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

Rhizoctonia solani is one of the most devastating pathogens. R. solani AG-1 IA causes sheath blight in rice, maize, and other Gramineous plants. Accurate identification is essential for the effective management of this pathogen. In the present study, a set of four primers were designed viz. RSPG1, RSPG2, RSPG4, and RSPG5 for polygalacturonase (PG) gene, an important virulence factor in phytopathogenic fungi. All four primer sets showed specific amplification of 300 bp (RSPG1F/R), 375 bp (RSPG2F/R), 500 bp (RSPG4F/R) and 336 bp (RSPG5F/R) amplicons. q-PCR detection using each primer sets could detect up to 10 pg of DNA. We also designed six primers (RS_pg_F3_1/RS_pg_B3_1, RS_pg_FIP_1.1/RS-pg_BIP_1.1, and RS_pg_LF_1/RS_pg_LB_1) for PG gene. Further, a colorimetric LAMP assay developed yielded visual confirmation of the pathogen within 45 min of sample collection when coupled with rapid high throughput template preparation method (rHTTP) from infected samples. The sensitivity of the LAMP assay was as low as 1.65 fg/µl of template DNA and could effectively detect R. solani AG-1 IA from diseased plant tissues and soil samples. The LAMP assay was highly specific for R. solani as it did not show any amplification with other AG groups of R. solani and closely related fungal and bacterial outgroups. This study will help in designing an effective point of care diagnostic method for early monitoring of R. solani and thereby planning timely preventive measures against the pathogen.

few reports on development of LAMP assays for detection of R. solani and R. zeae causing soybean seedling blight and charcoal rot. Majority of these reports on specific detection of R. solani have employed rDNA internal transcribed spacer (ITS) region. Apart from ITS regions, other genomic regions or genes like noxB, calmodulin, CYP51C, etc. could also be used for specific detection of fungal pathogens [18][19][20] .
Polygalacturonase (PG) enzymes are members of glycosyl hydrolase family 28 and catalyze the random hydrolysis of 1,4-α-d-galacturosiduronic linkages in pectate and other galacturonans. It is an important virulence factor in many phytopathogenic fungi [21][22][23][24] . This enzyme has been exploited as a marker for grouping and detection of R. solani. MacNish et al. used pectic zymograms for characterization of R. solani AG-8 isolates 25 . Pectic zymogram variations have also been used for characterization and grouping of R. solani AG-4 infecting beans and R. solani AG 1 isolates infecting tobacco 26,27 . Despite its association with virulence and prior use for fungal detection, no molecular marker is available for this important enzyme. Development of diagnostic marker using such genes can be very useful to monitor virulent isolates of R. solani which can help in its effective and timely management. To the best of our knowledge, very little effort has been made to develop diagnostic markers for R. solani AG-1 IA isolates mainly infecting rice.
In the present study, we developed a polygalacturonase gene based diagnostic PCR assay for detection of R. solani AG1 IA isolates infecting rice and validated its utility in disease detection in infected samples. Recently, our group reported a simple protocol (Rapid high throughput template preparation method: rHTTP method) to prepare PCR ready templates for a wide range of plants 28 which could be coupled to molecular diagnostics. Therefore, to further develop a simple strategy for on-field detection of the pathogen, a LAMP based assay (coupled with rHTTP method) was designed and compared to the conventional PCR based diagnostics.

Materials and methods
Field sampling. For fungal isolation. Infected leaf samples (rice and maize) were collected from Punjab and Uttar Pradesh ( Table 1). All samples were collected in polypropylene bags and stored at 4 °C prior to use. Isolation and morphological identification of R. solani AG-1 IA isolates from diseased samples. Infected tissue parts were cut (~ 1 cm), sterilized in 1% (w/v) sodium hypochlorite solution and placed on water agar for initial isolation. The pure cultures were characterized morphologically based on their appearance and sclerotial features.

