Fifty S. oryzae isolates were isolated from rice grain samples which were randomly collected from farmer’s field, storage godowns and research centres from different locations of Tamil Nadu, India, labeled appropriately and brought to the laboratory for further studies. Each isolates were incubated at 27 ± 2°C for 10- 20 days on potato dextrose agar (PDA) media. For extracting DNA from fungal mycelia, cultures were grown in a 250 ml conical flask with 100 ml of potato dextrose broth that had been autoclaved at 121°C and 15 psi. The fungal mycelial discs (4 no.) were then inoculated into flasks and incubated at 27 ± 2°C for 7-10 days. The mycelial mat was taken from the broth and dried on filter paper. The modified CTAB method was used to extract total genomic DNA (Doyle, 1991). A Nano-drop Spectrophotometer (ND-1000) was used to measure the concentration and purity of each genomic DNA, and the quality of all extracted DNA was determined by measuring its absorbance at 260 and 280 nm. Agarose gel electrophoresis was done to confirm DNA concentrations. On an agarose gel with a 1.2% (w/v) concentration and 0.1 µg/mL of ethidium bromide, ten microliters of purified DNA were run. The Biorad Gel Doc was used to visualize the DNA. The DNA was kept at -20°C for further use.

PCR analysis was carried out in a 25 µL reaction mixture containing 1 µL of genomic DNA, 12.5 µL of 2X Master mix (RR310 EmeraldAmp), and 2.5 µL of both forward and reverse primer (ITS 1F and ITS 4R). Nuclease-free water was added to bring the total amount to 25 µL. DNA amplification was accomplished in an Eppendorf thermocycler (Germany) under the following cycling conditions: primary denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 30 s; annealing at 55°C for 30 s; elongation at 72°C for 60 s and terminal elongation 72°C for 10 min. The DNA bands were separated by gel electrophoresis in a 1.2% agarose (Himedia) gel stained with 0.1 µg/mL of ethidium bromide. The UV gel documentation system was used to visualize the DNA bands resolved on agarose gel. By contrasting the amplicons with a 100 bp DNA ladder (3422A Takara) on an agarose gel, the amplicons sizes were estimated. The amplified PCR product was sequenced at Biokart India Pvt. Ltd. (Bangalore, India) by Sanger’s dideoxy method. The sequences were compared with other ITS region of S. oryzae available at NCBI database to assess for their variability ( Supplementary Table 1 ).

The software tool PRIMER EXPLORER V5 (http://primerexplorer.jp/e/) which is accessible on the Eiken Genome website was used to design LAMP primers. A critical part in LAMP primer designing is the selection of a potential target gene with high nucleotide variability among related species. LAMP primers were designed by utilizing the β-tubulin gene of S. oryzae. A set of six primers was designed by using the sequences retrieved from NCBI GenBank (Accession number MZ614962.1). Each parameter was set to its default value. The primer sequences utilized in this experiment are furnished in Table 1 . Figure 1 depicts LAMP primer’s structure and its complement target DNA used in this work. In addition, β-Tubulin sequences of S. oryzae strain Sor-3 (MZ614962.1), S. oryzae strain Sor-2 (MZ614961.1),

S. oryzae strain Sor-1 (MZ614960.1), S. strictum (KM249114.1), S. kiliense (MZ833455.1) and S. spirale (LC464483.1) were acquired from the NCBI website (http://www.ncbi.nlm.nih.gov) to verify the specificity of primers against other three Sarocladium spp. ( Figure 2 ). Oligo Evaluator™ (Sigma Aldrich) was employed to verify developed primers for secondary structure formation (hairpins, palindromes, repetitions, dimers, cross dimers and runs), primer dimerization (primer-dimer), % GC and GC clamp (https://www.sigmaaldrich.com) (Zaczek-Moczydłowska et al., 2020). The specificity of each primer was confirmed by comparing the primer sequences with the NCBI GenBank nucleotide and genome databases by employing the BLASTn programme (Altschul et al., 1990).

