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Gene Genealogies Reveal High Nucleotide Diversity and Admixture Haplotypes Within Three Alternaria Species Associated with Tomato and Potato

    Affiliations
    Authors and Affiliations
    • Tika B. Adhikari1 †
    • Thomas Ingram1
    • Dennis Halterman2
    • Frank J. Louws1 3 †
    1. 1Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695
    2. 2United States Department of Agriculture-Agricultural Research Service, Vegetable Crops Research Unit, Madison, WI 53706
    3. 3Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695
    Published Online:18 Jun 2020https://doi.org/10.1094/PHYTO-12-19-0487-R

    Abstract

    Early blight (EB) and leaf blight are two destructive diseases of tomato in North Carolina (NC), caused by Alternaria linariae and A. alternata, respectively. During the last decade, EB caused by A. solani has increased in potato-producing areas in Wisconsin (WI). We collected 152 isolates of three Alternaria spp. associated with tomato and potato in NC and WI and used the gene genealogical approach to compare the genetic relationships among them. Two nuclear genes: the glyceraldehyde-3-phosphate dehydrogenase (GPDH), RNA polymerase second largest subunit (RPB2), and the rDNA internal transcribed spacer (ITS) region of these isolates were sequenced. Besides, sequences of the GPDH locus from international isolates described in previous studies were included for comparison purposes. A set of single nucleotide polymorphisms was assembled to identify locus-specific and species-specific haplotypes. Nucleotide diversity varied among gene sequences and species analyzed. For example, the estimates of nucleotide diversity and Watterson’s theta were higher in A. alternata than in A. linariae and A. solani. There was little or no polymorphisms in the ITS sequences and thus restricted haplotype placement. The RPB2 sequences were less informative to detect haplotype diversity in A. linariae and A. solani, yet six haplotypes were detected in A. alternata. The GPDH sequences enabled strongly supported phylogenetic inferences with the highest haplotype diversity and belonged to five haplotypes (AaH1 to AaH5), which consisted of only A. alternata from NC. However, 13 haplotypes were identified within and among A. linariae and A. solani sequences. Among them, six (AsAlH1 to AsAlH6) were identical to previously reported haplotypes in global samples and the remaining were new haplotypes. The most divergent haplotypes were AaH1, AsAlH2/AsAlH3, and AsAlH4 and consisted exclusively of A. alternata, A. linariae, and A. solani, respectively. Neutrality tests suggested an excess of mutations and population expansion, and selection may play an important role in nucleotide diversity of Alternaria spp.

    Tomato (Solanum lycopersicum L.) and potato (Solanum tuberosum L.) represent important vegetables in North Carolina (NC) and Wisconsin (WI), respectively. Most of the tomato production is in western NC and sporadic in eastern NC and the Piedmont region. On the other hand, potato is a major vegetable in WI, which is the third largest potato-producing state in the country (USDA-NASS 2018). Commercial potato cultivars grown in WI are Russet, White, Red, Yellow, Blue, and Fingerling. Different Alternaria species (spp.) were found to be associated with early blight (EB) of tomato and potato (Rotem 1994; Simmons 2000). Based on morphology, Alternaria specimens from Solanaceae were revised into several new species including A. grandis (Simmons 2000, 2007). Besides A. solani sensu stricto (Ell. and Mart.) Jones and Grout and A. grandis from potato, a new species called A. tomatophila was identified to cause EB on tomato (Simmons 2007). Later, this species was named A. linariae (Neerg.) Simmons infecting Solanaceae, Cucurbitaceae, and Scrophulariaceae (Woudenberg et al. 2014). A. alternata sensu lato (Fr.) Keissler has been repeatedly isolated from Solanaceae with symptoms resembling that of EB. Although A. alternata causes brown leaf spot on potato (Ding et al. 2019; Droby et al. 1984) and leaf blight and stem canker on tomato (Grogan et al. 1975), both diseases pose a serious challenge for tomato and potato growers due to the lack of effective resistant cultivars (Adhikari et al. 2017).

    DNA sequencing approaches have provided data for both gene genealogies and phylogenetic analyses of genetic variations in different fungi (Brewer and Milgroom 2010; O’Donnell et al. 2000; Peever et al. 2002; Zaffarano et al. 2009). Gene genealogies have provided the evolutionary history and genetic relationships among and within populations in some fungi (Brewer and Milgroom 2010; O’Donnell et al. 2000; Zaffarano et al. 2009) including A. alternata from citrus (Peever et al. 2002). Multigene phylogenies using ribosomal DNA and the nuclear genes such as glyceraldehyde-3-phosphate dehydrogenase (GPDH), internal transcribed spacer (ITS), translation elongation factor 1 (EF-1α), and the gene encoding the allergenic protein alt a 1 (Alt a 1) have been used to determine phylogenetic relationships among isolates of A. solani and A. alternata from tomato and potato from Brazil and WI (Ding et al. 2019; Lourenço et al. 2009). Leaf blight and EB, caused by A. alternata and A. linariae (syn. A. tomatophila), respectively, are destructive diseases of tomato in the United States (Jones et al. 2014). A. linariae is considered a primary pathogen causing EB on tomato (Woudenberg et al. 2014), but it can also infect potato (Ayad et al. 2017). Similarly, EB is a major disease of potato and is spread worldwide including in the United States (Pryor and Gilbertson 2000).

    Tomato is an important vegetable and widely cultivated in western NC. A. linariae is a serious threat to tomato production, resulting in severe economic losses in the Southeastern United States including NC (Kuhar et al. 2019). Unlike A. linariae, A. alternata on tomato has been a neglected pathogen and is poorly understood in NC. Although these pathogens were common in NC, the currently available knowledge on molecular diversity of Alternaria spp. from tomato is limited. The outbreaks of brown spot, caused by A. alternata and EB caused by A. solani on potato have increased recently in WI (Ding et al. 2019; Weber and Halterman 2012). However, it is unknown whether recurrent disease epidemics in tomato and potato in NC and WI are caused by the same or different haplotypes within each species of Alternaria. To address this question, we used coalescent genealogical approaches to obtain phylogenetically informative data derived from DNA sequences of Alternaria spp. using two nuclear loci: GPDH, RNA polymerase second largest subunit (RPB2) (Liu and Hall 2004), and the ITS region.

