Current and emerging trends in techniques for plant pathogen detection

Abstract

Plant pathogenic microorganisms cause substantial yield losses in several economically important crops, resulting in economic and social adversity. The spread of such plant pathogens and the emergence of new diseases is facilitated by human practices such as monoculture farming and global trade. Therefore, the early detection and identification of pathogens is of utmost importance to reduce the associated agricultural losses. In this review, techniques that are currently available to detect plant pathogens are discussed, including culture-based, PCR-based, sequencing-based, and immunology-based techniques. Their working principles are explained, followed by an overview of the main advantages and disadvantages, and examples of their use in plant pathogen detection. In addition to the more conventional and commonly used techniques, we also point to some recent evolutions in the field of plant pathogen detection. The potential use of point-of-care devices, including biosensors, have gained in popularity. These devices can provide fast analysis, are easy to use, and most importantly can be used for on-site diagnosis, allowing the farmers to take rapid disease management decisions.

Keywords: PCR-based detection; agriculture; biosensors; cultivation-based detection; immunologica detection; pathogen detection; plant pathogens; sequencing-based detection.

Figure 1

Schematic overview of plant pathogen detection techniques discussed in this review, including non-invasive monitoring, cultivation-based and immunological techniques, nucleic acid amplification and hybridization techniques, DNA sequencing techniques, and biosensors.

Figure 2

Schematic overview of the different scales at which non-invasive spectral and optical techniques of plant parts, plants, and entire fields can be used to detect biotic stresses in plants (Adapted from Singh et al., 2021).

Figure 3

(A) Schematic overview of a sandwich ELISA assay. The wells are coated with an antibody which targets the antigen. The antigen of interest in the sample is then added and binds to the capture antibody. Next, the detection antibodies are added, which target a different epitope of the antigen. The detection antibodies are labeled with an enzyme, capable of converting a colorless substrate to generate a colorimetric signal. ELISA reactions are typically performed in a 96-well plate. (B) Schematic overview of the working principle of the lateral flow immunoassay (LFIA). After application of the sample on the sample pad, it flows in the direction of the absorbent pad due to capillarity and passes through the conjugate release pad, where the labelled detector antibodies can bind to the target analyte. Next, the sample will continue to flow towards the test and control lines, where the analyte (coupled to the detector antibody) will bind to specific (secondary) antibodies immobilized in the test zone. The excess of unbound detector antibodies will flow towards the control zone, where they are bound to immobilized antibodies specific for the detector antibody. Aggregation of the labelled detector antibodies in both test and control zone can be visually observed as illustrated on the right-hand side of the figure (Adapted from Hsiao et al., 2021). (C) Schematic illustration of how the oligonucleotide sequence self-hybridizes into its functional conformation. In its functional conformation, the aptamer is able to bind to its target antigen (Ag) (Adapted from Sun et al., 2014).

Figure 4

Schematic representation of a digital droplet PCR workflow. From left to right: The DNA sample is prepared by generating water-in-oil droplets containing template and the necessary PCR reagents and dyes (1); The droplets are thermally cycled until the PCR reactions reach their end-point (2); The presence of an amplicon (and hence target DNA in the sample) in each droplet is visualized by dsDNA binding dye or by sequence-specific probes and is detected in a microfluidics device (3). Fluorescent signals are processed to detect and quantify the number of pathogens in the sample (4) (Adapted from Kokkoris et al., 2021).

Figure 5

Schematic illustration of the working principle of isothermal amplification methods LAMP and RPA. (A) In LAMP, the use of self-complementary forward and reverse primers results in the formation of a characteristic dumbbell-like structure. This serves as a template for isothermal amplification that is initiated by annealing of a mixture of self-complementary primers. This results in the generation of an increasing number of additional priming sites, ultimately leading to concatamers of the dumbbell DNA structure. (B) On the right hand side the RPA mechanism is shown, with the recombinase enzymes that bind the forward and reverse primers, which subsequently scans the template DNA for complementary sites. Upon finding the complementary site, the primer binds to its complementary sequence though strand invasion. The polymerase generates a new complementary DNA strand starting from the primers, thereby displacing the original DNA strand. The use of a strand-displacing DNA polymerase avoids the need for denaturation. (Adapted from Lmstanfield (2014), Lmstanfield at English Wikipedia, CC BY-SA 3.0 , via Wikimedia Commons and Obande and Singh, 2020).

Figure 6

Schematic presentation of the working principle of a biosensor based on RPA amplification to detect plant pathogens. The DNA extracted from a(n) (infected) plant sample is subjected to RPA amplification. The reverse primer is labelled with biotin, while the forward primer contains a 5’ addition complementary to oligonucleotide probes that are bound to gold particles. Should the sample contain the target pathogen (indicated by (+) in the figure), amplification will occur, resulting in amplicons labeled with a biotin label on one end and a DNA sequence complementary to the probes on the gold particles on the other end. The amplified product is incubated together with streptavidin magnetic beads and gold nanoparticles coated with capture probes, and will form a complex if amplification of the target sequence occurred. If the sample contains no target DNA (indicated by(-)), no labeled amplicons are formed. After magnetic separation of the complex, the complex is dissociated by heat treatment, resulting in the release of the gold nanoparticles. The gold nanoparticles will be deposited on an electrode surface, and through differential pulse voltammetry a characteristic signal is obtained, which indicates the presence of a pathogen (adapted from Lau et al., 2017).