An Accurate, Affordable, and Precise Resazurin-Based Digital Imaging Colorimetric Assay for the Assessment of Fungicide Sensitivity Status of Fungal Populations

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Silva, Tatiane Carla ; Moreira, Silvino Intra ; Assis Jr, Fabio Gomes ; Campos Vicentini, Samara Nunes ; Silva, Abimael Gomes ; Kitayama de Oliveira, Tamiris Yoshie ; Christiano Jr, Felix Sebastiao ; de Paiva Custodio, Adriano Augusto ; Leite Jr, Rui Pereira ; de Godoy Gasparoto, Maria Candida ; de Jesus Jr, Waldir Cintra ; Ceresini, Paulo Cezar

• Document type: Journal article
• Source: AGRONOMY-BASEL; v. 13, n. 2, p. 19-pg., 2023-02-01.

Abstract

This study aimed at the development and validation of an accurate, more affordable, and precise digital imaging resazurin-based fungicide sensitivity colorimetric assay (COL-assay) for fungal plant pathogens from the genera Mycosphaerella and Pyricularia. This proposed digital imaging assay was based on colorimetric estimates of resazurin reduction, which was used as a metabolic indicator of fungal respiration activity on microplate cultures. As fungal model systems, we used the yellow and black Sigatoka pathogens [Mycosphaerella musicola (Mm) and M. fijiensis (Mf), respectively] and the wheat blast pathogen, Pyricularia oryzae Triticum lineage (PoTl), which were previously characterized for QoI, DMI, and SDHI fungicide sensitivity. We then compared the classical spectrophotometry detection assay (SPEC-assay) with the proposed COL-assay based on the analyses of digital images of the microplates' cultures captured with mobile phone cameras on a handmade trans-illuminator built for poorly equipped labs. Qualitatively, in terms of accuracy, there was full correspondence between the SPEC-assay and the COL-assay according to the fungal EC50 or the relative growth classes on QoI, SDHI, and DMI fungicides for both Mycosphaerella and Pyricularia pathogens. We also observed a strong to very strong correlation coefficient between the COL-assay and the SPEC-assay fungicide sensitivity values for the QoI azoxystrobin, the SDHI fluxapyroxad, and the DMI tebuconazole. Our conclusion was that the COL-assay had a similar accuracy as the SPEC-assay (i.e., resulted in similar fungicide-sensitivity categories for both resistant or sensitive fungal isolates) and high precision. By openly sharing here the COL-assay's full methodology, and the blueprints of the handmade trans-illuminator, we foresee its adoption by poorly equipped labs throughout the country as an affordable venue for monitoring the fungicide resistance status of populations of important fungal plant pathogens such as M. fijiensis, M. musicola, and P. oryzae Triticum and Oryza lineages. (AU)

• FAPESP's process: 20/07611-9 - Development of a joint platform for disease epidemics monitoring and fungicide resistance detection to foster a smart program for management of wheat blast in Paraná and São Paulo states
• Grantee: Paulo Cezar Ceresini
• Support Opportunities: Regular Research Grants
• FAPESP's process: 21/03402-9 - Multi-user Equipment (MUE) approved in the grant 20/07611-9: Real Time Quantitative PCR (qPCR) Instrument
• Grantee: Gabriel Madoglio Favara
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 18/21197-0 - Novel real-time disease surveillance and fungicide resistance monitoring tools to foster a smart and sustainable crop protection platform in Brazil
• Grantee: Paulo Cezar Ceresini
• Support Opportunities: Regular Research Grants
• FAPESP's process: 19/12509-1 - Novel real-time disease surveillance and fungicide resistance monitoring tools to foster a smart and sustainable crop protection platform in Brazil
• Grantee: Silvino Intra Moreira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/50456-1 - Understanding the evolution of fungicide resistance in field populations of the wheat blast pathogen from Brazil: can we learn lessons for future disease management?
• Grantee: Paulo Cezar Ceresini
• Support Opportunities: Regular Research Grants