    The main objective of this study was to compare the relative level of DNA polymorphisms of GPDH, ITS, and RPB2 within each species and determine haplotype diversity and infer phylogenetic relationships of these isolates with publicly available sequences of the GPDH gene of Alternaria spp. from different countries. GPDH sequences were selected because this nuclear gene has been used previously to analyze the phylogenetic analysis of A. alternata and A. solani, respectively (Ding et al. 2019; Lourenço et al. 2009). A haplotype herein is defined as a set of DNA variations or single nucleotide polymorphisms (SNPs) found on the gene regions (Rebbeck et al. 2004). The findings of this study are important for understanding the phylogenetic relationships among isolates of Alternaria spp. recovered from tomato and potato and for devising disease resistance breeding programs.

    MATERIALS AND METHODS

    Sample locations and collection.

    Tomato is mainly grown in NC, and leaf blight symptoms caused by A. alternata and A. linariae in tomato were found at late and early stages of plant growth, respectively. The northeast and central NC (∼100 m above sea level) regions have a warm humid and coastal climate and mild winters. A. alternata was found to be sporadic in these regions in NC, and we intentionally sampled this species from the northeast and central region between 2012 and 2014 (Table 1). In the case of A. alternata, samples from Stokes County were mainly collected from heirloom tomatoes grown on an organic farm while samples from Pender and Sampson counties were collected from commercial hybrids. The western NC represents high elevations (>600 m above sea level) and has a temperate climate with cold winters and rainy summers. A. linariae was endemic in western NC and leaf samples were intentionally collected from major tomato-growing counties during the early season in 2014. The EB symptoms showing circular lesions with concentric rings surrounded by a yellow hallow caused by A. linariae were commonly found in hybrid tomato and sampled from five counties: Haywood, Henderson, Macon, Madison, and Swain using a stratified random sampling approach (Cochran 1977). Up to four young leaflets with EB lesions were sampled from each plant and ranged from 20 to 60 samples per county. The procedures for isolation of A. alternata and A. linariae from tomato have been described previously (T. B. Adhikari, S. Timilsina, K. Bhattarai, I. Meadows, D. Halterman, and F. J. Louws, unpublished data).

    TABLE 1. Identity, host plant, geographic location of origin, accession number, and isolates of Alternaria species used in this studya

    Weather conditions in WI typically include extreme cold winters and warm summers and tomato are rarely grown in WI. A. alternata isolates were isolated from potato in WI but they only considered A. alternata as an opportunistic pathogen (Weber and Halterman 2012). EB caused by A. solani was an endemic disease mainly in Waushara County, WI. Isolation and purification of A. solani isolates were performed previously (Weber and Halterman 2012). In all, 152 single-spore isolates of Alternaria spp. were collected and used in this study (Table 1). Population herein is referred to as samples or isolates collected from a location or county.

    Morphological characterization.

    Each isolate was transferred to acidified potato dextrose agar (A-PDA) medium (4 g of potato starch, 20 g of dextrose, and 15 g of agar/liter of distilled water and amended with two antibiotics: ampicillin [0.06 g/liter] and rifampicin [0.024 g/liter]). Morphological characters (e.g., conidia type, septate, presence and number of beaks) of the isolates were examined under a binocular stereomicroscope (Leica Microsystems, Wetzlar, Germany). Species identification based on morphology was performed as described previously (Simmons 2007; Woudenberg et al. 2014).

    Molecular characterization.

    Each culture was transferred to 50 ml of Difco potato sucrose broth medium in Erlenmeyer flasks that were continuously agitated at 110 rpm at room temperature. After 7 days, mycelium was transferred to 2-ml centrifuge tubes and lyophilized. To obtain the fine powder, the lyophilized-mycelium tissues were ground using a microtube homogenizer (Model D1030-E, Beadbug, Benchmark Scientific Inc., Edison, NJ) at 400 rpm for 3 min. Genomic DNA was extracted using the DNeasy Plant Mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The DNA concentration was quantified using a spectrophotometer (ND-1000, NanoDrop Technologies Inc., Wilmington, DE) and adjusted to a concentration of DNA at 10 ng/µl for each isolate. In the previous study (Gannibal et al. 2014), OAsF7 and OAsR6 primers were used to differentiate morphologically similar large-spores Alternaria spp. such as A. solani and A. linariae. We used A. solani-specific primers OAsF7 (5′-CGACGAGTAAGTTGCCCTCA-3′) and OAsR6 (5′-TGTAGGCGTCAGAGACACCATT-3′), which amplified the Alt a1 genomic region only in A. solani, and another primer set, OAtF4 (5′-TGCGGCTTGCTGGCTAAGGT-3′) and OAtR2 (5′-CAGTCGATGCGGCCGTCA-3′), which amplified DNA fragment from the calmodulin encoding gene of A. linariae and some other large-spored Alternaria species excluding A. solani (Gannibal et al. 2014).

    PCR assays and sequencing.

    Three nuclear loci: GPDH, RPB2, and the ITS region were selected because some of these genes were used previously to compare patterns of genetic variation of A. solani from tomato and potato in Brazil (Lourenço et al. 2009) and A. solani and A. alternata from potato in WI (Ding et al. 2019). Partial sequences of GPDH, RPB2, and the ITS region were amplified using primer sets GPD-F/GPD-R (Berbee et al. 1999), RPB2-F/RPB2-R (Woudenberg et al. 2015), and ITS4 and ITS5 (White et al. 1990), respectively. A 25-µl mixture was used for the amplification and each PCR contained 8.5 µl of sterile distilled water, 2 µl of 10 ng/µl genomic DNA, 1 µl each of 10 µM reverse and forward primer, and 12.5 µl of GoTaq Green PCR mix (Promega Inc., Madison, WI). The amplification with GPDH and ITS primer sets was performed individually using the same program: 95°C for 30 s, followed by 44 cycles: 94°C for 15 s, 50°C for 30 s, and 68°C for 3 min; and final elongation of 72°C for 15 min. The amplification of RPB2 was the following: 95°C for 30 s; 30 cycles: 94°C for 15 s, 60°C for 30 s, 68°C for 3 min and final extension 72°C for 7 min. The quality of the PCR product of each sample and fragment of the expected size was confirmed by electrophoresis on a 1.5% agarose gel (wt/vol). PCR product purification was done using ExoSAP-IT PCR product cleanup reagent (Affymetrix, Inc., Santa Clara, CA). Six microliters of PCR product and 1 µl of 10 µM reverse or forward primer were added to each reaction and sent for sequencing at Genomic Sciences Laboratory, North Carolina State University, Raleigh, NC.