Development and validation of diagnostic markers. Primer designing and conventional PCR assay
optimization. Available nucleotide sequences of polygalacturonase gene (HQ197936, HQ197944, FJ544456, KP896519, KP896521, KP896522) of R. solani AG-1 IA were downloaded from NCBI database and aligned with ClustalW in MEGA 7.0 29 . From the aligned sequences, conserved regions were identified. Four sets of forward and reverse primers were picked manually from these conserved regions (Table 2). Self complimentary and hairpin formation for the primer sets were checked using OligoCalc program 30 . Specificity of the primer sets were checked using primer BLAST tool (https ://www.ncbi.nlm.nih.gov/tools /prime r-blast /index .cgi) available at National Center for Biological Information (NCBI). Annealing temperatures for the primer sets were determined using gradient PCR. PCR assays were performed in a total volume of 25 μl by mixing 10 × PCR buffer (2.5 μl), dNTPs (1.5 μl, 50 µM each), 1 μl (10 mM) each of forward and reverse primer, 1.0 U of Taq DNA polymerase (Genei, Bangalore, India), and 2 µl of template DNA (~ 50 ng). PCR was performed in a G Storm GS4 thermal cycler (Somerset, UK) with initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 1 min, annealing for 1 min (RSPG1F/R, 2F/R and 4F/R-52.4 °C, RSPG5F/R-54 °C) (Here we defined these four primer sets as RSPG q-PCR primer sets), extension at 72 °C for 1 min and the final extension of 72 °C for 10 min. PCR products were electrophoresed on 1.5% agarose gel and the photographs were documented in a gel documentation system (Universal Hood 2, BioRad, USA). PCR products (amplified with RSPG2F/R) of 14 random isolates were purified using Wizard SV Gel and PCR Clean up System (Promega, USA) following manufacturers protocol and sequenced from Eurofins Scientific, India. Identity of the sequences was verified through performing BLAST search at NCBI (Supplementary Table S1). The sequences were deposited in NCBI Genbank as accession numbers MT882056-MT882069.
Microbial cultures for validation. Fungal and bacterial cultures procured from various sources (Table 3) were used for validation of PCR assay.
Molecular detection and sensitivity of the diagnostic markers using q-PCR. Molecular detection and sensitivity of the diagnostic markers were determined by q-PCR (G8830A AreaMx Real-Time PCR, Agilent Technologies, USA) using SYBR green I as fluorescent molecules. Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix was used for the reactions provided by Agilent Technologies, USA. The sensitivity assay for all the primers were performed using the protocol reported by Chakdar et al. (2019) with some modifications 20 . The real time qPCR assay was performed using all four RSPG primer sets (10 pmol/µl. q-PCR conditions were: initial heat activation at 95 °C for 10 min, followed by 40 cycles of amplification by 3 step cycling, denaturation was carried out at 95 °C for 5 s, annealing at (RSPG1F/R, 2F/R and 4F/R-52 °C, RSPG 5F/R-54 °C) for 30  www.nature.com/scientificreports/ 10 ng/µl, 1.0 ng/µl, 0.1 ng/µl and 0.01 ng/µl of DNA was prepared using nuclease free MiliQ water. A reference reaction setup was performed targeting actin gene. A no template control (NTC) was kept for all the experiments designed in order to check the specificity of the reactions. All dilutions of DNA (10-0.01 ng/µl) were used as template for performing the q-PCR experiments. All the experiments were carried out in triplicates. AreaMx V1.7 software was used to analyze the data generated. Efficiency of the q-PCR was calculated by plotting Ct values against -log (DNA concentration in g) and determining the regression equation.
LAMP assay validation, specificity, and sensitivity. LAMP assay standardization. Primers for the LAMP assay were designed using the polygalacturonase gene (NCBI accession number : MT882069) sequenced by using conventional PCR primer sets in this study. Primer Explorer v5 was used to design the primer sets (Eiken Chemical Co., Ltd., Tokyo, Japan) ( www.nature.com/scientificreports/ carried out as yellow colour development indicated positive reaction while red colour indicated no reaction. The amplified LAMP products were further observed on 2% agarose gel with EZ-Vision One as DNA dye (Amresco, USA) to confirm amplification (Fig. 4).
Sensitivity of LAMP assay. The sensitivity of the LAMP assay was determined using different R. solani AG-1 IA DNA concentrations in descending order by 10-fold serial dilution with sterilized double-distilled water from 165 ng/µl to 1.65 fg/µl. Serially diluted DNA (3 µl each) was used as template DNA in the LAMP reaction to quantify its sensitivity in a thermal cycler at a uniform temperature of 65 °C for 30 min.
Specificity of LAMP assay. To check the specificity, fungal and bacterial cultures listed in Table 3 were used. A "no template control" was kept in all experimental set ups and the experiments were repeated three times.
In vitro and pot experiments for validation of LAMP assay. Leave sheaths of rice were infected using virulent strains of R. solani AG-1 IA both by moist chamber technique and rice plants grown in pots in order to check early detection of this pathogen 34 . In case of a moist chamber, leaves were cut to 2 cm in size, placed on a glass slide and the set up was kept on moist filter paper inside sterile glass petri dishes. Sclerotia was placed on the top of the leaves and the petri dishes were incubated at 28 ± 2 °C. While infecting leaves in a moist chamber, disease severity was kept at three levels (1 sclerotia-+, 2 sclerotia-++, and 3 sclerotia-+++) in order to test the applicability of LAMP assay with different inoculum loads of disease occurance. In case of rice plants in pots, the sclerotia was placed in the leaf sheath and covered with moist cotton to provide appropriate conditions for the infection process. The pots were maintained in net house conditions. All the healthy and infected samples were collected after 24 h to perform the LAMP assay.
Validation of LAMP assay using soil DNA. To determine the applicability of the LAMP method, soil samples were collected from rice fields at ICAR-IISS, Maunath Bhanjan, India. From 1 m 2 area, four samples each weighing 50 g were collected, dried under shade, finely ground using sterile mortar-pestle and sieved. 25 g from each of these four samples were pooled together. Total soil DNA was extracted from the pooled sample in six replications using a commercial kit (FastDNA Spin Kit for Soil, MP Biomedicals, USA) following minor modifications to manufacturer's protocol. DNA was quantified using Nano Drop and pooled DNA sample was used for the LAMP assay. The time for LAMP assay was optimized and the final assay conditions were 65 °C for 45 min. The rice field was monitored for disease incidence for one month after the collection of soil samples. Plant. Total genomic DNA from plant material was extracted according to DNA extraction methodologies described by Doyle and Doyle with modifications from the original method 32,33 . Leaf samples were washed and dried with paper towels to eliminate excess dirt. 0.5 g leaf tissue was used for DNA extraction. DNA extracted from infected and healthy leaf samples was used as template for specific detection of R. solani AG-1 IA in rice tissue. For all the LAMP assays, rHTTP method was followed for template preparation directly from infected and healthy tissues 28 .  Table S2). Distinct morphological features of the isolates established their identity as Rhizoctonia solani AG-1 IA. All the morphologically identified isolates were deposited in NAIMCC at ICAR-NBAIM, Mau as NAIMCC F-03220-03039 (Table 1).  (Fig. 1A-D). The results confirmed RSPG q-PCR primer sets markers as specific for R. solani AG-1 IA. Validation using live infected samples confirmed that the primers designed in this study could clearly detect R. solani AG-1 IA from the genomic DNA extracted from infected leaf sheath samples (Fig. 2). No amplification in healthy parts of the plant obtained confirmed the applicability of RSPG primers as diagnostic markers for the specific detection of phytopathogenic R. solani AG-1 IA.

