Development of Loop mediated isothermal ampli�cation (LAMP) assay for the detection of Magnaporthe oryzae causing blast in rice

Rice is one of the most important nourishment crops providing a quarter of calories consumption. It alone contributes 23 per cent of calories consumed by people all over the world. Rice blast pathogen is an important ascomycetes fungus which causes severe yield losses up to 100 per cent under favorable climatic conditions. A �eld survey on rice blast disease revealed that the disease incidence was ranged from 50.1% - 72.46% with the highest disease incidence of 72.46% at Coimbatore district, Tamil Nadu, India. Totally seven isolates of Magnaporthe oryzae were collected and the identity was con�rmed through morphological and molecular con�rmation. A loop-mediated isothermal ampli�cation assay was developed by targeting Pita 2 gene sequence of M. oryzae. The assay developed was more sensitive as it detected the genomic DNA of M. oryzae up to 10 fg. The speci�city of LAMP assay was proved by carrying out the assay with genomic DNA extracted from other fungal pathogens. Therefore, the LAMP assay developed will be helpful in rapid, speci�c and sensitive detection of rice blast pathogen at �eld level and will help in mitigating the disease incidence.


Introduction
Rice (Oryza sativa L.) is one of the most important cereal crops next to wheat and also most important staple food, which contributes approximately 30 per cent of nutritional intake (Gnanamanickam, 2009). It is widely grown in India, China and the rest of Asia where the maximum of 92 per cent of world's rice is grown. India is the second largest country in rice production next to China. The rice crop suffers from number of diseases, among the different diseases infecting rice, blast is one of the most destructive disease around the world. This disease was incited by a fungal pathogen called Magnaporthe oryzae (Synonym: Pyricularia oryzae Cavara) which results in extensive yield losses. Every year the rice crop will face severe yield losses up to 100% under favourable conditions (Liu et al., 2013). The pathogen M. oryzae is an ascomycetes fungus encompasses hundreds of races (pathotypes) around the world (Jia et al., 2014). The pathogen is quite capable to infect the rice plants at any stage of its growth period from seedling to grain formation and causes multifarious infection like leaf blast, collar rot, nodal blast and neck blast or panicle blast (Gowda et al., 2015). Therefore, it is necessary to identify the pathogen at earlier stage of its infection which helps to take up the timely management practices.
The identi cation of pathogens through morphometric analysis and molecular con rmation through nucleic acid based detection like PCR were time consuming, laborious and require skilled labours (Notomi et al., 2000). The sensitivity of the techniques was adversely affected by PCR inhibitors, particularly when inoculum levels are low or near detection limits. Loop mediated isothermal ampli cation assay (LAMP) is an novel detection technique, which was applied for the successful detection of several plant pathogens (Notomi et al., 2000, Thiessen et al., 2016, Tomlinson et al., 2010, Villari et al., 2017. This method requires DNA polymerase, and a set of four specially designed primers that recognize six different regions on the target DNA template. This method has been widely applied under eld condition for on-site detection, because of its low cost, high speci city, e ciency, simplicity of operation and rapidity (Niessen and Vogel, 2010). The entire reaction will be completed under isothermal condition in conventional water bath.
In order to mitigate the yield losses and to take up timely management practices, early detection and e cient diagnosis of plant diseases are needed. LAMP is a rapid technique emerging as a quick diagnostic tool for advanced detection and identi cation of plant diseases. While this helps to improve research on detection and reduces time for detecting plant pathogens during eld analysis. In this study, we have developed a LAMP assay protocol for rapid, early, speci c and sensitive detection of M. oryzae infecting rice for the rst time in India.

Materials And Methods
Survey and pathogen isolation A roving survey was conducted in Coimbatore and Erode districts of Tamil Nadu, India during Kharif season of 2018-19 in major rice growing areas to access the incidence of rice blast disease. The rice blast disease incidence was assessed by utilizing the scale of IRRI, 1996 as follows. 0-No lesion observed (Highly Resistant), 1-Small brown specks of pin point size (or) larger brown specks without sporulating centre (Resistant), 2-Small roundish to slightly elongated, necrotic gray spots, about 1-2 mm in diameter, with a distinct brown margin (Moderately Resistant), 3-Lesion type is the same as in scale 2, but signi cant numbers of lesions are on the upper leaves (Moderately Resistant), 4-Typical susceptible blast lesions of 3 mm or longer, infecting less than 4% of leaf area (Moderately Susceptible), 5-Typical blast lesions infecting 4-10% of the leaf area (Moderately Susceptible), 6-Typical blast lesions infecting 11-25% of the leaf area (Susceptible), 7-Typical blast lesions infecting 26-50% of the leaf area (Susceptible), 8-Typical blast lesions infecting 51-75% of the leaf area many leaves are dead (Highly Susceptible), 9-Typical blast lesions infecting more than 75% leaf area affected (Highly Susceptible). Finally, by using Mckinney (1923) formula, per cent disease index (PDI) of rice blast was calculated A total of seven rice blast diseased leaf samples were collected from Coimbatore and Erode districts of Tamil Nadu, India. A fungus was constantly isolated from the blast infected leaves by tissue segment method on PDA medium. The cultures were maintained at 4°C for further identi cation and characterization.

