Abstract
The cosmopolitan fungal genus Alternaria consists of multiple saprophytic and pathogenic species. Based on phylogenetic and morphological studies, the genus is currently divided into 26 sections. Alternaria sect. Alternaria contains most of the small-spored Alternaria species with concatenated conidia, including important plant, human and postharvest pathogens. Species within sect. Alternaria have been mostly described based on morphology and / or host-specificity, yet molecular variation between them is minimal. To investigate whether the described morphospecies within sect. Alternaria are supported by molecular data, whole-genome sequencing of nine Alternaria morphospecies supplemented with transcriptome sequencing of 12 Alternaria morphospecies as well as multi-gene sequencing of 168 Alternaria isolates was performed. The assembled genomes ranged in size from 33.3–35.2 Mb within sect. Alternaria and from 32.0–39.1 Mb for all Alternaria genomes. The number of repetitive sequences differed significantly between the different Alternaria genomes; ranging from 1.4–16.5 %. The repeat content within sect. Alternaria was relatively low with only 1.4–2.7 % of repeats. Whole-genome alignments revealed 96.7–98.2 % genome identity between sect. Alternaria isolates, compared to 85.1–89.3 % genome identity for isolates from other sections to the A. alternata reference genome. Similarly, 1.4–2.8 % and 0.8–1.8 % single nucleotide polymorphisms (SNPs) were observed in genomic and transcriptomic sequences, respectively, between isolates from sect. Alternaria, while the percentage of SNPs found in isolates from different sections compared to the A. alternata reference genome was considerably higher; 8.0–10.3 % and 6.1–8.5 %. The topology of a phylogenetic tree based on the whole-genome and transcriptome reads was congruent with multi-gene phylogenies based on commonly used gene regions. Based on the genome and transcriptome data, a set of core proteins was extracted, and primers were designed on two gene regions with a relatively low degree of conservation within sect. Alternaria (96.8 and 97.3 % conservation). Their potential discriminatory power within sect. Alternaria was tested next to nine commonly used gene regions in sect. Alternaria, namely the SSU, LSU, ITS, gapdh, rpb2, tef1, Alt a 1, endoPG and OPA10-2 gene regions. The phylogenies from the two gene regions with a relatively low conservation, KOG1058 and KOG1077, could not distinguish the described morphospecies within sect. Alternaria more effectively than the phylogenies based on the commonly used gene regions for Alternaria. Based on genome and transcriptome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of only 11 phylogenetic species and one species complex. Thirty-five morphospecies, which cannot be distinguished based on the multi-gene phylogeny, are synonymised under A. alternata. By providing guidelines for the naming and identification of phylogenetic species in Alternaria sect. Alternaria, this manuscript provides a clear and stable species classification in this section.
Key words: Alternaria alternata, Alternaria arborescens species complex, Multi-gene phylogeny, Transcriptome sequencing, Whole-genome sequencing
Introduction
Alternaria sect. Alternaria contains most of the small-spored Alternaria species with concatenated conidia. Almost 60 morphological or host-specific species can be assigned to this section, including the type species of the genus Alternaria, A. alternata (Woudenberg et al. 2013). Alternaria alternata is known as the cause of leaf spot and other diseases in over 100 host species of plants (Rotem 1994), but also as postharvest disease in various crops (Coates & Johnson 1997) and of upper respiratory tract infections and asthma in humans (Kurup et al. 2000). Other important plant pathogens in sect. Alternaria include A. longipes, the causal agent of brown spot of tobacco, A. mali, the causal agent of Alternaria blotch of apple, A. gaisen, the causal agent of black spot of Japanese pear and A. arborescens, the causal agent of stem canker of tomato. The first descriptions of the A. alternata, A. tenuissima, A. cheiranthi and A. brassicicola species-groups, based on sporulation patterns, were made by Simmons (1995). More recent molecular-based studies revealed that Alternaria species cluster in several distinct species clades, now referred to as sections (Lawrence et al. 2013, Woudenberg et al. 2013), which do not always correlate with the species-groups that were delineated based on morphological characteristics. Currently, 26 Alternaria sections are recognised based on molecular phylogenies (Woudenberg, 2013, Woudenberg et al., 2014 Grum-Grzhimaylo et al. 2015). So far, species within sect. Alternaria have been mostly described based on morphology and / or host-specificity; yet the molecular variation between them is minimal. The standard gene regions used for the delimitation of Alternaria species are not able to delineate species within sect. Alternaria (Peever et al., 2004, Andrew et al., 2009). Multiple molecular methods have been tested or proposed for distinguishing the small-spored Alternaria species, including random amplified polymorphic DNA (Roberts et al. 2000), amplified fragment length polymorphism (Somma et al. 2011), selective subtractive hybridisation (Roberts et al. 2012) and sequence characterised amplified genomic regions (Stewart et al. 2013a). However, none of these methods successfully distinguished all morphospecies described within sect. Alternaria.
