Chiang Mai Journal of Science

A Rapid and Field-Compatible Detection Platform for the AvrPi9 Gene in Magnaporthe oryzae Using Integrated DNA Extraction, RPA and CRISPR–Cas12a

1. INTRODUCTION

Rice (Oryza sativa L.) is the staple food for more than half of the world's population and is fundamental to global food security. Rice blast, caused by Magnaporthe oryzae, is among the most destructive rice diseases worldwide, causing severe yield losses under favorable environmental conditions [1,2]. In Thailand, the premium aromatic cultivar Khao Dawk Mali 105 (KDML105) is highly valued but remains highly susceptible to rice blast, emphasizing the need for effective disease surveillance and resistance management. The interaction between rice and M. oryzae follows the gene-for-gene model, in which host resistance (R) genes recognize corresponding pathogen avirulence (Avr) genes to trigger effector-triggered immunity (ETI) [3–6]. More than 40 R genes and 14 corresponding Avr genes have been identified in this pathosystem [7,8]. Among them, AvrPi9 is the cognate avirulence gene recognized by the broad-spectrum resistance gene Pi9 and is relatively conserved among Thai M. oryzae populations [9–11]. Consequently, monitoring the distribution of AvrPi9 provides valuable information for pathogen surveillance and strategic deployment of Pi9-mediated resistance in rice breeding programs. Conventional characterization of avirulence genes relies on pathogen isolation, pathogenicity assays, DNA extraction, PCR amplification, and sequence analysis. Although these approaches provide reliable identification and functional validation, they are labor-intensive, time-consuming, and dependent on specialized laboratory equipment [12–15]. These limitations highlight the need for rapid molecular tools that enable efficient monitoring of avirulence genes directly from infected samples [16].

Recent advances in simplified DNA extraction, isothermal amplification, and CRISPR-based detection have created new opportunities for rapid molecular diagnostics. Recombinase polymerase amplification (RPA) enables rapid DNA amplification under isothermal conditions with minimal instrumentation, while CRISPR–Cas12a provides highly specific sequence recognition for nucleic acid detection [17-21]. Integration of these technologies has enabled sensitive detection of several plant pathogens, although most reported assays rely on purified DNA templates and laboratory-based workflows.

In this study, we developed, to our knowledge, the first field-compatible molecular detection platform targeting AvrPi9 by integrating rapid crude DNA extraction with RPA and CRISPR–Cas12a detection. Unlike previously reported RPA–CRISPR/Cas12a assays, the workflow enables direct analysis of crude fungal mycelia and infected rice tissues without conventional DNA purification. It requires only simple heating devices and portable LED-based fluorescence visualization, making it suitable for decentralized and low-infrastructure diagnostic settings. Beyond AvrPi9 detection, this platform provides a practical framework for developing similar assays targeting additional avirulence genes to support pathogen surveillance, resistance gene deployment, and sustainable rice blast management.

2. MATERIALS AND METHODS

2.1 Plant Materials
Two-month-old greenhouse-grown plants of the Thai aromatic rice (Oryza sativa L.) cultivar Khao Dawk Mali 105 (KDML105) were used for all drop inoculation assays.

2.2 Fungal Materials
Two reference M. oryzae isolates were used throughout the study: isolate 10100 (AvrPi9-positive) as the positive control and isolate NYK56003 (AvrPi9-negative) as the negative control. Three additional Thai rice blast isolates (PL60011, SKA60001, and SKN60002.2), collected from infected rice fields in 2017, were included to evaluate the assay. Their AvrPi9 status was confirmed by PCR and sequencing before assay validation. All isolates were maintained on potato dextrose agar (PDA) at 28 °C, and fresh mycelia were used for DNA extraction.

