Early Detection of the Fungal Banana Black Sigatoka Pathogen Pseudocercospora fijiensis by an SPR Immunosensor Method

Abstract

Black Sigatoka is a disease that occurs in banana plantations worldwide. This disease is caused by the hemibiotrophic fungus Pseudocercospora fijiensis, whose infection results in a significant reduction in both product quality and yield. Therefore, detection and identification in the early stages of this pathogen in plants could help minimize losses, as well as prevent the spread of the disease to neighboring cultures. To achieve this, a highly sensitive SPR immunosensor was developed to detect P. fijiensis in real samples of leaf extracts in early stages of the disease. A polyclonal antibody (anti-HF1), produced against HF1 (cell wall protein of P. fijiensis) was covalently immobilized on a gold-coated chip via a mixed self-assembled monolayer (SAM) of alkanethiols using the EDC/NHS method. The analytical parameters of the biosensor were established, obtaining a limit of detection of 11.7 µg mL^-1, a sensitivity of 0.0021 units of reflectance per ng mL^-1 and a linear response range for the antigen from 39.1 to 122 µg mL^-1. No matrix effects were observed during the measurements of real leaf banana extracts by the immunosensor. To the best of our knowledge, this is the first research into the development of an SPR biosensor for the detection of P. fijiensis, which demonstrates its potential as an alternative analytical tool for in-field monitoring of black Sigatoka disease.

Keywords: Pseudocercospora fijiensis; black Sigatoka; immunosensor; plant pathogen; surface plasmon resonance.

Figure 1

(a) SPR optomechanical setup; (b) Scheme of the Flow cell mount and (c) Teflon cell with gold film adhered to prism; (d) connections to the injection system.

Figure 2

Schematic of the SPR immunoassay for detecting HF1 and illustrative representation of sensograms.

Figure 3

SPR response as a change in the reflectance during the immobilization process employing several antibody concentrations (10, 30, 50, 60 and 70 µg mL⁻¹) and two molar ratios of a MHDA: MUD (1:20 and 1:50). Each point represents the mean ±SD of three measurements.

Figure 4

(a) Real-time SPR sensorgram of anti-HF1 immobilization. Activation by EDC/NHS; then immobilization of the polyclonal antibodies and finally blocking of the sensor surface by ethanolamine and (b) Angular reflectance spectra measured from a sensor chip before (blue) and after the immobilization (red) of anti-HF1.

Figure 5

Real-time SPR sensograms for HF1 detection at different concentrations and calibration curves in PBS. Each point represents the mean ± SD of three replicates.

Figure 6

SPR sensograms evaluating the level of nonspecific signals due to matrix effects resulting from banana leaves extracts.