The terms forma specialis and pathotype have been used to describe isolates that are morphologically indistinguishable from A. alternata, but infect particular hosts. At least 16 different f. sp. epithets occur in the literature, of which most were raised to species level by Simmons (2007). Nishimura & Kohmoto (1983) proposed that Alternaria strains with identical morphology but producing different host-selective toxins (HST) should be defined as distinct pathotypes of Alternaria. Currently there are seven pathotypes of A. alternata described (Akimitsu et al. 2014), but this term is not widely adopted.
Because most morphospecies within sect. Alternaria cannot be distinguished based on sequences of standard housekeeping genes (Andrew et al. 2009), whole-genome sequencing technologies can be applied to search for genes, which can distinguish (most of) the described species (Lawrence et al. 2013). Since the introduction of next generation sequencing (NGS) many fungal genomes have become available for study, with the 1 000 fungal genomes project (Spatafora 2011) as a public stimulant for generating this kind of data. Currently there are two publicly available Alternaria genomes at NCBI (National Center for Biotechnology Information), namely A. brassicicola, sect. Brassicicola (BioProject PRJNA34523), and A. arborescens, sect. Alternaria (BioProject PRJNA78243).
In this study, whole-genome sequences of four Alternaria spp. from sect. Alternaria and five Alternaria spp. from five other sections were generated, and supplemented by transcriptome sequences of nine Alternaria spp. from sect. Alternaria and three Alternaria spp. from three other sections of Alternaria. Species were selected based on their phylogenetic position (Woudenberg et al. 2013) in such a way that they are representative of the genus Alternaria, from the sister section of sect. Alternaria, sect. Alternantherae (A. alternantherae), to the most distant section, sect. Crivellia (A. papaveraceae). Within sect. Alternaria, species were selected based on their economic importance. Based on the genome and transcriptome data, two gene regions with relatively low conservation, the eukaryotic orthologous group (KOG) protein loci, KOG1058 (96.8 % conservation) and KOG1077 (97.3 % conservation), were identified and tested for their potential discriminatory power within sect. Alternaria. Together with a standard multi-gene phylogeny of 168 Alternaria isolates based on sequences of parts of nine gene regions, namely the internal transcribed spacer regions 1 and 2 and intervening 5.8S nrDNA (ITS), the 18S nrDNA (SSU), the 28S nrDNA (LSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), translation elongation factor 1-alpha (tef1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG) and an anonymous gene region (OPA10-2), an attempt was made to create a clear and stable phylogenetic species classification in Alternaria sect. Alternaria.
Material and methods
Isolates
One-hundred-and-sixty-eight Alternaria strains, including 64 (ex-)type or representative strains, present at the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands, were included in this study (Table 1) based on the phylogenetic position derived from their ITS sequence. A “representative isolate” refers to the strain used to describe the species based on morphology in The Alternaria Identification Manual (Simmons 2007). Freeze-dried strains were revived in 2 mL malt / peptone (50 % / 50 %) and subsequently transferred to oatmeal agar (OA) (Crous et al. 2009). Strains stored in liquid nitrogen were transferred to OA directly from the −185 °C storage.
Table 1.
Isolates used in this study and their GenBank accession numbers.
ATCC: American Type Culture Collection, Manassas, VA, USA; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Personal collection of P.W. Crous, Utrecht, The Netherlands; DAOM: Canadian Collection of Fungal Cultures, Ottawa, Canada; DSM: German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany; E.G.S.: Personal collection of Dr. E.G. Simmons; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; HSAUP: Department of Plant Pathology, Shandong Agricultural University, China; IFO: Institute for Fermentation Culture Collection, Osaka, Japan; IHEM: Biomedical Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Brussels, Belgium; IMI: Culture collection of CABI Europe UK Centre, Egham UK; LCP: Laboratory of Cryptogamy, National Museum of Natural History, Paris, France; MAFF: MAFF Genebank Project, Ministry of Agriculture, Forestry and Fisherie, Tsukuba, Japan; MUCL: (Agro)Industrial Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Louvain-la-Neuve, Belgium; QM: Quarter Master Culture Collection, Amherst, MA, USA; VKM: All-Russian Collection of Microorganisms, Moscow, Russia.
T: ex-type isolate; R: representative isolate; Species names between parentheses refer to the former species name.
Bold accession numbers are generated in other studies; np: no product.
DNA and RNA isolation for NGS
The genomes of four Alternaria spp. from sect. Alternaria and five Alternaria spp. from five other sections (Table 2) as well as the transcriptome profiles of nine Alternaria spp. from sect. Alternaria and three Alternaria spp. representing three other sections of Alternaria were sequenced (Table 3). Species were selected based on their economic importance and their phylogenetic position, with the intention to be representative of the entire genus Alternaria with a focus on sect. Alternaria. Isolates were grown in malt peptone (MP) (Crous et al. 2009) supplemented with 1 × BME vitamin solution (Sigma-Aldrich® Chemie B.V., Zwijndrecht, The Netherlands) in a shaking incubator, at 25 °C, in the dark, for 3 d. When growth was observed, cultures were mixed in a blender and transferred to fresh MP with vitamin solution, and returned to the shaking incubator for another 2–3 d. When sufficient growth was observed, the mycelium was harvested with a Whatman No. 4 filter disk and a Buchner funnel, attached to a vacuum flask.