    Sequence analysis and alignments.

    Raw sequence data were visually evaluated for quality using Geneious ver. 11.1.4 (1 May 2018; Biomatters Ltd., Auckland, New Zealand). Consensus sequences were generated and aligned using eight iterations of MUSCLE (Edgar 2004). Base substitutions were grouped as phylogenetically informative or uninformative and each polymorphic site received a specific site number on the consensus sequence.

    Nucleotide diversity and haplotype assignment within species.

    The relative degree of DNA polymorphisms for each gene sequence data set was examined among isolates of each species. Nucleotide diversity at the three loci: GPDH, ITS, and RPB2 were determined using DNA Sequence Polymorphism software (DNASP) ver.6 (Rozas et al. 2017). To arbitrarily assign locus-specific and species-specific haplotype, sequence data sets were generated for each locus separately. Three data sets were mined to identify haplotype within species in this study: (i) AaH1 to AaH6 haplotype for RPB2 sequences for all 46 isolates of A. alternata; (ii) AaH1 to AaH5 haplotype for GPDH sequences for all 48 isolates of A. alternata; and (iii) AsAlH1 to AsAlH12 haplotype for GPDH sequences for all 106 isolates of A. solani and A. linariae. For comparisons, A. solani and A. linariae haplotypes AsAlH1 to AsAlH6 were like previously reported haplotype nomenclature (Lourenço et al. 2009). For each gene sequence data set, the SNAP Map program (Price and Carbone 2005) was used to collapse nucleotide sequences into haplotypes (Aylor et al. 2006). The SNAP Workbench Java program package was used to analyze gene genealogies and population parameters (Price and Carbone 2005). Compatibility matrices were developed for haplotypes that share a common ancestry to examine the overall or conflict among variable sites in DNA sequence alignments as described previously (Jakobsen et al. 1997). The population mutation parameter per nucleotide site Θ using Watterson’s (1975) Θw, based on the number of segregating sites (S), and the average pairwise nucleotide diversity (π) (Tajima 1989) were estimated using DNASP ver.5 (Librado and Rozas 2009) for each locus across isolates of each species.

    Tests of neutrality.

    Neutrality test statistics were estimated with Tajima’s D (TD) (Tajima 1989), Fu and Li’s D (FLD), Fu and Li’s F (FLF) (Fu and Li 1993), and Fu’s F (Fu 1997) using 5,000 permutations (Kimura 1980). Based on these tests, if only TD, FLD, and FLF were significant and Fu’s F was nonsignificant, this indicated background selection (Fu 1997). If only Fu’s F was significant, this suggested population growth. Likewise, when TD and Fu’s F test values were negative and nonsignificant, it indicated an excess of mutations. Positive values suggested a population division or background selection (Carbone et al. 2007). These four neutrality tests were calculated for all isolates of each species for each locus and the combined data of A. linariae and A. solani (N = 106).

    Phylogenetic inference.

    To determine genetic relationships among isolates within species, both ends of each gene sequences were trimmed to the same length before concatenating the sequences. The concatenation of the three gene sequences of each isolate was used for phylogenetic analysis. Phylogenetic relationships were analyzed using maximum likelihood (ML). For ML analysis, PAUP* ver. 4.b10 (Swofford 2004) was used with resampling estimated log-likelihood of 1,000 bootstrap replicates. Multilocus tree building was performed using the Tamura-Nei genetic distance model (Tamura and Nei 1993). Metadata were analyzed with the Tamura-Nei model using the Tree-Based Alignment Selector T-BAS v 2.0 (Carbone et al. 2017). Bootstrap analysis was used to determine the statistical support for each branch of trees generated with 1,000 replications. Phylogenetics was displayed in the circle tree format and the results were visualized as the outer rings of multigene ML phylogeny.

    Comparison of GPDH sequences between the current isolates of A. alternata and the global isolates.

    The GPDH gene sequences of 46 isolates of A. alternata from tomato in NC (Table 1) were compared with publicly available GPDH gene sequences of international isolates downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). For haplotype comparisons, sequences of 15 representative isolates of A. alternata from potato from the previous study of Ding et al. (2019) were retrieved. The isolates included were SAa1 (accession no. MG525459), SAa2 (MG525460), SAa3 (MG525461), SAa4 MG525462), SAa5 (MG525463), SAa6 (MG525464), SAa7 (MG525465), SAa8 (MG525466), SAa9 (MG525467), and SAa10 (MG525468) collected from Grand Marsh, Hancock, and Plover counties from WI in 2017, and Aa35 (MF279611), Aa52 (MF279619), Aa60 (MF279624), Aa70 (MF279631), and Aa79 (MF279640) collected from Hancock and Plover counties from WI in 2012. Also, sequences of the 17 international isolates of A. alternata collected from various host plants were downloaded from GenBank and included for comparisons (Table 1). These isolate sequences were from China (N = 8), Iran (N = 3), Italy (N = 1), Serbia (N = 3), and South Korea (N = 2). The results were visualized in T-BAS v.2.0 (Carbone et al. 2017).

    Comparison of GPDH sequences between the current isolates of A. linariae and A. solani and the global isolates.

