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 significant 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 identification 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 first 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 magnification 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 amplification 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 final extension for 10 minutes at 72°C. The PCR program for the amplification 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 final extension was done for 10 minutes at 72°C. The PCR amplified 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 MgSO4 in the reaction mixture, a total of five different concentrations of Mg2+ (2.00, 4.00, 6.00, 8.00 and 10.00 mM) were tested along with nuclease free water without MgSO4 (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 reconfirmed by assessment using HNB, EtBr, as well as resolving in 1.0 per cent agarose gel electrophoresis.
LAMP specificity assay
The specificity 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 specificity of the assay was assessed based on HNB-visualized color change, EtBr visualization and further confirmed with 1.0 per cent agarose gel electrophoresis.
LAMP sensitivity assay
The sensitivity of LAMP assay was evaluated by ten-fold serial dilution of purified 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 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 confirmed 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, 0.2 μM each of F3 and B3 primers, 0.4 μM each of the LF and LB primers, 1.4 mM each of dNTPs, 2.5 μl of 10X Thermophol reaction buffer, 4 mM MgSO4, 8U of Bst DNA polymerase (New England Biolabs, Ipswich, MA), 0.8 M betaine solution, 120 μM hydroxynaptholblue (HNB) indicator and 2.5 μl of DNA (~100 ng) template. The reaction tubes were incubated at 65°C for 60 minutes and terminated by thermal inactivation at 80°C for 2 minutes in eppendorf thermal cycler. The LAMP amplicons were visualized by different strategies: (a) visualization under hydroxy naphthol blue (HNB), (b) visualization under ethidium bromide (EtBr) and (c) 1.0 per cent agarose gel electrophoresis.