Table 2.
Assembly statistics of the Alternaria genomes.
| Species | Strain number(s) | Section | Sequencing method | Size (Mb) | Coverage (approx.) | % Repeats | % Identity | % SNPs2 |
|---|---|---|---|---|---|---|---|---|
| A. alternata | CBS 916.963 | Alternaria | Illumina | 33.3 | 40× | 1.4 | na3 | na3 |
| A. arborescens1 | E.G.S. 39.128 = CBS 102605 | Alternaria | – | 33.9 | – | 2.7 | 96.7 | – |
| A. citriarbusti (now A. alternata) | CBS 102598 | Alternaria | Ion Torrent | 34.8 | 38× | 1.7 | 98.1 | 1.4 |
| A. gaisen | CBS 118488 | Alternaria | Illumina | 35.2 | 182× | 1.8 | 96.7 | 2.8 |
| A. tenuissima (now A. alternata) | CBS 918.96 | Alternaria | Illumina | 33.5 | 260× | 1.4 | 98.2 | 1.5 |
| A. alternantherae | CBS 124392 | Alternantherae | Illumina | 35.0 | 210× | 16.5 | 89.3 | 8.0 |
| A. solani | CBS 109157 | Porri | Ion Torrent | 32.6 | 50× | 1.5 | 87.9 | 9.0 |
| A. avenicola | CBS 121459 | Panax | Illumina | 39.1 | 200× | 11.9 | 87.2 | 9.5 |
| A. infectoria | CBS 210.86 | Infectoriae | Illumina | 36.5 | 200× | 5.3 | 85.1 | 10.3 |
| A. papaveraceae | CBS 116607 | Crivellia | Illumina | 33.8 | 220× | 5.3 | 85.8 | 10.3 |
| A. brassicicola1 | ATCC 96836 = CBS 118699 | Brassicicola | – | 32.0 | – | 7.1 | 86.6 | – |
Publicly available genomes; A. arborescens downloaded from NCBI, A. brassisicola downloaded from JGI (http://genome.jgi-psf.org/Altbr1/Altbr1.home.html).
SNPs / covered base (>10×), duplicates removed.
Reference isolate.
Table 3.
Assembly statistics of the Alternaria transcriptome profiles.
| Species | Strain number | Section | % SNP2 |
|---|---|---|---|
| A. alternata | CBS 916.961 | Alternaria | 0.0 |
| A. arborescens | CBS 102605 | Alternaria | 1.8 |
| A. citriarbusti (now A. alternata) | CBS 102598 | Alternaria | 1.0 |
| A. citricancri (now A. alternata) | CBS 119543 | Alternaria | 0.9 |
| A. gaisen | CBS 118488 | Alternaria | 1.8 |
| A. mali (now A. alternata) | CBS 106.24 | Alternaria | 0.9 |
| A. tenuissima (now A. alternata) | CBS 918.96 | Alternaria | 0.8 |
| A. tomaticola (now A. alternata) | CBS 118814 | Alternaria | 0.9 |
| A. toxicogenica (now A. alternata) | CBS 102600 | Alternaria | 0.9 |
| A. alternantherae | CBS 124392 | Alternantherae | 6.1 |
| A. infectoria | CBS 210.86 | Infectoriae | 8.5 |
| A. papaveraceae | CBS 116607 | Crivellia | 8.4 |
Reference isolate.
SNPs / covered base (>10×), duplicates removed.
For isolating DNA, QIAGEN Genomic 100/G tips (QIAGEN Benelux B.V., Venlo, The Netherlands) were used and processed following the lysis protocol for tissue in the QIAGEN Blood & Cell Culture DNA kit. The following alternative steps, as suggested by the protocol, were followed. The mycelium, of which a maximum of 4 g (wet weight) was used, was grinded to a fine powder with liquid nitrogen in a pre-cooled mortar and pestle. Proteinase K stock solution was added to the solution, after which it was incubated for 2 h at 50 °C in a shaking incubator running at 700 rpm. Prewarmed QF buffer (50 °C) was used to elute the genomic DNA, and after precipitation the DNA was centrifuged at 4 °C for 20 min at 8 500 × g.
For isolating RNA, the QIAGEN RNeasy Midi kit was used following the protocol for isolation of total RNA from animal tissues including the optional on-column DNase digestion. For the disruption of the tissue and homogenisation of the lysate, the mortar and pestle with needle and syringe homogenisation method, as described in the protocol, was followed. All centrifuge steps are performed at room temperature at 4 000 × g. When necessary, a final standard LiCl purification was performed.
NGS
DNA sequence and RNA sequence library preparation (500 bp insert) for Illumina® sequencing and the sequencing itself (100-bp paired end reads) were performed at the Applied Biosystematics Group of Plant Research International (PRI, Wageningen).
