including small-scale farmers who may not have had the resources to perform such tests in the past.Furthermore, the benefits of this system extend beyond sunflower production.
The same principles can be applied to the detection of other plant pathogens. For instance, similar techniques could be developed to identify bacterial diseases such as those caused by Xanthomonas or viral infections like SARS-CoV-2 and Zika virus.
The flexibility of the RPA/CRISPR-Cas12a framework means that it has the potential to revolutionize pathogen detection across a broad spectrum of agricultural and even human health applications.
Future Applications and Scalability
Looking ahead, the adaptability of the RPA/CRISPR-Cas12a system paves the way for its use in a variety of diagnostic applications. Researchers believe that the framework can be modified to detect multiple pathogens by simply altering the primers and crRNA sequences.
This means that the technology could be expanded to cover a wide range of crop diseases, providing a universal platform for rapid diagnostics in agriculture.In addition to its agricultural applications, the technology holds promise for public health diagnostics.
The ability to quickly and accurately detect pathogens is crucial during outbreaks of diseases such as COVID-19 or Zika. In fact, similar systems have already been successfully used for the rapid detection of viruses, highlighting the potential for cross-sector benefits.
As the technology matures, it is likely that commercial kits will be developed, making the system even more accessible to end-users. These kits will allow for on-site testing without the need for extensive laboratory infrastructure, further democratizing access to advanced diagnostic tools.
The scalability of this technology is also an important consideration. As the global population grows and climate change alters agricultural landscapes, the need for rapid, reliable disease detection becomes ever more critical.
By providing a fast, cost-effective, and accurate method for diagnosing plant diseases, the RPA/CRISPR-Cas12a system could play a key role in ensuring food security.
The technology’s ability to detect pathogens at very low concentrations means that infections can be identified before they have a chance to spread widely, thereby minimizing crop losses and maintaining stable food supplies.
Conclusion
In conclusion, the RPA/CRISPR-Cas12a diagnostic system represents a major step forward in the field of plant disease management. By combining the rapid DNA amplification capabilities of RPA with the precise detection power of CRISPR-Cas12a, researchers have developed a tool that is both highly sensitive and exceptionally specific.
The system’s ability to detect as little as 0.1 pg/μL of D. helianthi DNA sets it apart from traditional methods, which require much higher concentrations of DNA for reliable detection. Moreover, the entire process is completed in just 45 minutes, offering an unprecedented speed that is essential for on-site applications in agriculture.
As we face the twin challenges of climate change and a rapidly expanding global population, innovations like the RPA/CRISPR-Cas12a system will become increasingly important. By enabling early detection of pathogens, this technology helps safeguard crops, stabilize food production, and ultimately contribute to global food security.
Power Terms
1. Diaporthe helianthi:
A fungal pathogen that causes “phomopsis stem canker,” a destructive disease in sunflowers. It infects stems, leaves, and seeds, leading to wilting, stem rot, and crop losses. Early detection is critical to prevent outbreaks in fields and during international seed trade. For example, this fungus caused a 40% yield loss in France in the 1980s.
2. Recombinase Polymerase Amplification (RPA):
A DNA amplification method that works at a constant temperature (37–42°C) without needing complex lab equipment. Unlike traditional PCR, RPA uses enzymes to copy DNA quickly (25–40 minutes). It’s vital for rapid, on-site pathogen detection, such as identifying D. helianthi in sunflower stems.
3. CRISPR-Cas12a:
A gene-editing tool repurposed here for diagnostics. Cas12a is an enzyme that cuts DNA when guided by a CRISPR RNA (crRNA). Once activated, it also indiscriminately shreds single-stranded DNA (ssDNA), producing detectable signals. This “collateral cleavage” helps confirm the presence of pathogens like D. helianthi.
4. Lateral Flow Assay (LFA):
A portable test strip (like a pregnancy test) that shows visual results. In this study, a “test line” appears if D. helianthi DNA is detected. It’s user-friendly for field use, requiring no lab tools. For example, a red or purple line confirms infection.
5. Calmodulin (Cal) Gene:
A gene used as a target to identify D. helianthi. Calmodulin proteins help regulate calcium levels in cells. The Cal gene is chosen because it’s unique enough to distinguish D. helianthi from similar fungi, ensuring accurate diagnosis.
6. Protospacer-Adjacent Motif (PAM):
A short DNA sequence (e.g., TTTN) required for CRISPR-Cas12a to bind and cut. Without PAM, Cas12a cannot recognize the target DNA. It acts like a “lock” that only the right “key” (crRNA) can open, ensuring precision in detecting D. helianthi.