2.3 Fungal Spore Induction and Drop Inoculation
Actively growing mycelia were transferred to oatmeal agar (OMA) plates and incubated at 28 °C for 7–10 days. After removal of aerial hyphae, cultures were exposed to black light at 25 °C for 4 days to induce sporulation. Conidia were harvested in sterile distilled water containing 0.02% Tween-20, filtered through sterile cheesecloth, and adjusted to 1 × 10⁵ conidia/mL. For inoculation, excised leaves from two-month-old KDML105 plants were placed on hydrophobic glass slides in a moist chamber with leaf ends embedded in 1% water agar. Leaves were wounded with a sterile pipette tip, inoculated with 25 µL of conidial suspension, incubated in the dark at 25 °C for 24 h, and then maintained under ambient laboratory conditions for 4 days. Three independent biological replicates were performed for each isolate.

2.4 DNA Extraction
Genomic DNA was extracted from fungal mycelia and infected rice leaf tissues (approximately 2 × 2 mm lesions) using either a modified cetyltrimethylammonium bromide (CTAB) method or a rapid extraction method. For the CTAB method, samples were ground in liquid nitrogen, followed by chloroform alcohol purification, isopropanol precipitation, ethanol washing, and DNA resuspension as previously described [22]. For the rapid extraction method, samples were incubated in extraction buffer (25 mM Tris-HCl, pH 8.7; 2.5 mM EDTA; 250 mM KCl; 0.05% Tween-20) at 65 °C for 15 min. The resulting supernatant was used directly as the DNA template [23]. DNA concentration and purity were measured using a NanoDrop spectrophotometer, and DNA integrity was evaluated by agarose gel electrophoresis.

2.5 PCR and RPA Amplification of AvrPi9
The AvrPi9 gene was amplified by polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) using gene-specific primers (Table 1). PCR was performed in a 50 µL reaction containing Taq DNA polymerase (Vivantis Technologies, Malaysia) with an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 5 min. RPA was performed using the TwistAmp® Basic kit (TwistDx, UK) following a modified manufacturer's protocol. Reactions were carried out in a final volume of 46.5 µL containing gene-specific primers, 50 ng genomic DNA, and magnesium acetate, followed by incubation at 37 °C for 20 min.

2.7 Limit of Detection of the RPA/CRISPR–Cas12a Assay
The CRISPR–Cas12a detection system, including the CRISPR RNA (crRNA) and fluorophore–quencher (FQ) reporter, was performed as previously described [24]. Analytical sensitivity was evaluated using fivefold serial dilutions of genomic DNA from isolate 10100 (500–0.03 ng/µL). RPA amplicons were subjected to CRISPR–Cas12a detection in 20 µL reactions containing crRNA, LbCas12a (New England Biolabs, USA), FQ reporter (Integrated DNA Technologies, USA), and 1 µL of amplification product. Reactions were incubated at 37 °C for 5 min, and endpoint fluorescence was visualized using a portable blue LED illuminator. The limit of detection (LoD) was defined as the lowest DNA concentration consistently producing visible fluorescence above the no-template control in three independent experiments.

2.8 Validation of the Field-Compatible Molecular Detection Platform Using Experimentally Infected Rice Leaves
DNA was extracted from two-month-old KDML105 rice leaves inoculated with M. oryzae isolate 10100 at 5 days post-inoculation (dpi) using the rapid extraction method. Crude extracts were directly subjected to RPA amplification followed by CRISPR–Cas12a detection, and endpoint fluorescence was visualized using a portable blue LED illuminator. To validate visual endpoint interpretation, fluorescence intensity was monitored over a 5-min detection period using a fluorescence reader and compared with visual observations. To minimize contamination, work surfaces and equipment were cleaned routinely, reagents were prepared as aliquots, and appropriate positive and negative controls were included in each experiment.

2.9 Validation of the Detection Platform Using Field-Collected Samples
To evaluate the applicability of the proposed platform under field conditions, rice leaves with typical blast symptoms were collected from three rice fields in Nakhon Pathom and Suphan Buri provinces, Thailand. Symptomatic samples were designated S1–S3, and healthy leaves collected from the corresponding fields (H1–H3) served as plant negative controls. DNA was extracted directly from symptomatic and healthy leaves using the rapid extraction method. Crude extracts were subjected to RPA amplification followed by CRISPR–Cas12a detection. Endpoint fluorescence was assessed visually using a portable blue LED illuminator, and representative images were captured with a gel documentation system. Fluorescence intensity was further quantified over a 5-min detection period to compare with visual endpoint interpretation.