The LAMP reagents were standardized in various concentrations ( Table 2 ) in order to determine the best conditions for the reaction. The final LAMP reactions were accomplished in a 25 µL LAMP reaction mixture that contained 1.5 µL of target DNA (~ 120 ng), 2.5 µL of Thermopol reaction buffer (10x), 1.4 mM of each dNTPs, 0.2 mM of both outer primers, 0.4 mM of both loop primers, 1.4 M of both inner primers, 1.2 M betaine, 8 mM MgSO₄, 2 U Bst polymerase (0.08 U/µL) and 120 µM hydroxyl naphthol blue. Instead of using a DNA template, the nuclease-free water served as the negative control. The reactions were performed in an Eppendorf thermal cycler for 30 min at a constant temperature of 65°C, and then the reaction was ceased by thermal denaturation for 2 min at 80°C.

A visual evaluation of the colour was done after termination. The quantity of amplification product as evaluated by 2% agarose gel electrophoresis was used to choose the optimal condition ( Supplementary Figure 1 ). Protocols with varying amplification temperature (56-70°C) and amplification time (10-150 min) were investigated to optimize the LAMP assay ( Supplementary Figure 2 ).

Genomic DNA that had been serially diluted ten times (100 ng/l to 1 fg/l) was used to assess the amplification potency and sensitivity of the LAMP primers. This experiment was carried out thrice ( Table 3 ; Figure 3 ).

Genomic DNA samples from 22 different phytopathogens ( Table 4 ), which were obtained from the Culture Collection Centre, Department of Plant Pathology at Tamil Nadu Agricultural University in Coimbatore, India were tested to ascertain the specificity of the LAMP assay ( Figure 4 ).

The LAMP assay technique developed was validated with different isolates of S. oryzae collected from various locations of Tamil Nadu ( Table 5 ; Supplementary Figure 3 ). As mentioned earlier, genomic template was extracted using a modified CTAB technique. The LAMP assay was carried out on the DNA of 50 fungal isolates, with nuclease-free water serving as a negative control. The LAMP reaction mixture was incubated at 65°C for 30 min, and then the reaction was ceased by heating it at 80°C for 2 min. The results were visualized by HNB dye, and the experiments were carried out thrice.

The LAMP assay was validated with field samples using naturally infected rice seeds and artificial pathogen inoculation in seeds. For naturally infected seeds, seeds from the infected panicle were collected from rice fields in the districts of Coimbatore, Trichy and Villupuram. Three samples were collected from each district, and apparently healthy seeds collected from the panicle showing no infection were kept as a control. Contrarily, for artificial inoculation, seeds were surface sterilized with a 3% sodium hypochlorite solution for 5 minutes, followed by three washes with sterile water. S. oryzae spore suspension was added to rice seeds 24 hours before DNA extraction and dried overnight to remove the excess spore suspension on seed surface. Using the CTAB method, genomic DNA was extracted from rice seeds. According to Liang et al., 2016, 2 mg of rice seeds were ground in a mortar and pestle with 150 µL of extraction buffer (200 mmol/L Tris-Cl (pH 8.0), 50 mol/L ethylenediaminetetraacetic acid (EDTA; pH 8.0), 2.8 mol/L NaCl, 4% CTAB, and 2% sodium dodecyl sulphate). The obtained genomic DNA sample was digested with 10 µg/mL of RNase to remove any RNA contamination and stored at -20°C. The 2 µL final solution was directly used as a DNA template for the LAMP assay.

Following the procedure mentioned in section 2.3, template DNA was extracted from the weeds randomly collected from bunds of sheath rot infected fields. The LAMP assay was carried out using templates following the previously stated optimal conditions. When testing rice seeds of different varieties, 2 µl of total genomic DNA was utilized for the assay while maintaining all other parameters unchanged.