DNA sequence library preparation for Ion Torrent™ sequencing was performed at the CBS. The Ion Torrent™ library preparation was carried out using the Ion Xpress™ Fragment Library Kit (Thermo Fisher Scientific, Bleiswijk, The Netherlands), with 180 ng of DNA. Adapter ligation, size selection and nick repair were performed as described in the Ion Torrent™ protocol using the Ion Xpress™ Plus Fragment Library Kit (Thermo Fisher Scientific), with a shearing time of 13 min. The 2100 Bioanalyzer system (Agilent Technologies Netherlands BV, Amstelveen, The Netherlands) and the associated High Sensitivity DNA Analysis kit (Agilent Technologies) were used to determine the quality and concentration of the libraries. The amount of library required for template preparation was calculated using the Template Dilution Factor calculation described in the protocol (DNA concentration diluted to 42 pM). Emulsion PCR and enrichment steps were carried out using the Ion PGM™ Template OT2 200 Kit (Thermo Fisher Scientific) and associated protocol. The enrichment percentage was determent via the Ion Sphere™ Quality Control Kit (Thermo Fisher Scientific) and was performed between the emulsion PCR and the enrichment step. Sequencing was performed using the Ion PGM™ Sequencing 200 Kit v. 2 (Thermo Fisher Scientific) with an Ion 318™ Chip Kit v. 2 (Thermo Fisher Scientific).
Genome assembly and mapping
De novo genome assembly of the Illumina® paired-end reads were quality-filtered and assembled using the A5 pipeline v. 13.01.2014 (Tritt et al. 2012) and de novo genome assembly of Ion Torrent™ reads was performed using Newbler v. 2.9 (454 Life Sciences, Roche Applied Science, Branford, CT, USA). Repeats in the assembled genomes were identified using de novo repeat detection with RepeatModeler (Smit & Hubley 2008) followed by genome-wide repeat annotation using RepeatMasker (Smit et al. 1996), combining the de novo repeats with previously described repeat families from RepBase Update (release 31-04-2014) (Jurka et al. 2005).
Whole-genome alignments were performed using NUCmer, part of the MUMmer v. 3.1 package (Kurtz et al. 2004), using the “mum” option to find matches unique in query and reference. Subsequently, the average identity of the aligned sequences was calculated using dnadiff, part of MUMmer v. 3.1.
Genomic variants were inferred using GATK v. 3.3 (DePristo et al. 2011). Briefly, genomic or transcriptomic reads were mapped against a reference genome (A. alternata CBS 916.96) using BWA (Li & Durbin 2009) using the BWA-MEM algorithm v. 0.7.5a-r405. Transcript reads were trimmed prior to mapping using fastx-tools. Duplicated reads were identified and marked using Picard tools (http://broadinstitute.github.io/picard). Using GATK, transcript reads were splitted into exons and overhangs were removed. Subsequently, transcript and genomic reads were locally realigned to minimise the number of mismatches over all reads. Afterwards, genomic variants (SNPs) were called using GATK's UnifiedGenotyper (standard call and emitting threshold of 20; haploid organisms), and the resulting SNPs were filtered based on quality (Qual = 50), depth (DP = 10) and allelic frequency (AF = 0.9).
Conserved eukaryotic orthologous group (KOG) proteins were identified using the Core Eukaryotic Genes Mapping Approach (CEGMA) pipeline (Parra et al. 2007). The conservation table was constructed from the five available genomes of sect. Alternaria to avoid alignment problems that could affect the conservation values.
The reference sequence alignment-based phylogeny builder (REALPHY) v. 1.09 (Bertels et al. 2014) was used to construct a phylogenetic tree based on the whole-genome and transcriptome reads and the previously assembled Alternaria genomes. Briefly, short reads (genome and transcriptome) as well as short sequence fragments (100 nt) derived from the previously assembled genomes were mapped against the reference genome (A. alternata CBS 916.96) using Bowtie2. Subsequently, polymorphic as well as non-polymorphic sites were filtered (per base quality [20], coverage [10] and polymorphism frequency [0.95]) and extracted. Only sites that were present in all species were retained. The derived pseudo-molecule was used to infer a maximum likelihood phylogenetic tree using PhyML using the generalised time reversible (GTR) nucleotide substitution model. The robustness of the phylogeny was assessed by 1 000 bootstrap replicates.