    GPDH gene sequences of the 59 isolates of A. linariae sampled from tomato in NC and 47 isolates of A. solani from potato were analyzed (Table 1). Additionally, six isolate sequences AS063_T (accession no. EU617542), AS307_P (EU61539), AS084_P (EU617510), AS069_T (EU17508), AS300_T (EU617537), and AS252_T (EU617532) of A. solani from Brazil were included for haplotype comparisons (Lourenço et al. 2009). For A. linariae, GPDH sequences downloaded from GenBank were from Algeria (N = 4) and Belgium (N = 1). For A. solani, GPDH sequences were from China (N = 1), Denmark (N = 1), Iran (N = 1), Netherland (N = 1), Pakistan (N = 1), and the United States (N = 10). Also, A. tomatophila (syn. A. linariae) isolate sequences used were one each from Australia, China, New Zealand, and Venezuela and two each from Russia and the United States. We also included sequences of A. porri and A. grandis from the United States (Table 1). FASTA format of each isolate was trimmed and aligned using Geneious ver. 11.1.4. Tree outputs were assembled for each isolate using the T-BAS v.2.0 as described previously (Carbone et al. 2017).

    Comparison of the RPB2 and GPDH locus-based haplotypes of Alternaria spp. identified in the current study with publicly available Alternaria whole-genome sequences.

    We retrieved publicly available whole-genome sequence data of three isolates: BMP 0185 (A. solani) (Dang et al. 2015), HWC-128 (A. solani; accession no. JRWV00000000), and SRC1lrK2f (A. alternata; accession no. GCA_001642055) were retrieved from GenBank and compared with the haplotypes identified in the current study. Sequence data were evaluated using Geneious ver. 11.1.4 (1 May 2018; Biomatters Ltd., Auckland, New Zealand) and aligned using MUSCLE (Edgar 2004). Base substitutions were between the RPB2 and GPDH locus-based haplotypes and whole-genome isolates were compared and grouped at a specific site on the consensus sequences.

    RESULTS

    Morphological characterization.

    Three Alternaria spp. can cause leaf blight symptoms with the same appearance of lesions in both tomato and potato in NC and WI. In total, 152 isolates were collected and identified as Alternaria spp. based on the microscopic examination of morphological traits. For purified isolates grown on A-PDA, colonies with aerial mycelium were dense and black in reverse. Dark brown conidia were small and large, and separated with or without a long beak. Based on the size of the conidia and the presence of beak, the isolates of Alternaria spp. were classified into two groups: small-spore and large-spore. All samples collected from the north and central NC were small-spored without beak and were identified as A. alternata. Similarly, all samples collected from the western NC were large-spored with long multiple beaks and were assigned to A. linariae, while all large-spored conidia with a single long beak collected from potato in WI were A. solani.

    Molecular characterization.

    As expected, the primer set OAsF7/OAsR6 amplified a 164 bp fragment from A. solani only, while OAtF4 and OAtR2 primers amplified a 438-bp DNA fragment from A. linariae (Gannibal et al. 2014). PCR assay based on DNA samples from tomato and potato confirmed the isolates to be A. linariae and A. solani, respectively.

    Tests of neutrality.

    Nucleotide polymorphisms analyzed through gene-by-gene comparisons in each species included 483 nucleotides for GPDH, 474 for ITS, and 669 for RPB2 and concatenated data for the three genes was 1,626 bp in length (Table 2). Intraspecific variability was detected in each species across the isolates analyzed. By comparisons across three species, 30, 31, and 59 nucleotide polymorphisms were identified in the GPDH, ITS, and RPB2 genes, respectively (Table 2). Nucleotide diversity (π) estimates in three species ranged from 0.00088 to 0.00264 for the GPDH gene; from 0 to 0.00019 for the ITS region, and 0 to 0.00674 for the RPB2 gene (Table 3). For the combined A. linariae and A. solani populations, π values for the GPDH, ITS, and RPB2 genes were 0.00278, 0.00004, and 0.01079, respectively. The GPDH and RPB2 genes had a higher number of segregating sites (S) and mean mutation parameters per site (Watterson’s Θw) than the ITS region (Table 3).

    TABLE 2. Variable bases in two nuclear genes, glyceraldehyde-3-phosphate dehydrogenase (GPDH) and RNA polymerase second largest subunit (RPB2), and the internal transcribed spacer (ITS) rDNA region were analyzed on the 152 isolates of three Alternaria spp. from tomato in North Carolina (NC) and potato in Wisconsin (WI)a

    TABLE 3. Estimates for neutrality tests and genetic diversity for three closely related Alternaria spp. collected from tomato in North Carolina (NC) and from potato in Wisconsin (WI)

    We used four neutrality tests to determine whether the data departed from an equilibrium model of neutral evolution and provided insight into an indication of recent population expansion. In A. alternata, most of the neutrality test values were negative for the GPDH locus and the ITS region (Table 3). Although FLD and FLF estimates were negative, these two values were significant (P ≤ 0.05) for the GPDH gene, suggesting an excess of recent mutations in the A. alternata population. In the case of A. linariae, TD, FLD, and Fu’s F values were negative for the RPB2 gene, and only the FLF estimate was positive. For A. solani, all four estimates were negative. However, FLF values were significant for both GPDH and ITS within selective species (Table 3). For the combined A. linariae and A. solani, TD and FLF statistics were positive and significant for the RPB2 gene. In contrast, all four neutrality test statistics (TD, FLD, FLF, and Fu’s F) were negative for both GPDH and ITS genes, but TD, FLD, and FLF values for the GPDH gene were significant (Table 3).

    Haplotype identification within species.

    For the ITS gene sequences, low nucleotide polymorphism was observed. Thus, it was less informative to identify haplotypes in all species tested. For A. alternata, the GPDH gene sequence data set yielded five haplotypes (AaH1, AaH2, AaH3, and AaH4, and one unique haplotype AaH5) of A. alternata with seven SNPs (Fig. 1; Table 4). Likewise, the GPDH gene data set yielded 12 haplotypes of A. solani and A. linariae with seven SNPs (Table 5; Fig. 1). Among them, six haplotypes (AsAlH7, AsAlH8, AsAlH9, AsAlH10, AsAlH11, and AsAlH12) were unique and new (Fig. 1). Intriguingly, a single SNP at position 20 led to an amino acid change from valine (Val) to leucine (Leu) in the isolates belonging to three haplotypes of A. linariae and A. solani: H7, H8, and H9 (Table 5). The RPB2 gene sequences had a lower number of polymorphic sites than the GPDH locus. Therefore, at least six haplotypes (H1 to H6) were detected with 15 SNPs in A. alternata for the RPB2 sequences (Table 6). Although 15 SNPs at the RPB2 sequences were found between A. linariae and A. solani, the identified nucleotide polymorphisms were not variable enough to allow for resolution to differentiate haplotypes within A. linariae or A. solani (Table 2).