In a PCR assay, fifty isolates of S. oryzae collected from diverse locations were amplified using the ITS 1 and 4 primers. The sequenced products were evaluated against other S. oryzae sequences found in the NCBI database. A similarity search was done in NCBI using the BLAST tool, the nucleotide sequence of the study isolate’s ITS region revealed 100% identity with other S. oryzae isolates and significantly very less similarity with other Sarocladium isolates.

Using the Primer explorer V5 software programme, at least five different primer sets were predicted for unique sequences and used to build LAMP assay specific for S. oryzae. The primers were designed to allow S. oryzae-specific amplification showed 100% identity with 100% query coverage for S. oryzae. The G values of the 3′ end were determined during the design of LAMP primers and were less than -4 Kcal/mole. One pair of primers with a stable DNA construct and no primer-dimer formation were seen from in silico evaluation of the five designed primer sets generated by the software. Eventually, a set of six primers with high species specificity and sensitivity that targeted the S. oryzae β-tubulin sequences were elected for future investigation. The potential for the development of very weak to moderate secondary structures were found for the outer primers (forward-F3 and reverse-B3), moderate to weak secondary structures were identified for inner primers (FIP and BIP) whereas no to strong secondary structures were identified for loop primers (LF and LB) ( Supplementary Table 2 ). The 3’ end of the primers was clamped with 2 to 3 guanine or/and cytosine bases ( Table 1 ).

In silico hybridization of the primers revealed that the following six primers annealed to unique locations in 713 bp of the specific β -tubulin target sequence: F3 (444-463 bp), F2 (465-483 bp), LF (484-505 bp), F1c (517-537 bp), B1c (553-574 bp), LB (576-594 bp), B2 (600-618 bp) and B3 (624-643 bp). In Figure 2 , binding positions of the primers were highlighted.

The critical components that could affect the effectiveness of the detection include the concentrations of MgSO₄, betaine, Bst polymerase, dNTPs, and HNB dye. Magnesium ions in particular have a significant impact on the activity of DNA polymerase and primer annealing (Saiki et al., 1988; Yeh et al., 2005). Reuter et al. (2020) reported that the colour shift in the LAMP assay is due to the chelation of Mg^{2 +} ions by dNTPs. During the LAMP assay, the development of sky-blue colour signifies the positive reaction in the suspect samples ( Supplementary Figure 1 ), whereas the violet colour signifies the negative reaction. The LAMP response was strongly impacted by the MgSO₄ concentration. Colour change was established as the major benchmark for the optimization of MgSO₄ concentration. The MgSO₄ at 8 mM Conc. exhibited a deep sky blue colour in the LAMP reaction. The intensity of the sky blue colour decreased with either an increase or decrease from the optimal MgSO₄ concentration. The intensity of sky blue decreased at concentrations of 4 mM, 6 mM, and 10 mM. Furthermore, the LAMP assay failed to produce colour at 0 mM and 2 mM concentration, indicating that MgSO₄ is highly imperative for the LAMP assay. The results were validated using 2% agarose gel electrophoresis. In the presence of 8 mM MgSO₄, a distinctive ladder-like pattern was visible; however, no such ladder-like pattern was found in the samples containing 0 mM, 2 mM, and 10 mM MgSO₄ ( Supplementary Figure 1A ). Similarly, betaine at 0.8 mM concentration generated a characteristic ladder-like pattern, but other concentrations did not, implying negative results ( Supplementary Figure 1B ). The LAMP assay results at varying concentrations of Bst DNA polymerase (0U, 2U, 4U, 6U, and 8U) implied amplification at all doses except at 0 U. The best amplification, however, was found at a concentration of 2U Bst polymerase ( Supplementary Figure 1C ). The amplification improved noticeably when the dNTPs dose increased from 0.6 mM to 1.4 mM, as evidenced by brighter bands on 2% agarose gels ( Supplementary Figure 1D ). Hydroxy naphthol blue (HNB), an indicator dye for LAMP-based detection was utilized for the LAMP assay to enable visualization in the current work. According to the result of a range of HNB dye concentration test, concentration less than 120 µM have an impact on the visual detection of LAMP results but have no impact on the ladder-like pattern on 2% agarose gel. However, at greater concentrations affected the visualization as well as the ladder-like pattern ( Supplementary Figure 1E ).

