Development and validation of novel diagnostic biosensor tools for Botrytis cinerea and Botrytis fabae on temperate legumes

Abstract

Temperate grain legumes are susceptible to attack by a large number of pathogens and of the necrotrophic foliar fungal pathogens, Botrytis cinerea and Botrytis fabae are capable of causing Botrytis Grey Mould (BGM) disease. Separately or within a complex, and within environmentally conducive seasons, these two fungi cause serious yield and quality losses. This occurs particularly in dense, early sown crops, when spring rainfall is high and there are prolonged wet periods. Disease management is largely reliant on multiple fungicide treatments since no high levels of genetic host resistance exist. Therefore, early diagnosis and quantitation of the causal pathogen species is crucial. In particular, information on their frequencies and locations of occurrence will enable better prediction of loss and inform on optimal temporal and spatial fungicide application. As an initial step, in Chapter 1, a comprehensive literature review was conducted to summarise the most desirable characteristics of a plant pathogen diagnostic tool. These were concluded to be highly specific, sensitive, easy to use, portable, reproducible and cost effective. To date, several immunogenic and molecular probe-type diagnostic methods have been developed for B. cinerea and none have been developed for B. fabae. However, the level of specificity of the existing B. cinerea diagnostic tools to discriminate between the two target Botrytis species and their usefulness in the paddock is unclear. Thus, the overall goal of this thesis was to develop and validate fast, accurate, affordable, species-specific and sensitive molecular biosensors for the causal agents of BGM; B. cinerea and B. fabae for use in informed disease management in temperate legume cropping in Australia. In Chapter 2, species-specific molecular probes were developed through alignment of existing genome sequences of B. cinerea and B. fabae in the NCBI databases to identify sites of ‘stable polymorphism’ among the two species. Potential probes were then tested for specificity using traditional polymerase chain reaction (PCR). Based on sequence alignments of multidrug resistance (MRR1) and necrosis and ethylene inducing protein (NEP1) genes. Subsequently, the MRR1Bc-f/MRR1Bc-r and NEP1Bc-f/NEP1Bc-r primer sets were designed to differentiate B. cinerea from B. fabae. These DNA probes were then validated for specificity and sensitivity using pure fungal target DNA and in traditional PCR were able to detect 10 pg of DNA (corresponding to 217 spores). When used within a qPCR, the sensitivity (threshold of detection) for both probes was increase to 100 fg (corresponding to ~2 spores). Then in Chapter 3, a gold nanoparticle-based PCR-free detection assay was developed for each of the target species using the same molecular probes. This device and the electrochemical detection protocol was ten times more sensitive than the qPCR method for both target species (able to detect 102 spores per mL of extracted plant sample). Using the newly developed nano-biosensor device, diagnosis and quantification of the target species was possible within 45 minutes from plant tissue sampling. The approach used inexpensive and portable screen-printed carbon electrodes (SPCEs) and biotinylated capture probes that were designed from the species-specific primer sequences identified in Chapter 2. Streptavidin coated dynabeads were modified with the biotinylated capture probes and were dispersed in a single stranded DNA sample population to isolate and purify the target pathogen DNA. The isolated target DNA was then directly adsorbed onto gold nanoparticles with ferrous oxide loading (AuNP Fe2O3NC) via a DNA–gold affinity interaction. These complexes were magnetically immobilised onto the SPCE and following a natural redox reaction, a chronocoulometric charge measurement was made that represented the presence and quantity of the target pathogen. The newly developed biosensors were subsequently validated in the laboratory using infected and uninfected plant tissues inoculated with B. cinerea or B. fabae at five concentrations from 10 spores to 105 spores/mL and collected at 24, 36 or 48 hours post inoculation (hpi), well before appearance of any visible symptoms. Finally, in Chapter 4, the biosensors were assessed for accuracy, sensitivity and portability under quasi-field conditions in shade house plots of lentil cultivars Bolt, Hurricane and Hallmark at the South Australian Research and Development Institute, Waite Campus Adelaide. Consistent with the results found in the laboratory environment in Chapter 3, the biosensors detected the pathogens in all three cultivars at 24 hpi and before visual appearance of the disease symptoms. This strongly indicated the useability of these two newly designed diagnostics for Point of contact (POC) application in the field. Further improvements for increased compactness and portability of the assay and biosensor device as well as considerations for broader industry validation are given in the general discussion Chapter 5.