PCR and sequencing
DNA extraction for gene sequencing was performed using the UltraClean™ Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. The SSU, LSU, ITS, gapdh, rpb2 and the tef1 gene regions were amplified and sequenced as described in Woudenberg et al. (2013) and the Alt a 1 gene as described in Woudenberg et al. (2014). The endoPG and OPA10-2 gene regions were amplified using the primers PG3 and PG2b and OPA10-2L and OPA10-2R (Andrew et al. 2009). For the KOG1058 and KOG1077 gene regions the primers KOG1058F2 (5′-GAG TCA CGT TAY CGC ASC-3′) and KOG1058R2 (5′-TGG CTK ACG GAR ACG-3′) and KOG1077F2 (5′-GGA GCA GTC GGG CAA CG-3′) and KOG1077R2 (5′-ATT CRT GTT GTA CRA TCG C-3′) were designed from the genomic data. The PCRs were performed in an Applied Biosystems® 2720 Thermal Cycler (Thermo Fisher Scientific), in a total volume of 12.5 μL. The PCR mixtures consisted of 1 μL genomic DNA, 1× NH4 reaction buffer (Bioline, Luckenwalde, Germany), 2 mM (endoPG, OPA10-2) or 1.6 mM MgCl2 (KOG1058, KOG1077), 20 μM of each dNTP, 0.2 μM of each primer and 0.5 U Taq DNA polymerase (Bioline). The PCR conditions consisted of an initial denaturation step of 5 min at 94 °C followed by 40 cycles of 30 s at 94 °C, 30 s at 50 °C and 30 s at 72 °C for endoPG, 35 cycles of 30 s at 94 °C, 30 s at 62 °C and 45 s at 72 °C for OPA10-2, and 35 cycles of 30 s at 94 °C, 30 s at 59 °C and 60 s at 72 °C for KOG1058 and KOG1077, and a final elongation step of 7 min at 72 °C. The PCR products were sequenced in both directions using the PCR primers and a BigDye® Terminator v. 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific), and analysed with an ABI Prism 3730xl DNA Analyser (Thermo Fisher Scientific) according to the manufacturer's instructions. Consensus sequences were computed from forward and reverse sequences using the BioNumerics v. 4.61 software package (Applied Maths, St-Martens-Latem, Belgium). All generated sequences were deposited in GenBank (Table 1).
Phylogenetic analyses
Multiple sequence alignments of individual data partitions were generated with MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html), and manually adjusted. The best nucleotide substitution model for each partition was determined with Findmodel (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). For the ITS and OPA10-2 partitions a K80 model with a gamma-distributed rate variation was suggested, for the SSU, LSU, tef1 and Alt a 1 partitions a HKY model, with gamma-distributed rate variation for LSU and Alt a 1, for the gapdh, rpb2 and KOG1077 partitions a TrN model with gamma-distributed rate variation and for the endoPG and KOG1058 partitions a GTR model with gamma-distributed rate variation. Bayesian analyses were performed with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist, 2001, Ronquist and Huelsenbeck, 2003) on the individual data partitions as well as the combined aligned dataset. The Markov Chain Monte Carlo (MCMC) analysis used four chains and started from a random tree topology. The sample frequency was set at 500 for the combined analysis and the less informative loci (SSU, LSU, ITS and tef1) and at 100 for the remaining loci. The temperature value of the heated chain was 0.1 and the run stopped when the average standard deviation of split frequencies fell below 0.01. Burn-in was set to 25 % after which the likelihood values were stationary. Tracer v. 1.5.0 (Rambaut & Drummond 2009) was used to confirm the convergence of chains. A maximum-likelihood analysis including 500 bootstrap replicates using RAxML v. 7.2.6 (Stamatakis & Alachiotis 2010) was additionally run on the combined aligned dataset. Sequences of A. alternantherae (CBS 124392) were used as outgroup. The resulting trees were printed with TreeView v. 1.6.6 (Page 1996) and, together with the alignments, deposited into TreeBASE (http://www.treebase.org).
Phylogenetic species recognition and naming in Alternaria sect. Alternaria
Individual gene trees were generated as described in the “Phylogenetic analyses” part above and examined manually. A species clade was only recognised as unique if it was well-supported and monophyletic with all of its included isolates in multiple single-gene phylogenies, and no incongruencies were observed in the other single-gene phylogenies, e.g. the included isolates clustered together in all single-gene phylogenies. Unique molecular markers for the recognised species, which separates them from the other species in sect. Alternaria, are described with the species below and listed in a table which can be downloaded from the CBS-KNAW website (www.cbs.knaw.nl/index.php/studies-in-mycology) or requested from the author. Unique fixed nucleotide positions were derived from the respective alignments of the separate loci deposited in TreeBASE based on a comparison of the sequences of all isolates from the specific species to the sequences of all isolates of the other recognised species within sect. Alternaria.
To further standardise the taxonomic terms used, the trinomial system introduced by Rotem (1994) is favoured. When differences in host affinity are observed within the isolates of one (of the above-defined) species, the third epithet, the forma specialis, defines the affinity to this specific host in accordance with the produced toxin causing this affinity. When different toxins are produced on the same host, but these toxins affect different host species, the term pathotype should be used in addition. All isolates which are not confined to specific hosts and / or toxins should retain only the binomial name until such specificity is found. For examples, please refer to the species notes under A. alternata below and to the Discussion.
