Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.

Significant advances have recently been made in our understanding of the genetic diversity in Verticillium. Five new Verticillium species were described [4], including V. isaacii and V. klebahnii that are morphologically indistinguishable from V. tricorpus, and V. alfalfae and V. nonalfalfae that resemble V. albo-atrum and cannot be differentiated based on morphology. Also, the relationship of V. dahliae to the diploid hybrid V. longisporum was clarified [47]. It was found that V. longisporum consists of at least three groups that evolved independently by hybridization involving two unknown species and two lineages of V. dahliae (Figure 1). The two unknown species have never been found except as parents of V. longisporum, and have informally been named Species A1 and Species D1, and the two V. dahliae lineages are referred to as V. dahliae lineages D2 and D3. The three groups or lineages of V. longisporum evolved by independent hybridization of Species A1 with Species D1, V. dahliae lineage D2 and V. dahliae lineage D3, respectively, and accordingly, are referred to as V. longisporum lineage A1/D1, V. longisporum lineage A1/D2 and V. longisporum lineage A1/D3, respectively.
We used DNA sequence data generated in conjunction with phylogenetic and taxonomic studies of Verticillium [4,47], and designed PCR assays for the identification of Verticillium species and V. longisporum lineages. The assays will be useful for diagnostics labs and research applications.

Results and Discussion
We designed eighteen PCR primers combined into eleven single-target (simplex) and four multi-target (multiplex) PCR assays for identification of all ten Verticillium species and V. longisporum lineages. PCR primer design was based on DNA sequence data of 257 Verticillium isolates at five loci, which were previously identified to species using type material [4,47]. The targets of the eleven simplex PCR assays are shown in Figure 2, and included V. alboatrum, V. alfalfae, V. dahliae including V. longisporum lineage A1/D3 (Figure 1), V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two V. longisporum ancestors ( Figure 1). The reliability of the primer pairs was confirmed in PCR assays as described below, and various combinations of primer pairs were evaluated for simultaneous amplification of more than one target species or V. longisporum lineage in multiplex PCR assays. Four multiplex PCR assays containing between five and seven primers were able to reliably amplify separate templates of the following morphologically or ecologically similar groups of species: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; V. dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; V. isaacii, V. klebahnii and V. tricorpus.

Isolate Sampling for Primer Design and PCR Assay Validation
DNA sequence data of many isolates of Verticillium are available in GenBank. However, since a large proportion of DNA sequences in GenBank is derived from isolates that are not correctly identified [48,49], we designed the species and lineage-specific primers based on a set of 1290 DNA sequences from two taxonomic and phylogenetic studies of type specimens of Verticillium to guarantee correct identification [4,47]. The sequences were from five loci of 257 Verticillium isolates and a Gibellulopsis nigrescens negative control. The largest proportion of isolates, 196 out of 257, was from V. dahliae and its close relative V. longisporum. The numbers for the other species ranged from four to fourteen as shown in Table 1. The DNA sequences retrieved for primer design represented the total genetic diversity at each of the five loci for all species, and were derived from one to eight isolates depending on the species (Table 1). The sequences were aligned separately for each locus, and PCR primers were designed as described in the Materials and Methods ( Table 2). All alignments with primer sites are provided (Alignments S1, S2, S3, S4, S5).
The isolates employed for the validation of the PCR assays included Verticillium and Gibellulopsis strains used for primer design [4,47], in addition to a Musicillium theobromae negative control, twelve uncharacterized isolates of V. dahliae, V. isaacii and V. klebahnii, and V. longisporum. Primer pairs were first evaluated in simplex PCR assays using different numbers of isolates as positive and negative controls for each primer pair as shown in Table 3, depending on the genetic diversity of the target species, the numbers of isolates available, and the numbers of non-target species with similar primer sites as described in the Materials and Methods. The PCR conditions are detailed in Table 4. The PCR banding patterns for the simplex assays are shown in Figure 3, and the results from PCRs with additional isolates are in Figure S1 and Figure S2.
All multiplex PCR assays were validated with a set of 26 representative isolates ( Table 5). The PCR conditions are described in Table 6 and the PCR banding patterns for the multiplex assays are shown in Figure 4, and in Figures S2 and S3  for additional isolates. The total number of isolates involved in the validation of each primer pair in both simplex and multiplex PCR assays varied from 27 to 43 as summarized in Table S1.