Molecular detection and sensitivity of diagnostic markers by q-PCR.
When the DNA extracted from leave sheaths with early symptoms (tiny brown spots) indicating probable R. solani infection, was subjected to real time PCR using RSPG q-PCR primer sets, amplification was observed till 0.01 ng/µl (Fig. 3). All the primers could detect 0.01 ng DNA/µl with efficiency ranging from 91 to 97.5% (Supplementary Table S3, File. [5][6][7][8]. No peaks were observed in no template control (NTC) indicating the specificity of the diagnostic markers. RSPG q-PCR primer sets gave good results with clear fluorescence peaks. The fluorescent peaks corresponding to the amplicons were centered around 84 °C (Supplementary Fig. S1).
Validation and specificity of the LAMP assay for R. solani AG-1 IA detection. The primer set designed for the study targeted polygalacturonase gene sequenced during the study (Supplementary Fig. S2). The diagnostic marker, once validated, was used for designing the LAMP primers. In silico primer BLAST results for all the primers gave specific hits to R. solani AG-1 IA. The R. solani AG-1 IA isolates used in the study showed positive reactions with the RSPG LAMP primer sets, but did not amplify in other tested AG groups (AG 1-IB, 2-2IIIB, www.nature.com/scientificreports/ 3, 7 & 8) and other major rice pathogens (Fig. 4, Supplementary Figs. S3,S4). The LAMP assay and conventional PCR showed the same specificity when validated with other prominent rice pathogens including cross kingdom specificity. The assay could detect the target gene in 1.65 fg/µl of the template DNA (Fig. 5). rHTTP method was used for the template preparation from diseased plant tissues having sheath blight symptoms. The template was then tested for LAMP assay. Thus, the total time taken for the assay was 45 min (rHTTP: 15 min and LAMP: 30 min).  www.nature.com/scientificreports/ The LAMP assay was also able to detect the pathogen from artificially infected leaf samples with varying degree of disease severity appeared after 24 h of inoculation (Fig. 6). The primer sets were highly specific as it could differentiate between the healthy and infected plant tissue samples showing initial symptoms collected from pot trials just 24 h after the inoculation (Fig. 7). The results obtained highlighted the fact that the LAMP assay developed in this study could efficiently and accurately detect the disease as soon as the pathogen gets entry to the plant. The assay was further validated using DNA from soil samples from rice fields. The extracted soil DNA samples were quantified (160-180 ng/µl). The LAMP assay gave good results when the assay time was of 45 min for the visual confirmation in the case of soil DNA as template (Fig. 7, Supplementary Fig. S5). The fields from which the soil samples were taken had severe symptoms of Sheath Blight Disease (SBD) when observed after one month of collection of soil samples (Fig. 7c).

