Monitoring Resistance Using Molecular Methods

Abstract

Although cultural methods and bioassays remain a cornerstone in the detection and monitoring of fungicide resistance, molecular diagnostics are playing an important part in many aspects of fungicide resistance research. Molecular methods can avoid the need to isolate pathogens and detect resistance alleles at lower frequencies and are less resource-intensive than cultural methods and bioassays. Molecular diagnostics generally identify single-nucleotide polymorphisms (SNPs) and so require that point mutations causing resistance are known. The polymerase chain reaction (PCR) is at the core of most methods described here, in which detection is based on hybridization or amplification with allele-specific probes or primers, use of restriction enzymes or sequencing. Results may be presented just as the presence or absence of resistance, but using fluorescent markers, and following hybridization or amplification in real time, allows quantification of the frequency of resistance mutations within pathogen populations. The complexity and equipment requirements for many of these methods prevent the use in small laboratories lacking PCR equipment and expertise. Unlike Taq polymerase used in PCR, Bst polymerase amplifies DNA without denaturation, so that, using specific primer sequences, SNPs can be detected isothermally using just a water bath and a single temperature. Known as loop-mediated isothermal amplification (LAMP), it offers the potential for accurate and rapid detection of resistance in field samples, even for small diagnostic laboratories. Because the ``on-site'' methods are still insufficient, molecular diagnostics have not yet proved useful to growers in guiding in-season antiresistance strategies, but further developments in LAMP may overcome this limitation.