Identifying Verticillium Species and Setting Up PCR Assays
The deployment of the PCR assays described here assumes identification of Verticillium at the genus level using morphological characters, which can be challenging. If a fungal culture isolated from an agricultural substrate contains thick-walled, darkpigmented resting structures, and long, narrow conidiogenous cells arranged in whorls along the main axis of the conidiophore, chances are high that it is Verticillium [4]. However, there are exceptions. Gibellulopsis nigrescens and Musicillium theobromae are associated with plants, resemble Verticillium species in terms of conidiophore and resting structure morphology, but are phylogenetically distinct and belong to different genera [5]. Also, numerous other unrelated fungi have conidiophores suggestive of Verticillium [50,51,52]. Should all the PCR assays fail and positive control isolates are not available, confirmation of genus identity can be performed by sequencing the ITS region and undertaking a nucleotide BLAST search at GenBank, or preferably, phylogenetic analyses with a Verticillium ITS dataset that contains ex-type sequences [4], available from TreeBASE at www.treebase.org [53].
To help select the most appropriate PCR assay to use, a morphology-based key is available for preliminary identification to species or species groups [4]. For identification of V. dahliae and V. longisporum, the V. dahliae -V. longisporum multiplex assay should generally be used, because it is the only one of the assays presented here that is able to distinguish V. dahliae from all V. longisporum lineages. The V. dahliae simplex assay in Table 4 also amplifies isolates of the V. longisporum lineage A1/D3 (Figure 3), and can thus lead to false positive results.
Verticillium dahliae and V. longisporum are the most difficult Verticillium species to identify by PCR assay, because V. dahliae is the parent of two of the three V. longisporum lineages, V. longisporum lineage A1/D2 and V. longisporum lineage A1/D3 ( Figure 1). Due to the high genetic similarity between V. longisporum and V. dahliae, PCR primers specific to V. dahliae protein-coding genes will in most cases amplify the orthologs in V. longisporum lineages A1/D2 and A1/D3. Our multiplex PCR assay differentiates V. dahliae from V. longisporum by targeting the Species A1 EF allele that is unique to V. longisporum. Verticillium longisporum lineage A1/D1 is differentiated from the other lineages by an amplicon of the Species D1 GPD allele, and V. longisporum lineage A1/D3 is the only lineage that has an ITS region derived from V. dahliae. The other two lineages' ITS regions are from Species A1. Due to concerted evolution, all of the V. longisporum lineages appear to have just one type of ITS region [47]. Thus, the V. longisporum lineage A1/D1 PCR banding pattern Table 1. Numbers of isolates used for primer design in relation to numbers of isolates and genetic diversity of each Verticillium species in Inderbitzin et al. [4,47].