3. RESULTS AND DISCUSSION

3.1 Optimization of DNA Extraction Methods
To evaluate whether the rapid extraction method generated amplification-compatible DNA for the proposed detection platform, a modified CTAB method and a rapid extraction method were compared using Magnaporthe oryzae mycelia. DNA concentration and purity were measured by NanoDrop spectrophotometry, and DNA integrity was assessed by agarose gel electrophoresis. The modified CTAB method produced higher apparent nucleic acid concentrations and purity than the rapid extraction method, with distinct high-molecular-weight DNA bands visible on agarose gels (Figure 1). In contrast, rapid extraction yielded lower apparent nucleic acid concentrations, reduced OD260/230 ratios, and faint DNA bands, indicating the presence of co-extracted contaminants. Because RNA removal was not performed, residual RNA and other co-extracted molecules likely contributed to the absorbance-based concentration values.

Despite these differences, DNA obtained by the rapid extraction method consistently supported downstream PCR, RPA, and CRISPR–Cas12a detection (Sections 3.2–3.5), demonstrating that crude extracts were fully compatible with the proposed detection platform. These results indicate that spectrophotometric concentration and purity alone are not reliable indicators of DNA suitability for amplification-based assays. The rapid extraction method required only a single incubation at 65 °C for 15 min without conventional DNA purification, substantially simplifying sample preparation. Similar rapid fungal DNA extraction approaches have been reported previously [23, 25-27].

These results demonstrate that the rapid extraction method generates DNA suitable for both PCR and RPA. Although PCR benefited from higher-quality CTAB-extracted DNA, RPA remained robust using crude extracts, consistent with its reported tolerance to inhibitors commonly present in fungal and plant tissues [28,29]. These findings further support the integration of rapid DNA extraction with RPA for the proposed field-compatible molecular detection platform.

This LoD lies within the low nanogram range after RPA amplification. Although lower limits have been reported for other RPA–CRISPR/Cas12a systems (e.g., 10 pg/µl for Phytophthora sojae [30] and 10² copies/µl for Xanthomonas oryzae pv. oryzae [31]), comparisons are constrained by differences in target copy number, genome size, assay chemistry, and detection formats. Here, AvrPi9 is a single-copy gene in the ~40 Mb genome of M. oryzae [32], requiring prior amplification for reliable detection.

Unlike assays optimized for maximal sensitivity using purified DNA or real-time fluorescence detection, this platform prioritizes rapid crude extraction, unpurified samples, endpoint readout, and operational simplicity. Consistently, our previous assay detected AvrPi9 at 1.9 ng/µl using purified RPA products [24], indicating that sensitivity is strongly influenced by sample preparation and detection mode. Despite this trade-off, the achieved sensitivity enables direct detection from crude fungal and infected rice leaf extracts without DNA purification. Overall, the assay balances sensitivity, speed, and simplicity, supporting decentralized pathogen detection and resistance screening.

Clear fluorescence was observed in the positive control, with no signal in the negative control. All four AvrPi9-positive infections produced positive fluorescence, whereas mock-inoculated leaves and the no-template control showed no signal (Figure 4B). These results confirm specific detection of AvrPi9 directly from infected tissues without interference from plant-derived background DNA. Direct detection from crude extracts demonstrates compatibility of the rapid DNA extraction method with RPA–Cas12a workflow. Despite potential plant-derived inhibitors, no reduction in assay performance was observed, indicating robustness for direct tissue analysis. The absence of signals in negative controls further confirms assay specificity.