    Fig. 1.

    Fig. 1. Identification and distribution of haplotypes (H) of Alternaria spp. analyzed using the single-copy two nuclear genes: the glyceraldehyde-3-phosphate dehydrogenase (GPDH) and RNA polymerase second largest subunit (RPB2). In all, 152 isolates of three closely related Alternaria spp. collected from the sampled counties in tomato- and potato-producing area (red spots on the map) in North Carolina (NC) and Wisconsin (WI). Numbers in pie charts indicate the frequency of isolates from each haplotype from each species of Alternaria.

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    TABLE 4. Nucleotide polymorphisms in the glyceraldehyde-3-phosphate dehydrogenase (GPDH) among haplotypes of Alternaria alternata isolates (N = 46) collected from tomato in North Carolina

    TABLE 5. Nucleotide polymorphisms in the glyceraldehyde-3-phosphate dehydrogenase (GPDH) locus among haplotypes of Alternaria linariae (N = 59) and A. solani (N = 47) collected from tomato and potato in North Carolina and Wisconsin, respectively

    TABLE 6. Nucleotide polymorphisms in the RNA polymerase second largest subunit (RPB2) locus among haplotypes of Alternaria alternata (N = 46) collected from tomato in North Carolina

    For the GPDH gene sequences, AaH1 to AaH5 were more common in A. alternata than other haplotypes in all counties in NC sampled. However, AaH1 (N = 25) was the most frequent haplotype in Stokes County followed by Henderson and Sampson counties (Supplementary Table S1; Fig. 1). The number of isolates detected in each haplotype in the other counties was low (between 0 and 3). One unique haplotype was detected in Henderson and Stokes counties (N = 4). Based on the RPB2 gene, six haplotypes (AaH1 to AaH6) and one unique haplotype were found in A. alternata from NC (Supplementary Table S2; Fig. 1). In Stokes County, haplotype AaH3 was common (N = 10) followed by AaH5 (N = 6) and AaH4 (N = 5). In other counties, the distribution of isolates in each haplotype was low ranging from 0 to 3.

    In A. solani, AsAlH4 (N = 42) was exclusively found in Waushara County, WI (Supplementary Table S3; Fig. 1) followed by AsAlH12 (N = 2). Only one unique haplotype (N = 3) was found. However, no other haplotypes were detected in Waushara County, WI. In A. linariae, haplotypes AsAlH2/H3 were found in Haywood (N = 7), Macon (N = 8), and Madison (N = 4) counties (Supplementary Table S3; Fig. 1). Similarly, haplotype AsAlH7 was detected in Haywood (N = 1), Madison (N = 1), and Swain (N = 8) counties. Haplotype AsAlH8 was rare but it was found only in Madison and Swain counties. Likewise, haplotypes H9 and H10 were detected only in Madison and Macon counties. Haplotype AsAlH11 was present in Haywood and Madison counties. Interestingly, haplotypes AsAlH4 and AsAlH12 were absent in A. linariae in NC. One unique haplotype was detected in Haywood (N = 2), Macon (N = 3), and Swain (N = 1) counties (Supplementary Table S3; Fig. 1). At the RPB2 locus, 15, 43, and 44 nucleotide variations occurred between A. linariae and A. solani, A. linariae and A. alternata, and A. solani and A. alternata, respectively (Supplementary Table S4). Interestingly, all isolates of A. linariae analyzed had similar SNPs at the RPB2 region and had a poor resolution to distinguish isolates within this species. Similarly, for A. solani isolate sequences were too conserved and did not distinguish isolates within A. solani. However, there was an apparent separation of A. linariae isolates and those of A. solani isolates based on the metadata profile of the sequences mined from the RPB2 locus (Table 2; Supplementary Table S4).

    Gene genealogy.

    The rooted trees generated from the RPB2 gene sequences revealed that haplotype networks showed mutations along with clades specific to A. alternata; however, these gene sequences were not variable enough to allow for resolution among the isolates within A. solani and A. linariae to form discrete subbranches (Fig. 2A). Based on the RPB2 locus sequence, high bootstrap score (97%) supported haplotypes AaH4, AaH5, and AaH6 network and appeared to be independent; however, the remaining haplotypes (bootstrap score >58%) AaH1, AaH2, and AaH3 were likely divergent from other haplotypes. Sequences of the two representative isolates of A. linariae (isolate 1) and A. solani (P-2) were located in two branches but had no discernible bootstrap score (100%) between these two isolates. The patterns of haplotype branches based on ML analysis for the GPDH gene sequences broadly separated three species into two clades (Fig. 2B). The top clade contained the A. alternata haplotypes: (AaH1 to AaH4 [bootstrap score >74%]) while another clade consisted of the two haplotypes (AsAl12 and AsAlH4 [score 100%]) of A. solani and six new haplotypes (AsAl H7 to AsAl H11 located at the bottom belonging to A. linariae) (Fig. 2B).

    Fig. 2.

    Fig. 2. Phylogenetic relationships of Alternaria alternata (Aa), A. solani (As), and A. linariae (Al) of haplotypes based the sequences of the A, RNA polymerase second largest subunit (RPB2) and B, glyceraldehyde-3-phosphate dehydrogenase (GPDH). Each number indicates a representative isolate from each haplotype, and letter before isolate number refers to host: T = tomato and P = potato from which isolate was collected. All rooted maximum-likelihood trees are drawn to scale with branch lengths proportional to the substitutions per site. Bootstrap support was calculated from 1,000 replicates and values >50% are indicated above the branches.

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    Phylogenetic analysis.

    In the previous studies (Ding et al. 2019; Woudenberg et al. 2015), single-gene phylogenetic analyses could not separate isolates of small-spored Alternaria into groups. In this study, phylogenetic analyses based on the concatenation of three nuclear loci (GPDH, ITS, and RPB2) sequences showed evolutionary diversity within each species (Fig. 3). ML analysis of A. alternata isolates placed them into few clades. Although the tree topologies identified eight haplotypes (Fig. 2B), combined sequence data within isolates of A. linariae and A. solani appeared to represent monophyletic species with 100% bootstrap value (Fig. 3). No association between haplotype or species and host geographic locations was observed. Using the multigene phylogeny analyses, there was no cluster detected by location in A. alternata isolates collected from three locations: Grand Marsh, Hancock, and Plover from potato in WI (Ding et al. 2019). A. alternata genotype 1A was predominantly found in most locations sampled from potato in WI.