Discussion
Accurate identification of R. solani AG-1 IA is essential for its effective management. Excessive and inappropriate use of anti-fungal agents not only hampers the crop production but also damage soil health jeopardizing human health and livestock eventually 35 . SBD of rice hampers the production of rice worldwide ranging from 8 to 50% loss in yield 36 . Many studies are being carried out to increase the resistance of plants against sheath blight but it also involves precise detection of the pathogen 36,37 . www.nature.com/scientificreports/ There have been efforts for molecular detection of R. solani but no study is reported till date of using a virulence factor as the basis of molecular detection of the pathogen. Polygalacturonase encoding genes form the basis for the infection process of R. solani among its various pathotypes 6,38 . This study used PG gene to design four sets of primers and thus the detection is based on virulence factor of the pathogen. The primer sets were tested with the sheath samples collected from farmer's field where chances of mixed infection remained in a high proportion. Specific bands were seen which detect R. solani AG-1 IA among other close relatives of fungal kingdom. These molecular markers can be used for detection of R. solani AG-1 IA infection and thus effective use of fungicides can be monitored and will definitely help to devise control strategies for reducing economic losses.
In this study, a total 51 R. solani AG-1 IA isolates were obtained from diverse agro-ecological regions of India. An important virulence factor i.e., polygalacturonase gene based diagnostic markers were validated on these isolates of R. solani AG-1 IA. PCR based detection of pure cultures of R. solani AG-1 IA isolates showed positive amplification while no amplification was obtained for other fungi or bacteria indicating its specificity for R. solani AG-1 IA. Further, the designed primer sets could clearly distinguish between healthy and R. solani infected leaf samples and even detect the presence of pathogen in samples. Earlier, efforts have been made to detect R. solani with distinct pathotypic and genetic diversity. Majority of the studies carried out yet reported no significant relationships between genetic diversity and aggressiveness or geographic origin among populations of R. solani AG-1 IA 39,40 . Virulence is independent of molecular variation, and the high levels of genetic recombination may also contribute to new genotypes with a high degree of variation in virulence 41,42 .
As use of PCR based markers is not easy and convenient in the fields, it was required to go for isothermal amplifications which are more convenient to be used in fields. Earlier, Lu et al. reported an ITS gene based LAMP assay to detect R. solani causing soybean seedling blight 17 . The assay took more than 2 h and the DNA extraction was tedious and costly which is not suitable for developing point-of-care diagnostics. Patel et al. also targeted ITS region to detect R. solani AG-4 in Dypsis lutescens, Fittonia, Dianthus and Begonia 16 . Both the studies did not target SBD of rice and targeted the same region of the genome (ITS) for developing the LAMP assay. This study might be the first attempt to develop a LAMP based assay for the detection of R. solani AG-1 IA in rice. More importantly, visual confirmation provided by this method and the isothermal amplification property made this method more convenient for pathogen detection. The assay was first optimized using fungal DNA from pure cultures of isolates. In case of detection from live infected tissues (both in vitro and pot trial infections) rHTTP method was employed for template preparation 28 . The integration of rHTTP and LAMP assay approach required a total of only 45 min from sample preparation to pathogen detection. The rHTTP method uses a single temperature during the entire template preparation method, hence the whole experimental set up (right from sample collection to detection) can be performed in any portable water bath or hot plate. LAMP was more simple, cost effective and required less complex instrumentation systems. Moreover, it could detect the pathogen as early as 24 h after the infection (Fig. 7b). Hence, this would be the most appropriate method for on field detection studies. Studies have been reported which have highlighted the use of LAMP based assays for the detection of important phytopathogens like Rhizoctonia bataticola 43 , Talaromyces flavus 44 , Alternaria solani 45 , Didymella bryoniae, etc.
Further validation of the assay was done with soil samples collected from a rice field. Results indicated that the optimized assay could successfully detect the presence of pathogen from soil samples although it took a little more time for optimal amplification. The sampling site was marked and kept under observation. As our assay results suggested, we observed devastating symptoms of R. solani AG-1 IA infection in the rice plants one month after the sampling date (Fig. 7c). This observation substantiates the potential use of this LAMP based assay for early monitoring sheath blight of rice. Reports on biological control of sheath blight suggest that control measures, if backed up by affordable point of care diagnostic methods as achieved in this study, may be the possible solution for farmers who suffer heavy losses each year due to SBD 46,47 .

Conclusion
The main features of the developed method are: (1) colorimetric detection, (2) requires less time (45 min), (3) minimum instrumentation required, (4) detection at early stages of the disease, (5) applicable for environmental samples including diseased tissues and soil, (6) coupling with rHTTP further reduces cost, time and effort. With these results in place, we propose this LAMP assay to be used in order to plan effective management strategies so that the damage caused by this phytopathogen can be minimized. Although, the conventional PCR based assays were also quite sensitive but their requirement for instrumentation and lack of visual confirmation make them unsuitable for use in field conditions.