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. 2022 Nov 21:13:1077328.
doi: 10.3389/fpls.2022.1077328. eCollection 2022.

A colorimetric hydroxy naphthol blue based loop-mediated isothermal amplification detection assay targeting the β-tubulin locus of Sarocladium oryzae infecting rice seed

R Logeshwari  1 , C Gopalakrishnan  1 , A Kamalakannan  1 , J Ramalingam  2 , R Saraswathi  3
Affiliations

A colorimetric hydroxy naphthol blue based loop-mediated isothermal amplification detection assay targeting the β-tubulin locus of Sarocladium oryzae infecting rice seed

R Logeshwari et al. Front Plant Sci. .

Abstract

Sarocladium oryzae is a widely prevalent seed-borne pathogen of rice. The development of a rapid and on-site detection method for S. oryzae is therefore important to ensure the health of rice seeds. Loop-mediated isothermal amplification (LAMP) is ideal for field-level diagnosis since it offers quick, high-specific amplification of target template sequences at a single temperature. We designed primers based on the β-tubulin region of S. oryzae. The LAMP technique devised was extremely sensitive, detecting the presence of the S. oryzae template at concentrations as low as 10 fg in 30 minutes at 65°C. The assay specificity was confirmed by performing the experiment with genomic DNA isolated from 22 different phytopathogens. Through the addition of hydroxy naphthol blue in the reaction process prior to amplification, a colour shift from violet to deep sky blue was seen in the vicinity of the target pathogen only. Finally, the LAMP assay was validated using live infected tissues, weeds and different varieties of seeds collected from different locations in Tamil Nadu, India. If developed into a detection kit, the LAMP assay developed in this study has potential applications in seed health laboratories, plant quarantine stations, and on-site diagnosis of S. oryzae in seeds and plants.