    Fig. 3.

    Fig. 3. The phylogenetic tree derived from a concatenated sequence made up of glyceraldehyde-3-phosphate dehydrogenase (GPDH) and RNA polymerase second largest subunit (RPB2), and the rDNA internal transcribed spacer (ITS) region of the 152 isolates of Alternaria alternata (N = 46), A. linariae (N = 59), and A. solani (N = 47) isolates from North Carolina and Wisconsin. Innermost ring: host plant from which isolates were collected and each isolate designation is connected with principal component value. Letter before isolate number refers to host: T = tomato and P = potato from which collected; ring 2: fungal species A. alternata, A. linariae, and A. solani; and ring 3: state or county from which the isolate was sampled. Bootstrap values >50 are labeled along branches.

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    Comparison between Alternaria spp. GPDH locus-based haplotypes of the current study and global isolates.

    The phylogenetic relationships among global isolates of Alternaria spp. were inferred by analysis of GPDH sequences because both ITS and RPB2 sequences in the data set were too invariable for accurate phylogeny. Therefore, we compared the GPDH sequences of 46 isolates of A. alternata with 22 global isolate sequences from different countries. Similarly, sequences of 59 isolates of A. linariae from tomato and sequences of 47 isolates of A. solani with 26 sequences from the previous studies (Ding et al. 2019; Lourenço et al. 2009) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/) were downloaded. A. alternata metadata revealed that haplotype AaH1 exhibited the highest frequency within the GPDH sequences from NC and other countries and formed similar network topologies (Fig. 4). On the other hand, haplotypes AaH3, AaH5, AaH9, andAaH10 exhibited the lowest frequency among the GPDH sequences from tomato in NC. These haplotypes were not present in other countries. Of the GPDH sequences of the 15 isolates of A. alternata from potato in WI of the study of Ding et al. (2019) that we compared, three isolates (Aa3, Aa52, and Aa70 sequences) appeared to be identical to sequences of our five isolates (T9, T16, T36, T71, and T77) of A. alternata from tomato in NC. Based on the mutations, six haplotypes of A. solani were inferred for the GPDH locus in the previous study (Lourenço et al. 2009). Among these haplotypes, H2, H3, H4, and H5 were detected in tomato isolates while haplotypes H1 and H6 were found in potato isolates. Based on the frequency from where they originated, haplotype AsAlH4 described here appeared to represent the most important GPDH haplotype within sequences of A. solani isolates from WI and global isolates including Australia, Algeria, Brazil, China, New Zealand, and Russia (Table 1; Fig. 5). The most common haplotype AsAlH2/H3 was found within A. linariae from tomato in the current study and was also exclusively present in Algeria, Belgium, China, Demark, Iran, Netherland, Pakistan, Russia, and several other states (e.g., IN, PA, and AZ) of the United States. (Table 1). The results of this study suggest that two haplotypes (AsAlH2/H3 and AsAlH4) could be widely distributed clones in tomato- and potato-growing regions.

    Fig. 4.

    Fig. 4. The maximum-likelihood phylogenetic trees of the 46 isolates of Alternaria alternata sampled from tomato and potato produced from North Carolina and Wisconsin, and the representative isolates from different countries were derived using the Tamura-Nei DNA substitution model on the glyceraldehyde-3-phosphate dehydrogenase (GPDH) locus sequence alignment. For comparison purposes, sequences of the global isolates of A. alternata were obtained from the GenBank BLASTn search engine of the National Center for Biotechnology Information. Bootstrap values (>50%) are shown above the branches. GPDH haplotypes were determined by differences at variable sites, and dashed lines connect individual isolates. Innermost ring 1: principal component analysis, isolate designation and accession numbers from GenBank, and country of origin from which these isolates were collected. Letter before isolate numbers refers to tomato (T) isolates from which isolates were collected and sequenced; ring 2: haplotypes of A. alternata; and ring 3: host plants from which these isolates were sampled. The dotted lines within the ring are cosmetic, and solid lines refer to high nucleotide polymorphisms.

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    Fig. 5.

    Fig. 5. The maximum-likelihood phylogenetic trees of the 59 and 47 isolates of Alternaria linariae and A. solani sampled from tomato and potato production regions from North Carolina and Wisconsin, respectively, and representative isolates derived using the Tamura-Nei DNA substitution model on the glyceraldehyde-3-phosphate dehydrogenase (GPDH) locus sequence alignment. For comparison purposes, sequences of the global isolates of A. linariae, A. solani, A. grandis, and A. porri were obtained from the GenBank BLASTn search engine of the National Center for Biotechnology Information. Bootstrap values (>50%) are shown above the branches. GPDH haplotypes were determined by differences at variable sites, and dashed lines connect individual isolates. Innermost ring 1: Alternaria spp., principal component analysis, and isolate designation and accession numbers from GenBank, and A. linariae and A. solani. Letter before isolate numbers refers to potato (P) isolates from which isolates were collected and sequenced. Ring 2: haplotype; ring 3: host plant of origin from which these isolates were sampled, and ring 4: country of origin from which these isolates were collected. The dotted lines within the ring are cosmetic, and solid lines represent high nucleotide polymorphisms.

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    Comparison of the RPB2 and GPDH locus-based haplotypes of Alternaria spp. with publicly available Alternaria whole-genome sequences.