Morphological and molecular characterization of M. oryzae
The actively growing mycelia were taken from the edge of 9 days old mother cultures of each isolate placed on PDA medium. The radial growth of different isolates was measured daily from the rst day after inoculation until maximum growth on the Petri dishes. Radial growth of the isolates was compared on the 10th day after inoculation. The length and breadth of the conidia of seven M. oryzae isolates were measured using a light microscope of 400X magni cation and photographed in Image Analyser. Molecular characterization of rice blast pathogen was done using fungal culture of all seven isolates. A fresh fungal culture from each isolates was inoculated into 100 ml of PDA broth. A 100 g of 14 day's old dried fungal mat was harvested from the broth and subjected to genomic DNA isolation by Cetyl Trimethyl Ammonium Bromide (CTAB) method. The genomic DNA was checked by gel electrophoresis and DNA concentrations of the samples were determined using a spectrophotometer (Nanodrop, ND-1000, Wilmington, DE) and stored at -20°C for further use. The conventional PCR was performed at 20 μl mixture containing 2 μl of genomic DNA (~ 50 ng/ μl), 10 μl of TaKaRa master mix (2 X concentration) and 2 μl of each forward and reverse primers (20 pmol). The reaction was carried out in eppendorf thermocycler. The PCR ampli cation of ITS region consisted of an initial denaturation of 4 minutes at 94°C followed by 40 cycles of 2 minutes of denaturation at 94°C, 45 seconds of annealing at 53 °C, 2 minutes of extension at 72°C and a nal extension for 10 minutes at 72°C. The PCR program for the ampli cation of Pot 2 transposon region consisted of an initial denaturation of 4 minutes at 94°C followed by 40 cycles of 45 seconds of denaturation at 94°C, 45 seconds of annealing at 55°C, and 45 seconds of extension at 72°C. The nal extension was done for 10 minutes at 72°C. The PCR ampli ed products were visualized under UV and the images were documented with an Alpha Imager EC (USA).

LAMP primers designing
The Pita 2 gene of M. oryzae was selected as a target site for designing LAMP primers. The primer sequences were designed using Primer Explorer version 5.0 software on the Eikon Genome site. All parameters viz., GC content, melting temperature, distance between the primer ends were as per the default setting.
Optimization of LAMP reaction LAMP assay was performed using Magnaporthe oryzae DNA as template to determine the optimum reaction temperature and time. To determine the optimum temperature, the assay was tested with a range of temperature (56 to 68 °C) using pure DNA of M. oryzae and to determine the optimum time, the assay was tested with a range of time (30 to 120 minutes). The reaction was terminated by heat inactivation at 80 °C for 2 minutes.
To optimize the concentration of MgSO 4 in the reaction mixture, a total of ve different concentrations of Mg 2+ (2.00, 4.00, 6.00, 8.00 and 10.00 mM) were tested along with nuclease free water without MgSO 4 (negative control). The concentration of other components such as LAMP primers, Thermophol reaction buffer, dNTPs, Bst DNA polymerase, betaine, hydroxynaptholblue (HNB) indicator, DNA template and water in LAMP assay was kept constant. The tubes containing 25 μl reaction mixtures were incubated at 65°C for 60 minutes and reaction was terminated by heat inactivation at 80°C for 2 minutes. The results were recon rmed by assessment using HNB, EtBr, as well as resolving in 1.0 per cent agarose gel electrophoresis.

