A rapid colorimetric LAMP assay for detection of *Rhizoctonia solani* AG-1 IA causing sheath blight of rice

Rice sheath blight, caused by the ubiquitous fungus *Rhizoctonia solani*, is a major threat to rice production globally, particularly in South Asian countries. Sheath blight can result in significant yield losses, ranging from 10–25% and even up to 50% under favorable conditions and susceptible varieties. In India alone, losses have reached 54.3%. The cultivation of high-yielding, semi-dwarf rice varieties, often requiring higher nitrogen fertilizer inputs, has exacerbated the problem. Existing diagnostic methods, relying on culture and morphology, are time-consuming and require expertise. Molecular methods offer improved speed and accuracy. Previous studies have developed PCR assays for *R. solani* detection, often targeting the rDNA internal transcribed spacer (ITS) region or other genes such as *nox*B, calmodulin, and CYP51C. However, these methods may not be suitable for point-of-care diagnostics in resource-limited settings. Loop-mediated isothermal amplification (LAMP) is a visual detection method that offers advantages for field use due to its simplicity and speed. Polygalacturonase (PG) enzymes are important virulence factors in many phytopathogenic fungi, including *R. solani*. Although pectic zymograms have been used to characterize *R. solani*, no molecular marker based on PG genes has previously been developed. This study aims to develop a rapid, sensitive, and specific diagnostic assay for *R. solani* AG-1 IA, a major rice pathogen, by utilizing the PG gene and a LAMP-based approach, coupled with a recently developed rapid high-throughput template preparation method (rHTTP). This approach promises to provide a simple, cost-effective, and field-applicable tool for early disease monitoring.

Several studies have explored molecular methods for *Rhizoctonia solani* detection. Mazzola et al. (1996) made early attempts to design molecular markers for *R. oryzae*. Lees et al. (2002) developed a sensitive PCR assay for *R. solani* AG-3. Woodhall et al. (2013) reported a quantitative real-time PCR assay for *R. solani* AG3-PT. Loop-mediated isothermal amplification (LAMP) has emerged as a reliable method for pathogen detection, with some reports on its application for *R. solani* and *R. zeae*. However, many of these studies have used the rDNA internal transcribed spacer (ITS) region. Polygalacturonase (PG) genes, involved in pectin degradation and virulence, represent a potential target for pathogen-specific detection. While pectic zymograms have been employed for *R. solani* characterization, no molecular marker based on PG genes was available prior to this study. This gap in the literature highlights the need for a novel, efficient diagnostic tool.

The study involved several steps: 1. **Field sampling:** Infected rice and maize leaf samples were collected from various locations in India. 2. **Fungal isolation and identification:** *R. solani* AG-1 IA isolates were isolated and characterized morphologically. 3. **Primer design and PCR assay optimization:** Primers targeting the polygalacturonase (PG) gene were designed, and PCR conditions were optimized. Four primer sets (RSPG1F/R, RSPG2F/R, RSPG4F/R, RSPG5F/R) were designed for conventional PCR and q-PCR. Specificity was checked using Primer BLAST. The sensitivity was determined using serial dilutions of DNA. 4. **Quantitative PCR (q-PCR):** The sensitivity of the four RSPG primer sets was determined by q-PCR using serial dilutions of *R. solani* DNA. The amplification efficiency was calculated. 5. **LAMP assay development:** Six primers (RS_pg_F3_1/RS_pg_B3_1, RS_pg_FIP_1.1/RS-pg_BIP_1.1, RS_pg_LF_1/RS_pg_LB_1) for LAMP assay were designed using Primer Explorer v5. Specificity was tested against various fungal and bacterial cultures. 6. **LAMP assay validation:** The sensitivity of the LAMP assay was determined by using serial dilutions of *R. solani* AG-1 IA DNA. Specificity was evaluated against other fungal and bacterial species. 7. **In vitro and pot experiments:** Rice leaves were infected with *R. solani* AG-1 IA, and the LAMP assay was used for early detection. 8. **Soil DNA extraction and LAMP assay:** Soil samples from rice fields were collected, DNA was extracted, and the LAMP assay was performed on extracted DNA. The time for LAMP assay was optimized and final assay condition was 65°C for 45 min. 9. **Genomic DNA isolation:** Genomic DNA was extracted from fungal cultures and plant materials using established protocols, with some modifications. 10. **Template Preparation**: The rapid high throughput template preparation method (rHTTP) was used for DNA preparation from infected tissues.

The study successfully developed a rapid colorimetric LAMP assay for the detection of *Rhizoctonia solani* AG-1 IA. The four primer sets designed for conventional PCR and q-PCR showed specific amplification of the PG gene, with amplicon sizes of 300 bp, 375 bp, 500 bp, and 336 bp, respectively. q-PCR analysis demonstrated high sensitivity, capable of detecting down to 0.01 ng/µl of DNA with amplification efficiency ranging from 91% to 97.5%. The LAMP assay, utilizing six newly designed primers, displayed exceptional sensitivity, detecting as low as 1.65 fg/µl of template DNA. This high sensitivity was maintained even when using DNA extracted from infected plant tissues, with a total assay time of only 45 minutes, including the rapid template preparation step (rHTTP). Crucially, the LAMP assay exhibited high specificity, with no amplification observed in other *R. solani* AG groups or in closely related fungal and bacterial species. The assay successfully detected the pathogen in artificially infected leaf samples with varying degrees of disease severity, as early as 24 hours post-inoculation, and in field soil samples. The use of rHTTP made the entire process (sample collection to detection) extremely simple, time efficient and cost effective, with the procedure feasible even in settings lacking sophisticated laboratory equipment. The field soil samples which showed the positive results through LAMP assay developed severe symptoms of sheath blight disease when monitored after one month indicating the potential for early disease detection using this assay.

This study addresses the need for a rapid and field-deployable diagnostic tool for *Rhizoctonia solani* AG-1 IA, a major rice pathogen. The successful development of a colorimetric LAMP assay targeting the polygalacturonase (PG) gene, a virulence factor, provides a significant advancement over existing methods. The high sensitivity and specificity of the assay, coupled with its short turnaround time and minimal instrumentation requirements, make it particularly suitable for point-of-care diagnostics. The integration of the rHTTP method further enhances the assay's practicality and cost-effectiveness, making it ideal for use in resource-limited agricultural settings. The ability to detect the pathogen as early as 24 hours after infection enables timely intervention and disease management strategies. This study highlights the importance of using virulence factors as targets for diagnostic assays, as they offer greater specificity and relevance to disease progression. The results of this study have significant implications for the effective management of rice sheath blight and the reduction of economic losses associated with this disease.

This study successfully developed a rapid, sensitive, and specific colorimetric LAMP assay for the detection of *Rhizoctonia solani* AG-1 IA in rice. The assay's speed (45 minutes), simplicity, low cost, and visual readout make it highly suitable for on-field diagnostics and early disease management. The integration with the rHTTP method enhances efficiency and accessibility. Further research could focus on developing a user-friendly, portable device for wider implementation of this assay and evaluating its effectiveness in various agro-ecological conditions.

While this study demonstrates the effectiveness of the LAMP assay in detecting *R. solani* AG-1 IA, certain limitations should be considered. The study primarily focused on isolates from specific regions of India, so further validation is needed with isolates from diverse geographical locations and across wider environmental conditions. The artificial infection experiments could be further expanded to include a larger set of field conditions and natural infections, and the sensitivity of LAMP assay in soil DNA could be improved by changing the extraction methodology or optimization of LAMP assay reaction condition.