Compared with previous RPA–CRISPR/Cas12a-based plant pathogen diagnostics that often rely on purified DNA and laboratory instrumentation [33–35], the present platform integrates crude extraction, isothermal amplification, and endpoint detection in a simplified workflow. Although some systems achieve higher sensitivity, they typically require more complex sample processing. Here, the focus is on rapid, extraction-minimal, and field-compatible detection while maintaining adequate analytical performance. These features support the use of this platform for decentralized diagnostics and rapid monitoring of AvrPi9 in rice blast surveillance and resistance breeding programs.

The assay is intended as a rapid screening tool rather than a replacement for full disease diagnosis, which still requires pathogen isolation, morphological and molecular characterization, and pathogenicity testing. Field samples used here reflected typical breeding and surveillance materials with lesion scores of 1–4 [36]. The assay detects AvrPi9 sequences but cannot distinguish functional alleles from nonfunctional variants outside primer/crRNA regions; thus, sequencing and pathogenicity assays remain necessary. Its diagnostic relevance is highest in systems involving Pi9-mediated resistance. Nevertheless, the workflow is readily adaptable to other Avr genes by redesigning primers and crRNAs, enabling future multiplex surveillance of Avr–R interactions. Larger field-scale validations across regions and pathogen populations are needed to further confirm robustness.

Although a formal cost analysis was not performed, the estimated reagent cost is ~US$10.84 per sample. In contrast, conventional CTAB extraction, PCR, electrophoresis, and sequencing require longer processing times and laboratory infrastructure. The proposed assay is completed within ~40–45 min using isothermal conditions and simple heating and LED-based visualization. Thus, “field-compatible” here refers to low-infrastructure deployment rather than fully equipment-free use.

Overall, the integrated crude extraction–RPA–CRISPR/Cas12a platform enables rapid detection of AvrPi9 directly from infected tissues without DNA purification, reducing time and complexity while maintaining reliability. This approach provides a practical framework for extending RPA–CRISPR diagnostics to other avirulence genes, supporting Avr–R surveillance and more informed resistance deployment in rice breeding and disease management.

4. CONCLUSIONS

This study presents an integrated detection platform combining rapid crude DNA extraction, RPA, and CRISPR–Cas12a for rapid identification of AvrPi9 in Magnaporthe oryzae. The workflow enables direct analysis of fungal mycelia, experimentally infected rice tissues, and field-collected samples without DNA purification, thereby reducing time, procedural complexity, and equipment requirements. Despite the use of crude extracts, the system achieved specific AvrPi9 detection with low-nanogram sensitivity after RPA amplification. Validation using both experimental and natural infections confirmed compatibility with plant-derived materials and suitability for low-infrastructure diagnostic settings. While intended as a rapid first-line screening tool rather than a replacement for full pathogen diagnosis, it supports surveillance of AvrPi9 distribution and assists rice blast resistance breeding programs. Beyond AvrPi9, the platform provides a general framework for developing additional RPA–CRISPR/Cas12a assays targeting other avirulence genes, and future multiplex adaptation could further enable molecular surveillance of Avr–R interactions to support more informed resistance deployment and sustainable rice blast management.

ACKNOWLEDGEMENTS

This research was funded by Kasetsart University Research and Development Institute, KURDI, FF(KU)5.69 and Science Achievement Scholarship of Thailand (SAST).

AUTHOR CONTRIBUTIONS

Atcharapohn Jai-uean: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing – Original draft. Pattavipha Songkumarn: Supervision, Writing – Review and Editing. Tanee Sreewongchai: Supervision, Writing – Review and Editing. Nonglak Parinthawong: Resources, Investigation, Validation. Jyh Jian Chen: Methodology, Validation. Chatchawan Jantasuriyarat: Conceptualization, Supervision, Project administration, Funding acquisition, Writing – Review and Editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DECLARATION OF USE OF GENERATIVE AI

During the preparation of this manuscript, the authors used a generative AI tool for language refinement and improving clarity. The authors have carefully reviewed and edited the manuscript and take full responsibility for its content.

FUNDING

This research was financially supported by the Kasetsart University Research and Development Institute (KURDI): FF(KU)5.69.

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