    For both genes, the haplotypes identified in the current study using our approach were compared with publicly available A. alternata and A. solani whole-genome sequences. For comparing the RPB2 locus sequences, we identified six haplotypes of the A. alternata and these haplotype sequences had little or no (0 to 8) nucleotide differences between them and their respective isolates (Supplementary Table S5). Importantly, A. alternata haplotype AaH3 identified from the RPB2 gene sequence corresponded perfectly with the reference genome sequence of A. alternata isolate SRC1lrK2f (Supplementary Table S5). For the RPB2 locus, A. solani representative isolate P2 sampled from a potato field in WI was identical to the genome sequence of A. solani isolate BMP 0185 from Arkansas of the United States. However, there were three nucleotide differences between A. solani isolate P2 and another genome sequence of A. solani isolate HWC 168 from China (Supplementary Table S5). Our results show 47 to 50 nucleotide differences between A. alternata isolate SRC1lrK2f versus A. solani and A. linariae, and between A. solani isolate BMP 0185 versus A. alternata haplotypes.

    For the GPDH locus, maximum nucleotide differences resulted in haplotypes of A. alternata when compared with the reference genome of A. solani isolate BMP 0185 and isolate HWC 168 or when A. solani and A. linariae haplotypes were compared with the reference genome of A. alternata isolate SRC1lrK2f (Supplementary Table S6). However, in each of the haplotypes corresponding to A. solani and A. linariae, there were one to two nucleotide differences. With the GPDH locus-based sequence dataset, A. solani and A. linariae haplotype P1-AsAlH4 was identical with the genome of A. solani isolate BMP 0185 and isolate HWC 168 (Supplementary Table S6). Similarly, A. alternata haplotype T-5AaH1 corresponded perfectly with the reference genome of A. alternata isolate SRC1lrK2f.

    Sequence data availability.

    The DNA sequences reported in this study have been deposited in GenBank under the accession numbers MK928512 to MK928663 (ITS), MK928664 to MK928815 (GPDH), and MK928816 to MK928967 (RPB2). Sequences of publicly available Alternaria whole-genome sequences of the three isolates used in this are available from GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

    DISCUSSION

    One of the primary objectives of the current study was to use the gene genealogical approach to improve our understanding of the genetic diversity of economically important members of the Alternaria spp. Toward this end, we analyzed 152 isolates of Alternaria spp. sampled from major tomato and potato growing areas in NC and WI. We also included DNA sequence data per isolate used in previous studies (Ding et al. 2019; Lourenço et al. 2009; Woudenberg et al. 2014). As a result, phylogenetic analysis of a single-gene data set was informative to provide the haplotype diversity and evolutionary relationships between species of Alternaria spp. As noted in published phylogenetic studies (Ding et al. 2019; Lourenço et al. 2009; Woudenberg et al. 2015), our results also indicate that nucleotide diversity was lower for the ITS sequences than the RPB2 and GPDH sequences. Although the RPB2 sequences were too conserved and did not distinguish among individuals within A. linariae or within A. solani, nucleotide substitution sites or SNPs were useful to allow delineation at the species level. The GPDH sequences outperformed the other two genes in providing superior resolution for haplotype placement and phylogenetic inference. For A. linariae or A. solani, sequences of haplotypes AsAlH1 to AsAlH6 were identical to previously reported haplotypes from Brazil (Lourenço et al. 2009). The remaining AsAlH7 to AsAlH12 were unique and new haplotypes. Our results suggest that A. linariae or A. solani contain new and previously reported haplotypes that are capable of causing disease epidemics in tomato- and potato-producing areas in NC and WI.

    Low level of genetic diversity was found for Alternaria spp. in this study. Based on the ITS locus, Lourenço et al. (2009) also reported a low level of diversity for isolates of A. solani collected from tomato and potato in Brazil. Similarly, ITS locus was less informative and resulted in poor resolution in Fusarium spp. (O’Donnell et al. 2013; Ramdial et al. 2017). In contrast, the GPDH gene sequences were useful and informative to compare the genetic diversity and genetic relationships of Alternaria spp. in previous studies (Ding et al. 2019; Lourenço et al. 2009). In the case of A. linariae or A. solani, 13 haplotypes (including unique haplotypes) were identified, and six of them were identical to previously reported haplotypes (Lourenço et al. 2009). The remaining haplotypes were new. Our findings based on the consensus sequence across the GPDH locus suggest that the two most frequently diverse haplotypes, AsAlH2/AsAlH3 and AsAlH4, were associated with A. linariae and A. solani in the United States. Interestingly, all global isolates of A. linariae and A. solani included in this study share these haplotypes. Our findings support previous results showing that the majority of gene diversity may be distributed within individuals and populations are not geographically differentiated (Linde et al. 2002; McDonald et al. 1999). We found seven new haplotypes (AsAlH7 to AsAlH13) that were not present in previously reported haplotypes of A. solani (Lourenço et al. 2009). Among these haplotypes, AsAlH2/AsAlH3 and AsAlH4 have been mainly detected in A. linariae isolates from tomato in NC and in A. solani isolates from potato in WI, respectively, but formed a single clade in the neighbor-joining trees. These two haplotypes also displayed closer proximity to the clusters that contain haplotypes AsAlH7, AsAlH8, AsAlH9, AsAlH10, and AsAlH11 of A. linariae and one haplotype AsAlH12 of A. solani. Although population differentiation according to the host of origin in this study was not possible due to limited sample size and no overlap between tomato and potato, the haplotype association with host species has been demonstrated in the previous study (Lourenço et al. 2009). For example, the GPDH locus-based haplotypes (H1, H2, H4, and H5) were mainly of tomato isolates, whereas two haplotypes (H6 and H7) were commonly detected in potato isolates from Brazil (Lourenço et al. 2009). Future research will need to collect additional samples from different hosts, geographic locations, and years, and a more detailed assessment involving multigene sequence analysis may provide insight into the genetic diversity of Alternaria spp. across the United States.

    Based on combined sequences analysis of four genes (Alt a1, GPDH, ITS, and translation elongation factor), Ding et al. (2019) reported five genotypes of A. alternata from potato sampled in WI. In our study, we also included the 15 isolate sequences of A. alternata from potato from the previous study (Ding et al. 2019) for comparisons. Of the 15 isolate sequences analyzed, 12 isolates belonged to genotype I in the previous study and were haplotype AaH1 in this study. Similarly, one isolate was genotype III and two isolates were genotype V. Although we found the GPDH gene sequence similarities between the isolates of A. alternata from potato sampled in WI and the isolates of A. alternata from tomato sampled in NC, the haplotype nomenclature in the previous study was based on multigene analysis (Ding et al. 2019). However, in our analysis, it was based on single-copy GPDH gene sequences. Haplotype networks of A. alternata from tomato are considered to be a conservative exploration of understanding the phylogenetic hypothesis that described the relationships among closely related species such as those of A. alternata from potato between the different geographic origin (NC versus WI) and isolates infecting different host plant of origin (tomato versus potato).