A short range of temperatures (56°C to 70°C) ( Supplementary Figure 2A ) and minimum time (10-150 min) ( Supplementary Figure 2B ) were used to accurately assess the colour intensity using template DNA from the S. oryzae to find the optimum temperature and reaction time for the assay. The assay performed well at various temperatures. The outcomes were also evaluated on a 2% agarose gel. Positive results were seen after 30 min of incubation; however, no amplification was seen during the incubation period of 10 or 20 min. In case of different temperature range tested, positive results were obtained in all the cases but with varying range of colorimetric reaction and degree of amplification. However, conspicuous amplification and colorimetric reaction was seen at a temperature of 65°C. Therefore, S. oryzae was best detected at 65°C for 30 minutes under optimal circumstances.

After establishing that the primer targeting the β-Tubulin gene became unique for S. oryzae, the lowest detection limit was interpreted with the use of 10-fold serial dilutions of S. oryzae DNA (100 ng to 1 fg). The least possible detection limit for S. oryzae as per reaction within 30 min incubation was 10 fg and was confirmed by colouration shift from violet to sky blue and reconfirmed by way of characteristic ladder-like pattern on 2% gel electrophoresis ( Figure 3 ; Table 3 ). This experiment was repeated thrice.

S. oryzae isolate and non-target DNA samples from 22 different phytopathogens listed in Table 5 were used to test the primers’ specificity. The LAMP assay successfully identified S. oryzae, as shown by a colour change from violet to sky blue in the reaction solution, and did not cross-react with non-target phytopathogens, which retained their original violet colour. The specificity of the S. oryzae LAMP assay was validated by electrophoresis on 2% agarose gels stained with ethidium bromide. A characteristic ladder-like pattern was observed in reactions with genomic DNA from S. oryzae but not with DNA from other non-target phytopathogens. The findings demonstrated the great specificity of the newly designed LAMP primer targeting the β-Tubulin gene in identifying S. oryzae ( Figure 4 ; Table 4 ).

Primarily based on the colour development, the LAMP assay was initially optimized for S. oryzae pathogen to confirm the facilitative evaluation of a reaction with different isolates collected from different locations of Tamil Nadu. And it was reconfirmed by subjecting the samples to 2% agarose gel electrophoresis. A deep sky blue colour was visualized from the tubes retaining template DNA and HNB indicator dye. Meanwhile the violet colour indicated the absence of S. oryzae in the tubes with nuclease-free water ( Supplementary Figure 3 ).

The initial validation of the LAMP assay technique was performed on the S. oryzae culture sample. 30 samples were collected from several farmers’ fields and seed godowns for implementation at the field level. The LAMP assay was validated for field samples in each location using uninfected sample as a healthy control. The seeds from the infected panicle and the seeds with discolouration showed a positive reaction (deep sky blue colour), whereas the samples without infection showed a negative reaction (violet colour). In case of artificially inoculated seed samples, those seeds which are inoculated with pathogen showed colour development whereas, those which are surface sterilized didn’t developed colour resulting in negative reaction. Similar negative results were seen in nuclease-free water, which served as the negative control ( Figure 5 ).

When weed samples were evaluated utilizing Tub LAMP primer sets, all weed species and rice sheath rot infected samples generated positive outcomes. However, the degree of amplification varied among the weed species. No amplification was observed in NTC ( Figure 6 and Table 6 ).

Rice seeds from 11 of 13 rice varieties have showed positive reaction for S. oryzae ( Table 7 ). Two varieties namely, TRY 43 and RNR 15048 did not exhibit amplification ( Figure 7 ) along with NTC.