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Expression of MfAvr4 in banana leaf sections with black leaf streak disease caused by Mycosphaerella fijiensis: a technical validation

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Abstract

The objective of this study was to validate the use of banana leaf sections as a technique to study the molecular interaction between Mycosphaerella fijiensis and Musa spp. without the interference of biotic and abiotic factors that commonly occur under field conditions. The growth of M. fijiensis in banana leaf sections was evaluated and compared with the growth of the fungus in leaves under field conditions. Growth comparison was carried out through the absolute quantification by real-time PCR of a segment of the β-tubulin gene of M. fijiensis. Validation of the banana leaf sections technique consisted in monitoring M. fijiensis MfAvr4 gene expression and its relative quantification by real-time PCR in banana leaf sections. With this technique, it was shown that the growth of M. fijiensis and MfAvr4 gene expression were similar to those observed in infected leaves in the field. These quantitative real-time PCR results support the suitability of using banana leaf sections for molecular studies of gene expression in M. fijiensis-Musa spp. interactions.

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Acknowledgments

We thank C. Ortiz, R. Grijalva, R. Escobedo, R. Ku, M. Mahendhiran, S.R. Peraza, Y. Sánchez, J.H. Ramírez, I. Córdova, R. Vázquez, M. Tzec and R. Sulub for their technical support. The research was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico; grant 58869), a fellowship to A. Canché (240168), and by the Fondo Institucional de Fomento Regional Para el Desarrollo Científico, Tecnológico y de Innovación (FORDECYT, Mexico; grant 116866).

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Authors and Affiliations

  1. Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A.C., Calle 43 No. 130 Chuburná de Hidalgo, CP 97205, Mérida, Yucatán, México

    Cecilia Mónica Rodríguez-García, Abril Diane Canché-Gómez, Luis Sáenz-Carbonell, Leticia Peraza-Echeverría, Blondy Canto-Canché & Santy Peraza-Echeverría

  2. Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, A.C., Calle 43 No. 130 Chuburná de Hidalgo, CP 97205, Mérida, Yucatán, México

    Ignacio Islas-Flores

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  1. Cecilia Mónica Rodríguez-García
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Correspondence to Cecilia Mónica Rodríguez-García.

Electronic Supplementary Material

Fig5

Figure SM1 Standard curve regression between the threshold cycle (Ct) values for the β-tubulin gene and the log10 of M. fijiensis DNA. Serial dilutions (1:10) of gDNA (100 ng/ μL) from mycelia of M. fijiensis were prepared to construct the standard curve. Black circles represent the mean of data used to calculate the PCR efficiency value using the formula: 10(−1/ slope) − 1 (GIF 14 kb)

High resolution image (TIFF 1524 kb)

Fig6

Figure SM2 Schematic representation of the validation of inter-assay using a single sample naturally infected (stage 5) on different days. Five replicates of the same DNA sample were analysed in five repeated experiments; different letters indicate statistically significant differences (P < 0.05) determined by ANOVA and Fisher tests (GIF 21 kb)

High resolution image (TIFF 2820 kb)

Fig7

Figure SM3 Schematic representation of the validation of inter-sample using samples naturally infected (stage 3). Three different gDNA samples of these banana leaves were analysed in a single run. Different letters indicate statistically significant differences (P < 0.05) determined by ANOVA and Fisher tests (GIF 13 kb)

High resolution image (TIFF 1987 kb)

Fig8

Figure SM4 Standard curve regression between the threshold cycle (Ct) values for the β-tubulin (a) and MfAvr4 (b) genes and the log10 of M. fijiensis, to validate the relative quantification in artificially infected banana leaf sections, and in naturally infected banana leaves. Serial dilutions (1:10) of cDNA (200 ng/μL) from mycelia of M. fijiensis were prepared to construct the standard curve. Black circles represent the mean of data used to calculate the PCR efficiency value using the formula 10(−1/ slope) − 1 (GIF 24 kb)

High resolution image (TIFF 2104 kb)

ESM 1

(DOCX 115 kb)

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Rodríguez-García, C.M., Canché-Gómez, A.D., Sáenz-Carbonell, L. et al. Expression of MfAvr4 in banana leaf sections with black leaf streak disease caused by Mycosphaerella fijiensis: a technical validation. Australasian Plant Pathol. 45, 481–488 (2016). https://doi.org/10.1007/s13313-016-0431-6

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