Results
NGS
Nine Alternaria (morpho)species were sequenced using Ion Torrent™ or Illumina® sequencing technologies, yielding between 38× and >260× average genome coverage (Table 2). The assembled genomes ranged in size from 33.3–35.2 Mb within sect. Alternaria and from 32.0–39.1 Mb for all Alternaria genomes (Table 2). To characterise the assembled genomes, the repetitive complement of each individual genome was identified and classified using a combination of de novo prediction and identification of known repetitive elements. Surprisingly, the number of repetitive sequences differed significantly between different Alternaria genomes. Within sect. Alternaria, the number of repetitive sequences is relatively low; only 1.4–2.7 % of each genome was classified as repetitive (Table 2). In contrast, A. avenicola and A. alternantherae carry significantly higher percentages of repetitive elements, >10 % and >15 %, respectively (Table 2).
To assess the genomic differences between the included species, whole-genome alignments to the reference genome of A. alternata (CBS 916.96) were performed. These alignments revealed 96.7–98.2 % genome identity within sect. Alternaria compared to 85.1–89.3 % genome identity between isolates from other sections with A. alternata. Furthermore, the number of single nucleotide polymorphisms (SNPs) between the different species were assessed by mapping genomic reads to the reference genome of A. alternata (CBS 916.96). Between isolates from sect. Alternaria, 1.4–2.8 % SNPs were observed, while the percentage of SNPs found in isolates from different sections was considerably higher, ranging from 8.0–10.3 % (Table 2).
To further characterise the genus, deep transcriptome sequences of 12 isolates were derived that were mapped to the reference isolate of A. alternata (CBS 916.96). In this case, 0.8–1.8 % SNPs among the isolates from sect. Alternaria were observed, while the isolates from other sections displayed 6.1–8.5 % SNPs (Table 3).
Marker genes with potential discriminatory power were identified by predicting a set of conserved eukaryotic genes (KOG) in the genomes of the five assembled sect. Alternaria genomes using the CEGMA pipeline. Out of 380 included KOGs, 326 (86 %) had a conservation level of ≥98 %. Therefore, we focused on the 25 KOGs with the lowest degree of conservation, ranging from 83.0–97.3 %, and evaluated their discriminatory power. KOGs that were not able to distinguish all morphospecies included in the whole-genome and transcriptome sequencing were immediately rejected. Primers spanning the first 5 introns of KOG1058 and KOG1077 were designed (see the “PCR and sequencing” part of the “Material and Methods”). These proteins were found on place 16 and 23 in the conservation table and both act in the vesicle coat complex, although in different systems; namely COPI versus AP-2.
The pseudo-molecule derived from the whole-genome and transcriptome reads with REALPHY contained 1 750 944 nt. The topology from the REALPHY phylogeny (Fig. 1) corresponds to the multi-gene phylogeny based on a five-gene combined dataset (fig. 3 in Lawrence et al. 2013) and a three-gene combined dataset (fig. 1 in Woudenberg et al. 2013). Section Alternantherae and sect. Porri are the sister sections of sect. Alternaria, while sect. Infectoriae and sect. Crivellia, are the most distant sections (Fig. 1).
Fig. 1.
PhyML tree based on the whole-genome and transcriptome reads of 15 Alternaria species using REALPHY. The bootstrap support values are given at the nodes; thickened lines indicate a fully supported node. The grey box represents species which are now synonymised under A. alternata. The tree was rooted to A. papaveraceae (CBS 116607).
Gene-based phylogeny and identification
From the 168 isolates included in the multi-gene phylogeny, the amplification and / or sequencing of two isolates for the rpb2 gene, three for the Alt a 1 gene, one for the endoPG gene and four for the OPA10-2 regions failed (Table 1); these genes were included as missing data in the combined analysis. The aligned sequences of the SSU (1 021 aligned characters), LSU (849 aligned characters), ITS (523 aligned characters), gapdh (579 aligned characters), tef1 (241 aligned characters), rpb2 (753 aligned characters), Alt a 1 (473 aligned characters), endoPG (448 aligned characters) and OPA10-2 (634 aligned characters) gene regions contained 6, 9, 27, 60, 42, 87, 110, 59 and 123 unique site patterns, respectively. Because of the low informative value of the SSU and LSU sequences (6 / 9 unique site patterns out of 1 021 / 849 aligned characters) these genes were excluded from the multi-gene phylogeny. The multi-gene phylogeny based on the remaining seven gene regions contained 3 651 characters including alignment gaps, which, after discarding the burn-in phase, resulted in a 50 % majority rule consensus tree based on 15 002 trees from two runs (Fig. 2).
Fig. 2.
Bayesian 50 % majority rule consensus tree based on the ITS, gapdh, tef1, rpb2, Alt a 1, endoPG and OPA10-2 sequences of 168 Alternaria strains. The Bayesian posterior probabilities >0.75 (PP) and RAxML bootstrap support values >65 (ML) are given at the nodes (PP / ML). Thickened lines indicate a PP of 1.0 and ML of 100. Species names between parentheses represent synonymised species names. Ex-type strains are indicated with T and representative strains with R. The ex-type strains of here recognised species are printed in bold face. The tree was rooted to A. alternantherae (CBS 124392).