Species/Totals
Isolates/species A Isolates representing intraspecific diversity B Isolates used for primer design C V. albo-atrum Total 257 30 34 A Number of isolates in each species [4,47]. B Number of isolates representing the genetic diversity across ACT, EF, GPD, ITS and TS [4,47]. C Number of isolates used for primer design, for details see Table S4. Selected isolates represent the genetic diversity of each species at ACT, EF, GPD, ITS and TS [4,47]. D The V. longisporum isolates with the best sequencing coverage were used at each locus (Table S4) Figure 4). The V. longisporum lineage A1/D2 banding pattern is identical to the pattern expected for Species A1. However, Species A1 has never been found and is only known as one of the parents of V. longisporum [47]. PCR assays for the identification of V. dahliae and V. longisporum have previously been published, including the assay by Karapapa and Typas [43] who used the presence of a 839-bp intron in the nuclear SSU rRNA gene as a marker for V. longisporum. In agreement with the ITS data, V. longisporum lineage A1/D3 also lacks the SSU intron ( Figure 5), and based on Karapapa and Typas' assay, V. longisporum lineage A1/D3 would thus be identified as V. dahliae. A similar problem exists for V. dahliae diagnostic assays that target the ITS region [40], or protein coding genes which might falsely identify V. longisporum lineages A1/D2 and A1/D3 as V. dahliae [42]. However, V. dahliae and V. longisporum tend to have different host ranges, and in many cases, a V. dahliae assay that excludes all V. longisporum lineages may not be necessary.
Details of all fifteen PCR assays designed in this study for identification and differentiation of Verticillium species, including primers and other PCR conditions, are listed in Table 4 and Table 6. The setup of the multiplex PCR assays involves preparation of a primer master mix from 100 mM primer stocks as detailed in Tables 7,8,9,10. Screening for the Unknown V. longisporum Parents Species A1 and Species D1 The two informally named Species A1 and Species D1 [47] have never been found and are only known as parents of V. longisporum (Figure 1). Since neither the morphology nor ecology of Species A1 and Species D1 is known, and the two species may resemble V. longisporum and V. dahliae morphologically, PCR assays provide an opportunity to screen existing or new collections for isolates of Species A1 and Species D1. With the V. dahliae -V. longisporum multiplex PCR assay (Table 6), the Species D1diagnostic PCR banding pattern is expected to consist of only the 1020-bp Species D1 band, and the Species A1 banding pattern would be identical to the banding pattern of V. longisporum lineage A1/D2 that comprises one 310-bp Species A1 band (Figure 2, Figure 4). To differentiate Species A1 from Verticillium longisporum lineage A1/D2, PCR reactions targeting protein coding genes, for Target isolates served as positive controls for the respective species and lineage specific primer pairs. Strain numbers were compiled from Figure 3 and Figure S2. B Negative control isolates were selected to represent all species that differed from the respective target species by four or fewer substitutions at the more divergent primer site, or were randomly selected if all non-target species differed by more than four substitutions from the target species (Table S4). Strain numbers were compiled from Figure 3 and Figure S2. C PCR primer pair targeting this species was not used in any multiplex PCR assay and was thus also tested with the set of 26 isolates in Table 5   The PCR program consisted of a 2 min initial denaturation step at 94uC, 32 or 35 cycles of 10 sec at 94uC, 20 sec at the PCR assay-dependent annealing temperature, and 1 min at 72uC, followed by a final extension of 7 min at 72uC. PCR reactions were set up at room temperature under sterile conditions and run immediately, or were stored in a freezer.

DNA Sequence Data
A total of 104 DNA sequences from ten Verticillium species and Gibellulopsis nigrescens were retrieved from GenBank or the Broad Institute website (Table S2). Ninety-five of the Verticillium sequences were from 34 isolates that represented the genetic diversity at five loci, including the ribosomal internal transcribed spacer (ITS) region, actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS), in the ten Verticillium species in Inderbitzin et al. [4,47]. Four Verticillium sequences from Klosterman et al. [54] and Pramatef-taki et al. [55] provided DNA sequencing coverage for nonspecific primers outside of the regions sequenced by Inderbitzin et al. [4,47].
The ITS sequence of Musicillium theobromae strain PD686 (CBS 110322), an additional negative control [5], was generated using primers ITS1-F [56] and ITS5 [57] with settings described in Inderbitzin et al. [47]. The ITS sequence was submitted to GenBank as JQ621980. The species identification of M. theobromae strain PD686 was based on GenBank ITS BLAST hits [58], and is thus tentative.

DNA Sequence Alignments and Primer Design
DNA sequences were aligned separately for each locus (Alignments S1, S2, S3, S4 and S5) using CLUSTAL X version 2.0 [59,60], and eighteen primers were designed manually in Geneious Pro version 4.8.5 [61] (Table S3). Primer specificity was achieved by maximizing the number of mismatches between a primer's 39-end and homologous sites in non-target lineages [62].