Keywords: HNB dye; Loop-mediated isothermal amplification; Oryza sativa; Sarocladium oryzae; seed; β-tubulin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Diagrammatic illustration of in silico hybridization of LAMP primers positioned within the β-tubulin (713 bp) domain of the pathogen Sarocladium oryzae visualized using SnapGene viewer® 6.1.1. Outer primers: F3 and B3 (blue), loop primers: LF and LB (purple), inner primers: F2 and F1c (Red) and B1c and B2 (green).
Figure 2
Figure 2
Comparison of the β-tubulin region of S. oryzae with other Sarocladium spp.
Figure 3
Figure 3
Sensitivity of LAMP assay using ten-fold serially diluted genomic DNA (from 100ng to 1 fg) from Sarocladium oryzae isolate OM841519 as template and nuclease free water as negative control. Upper panel in the figure shows agarose gel electrophoresis results whereas lower panel shows reaction tubes for colorimetric detection. M- 100bp Ladder; Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1 pg; Lane 7: 100 fg; Lane 8: 10fg; Lane 9: 1 fg; Lane 10: Nuclease free water (Control).
Figure 4
Figure 4
Specificity of LAMP assay using Sarocladium oryzae isolate OM841519 as template and nuclease free water as negative control. Upper panel in the figure shows agarose gel electrophoresis results whereas lower panel shows reaction tubes for colorimetric detection. M- 100bp Ladder; Lane 1: Sarocladium oryzae; Lane 2: Sarocladium attenuatum; Lane3: Sarocladium strictum; Lane 4: Bipolaris oryzae; Lane 5: Magnaporthe oryzae; Lane 6: Rhizoctonia solani; Lane 7: Ustilaginoidea virens; Lane 8: Exserohilum rostratum; Lane 9: Simplicillium obclavatum; Lane 10: Pyricularia grisea; Lane 11: Helminthosporium turcicum; Lane 12: Macrophomina phaseolina; Lane 13: Sclerotium rolfsii; Lane 14: Sclerotinia sclerotiorum; Lane 15: Colletotrichum gloeosporioides; Lane 16: C. scovillea; Lane 17: C. truncatum; Lane 18: Fusarium oxysporum f. sp. cubense; Lane 19: Phytophthora infestans; Lane 20: Puccinia arachidis; Lane 21: Uncinula nector; Lane 22: Pantoea dispersa; Lane 23: Ralstonia solanacearum; Lane 24: Nuclease free water (Negative control).
Figure 5
Figure 5
Validation of LAMP assay with S. oryzae infected field samples. Bright sky blue colour represents positive reaction while the violet colour indicates the negative reaction. Upper panel in the figure shows agarose gel electrophoresis results whereas lower panel shows reaction tubes for colorimetric detection. M- 100bp Ladder; Lane 1: Sarocladium oryzae isolate OM841519 (Positive control); Lane 2: Coimbatore infected sheath sample 1; Lane 3: Coimbatore infected sheath sample 2; Lane 4: Coimbatore infected sheath sample 3; Lane 5: Coimbatore healthy sheath sample; Lane 6: Coimbatore infected seed sample 1; Lane 7: Coimbatore infected seed sample 2; Lane 8: Coimbatore infected seed sample 3; Lane 9: Coimbatore healthy seed sample; Lane 10: Villupuram infected sheath sample 1; Lane11: Villupuram infected sheath sample 2; Lane 12: Villupuram infected sheath sample 3; Lane 13: Villupuram healthy sheath sample; Lane 14: Villupuram infected seed sample 1; Lane 15: Villupuram infected seed sample 2; Lane 16: Villupuram infected seed sample 3; Lane 17: Villupuram healthy seed sample; Lane 18: Sarocladium oryzae isolate OM841519 (Positive control); Lane 19: Trichy infected sheath sample 1; Lane 20: Trichy infected sheath sample 2; Lane 21: Trichy infected sheath sample 3; Lane 22: Trichy healthy sheath sample; Lane 23: Trichy infected seed sample 1; Lane 24: Trichy infected seed sample 2; Lane 25: Trichy infected seed sample 3; Lane 26: Trichy healthy seed sample; Lane 27: Sarocladium oryzae isolate OM841519 (Positive control); Lane 28: Artificially inoculated seed sample 1; Lane 29: Artificially inoculated seed sample 2; Lane 30: Artificially inoculated seed sample 3; Lane 31: Artificially inoculated seed sample 4; Lane 32: Artificially inoculated seed sample 5; Lane 33: Surface sterilized uninoculated seed sample; Lane 34: Nuclease free water (Negative Control).
Figure 6
Figure 6
Validation of LAMP assay with S. oryzae infected paddy and weed samples. Bright sky blue colour represents positive reaction while the violet colour indicates the negative reaction. Total DNA isolated from fungi was used as the positive control. Upper panel in the figure shows agarose gel electrophoresis results whereas lower panel shows reaction tubes for colorimetric detection. M- 100bp Ladder; Lane 1: Oryza sativa; Lane 2: Echinchloa crusgalli: Lane 3: Cyperus difformis; Lane 4: Eleusine indica; Lane 5: Dactyloctenium aegyptium; Lane 6: Cyperus iris; Lane 7: Eragrostis amabilis; Lane 8: Leersia hexandra; Lane 9: Phalaris minor; Lane 10: Brachiaria mutica; Lane 11: Sarocladium oryzae isolate OM841519 (Positive control); Lane 12: Nuclease free water.
Figure 7
Figure 7
Validation of LAMP assay with naturally infected seeds of different varieties. Bright sky blue colour represents positive reaction while the violet colour indicates the negative reaction. Upper panel in the figure shows agarose gel electrophoresis results whereas lower panel shows reaction tubes for colorimetric detection. M- 100bp Ladder; Lane 1: Sarocladium oryzae isolate OM841519 (Positive control); Lane 2: Co 39; Lane 3: Co 51; Lane 4: Co 52; Lane 5: Co 53; Lane 6: Co 54; Lane 7: Co 55; Lane 8: ADT 37; Lane 9: ADT 43; Lane 10: RNR 15048; Lane11: TN 1; Lane 12: TPS 5; Lane 13: TRY 43; Lane 14: ASD 16; Lane 15: Nuclease free water (Negative Control).
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