LAMP speci city assay
The speci city of LAMP assay was determined using the total genomic DNA isolated from M. oryzae and other plant pathogens like Helminthosporium oryzae infecting rice (Brown spot), Plasmopara viticola infecting grapes (Downy Mildew), Erysiphe necator infecting grapes (Powdery mildew), Fusarium oxysporum f. sp. cubense infecting banana (Panama wilt), Colletotrichum capsici infecting chilli (Chilli anthracnose), Pernoscleropsora sorghi infecting sorghum (Sorghum downy mildew) and Sclerospora graminicola infecting bajra (Cumbu downy mildew) along with nuclease free water serves as negative control. The reaction mixtures (25 μl) with template DNA of different fungal pathogens in each tube were incubated at 65 °C for 60 minutes and reaction terminated by heat inactivation at 80 °C for 2 minutes. The speci city of the assay was assessed based on HNBvisualized color change, EtBr visualization and further con rmed with 1.0 per cent agarose gel electrophoresis.

LAMP sensitivity assay
The sensitivity of LAMP assay was evaluated by ten-fold serial dilution of puri ed genomic DNA of M. oryzae from 100 nano gram to 1 femto gram (100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg). The 25 μl of reaction mixtures with different concentration of serially diluted genomic DNA of M. oryzae in each tube Page 5/22 were incubated at 65°C for 60 minutes and reaction was terminated by heat inactivation at 80°C for 2 minutes.
The sensitivity of the assay was assessed based on HNB-visualized color change, EtBr visualization and further con rmed with 1.0 per cent agarose gel electrophoresis.

Detection of M. oryzae by LAMP
The LAMP assay was performed in a 25 μl reaction mixture containing 1.4 μM each of the FIP and BIP primers, 0.

Survey and pathogen isolation
Field survey results indicated that the highest incidence of rice blast infection was observed in Poosaripalayam village of Coimbatore district with PDI of 72.46%, followed by 67.75%, 60.63% in Booluvampatti and Bhavanisagar, respectively. The Gobichettipalayam village of Erode district showed the lowest incidence of 50.10% (Table 1). A fungus was constantly isolated from the rice leaf samples showing spindle shaped lesions on PDA medium. A total of seven isolates were isolated from infected leaf samples and stored at 4°C for characterization ( Figure 1).

Morphological and molecular characterization of M. oryzae
The fungus mycelium was hyaline, septate and branched. The conidiophores were thin walled, slender and unbranched. The fungus produced pyrifom conidia with 2 septation and hilum at base ( Figure 2). The genomic DNA samples of M. oryzae obtained from mycelium of all the seven isolates were subjected to PCR ampli cation using universal primers ITS 1 and ITS 4. PCR ampli cation yielded a fragment of expected amplicon size of 550 bp ( Figure 3). The ampli ed products of all the isolates was sequenced at Euro ns genomics India Pvt. Ltd. and con rmed as M. oryzae by comparing with the sequences already deposited in NCBI database. The comparison of nucleotide sequences showed an identity of 97-100% with the M. oryzae isolates available in GenBank (CP034204, MH859782, MF583153, LK932250, MF583148, KJ522980 and KM816801). The sequences of all seven isolates were deposited in GenBank and accession numbers were obtained ( Table 2). The genomic DNA samples were further ampli ed using species speci c primer set targeting Pot 2 transposon gene. The PCR ampli cation generated an amplicon size of approximately 680 bp for all the seven isolates authenticating the fungus as M. oyzae (Figure 4).

LAMP primers designing
The Pita 2 gene sequence (GenBank accession no: AB607344) of M. oryzae was selected as the target region for designing of LAMP primers. We hypothesized that targeting the speci c gene sequence (Pita 2) through LAMP assay may be more appropriate for accurate detection with 100 per cent e ciency. There are totally six primers (inner primers (FIP and BIP), outer primers (F3 and B3) and Loop primers (F loop and B loop)) designed using Primer Explorer v.5 software. The designed primers were projected in Table 3.

LAMP optimization
The optimum LAMP reaction conditions such as temperature and time for the detection of Magnaporthe oryzae were determined. Among the ve different temperatures tested, positive reaction of sky blue color change was observed at almost all the temperature except at 56°C. However, the ampli cation e ciency exhibited a strong increase at 65°C, where the discrete ladder like pattern was more consistent and emitted a strong uorescence under EtBr visualization (Table 4 and Figure 5). When the LAMP was performed at 65°C with range of test time, colour change was observed at two different temperatures. However, the ampli cation e ciency exhibited a strong increase at 60 minutes of incubation time under HNB visualization, EtBr uorescence and the discrete ladder like pattern was more consistent at the same test time (Table 5 and Figure 6).
Among ve different concentrations of Mg 2+ (2.00, 4.00, 6.00, 8.00 and 10.00 mM) tested, the colour change was observed only at 4.00, 6.00 and 8.00 mM of Mg 2+ . However, the strong sky blue colour development was observed