    We analyzed the genetic variability of Alternaria spp. using two diversity estimates (S and π) (Nei 1973) and assessed simple models of Watterson’s Θw (Watterson 1975) and four neutrality tests: D, FLD, FLF, and Fu’s F (Carbone et al. 2007; Fu 1997; Fu and Li 1993; Tajima 1989). Data indicate greater π and Watterson’s Θw estimates in A. alternata than estimates in A. linariae and A. solani isolates. In general, a higher degree of nucleotide diversity is expected in an ancestral population (Nei 1973). Comparatively, S values for all three genes (GPDH = 9, ITS = 2, and RPB2 = 18) were higher in A. alternata than A. linariae and A. solani and can explain the differences in nucleotide diversity in these populations. Four neutrality test estimates varied with Alternaria spp. and the nuclear genes analyzed, and most values were not significant. All negative values for the three genes indicated an excess of recent mutation and population expansion in Alternaria spp. The mutation is one of the principal evolutionary mechanisms, which causes a heritable change in the nucleotide sequence in several fungi (Hartl and Clark 2007). We hypothesize that new haplotypes AsAlH7 to AsAlH13 have emerged independently in each population. Our data indicate Tajima’s D value was positive, but Fu’s F was nonsignificant for the GPDH gene in A. linariae. This result is likely due to balancing selection where the polymorphic alleles are in a position to diversify by additional mutations (Tajima 1989). In this study, A. alternata and A. linariae were sampled from tomatoes from limited geographic areas in NC between 2012 and 2014 while A. solani samples were collected from a potato from a single location in WI during the 2008 season. Thus, we were unable to compare haplotypes over time and space or determine whether some historical events had favorable or unfavorable effects on estimating the number of population genomic parameters for the respective pathogen populations.

    Morphological characterization of Alternaria large-spored species associated with EB of tomato and potato appeared to be insufficient to distinguish between A. solani and A. linariae (Woudenberg et al. 2014). Importantly, large-spored A. linariae has been isolated from Solanaceae vegetables with symptoms resembling that of EB at the early stages of infection (Ayad et al. 2017). Interestingly, A. linariae was not only specific to tomato but it has also been isolated from potato (Ayad et al. 2017; Bessadat et al. 2016; Rodrigues et al. 2010; Simmons 2000). Since A. solani and A. linariae are the most common species and widely distributed in tomato- and potato-producing regions, molecular markers are necessary to identify and differentiate these species. The RPB2 gene encodes a protein with a modest rate of evolutionary change and its polypeptide sequence has been used for phylogenetic studies in green plants (Denton et al. 1998) and ascomycetes, including lichen-forming taxa (Liu and Hall 2004). In the current study, comparative genealogy analyses indicate that gene variations exist in these closely related Alternaria isolates, suggesting that these three Alternaria spp. might have diverged in the genome evolution. In the previous studies, both GPDH and RPB2 genes have been used to compare sequence variation in agriculturally important Fusarium species complex (O’Donnell et al. 2000, 2013) and several Alternaria spp. (Woudenberg et al. 2015; 2014). In particular, we also found that the RPB2 locus sequences provided the most conclusive and specific evidence to delineate two closely related species: A. linariae and A. solani. We reasoned that both A. linariae and A. solani have identical asexual conidia morphology (Woudenberg et al. 2014) and can cause EB disease in tomato and potato (Ayad et al. 2017; Bessadat et al. 2016). Our result suggests that the development of molecular markers from the RPB2 genomic region can be useful to differentiate A. linariae and A. solani.

    All tomatoes grown in NC are for fresh market. Most of the tomato industries use conventional management practices and utilize the plasticulture system that includes the use of fumigants and conventional fungicides. However, there is a trend of growing organic tomatoes in NC. Tomato cultivars (hybrids, determinate, semideterminate, indeterminate, and heirloom) are diverse, with different combinations of fruit size, growth habit, and disease resistance (https://plantpathology.ces.ncsu.edu/pp-vegetables/pp-vegetables-tomatoes/). The isolates of A. solani and A. linariae were collected in the early season, while isolates of A. alternata were collected from tomato later of the season. EB epidemics on tomato caused by A. linariae in western NC are recurrent and could be due to planting susceptible tomato cultivars or hybrids, change in virulence of the pathogen, conducive weather parameters, and climate changes (Anderson et al. 2004; Stukenbrock and Bataillon 2012; Stukenbrock and McDonald 2008; Sumabat et al. 2018). In this study, A. solani isolates were collected from diverse potato breeding lines and sampled from only one location in WI. We found that the majority (>91%) of the isolates exhibited haplotype AsAlH4. In the previous study, Ding et al. (2019) analyzed 33 isolates of A. solani sampled from Grand Marsh, Hancock, and Plover, which were different sampling sites than in our study. Based on multigene phylogeny, they also found only one genotype of A. solani in different potato fields in WI. It appeared that A. solani isolates with identical (GPDH, ITS, and RPB2) marker sequences were well established in these environments and can cause disease epidemics in potato in WI.

    In conclusion, we used DNA sequencing at multiple loci and genealogical approaches to provide new insights into the population genetic structure of Alternaria spp. This study revealed the presence of previously reported haplotypes and new haplotypes in tomato- and potato-producing areas in NC and WI. Both A. linariae and A. solani can cause similar lesions on leaves and have been isolated from single symptomatic leaf tissue of tomato and potato (Ayad et al. 2017; Bessadat et al. 2016; Simmons 2007). SNP-based diagnostic molecular markers can be developed from sequences of the RPB2 gene and used to differentiate these two species.

    ACKNOWLEDGMENTS

    We thank Inga Meadows for helping with isolate collection; and Audrey Djunaedi, Stella Chang, and Virginie Rumsch for assisting in the laboratory.

    The author(s) declare no conflict of interest.

    LITERATURE CITED

    The author(s) declare no conflict of interest.