The alignments of the additional gene regions that were sequenced, KOG1058 and KOG1077, consisted of 921 and 781 aligned characters, respectively, of which 118 and 78 were unique site patterns. The amplification and / or sequencing of the KOG1077 gene failed in six of the 49 isolates, representing the species A. alstroemeriae, A. iridiaustralis and A. jacinthicola (Table 4). Since the KOG1077 sequences could not separate A. longipes from A. gossypina, no further effort was put in optimising the primers to obtain the missing data.
Table 4.
Comparison of gene ability to distinguish species in sect. Alternaria.
 |
Although the single-gene phylogenies are not fully congruent in terms of species resolution (see TreeBASE), 11 clades can be distinguished consistently within the single-gene phylogenies and in the multi-gene phylogeny (Fig. 2). Eight of those are single species clades representing A. alstroemeriae, A. betae-kenyensis, A. eichhorniae, A. gaisen, A. iridiaustralis, A. jacinthicola, A. longipes, and A. tomato. Three further clades constitute numerous morphospecies, which are synonymised here under A. burnsii, A. gossypina and the A. arborescens species complex (AASC). However, the majority of the isolates (105 / 168), representing 35 morphospecies, do not form clear phylogenetic clades. The subclades that are formed by these isolates are incongruent between the different gene regions sequenced; no two genes show the same groupings from any of the 100 plus isolates. These morphospecies are synonymised below under A. alternata.
None of the genes sequenced in this study enabled us to distinguish all of the phylogenetic species recognised here on its own (Table 4). The commonly used gapdh sequence could distinguish all species, except the A. arborescens species complex (AASC), from A. alternata. Five genes, namely rpb2, OPA10-2, Alt a 1, endoPG and KOG1058, could separate all species from A. alternata, but failed to separate different pairs of other species from one another (see Table 4). The SSU, LSU and ITS genes were least successful in separating the species accepted in this study. The unique fixed nucleotides per gene region are provided below under the treatment of each species, and are summarised in a table which can be downloaded from the CBS-KNAW website (www.cbs.knaw.nl/index.php/studies-in-mycology) or requested from the author.
Phylogenetic species in sect. Alternaria
Alternaria alstroemeriae E.G. Simmons & C.F. Hill, CBS Biodiversity Ser. (Utrecht) 6: 444. 2007.
Specimens examined: Australia, from leaf of Alstroemeria sp. (Alstroemeriaceae), Jul. 2005, C.F. Hill, culture ex-type CBS 118809 = E.G.S. 52.068. USA, California, Sacramento, from leaf spot of Alstroemeria sp., before Apr. 2002, D. Fogle, CBS 118808 = E.G.S. 50.116.
Unique fixed nucleotides: gapdh position 485 (T); rpb2 position 162 (G); tef1 position 52 (C), 143 (C), 165 (T), 205 (G); OPA10-2 position 120 (T), 151 (T), 303 (G), 318 (G), 330 (C), 390 (G), 417 (C), 486 (G); Alt a 1 position 157 (T), 178 (T), 404 (A); endoPG position 37 (A), 46 (C), 316 (T); KOG1058 position 51 (C), 514 (T), 533 (C).
Alternaria alternata (Fr.) Keissl., Beih. Bot. Centralbl., Abt. 2, 29: 434. 1912.
Basionym: Torula alternata Fr., Syst. Mycol. (Lundae) 3: 500. 1832. (nom. sanct.)
= Alternaria tenuis Nees, Syst. Pilze (Würzburg): 72. 1816 [1816–1817].
= Helminthosporium tenuissimum Kunze ex Nees & T. Nees, Nova Acta Acad. Caes. Leop.-Carol. German. Nat. Cur. 9: 242. 1818.
≡ Macrosporium tenuissimum (Nees & T. Nees) Fr., Syst. Mycol. 3: 374. 1832. (nom. sanct.)
≡ Clasterosporium tenuissimum (Nees & T. Nees: Fr.) Sacc., Sylloge Fungorum (Abellini) 4: 393. 1886.
≡ Alternaria tenuissima (Nees & T. Nees: Fr.) Wiltshire, Trans. Brit. Mycol. Soc. 18: 157. 1933.
= Macrosporium fasciculatum Cooke & Ellis, Grevillea 6: 6. 1877.
≡ Alternaria fasciculata (Cooke & Ellis) l.R. Jones & Grout, Bull. Torrey Bot. Club 24: 257. 1897.
= Macrosporium caudatum Cooke & Ellis, Grevillea 6: 87. 1878.
≡ Alternaria caudata (Cooke & Ellis) E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 496. 2007.
= Macrosporium maydis Cooke & Ellis, Grevillea 6: 87. 1878.
= Macrosporium inquinans Cooke & Ellis, Grevillea 7: 39. 1878.
= Macrosporium meliloti Peck, Rep. (Annual) NewYork State Mus. Nat. Hist. 33: 28. 1880.
= Macrosporium erumpens Cooke, Grevillea 12: 32. 1883.