7. Collateral Cleavage Activity:
A unique feature of Cas12a where, after binding to target DNA, it non-specifically cuts nearby ssDNA. This activity releases fluorescent or colored markers (e.g., FAM molecules) to signal pathogen presence. It amplifies detection sensitivity.
8. Isothermal Nucleic Acid Amplification:
DNA copying at a single temperature (no need for heating/cooling cycles). RPA is an example. It’s faster and cheaper than PCR, making it ideal for field use. For instance, farmers can test crops without a lab.
9. Real-Time PCR:
A lab-based DNA detection method that quantifies pathogens using fluorescent dyes. While sensitive, it requires expensive machines and trained technicians. In this study, RPA/CRISPR was 100x more sensitive than real-time PCR.
10. Fluorescence Detection:
A method where light emitted by molecules (e.g., FAM) signals pathogen DNA. When Cas12a cuts FAM-tagged ssDNA, it releases a fluorescent glow visible under blue light. This is used in labs or portable devices for precise results.
11. Nucleic Acid Detection:
Identifying DNA/RNA to diagnose pathogens. Techniques like RPA and CRISPR-Cas12a target unique genetic sequences (e.g., Cal gene) to confirm infections. It’s faster than growing fungi in labs.
12. Pathogen Quarantine:
Isolating infected plants or seeds to stop disease spread. Rapid tests like RPA/CRISPR help inspectors at ports detect D. helianthi in imported sunflower seeds, protecting local crops.
13. Fungal Pathogens:
Disease-causing fungi like D. helianthi. They harm crops, reduce yields, and threaten food security. Early detection tools are critical for managing outbreaks in agriculture.
14. Magnetic Bead DNA Extraction:
A method to purify DNA using tiny magnetic particles that bind genetic material. It’s quick and efficient, allowing farmers or inspectors to prepare samples on-site without labs.
15. Sensitivity (Diagnostics):
The lowest amount of pathogen a test can detect. The RPA/CRISPR system here detects 0.1 pg/μL of DNA (1–14 copies), making it highly sensitive for early infection stages.
16. Specificity (Diagnostics):
A test’s ability to avoid false positives. The RPA/CRISPR system only reacts to D. helianthi DNA, not other fungi like Sclerotinia, ensuring accurate diagnosis.
17. crRNA (CRISPR RNA):
A guide RNA that directs Cas12a to the target DNA sequence (e.g., the Cal gene). It ensures the enzyme cuts only D. helianthi DNA, avoiding mistakes.
18. Single-Stranded DNA (ssDNA):
DNA with one strand, not two. In this study, ssDNA labeled with fluorescent (FAM) or biotin molecules is cut by Cas12a to produce detection signals.
19. Quencher Molecules (e.g., BHQ1):
Chemicals that block fluorescence until Cas12a cuts the DNA. For example, BHQ1 “silences” FAM until cleavage, allowing a fluorescent signal only when D. helianthi is present.
20. Gold Nanoparticles (GNP):
Tiny gold particles used in lateral flow strips. They bind to FAM tags, creating a visible line (test line) when D. helianthi DNA is detected.
21. Streptavidin:
A protein in lateral flow strips that captures biotin-labeled molecules. It creates a “control line” to confirm the test worked, even if no pathogen is found.
22. Acupuncture Inoculation Method:
A technique where sunflower stems are pricked with a needle dipped in D. helianthi spores. It mimics natural infection to study disease progression in labs.
23. Days Post Inoculation (dpi):
Time elapsed after infecting a plant. For example, symptoms in sunflowers worsen from 5 to 15 dpi, helping researchers track how quickly the test detects the fungus.
24. Internal Transcribed Spacer (ITS):
A DNA region used to identify fungi. While common, it’s less specific for closely related species like D. helianthi, so the Cal gene was chosen instead.
25. Translation Elongation Factor-1α (EF1-α):
Another gene tested for detecting D. helianthi. However, the Cal gene showed higher sensitivity in this study, making it a better diagnostic target.
Reference:
Kuang, R., Lei, R., Li, M. et al. Rapid diagnosis of Diaporthe helianthi in sunflower using RPA/CRISPR-Cas12 and lateral flow assay. Phytopathol Res 7, 17 (2025). https://doi.org/10.1186/s42483-024-00310-4
