The objective of this study was to test the accuracy of the SNP-based assay to identify the four common crop-associated Sclerotinia
species. The assay accurately identified 96% of all Sclerotinia
isolates, as 46 of the total 48 isolates positively hybridized to their respective species-specific SNP probes (Fig. ), and these results were consistent both with the additional calmodulin sequence and with the Southern hybridizations using probe pMF2. The two isolates with inconclusive hybridization results were isolates 02-26 and 3-A5, from Finland, originally identified as being S. trifoliorum
by the collectors (Table ). Neither isolate produced sclerotia under our growing conditions, although a loss of sclerotial production is not uncommon with prolonged serial culture of Sclerotinia
). For example, T4, one of the sequenced strains in the Botrytis cinerea
genome project, does not produce sclerotia, yet in whole-genome comparisons to the other reference strain, B05.10, it is indisputably B. cinerea
). The RFLP phenotypes from DNAs of 02-26 and 3-A5 were considerably different from those of S. trifoliorum
isolates, yet these isolates had RAS sequences that were identical to those of all other isolates of S. trifoliorum
(data not shown).
Because mutations continually arise spontaneously in populations, no sample can census all real or potential intraspecific variation in Sclerotinia sclerotiorum
, S. minor
, S. trifoliorum
, or Sclerotinia
species 1 that may be encountered in nature, nor can it census all emerging or new, undescribed species that may exist in crops. Our SNP-based assay can detect genetic variants within the four crop-infecting species of Sclerotinia
that may represent mutations within populations or emerging or cryptic species. The sample in the present study revealed two genetic variants within S. trifoliorum
. The sample of 465 isolates from which the loci for the present study were selected revealed two “cryptic” species, one of which is Sclerotinia
species 1 and the other of which was reported to be S. sclerotiorum
but is actually a newly discovered species in another genus, Dumontinia
). Species other than the four species commonly associated with crops might be expected among isolates from wild hosts or understudied geographical areas. A failure to hybridize to one of the diagnostic probes could indicate a genetic variant within one of the four species. Failure to hybridize to the suite of probes that are diagnostic for a species could indicate a genetic variant within the four species or two additional possibilities: (i) the isolate represents a rare but previously described species, or (ii) the isolate might represent a new species. A survey of species epithets associated with the host or location of interest, and the sequencing of herbarium specimens that would establish the connection between the contemporary isolate and the name in the literature, would be required to distinguish the former from the latter.
The SNP-based hybridization method is faster and more robust than the rDNA-RFLP hybridization method and is amenable to the high-throughput screening of samples that might be required for an epidemiological study, in which there could be a high likelihood of a repeated sampling of one or two species. The RFLP method requires a 6-h restriction enzyme digestion of whole genomic DNA, followed by gel electrophoresis for 12 h to ensure the separation of the fragments. A maximum of 20 DNA samples, plus references, can be loaded onto a membrane with acceptable imaging. In contrast, the SNP-based method uses PCR amplification that yields high copy numbers of the desired sequence with a high affinity for the probe. The gel electrophoresis step is short, 15 to 20 min, compared to the RFLP method. With the SNP diagnostic method, hundreds of sample DNAs can be spotted onto one membrane, allowing high-throughput screening of samples. Although the RFLP method requires only one hybridization step, it is 16 to 24 h long, while SNP-based hybridizations take only 3 h per probe. Finally, hybridization results with the SNP diagnostic system are robust even under variable conditions. In the RFLP method, in which the template is restriction-digested whole genomic DNA, there are often differences in signal intensities among hybridizing bands, ranging from very light to highly saturated. Two or more exposure times for a single blot may be required to identify real, repeatable bands worthy of scoring. The dot blot format of our diagnostic-screen Southern blots is chemically quite different from that used for the RFLP screen, as the high copy number of PCR-amplified DNA sequences provides a template for which the probes will have a high affinity, resulting in a bright signal that requires little interpretation regardless of exposure time.
We propose that the SNP-based assay be conducted as outlined in Fig. . At least two species-specific SNP probes should be used for positive identification, and use of the entire suite of probes is the best approach for addressing concerns over false-positive or -negative scoring. Comparing all probes and examining them for cross-hybridization with more than one species-specific diagnostic SNP (Fig. ) would be evidence of a false-positive reaction (not observed in the present study). Concerns over false-negative scoring can be addressed by using both sets of probes; as observed in the present study, a failure to hybridize to both sets of probes is unlikely without actual sequence variation consistent with a true negative result. Although we used a radioactive label, probes can also be ordered with a nonradioactive label, such as biotin, in large quantities and stored indefinitely. The SNP-based assay outlined here is an effective and rapid method for identifying the four described crop-associated Sclerotinia species. Importantly, this assay indicates with confidence when an isolate is not one of the described species. The SNP diagnostic system may be particularly useful for epidemiological studies with high-throughput requirements, especially when two or more species are mixed in plants or within fields.