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. 2022 Aug 18:13:936766.
doi: 10.3389/fpls.2022.936766. eCollection 2022.

Colorimetric loop-mediated isothermal amplification assay for detection and ecological monitoring of Sarocladium oryzae, an important seed-borne pathogen of rice

Prassan Choudhary  1 , Sanjay Kumar Goswami  1   2 , Hillol Chakdar  1 , Shaloo Verma  1 , Shobit Thapa  1 , Alok Kumar Srivastava  1 , Anil Kumar Saxena  1
Affiliations

Colorimetric loop-mediated isothermal amplification assay for detection and ecological monitoring of Sarocladium oryzae, an important seed-borne pathogen of rice

Prassan Choudhary et al. Front Plant Sci. .

Abstract

Accurate and timely disease detection plays a critical role in achieving sustainable crop protection. Globally, rice has been a staple crop for centuries plagued by the diseases that greatly hamper its productivity. Sheath rot, an emerging disease of rice caused by the seed-borne pathogen Sarocladium oryzae, has reportedly caused heavy losses to agricultural produce in recent years. Our study has led to the development and validation of a LAMP assay for early detection of S. oryzae, the causal agent of sheath rot from the live-infected tissues, seeds, weeds, and environmental samples. The assay could detect as low as 1.6 fg/μl of the pathogen in 15 min. The assay was implemented to bio-surveil the presence of this pathogen by testing it on three weed species (Echinochloa colona, Echinochloa crus-galli, and Cyperus teneriffae) growing around the rice fields. The results showed the presence of the pathogen in two of the weed species viz. E. colona and E. crus-galli. The assay was used to test 13 different rice varieties for the presence of S. oryzae in seeds. In total, three of the varieties did not show the presence of S. oryzae in their seeds while the rest were found to harbor the pathogen. The developed assay can effectively be used to detect and screen the presence of S. oryzae in live samples including seeds and field soil.

Keywords: Sarocladium oryzae; bio-surveillance colorimetric LAMP assay for the detection of Sarocladium oryzae; isothermal amplification; sheath rot; weeds.

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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
Optimization and validation of LAMP assay. Yellow color indicated a positive reaction while red/pink color indicated no reaction. (A) LAMP assay optimized with pure fungal isolates with no template control. (B) M: 100 bp (Promega); 1: S. oryzae NAIMCC-F-01633; 2: No Template control. The assay time was kept at 15 minutes.
FIGURE 2
FIGURE 2
Specificity assay of the LAMP assay. (A) M: 100 bp (Promega); 1: S. oryzae NAIMCC-F-01630; 2: Rhizoctonia solani AG-1 IA isolate NAIMCC-F-03220; 3: R. solani AG-1 IB isolate M2; 4: R. solani AG 2-2IIIB isolate O1; 5: R. solani AG-8 isolate S1; 6: Magnaporthe oryzae isolate MG1; 7: R. oryzae-sativae isolate MTCC-9666; 8: Fusarium fujikuroi NAIMCC-F-03979; 9: Sclerotinia sclerotiorum NAIMCC-F-03341; 10: Trichoderma asperellum NAIMCC-F-03330; 11: F. oxyporum f. sp. lycopersici; 12: Alternaria alternata isolate NAIMCC-F-00067; 13: Ustilaginoidea virens NAIMCC-F-02995; 14: Helminthosporium oryzae NAIMCC-F-03040; 15: Pseudomonas plecoglossicida NAIMCC-B-00397; 16: No Template control; (upper panel: gel photograph; lower panel: colorimetric reactions in PCR tubes). (B) M: 100 bp (Promega); 1: S. oryzae NAIMCC-F-01631; 2: S. implicatum RPF 22 NAIMCC-F-04128; 3: Acremonium curvulum CABI-297016 NAIMCC-F-00022; 4: S. kiliense ATCC-14491/CABI-090242 (A. kiliense) NAIMCC-F-00028; 5: S. strictum ATCC-18941 CABI-230422 (A. strictum) NAIMCC-F-00053; and 6: No Template control.
FIGURE 3
FIGURE 3
Sensitivity of the LAMP assay when performed with 10-fold serial dilution of template DNA (S. oryzae NAIMCC-F-01630). M: 100 bp (Promega); 1: 160.4 ng/μl; 2: 160.4 × 10–1 ng/μl; 3: 160.4 × 10–2 ng/μl; 4: 160.4 × 10–3 ng/μl; 5: 160.4 × 10–4 ng/μl; 6: 160.4 × 10–5 ng/μl; 7: 160.4 × 10–6 ng/μl; 8: 160.4 × 10–7 ng/μl; 9: No template control; and L: 1 kb (Promega). Upper panel in the figure shows reaction tubes whereas lower panel shows agarose gel electrophoresis results.
FIGURE 4
FIGURE 4
The LAMP assay with infected rice leaves along with three weed species. M: 100 bp (Promega); 1: S. oryzae NAIMCC-F-01632; 2: S. oryzae infected tissue; 3: Cyperus teneriffae infected tissue; 4: Echinochloa crus-galli infected tissue; 5: E. colona infected tissue; and 6: No template control; L: 1 kb (Promega). Middle panel in the figure shows agarose gel electrophoresis results, lower panel shows diseases severity in the samples and upper panel shows reaction tubes.
FIGURE 5
FIGURE 5
M: 100 bp (Promega); 1: artificial inoculation with S. oryzae NAIMCC-F-01631; 2: artificial inoculation with S. oryzae NAIMCC-F-01634; 3: Pusa Basmati 1121; 4: RNR 2415; 5: Rajendra sweta; 6: Swarna MTU 7024; 7: Kalanamak BK-102; 8: TKM 13; 9: Kalanamak KN 3; 10: BPT 5204; 11: Saryu 52; 12: CO- 51; 13: Kalanamak 101; 14: Swarna sub 1; 15: Rajendra kasturi; and 16: No template control.
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