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

Rice is a major cereal crop worldwide, particularly in South Asia, and suffers significant yield losses from diseases, notably sheath blight (SBD), the second most devastating rice disease. Rhizoctonia solani, a ubiquitous pathogen of cereals and grasses, can cause 10–25% yield losses, and up to 50% under conducive environments and susceptible varieties. The increased cultivation of high-yielding semi-dwarf rice with high nitrogen inputs has intensified sheath blight incidence. R. solani produces virulence factors, including toxins and cell wall-degrading enzymes. Effective disease management requires simple, specific, and rapid diagnostics to detect the pathogen in crops and soils and to inform management thresholds. Traditional culture/morphology-based identification is slow and requires expertise. Molecular assays have been developed for other R. solani AG groups, often targeting the rDNA ITS region; however, alternative gene targets, such as noxB, calmodulin, and CYP51C, can improve specificity. Polygalacturonase (PG), a key virulence factor in many phytopathogenic fungi, has been used for biochemical grouping of R. solani but lacks molecular diagnostic markers. This study aimed to develop PG gene-based PCR and a rapid colorimetric LAMP assay, coupled with a simple template preparation method (rHTTP), for specific, sensitive, and field-deployable detection of R. solani AG-1 IA causing rice sheath blight.

Previous molecular detection of R. solani has focused on ITS-based PCR/qPCR assays for specific AG groups (e.g., AG-3 in potato; AG3-PT). LAMP assays have been reported for R. solani (soybean seedling blight) and R. zeae, but these typically target ITS and may be time-consuming with complex DNA extraction. Other gene targets (noxB, calmodulin, CYP51C) have improved specificity in fungal diagnostics. PG enzymes (glycosyl hydrolase family 28) are important virulence factors and have been exploited biochemically (pectic zymograms) for characterizing R. solani AGs (e.g., AG-8, AG-4, AG-1). Despite this, no molecular markers for PG have been available for R. solani AG-1 IA detection in rice. This gap motivated a PG-based molecular assay that could be more directly linked to virulence and useful for early detection and management planning.

Field sampling: Infected leaf samples (rice and maize) were collected from Punjab and Uttar Pradesh, India, for fungal isolation; additional healthy and infected rice leaf samples were collected for marker validation from fields in Mau, Uttar Pradesh and Cooch Behar, West Bengal. Isolation and identification: Infected tissues (~1 cm) were surface-sterilized in 1% sodium hypochlorite, placed on water agar for isolation, and pure cultures were characterized morphologically (colony/sclerotia features) as R. solani AG-1 IA. Fifty-one isolates were obtained and deposited at NAIMCC (ICAR-NBAIM). Primer design and conventional PCR: Polygalacturonase (PG) gene sequences of R. solani AG-1 IA (HQ197936, HQ197944, FJ544456, KP896519, KP896521, KP896522) were aligned (ClustalW, MEGA 7.0) to identify conserved regions. Four primer pairs (RSPG1F/R, RSPG2F/R, RSPG4F/R, RSPG5F/R) were designed; primer properties were checked (OligoCalc) and specificity verified (NCBI Primer-BLAST). PCR was optimized via gradient PCR. Reaction (25 µl): 10× buffer (2.5 µl), dNTPs (1.5 µl, 50 µM each), primers (1 µl each, 10 mM), Taq DNA polymerase (1 U), template DNA (~50 ng, 2 µl). Cycling: 95 °C 5 min; 35 cycles of 95 °C 1 min, annealing 1 min (RSPG1/2/4 at 52.4 °C; RSPG5 at 54 °C), 72 °C 1 min; final 72 °C 10 min. Amplicons were resolved on 1.5% agarose. Selected amplicons (RSPG2) were sequenced and deposited (MT882056–MT882069). Validation panel: Multiple fungal and bacterial outgroups and other R. solani AG groups were used for specificity testing (listed in Table 3). qPCR sensitivity: Real-time PCR (Agilent G8830A, SYBR Green) used Brilliant III Ultra-Fast SYBR Green Master Mix. Cycling: 95 °C 10 min; 40 cycles of 95 °C 5 s, annealing (RSPG1/2/4 at 52 °C; RSPG5 at 54 °C) 30 s, 72 °C 15 s; melt curve performed. Serial dilutions of R. solani genomic DNA (100 ng/µl to 0.01 ng/µl) were tested in triplicate; Ct vs −log DNA used to calculate efficiency. LAMP assay design and conditions: PG sequence (MT882069) was used to design LAMP primers with PrimerExplorer v5: RS_pg_F3_1/RS_pg_B3_1 (outer), RS_pg_FIP_1.1/RS-pg_BIP_1.1 (inner), RS_pg_LF_1/RS_pg_LB_1 (loop). Specificity was checked via Primer-BLAST. Colorimetric LAMP reactions (25 µl): 12.5 µl WarmStart Colorimetric LAMP 2× Master Mix (NEB), 2.5 µl primer mix, 3 µl template DNA, water to volume. Incubation at 65 °C for 30 min; positive reactions turned yellow; amplicons also checked on 2% agarose. LAMP sensitivity and specificity: Tenfold serial dilutions of template DNA from 165 ng/µl to 1.65 fg/µl (3 µl template per reaction) assessed detection limit at 65 °C for 30 min. Specificity tested across fungal and bacterial panels and other R. solani AGs. In vitro and pot infection validation: Rice leaf sheaths were inoculated with R. solani AG-1 IA (moist chamber and potted plants), with disease severity levels (+, ++, +++). Samples were collected 24 h post-inoculation for LAMP. Soil DNA validation: Rice field soil samples (composite from 1 m²) were processed; DNA extracted in six replicates (FastDNA Spin Kit for Soil), quantified (160–180 ng/µl), pooled, and used as template. LAMP incubation time optimized to 45 min for soil DNA. The field was monitored for one month post-sampling for SBD symptoms. DNA extraction: Fungal genomic DNA from 7-day cultures (per Kumar et al., with modifications). Plant DNA extracted per Doyle and Doyle (with modifications). For LAMP from tissues, the rHTTP method was used to prepare templates directly from infected/healthy tissues.