LAMP speci city assay
The speci city of LAMP assay was tested with 8 fungal pathogens with two indicator dyes like HNB and EtBr. The genomic DNA of fungal pathogens like Magnaporthe oryzae, Helminthosporium oryzae, Plasmopara viticola, Fusarium oxysporum f.sp. cubense, Colletotrichum capsici, Erysiphe necator, Pernoscleropsora sorghi and Sclerospora graminicola were subjected to LAMP assay. After incubation of LAMP reaction mixture at 65 °C for 60 minutes, a strong sky blue color development was observed only with M. oryzae DNA. However, LAMP reactions with the templates of other fungal isolates remained violet, indicating the absence of target DNA. A similar kind of results was obtained with the LAMP assay performed using EtBr indicator dye. The LAMP reaction with M. oryzae DNA developed a strong uorescence, whereas the LAMP reaction with other non target pathogens did not produce uorescence. The above results were con rmed using agarose gel electrophoresis. A discrete intense ladder like banding pattern was observed only in LAMP products with M. oryzae DNA. Ladder like banding pattern was absent in LAMP products with other fungal isolates and nuclease free water (negative control). This indicated that the developed LAMP assay was highly speci c for the detection of M. oryzae (Table 7 and Figure  8).

LAMP sensitivity assay
To determine the detection limit of LAMP assay, a sensitivity test was performed with different DNA concentration of M. oryzae with two indicator dyes namely HNB and EtBr. The results showed that the sky blue color development was visible in almost all the concentration of genomic DNA tested, except at 1 fg. A similar kind of results was obtained while performing the test with EtBr dye. LAMP reaction with EtBr produced uorescence from 100 ng to 10 fg except 1 fg of genomic DNA. The above results were con rmed using agarose gel electrophoresis assay where discrete ladder-like banding pattern was visible upto 10 fg DNA concentration. These results indicated that the developed LAMP assay could detect up to 10 fg of DNA of M. oryzae (Figure 9).

Detection of M. oryzae by LAMP
The LAMP assay speci c to M. oryzae was developed and tested against genomic DNA of seven isolates of M. oryzae study isolates with two indicator dyes such as HNB and EtBr. The strong sky blue color development was observed in all the tubes containing DNA of M. oryzae isolates. However, the tube with nuclease free water as a negative control did not showed any sky blue color development, indicating the absence of M. oryzae DNA.
A similar kind of results was obtained in LAMP reaction with EtBr indicator dye, where a strong uorescence was observed in all the tubes containing genomic DNA of M. oryzae. The absence of uorescence in the tube with nuclease free water indicated the absence of target DNA. The above LAMP products were con rmed with 1 per cent agarose gel electrophoresis assay. Agarose gel electrophoresis assay yielded a discrete ladder-like banding pattern for genomic DNA from M. oryzae isolates. Whereas no such ladder-like banding pattern was observed with nuclease free water ( Figure 10).

Discussion
M. oryzae causing rice blast disease is a major threat to rice cultivation at global level. A quick and on-site detection of pathogens will bring down the chances for the failure of rice crop thereby improves the yield. The conventional detection of rice blast pathogen based on symptoms and PCR which are time consuming and require skilled labor. LAMP assay is a rapid and more sensitive detection technique and it can be detected more number of samples at a time. Recently, many plant pathogens viz., Magnaporthe grisea, Alternaria solani, Plasmopara viticola, Erysiphe necator, Puccinia graminis f.sp. tritici has been successfully detected using LAMP assay. It has several advantages over conventional PCR assay such as ampli cation of DNA at very low concentration, speci c detection within 20 to 60 minutes, can be performed using water bath and visual interpretation of results with the help of indicator dyes. In present study, we strongly recommend LAMP assay for the on-site detection of rice blast pathogen, since it requires minimum quantity of DNA (10 fg) which is much lesser than that of the quantity required for conventional PCR. To the best of our knowledge, this is the rst report of application of LAMP assay in LAMP assay serves as a promising diagnostic kit, by forecasting the pathogen in advance. This will aid in designing of effective prediction models for the outbreak of diseases. Since, the early detection of the pathogen is achieved the protective measures can be taken much earlier by application of prophylactic fungicides.