Spatial Distribution of Single-Nucleotide Polymorphisms Related to Fungicide Resistance and Implications for Sampling

Abstract

Spatial distribution of single-nucleotide polymorphisms (SNPs) related to fungicide resistance was studied for Botrytis cinerea populations in vineyards and for B. squamosa populations in onion fields. Heterogeneity in this distribution was characterized by performing geostatistical analyses based on semivariograms and through the fitting of discrete probability distributions. Two SNPs known to be responsible for boscalid resistance (H272R and H272Y), both located on the B subunit of the succinate dehydrogenase gene, and one SNP known to be responsible for dicarboximide resistance (I365S) were chosen for B. cinerea in grape. For B. squamosa in onion, one SNP responsible for dicarboximide resistance (I365S homologous) was chosen. One onion field was sampled in 2009 and another one was sampled in 2010 for B. squamosa, and two vineyards were sampled in 2011 for B. cinerea, for a total of four sampled sites. Cluster sampling was carried on a 10-by-10 grid, each of the 100 nodes being the center of a 10-by-10-m quadrat. In each quadrat, 10 samples were collected and analyzed by restriction fragment length polymorphism polymerase chain reaction (PCR) or allele specific PCR. Mean SNP incidence varied from 16 to 68%, with an overall mean incidence of 43%. In the geostatistical analyses, omnidirectional variograms showed spatial autocorrelation characterized by ranges of 21 to 1 m. Various levels of anisotropy were detected, however, with variograms computed in four directions (at 0°, 45°, 90°, and 135° from the within-row direction used as reference), indicating that spatial autocorrelation was prevalent or characterized by a longer range in one direction. For all eight data sets, the β-binomial distribution was found to fit the data better than the binomial distribution. This indicates local aggregation of fungicide resistance among sampling units, as supported by estimates of the parameter θ of the β-binomial distribution of 0.09 to 0.23 (overall median value = 0.20). On the basis of the observed spatial distribution patterns of SNP incidence, sampling curves were computed for different levels of reliability, emphasizing the importance of sample size for the detection of mutation incidence below the risk threshold for control failure.

A pyrosequencing-based method to quantify genetic substitutions associated with resistance to succinate dehydrogenase inhibitor fungicides in Botrytis spp. populations

“…For comparison purposes, the SdhB fragment was sent to McGill University Génome Québec Innovation Centre for Sanger sequencing. In addition, the second series of swabs was analysed for boscalid resistance mutant profiles by using standard RFLP procedures as described by Van der Heyden et al (2014) …”

“…This raises the question of how to monitor fungicide resistance to determine this threshold, and how to scout resistance for early warning about possible disease control failure. The results of a recent study suggest that resistance management should be implemented at the regional level, which implies that a very large fungal population be assessed . Regardless of the implementation scale, the absence of sampling plans and of cost‐effective tools to estimate the percentage of resistant individuals in a population limits the implementation of effective resistance management strategies…”

“…These methods are time consuming, but they can be used to detect resistance caused by known and unknown genetic changes. Several molecular techniques, such as polymerase chain reaction (PCR), primer‐introduced restriction analysis–PCR (PIRA‐PCR), PCR–restriction fragment length polymorphism (PCR‐RFLP), allele‐specific PCR, allele‐specific real‐time PCR, and real‐time PCR, have been developed to facilitate resistance assessment . However, none of these molecular techniques allows quantitative analysis of the percentages of each genetic substitution (i.e.…”

“…The spatial distribution of two species of Botrytis ( cinerea and squamosa ) and of individuals carrying genetic substitution associated with two families of fungicide (SDHIs and dicarboximides) was recently investigated. Botrytis cinerea strains carrying H272R and H272Y genetic substitutions associated with SDHI resistance and B. cinerea and B. squamosa strains carrying the I86S genetic substitution associated with dicarboximide resistance were recently studied over a 2 year period in a vineyard and in two onion fields ( B. squamosa ) . The observed incidence of these genetic substitutions varied from 16 to 68%, with an overall mean of 43%.…”

“…The frequency of individuals carrying the different genetic substitutions followed a beta‐binomial distribution, indicating the presence of small‐scale spatial heterogeneity. Using the heterogeneity index derived from this study, sampling curves were computed for different levels of accuracy and incidence of genetic substitutions . For example, if the incidence of individuals carrying the targeted genetic substitution is 25% and a coefficient of variation of 10% in the estimation is acceptable, 81 and 51 sampling units, each one composed of ten samples, will be required if the aggregation is high or low respectively .…”