- Isolate collection and morphology: 51 R. solani AG-1 IA isolates were purified from rice and maize in Uttar Pradesh and Punjab; colonies ranged pale/yellowish/whitish brown, with varying sclerotia location/size. All isolates were deposited at NAIMCC (ICAR-NBAIM). - PG-based PCR markers: Four RSPG primer sets targeting the PG gene specifically amplified expected products in all 51 AG-1 IA isolates: 300 bp (RSPG1), 375 bp (RSPG2), 500 bp (RSPG4), and 336 bp (RSPG5). No amplification occurred with non-target fungi and bacteria (S. sclerotiorum, T. viride, F. oxysporum, A. alternata, C. capsici, B. subtilis, P. plecoglossicida), confirming specificity. - Validation on plant tissues: RSPG primers produced clear bands from genomic DNA of infected rice leaf sheaths but not from healthy tissues. - qPCR sensitivity and performance: All four RSPG primer sets detected template down to 0.01 ng/µl (10 pg/µl) with efficiencies of 91–97.5%; melt peaks centered around ~84 °C; NTCs were negative. - LAMP assay development and specificity: A colorimetric LAMP assay using six PG-targeted primers (RSPG LAMP set) showed positive reactions with R. solani AG-1 IA isolates and no amplification with other AG groups (AG-1 IB, 2-2IIIB, 3, 7, 8), R. oryzae-sativae, or major rice pathogens/fungal and bacterial outgroups, matching PCR specificity. - LAMP sensitivity and speed: Detection limit reached 1.65 fg/µl template DNA. Coupling rHTTP (15 min) with LAMP (30 min) enabled visible detection in 45 min total. - Early detection in planta: LAMP detected R. solani AG-1 IA from artificially infected leaf tissues with varying severity as early as 24 h post-inoculation and distinguished healthy from infected samples. - Soil DNA detection and field prediction: Soil DNA from a rice field yielded positive LAMP results (optimized 45 min incubation); one month later, the field showed severe sheath blight symptoms, supporting the assay’s utility for early field monitoring.

Targeting the polygalacturonase (PG) gene, a key virulence factor, enabled development of highly specific PCR and LAMP assays for R. solani AG-1 IA. Unlike many prior assays relying on the ITS region, PG-based markers may better discriminate pathotypes and are tied to pathogenicity. The RSPG primer sets provided specific and sensitive detection by conventional and real-time PCR, and the LAMP assay delivered comparable specificity with substantially faster turnaround and visual readout, facilitating field applicability. Integration with the rHTTP template preparation shortened sample-to-answer time to 45 minutes, requiring minimal equipment. The LAMP assay detected the pathogen from infected tissues within 24 hours of inoculation and from environmental (soil) DNA, demonstrating potential for early warning and guiding timely management interventions. Compared with previous LAMP methods for other hosts/AGs that were slower and extraction-intensive, this approach is simpler, cost-effective, and suitable for point-of-care diagnostics. The soil detection preceding visible field disease indicates promise for epidemiological surveillance and decision support in sheath blight management.

This study introduces PG gene-based diagnostic markers and a rapid colorimetric LAMP assay for specific detection of R. solani AG-1 IA in rice. The main advantages are colorimetric readout, rapid turnaround (45 min with rHTTP), minimal instrumentation, early detection capability (within 24 h post-infection), and applicability to environmental samples (plant tissues and soil). While conventional PCR assays were sensitive and specific, the LAMP assay’s simplicity and visual confirmation make it more suitable for field deployment. The assay can aid early monitoring and inform timely management strategies to reduce sheath blight losses. Future work could expand validation across broader geographies, seasons, and sample matrices, and integrate the assay into portable platforms for on-site surveillance.