Fungal Isolates, Cultures, DNA Extraction, PCR and Gel Electrophoresis
For information on all fungal isolates used in this study, see Inderbitzin et al. [4,47] [44], all retrieved from the Subbarao lab collection. All fungal isolates were derived from single conidia [4]. Culture conditions and DNA extraction protocols were as in Inderbitzin et al. [47]. PCRs were performed using GoTaq Colorless Master Mix (Promega Corp., Madison, WI, USA) in GeneMate 0.2 ml 8-strip PCR tubes (BioExpress, Kaysville, UT). Each PCR reaction comprised 10 ml template dilution containing 1, 10, or 100 ng DNA, 2.5 ml primer mixture (0.5 mM for each primer, except primers D3f and D3r that were 0.25 mM each when multiplexed) and 12.5 ml master mix, for a total volume of 25 ml. The PCR program consisted of a 2 min initial denaturation step at 94uC, 32 or 35 cycles of 10 sec at 94uC, 20 sec at the PCR assay-dependent annealing temperature, and 1 min at 72uC, followed by a final extension of 7 min at 72uC. PCR reactions were set up at room temperature under sterile conditions in a laminar flow hood wearing gloves and using plugged pipet tips to  Table 6. Details of Verticillium multiplex PCR assays, including target loci, primer pairs, DNA template concentrations, PCR annealing temperatures, numbers of PCR cycles, PCR product sizes, and agarose gel concentrations for gel electrophoresis.  (Table 4, Table 6). PCR product, 4-6 ml was loaded per well. A 2-log DNA ladder, 0.75 mg (New England Biolabs, Inc., Ipswich, MA) was loaded per well. Loading buffer contained xylene cyanol or bromophenol blue for small and large amplicons, respectively [64].

Validation of PCR Assays
Each PCR primer pair (Table 4) was initially validated in a simplex PCR assay that included one representative of the target species as a positive control, and negative controls that consisted of one representative of each species that differed from the target species by four or fewer substitutions at the more variable primer site ( Figure 3, Table S4). Further validation was performed with additional target and non-target isolates ( Figure S2). Each PCR primer pair was tested in at least three different PCR runs, except for the Species A1, Species D1 and V. dahliae primer pairs.
Validation of multiplex PCR assays ( Table 6) included two control isolates Gibellulopsis nigrescens strain PD595 [47] and the more distantly related Musicillium theobromae strain PD686 [5], and 24 Verticillium isolates representing the allelic diversity at ACT, EF, GPD, ITS and TS as determined by Inderbitzin et al. [4,47] ( Figure 4, Figure S3). Three strains represented V. dahliae lineages D2, D3 and the main group of V. dahliae [47], and one V. alfalfae isolate was included. Each multiplex PCR assay was run on three different PCR machines.
The V. dahliae -V. isaacii -V. klebahnii -V. tricorpus multiplex PCR assay was also validated with eleven genetically uncharacterized isolates from lettuce in California. These were V. dahliae The PCR results were confirmed by DNA sequencing with the respective species-specific primers ( Table 4, Table S3) followed by phylogenetic analyses using PAUP v.4.0b 10 [65] ( Figure S4). The methods used were as in Inderbitzin et al. [4,47]. Validation of the V. dahliae -V. longisporum multiplex PCR assay also included V. longisporum lineage A1/D1 strain PD725 from Steventon et al. [44] that was not included in Inderbitzin et al. [47].

Supporting Information
Table S1 Total counts of positive and negative control isolates used in the validation of Verticillium species and V. longisporum lineage specific primer pairs in both simplex and multiplex PCR assays. (DOCX)     [4,47]. Substitution numbers marked by an asterisk are inferred from DNA sequence alignments (Alignments S1, S2, S3, S4, S5), see Table S2 for accession numbers. The remaining substitution numbers are derived from single-locus phylogenetic trees in Inderbitzin et al. [4,47]. (DOCX) Alignment S1 FASTA text file with ACT alignment used for primer design, primer sites are indicated. Sequence accession numbers are given as part of sequence names for sequences in public databases. (TXT) Alignment S2 FASTA text file with EF alignment used for primer design, primer sites are indicated. Sequence accession numbers are given as part of sequence names for sequences in public databases. (TXT) Alignment S3 FASTA text file with GPD alignment used for primer design, primer sites are indicated. Sequence accession numbers are given as part of sequence names for sequences in public databases. (TXT) Alignment S4 FASTA text file with ITS alignment used for primer design, primer sites are indicated. Sequence accession numbers are given as part of sequence names for sequences in public databases. (TXT) Alignment S5 FASTA text file with TS alignment used for primer design, primer sites are indicated. Sequence accession numbers are given as part of sequence names for sequences in public databases. (TXT)