≡ Alternaria erumpens (Cooke) Joly, Le Genre Alternaria: 199. 1964.
= Macrosporium martindalei Ellis & G. Martin, Amer. Naturalist 18: 189. 1884.
≡ Alternaria martindalei (Ellis & G. Martin) Joly, Le Genre Alternaria: 209. 1964.
= Macrosporium polytrichi Peck, Rep. (Annual) NewYork State Mus. Nat. Hist. 34: 31. 1890.
= Macrosporium podophylli Ellis & Everh., Proc. Acad. Nat. Sci. Philadelphia 43: 92. 1891.
≡ Alternaria podophylli (Ellis & Everhart) Joly, Le Genre Alternaria: 212. 1964.
= Macrosporium seguierii Allescher, Hedwigia 33: 75. 1894.
= Macrosporium amaranthi Peck, Bull. Torrey Bot. Club 22: 493. 1895.
≡ Alternaria amaranthi (Peck) J. van Hook, Proc. Indiana Acad. Sci. 1920: 214. 1921.
= Alternaria citri Ellis & N. Pierce, Bot. Gaz. (Crawfordville) 33: 234. 1902.
= Alternaria ribis Bubák & Ranojević, Ann. Mycol. 8: 400. 1910.
= Alternaria mali Roberts, J. Agric. Res. 2: 58. 1914.
= Alternaria palandui Ayyangar, Bull. Agric. Res. Inst., Pusa 179: 14. 1928.
= Alternaria lini Dey, Indian J. Agric. Sci. 3: 881. 1933.
= Alternaria tenuissima var. godetiae Neerg., Trans. Brit. Mycol. Soc. 18: 157. 1933.
≡ Alternaria godetiae (Neerg.) Neerg., Aarsberetn. J. E. Ohlens Enkes Plantepatol. Lab. 10: 14. 1945.
= Macrosporium pruni-mahalebi Săvulescu & Sandu, Hedwigia 75: 228. 1935.
= Alternaria rumicicola R.L. Mathur, J.P. Agnihotri & Tyagi, Curr. Sci. 31: 297. 1962.
= Alternaria tenuissima var. verruculosa S. Chowdhury, Proc. Natl. Acad. Sci. India, Sect. B, Biol. Sci. 36: 301. 1966.
= Alternaria angustiovoidea E.G. Simmons, Mycotaxon 25: 198. 1986.
= Alternaria pellucida E.G. Simmons, Mycotaxon 37: 102. 1990.
= Alternaria rhadina E.G. Simmons, Mycotaxon 48: 101. 1993.
= Alternaria destruens E.G. Simmons, Mycotaxon 68: 419. 1998.
= Alternaria broussonetiae T.Y. Zhang, W.Q. Chen & M.X. Gao, Mycotaxon 72: 439. 1999.
= Alternaria citriarbusti E.G. Simmons, Mycotaxon 70: 287. 1999.
= Alternaria citrimacularis E.G. Simmons, Mycotaxon 70: 277. 1999.
= Alternaria dumosa E.G. Simmons, Mycotaxon 70: 310. 1999.
= Alternaria interrupta E.G. Simmons, Mycotaxon 70: 306. 1999.
= Alternaria limoniasperae E.G. Simmons, Mycotaxon 70: 272. 1999.
= Alternaria perangusta E.G. Simmons, Mycotaxon 70: 303. 1999.
= Alternaria tenuissima var. alliicola T.Y. Zhang, Mycotaxon 72: 450. 1999.
= Alternaria toxicogenica E.G. Simmons, Mycotaxon 70: 294. 1999.
= Alternaria turkisafria E.G. Simmons, Mycotaxon 70: 290. 1999.
= Alternaria sanguisorbae M.X. Gao & T.Y. Zhang, Mycosystema 19: 456. 2000.
= Alternaria platycodonis Z.Y. Zhang & H. Zhang, Flora Fungorum Sin., Alternaria: 66. 2003.
= Alternaria yali-inficiens R.G. Roberts [as ‘yaliinficiens’], Pl. Dis. 89: 142. 2005.
= Alternaria astragali Wangeline & E.G. Simmons, Mycotaxon 99: 84. 2007.
= Alternaria brassicinae E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 532. 2007.
= Alternaria citricancri E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 542. 2007.
= Alternaria daucifolii E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 518. 2007.
= Alternaria herbiphorbicola E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 608. 2007.
= Alternaria pulvinifungicola E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 514. 2007.
= Alternaria postmessia E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 598. 2007.
= Alternaria seleniiphila Wangeline & E.G. Simmons, Mycotaxon 99: 86. 2007.
= Alternaria soliaegyptiaca E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 506. 2007.
= Alternaria tomaticola E.G. Simmons & Chellemi, CBS Biodiversity Ser. (Utrecht) 6: 528. 2007.
= Alternaria vaccinii E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 432. 2007.
= Alternaria viniferae Yong Wang bis, Y.Y. Than, K.D. Hyde, X.H. Li, Mycol. Progr. 13: 1124. 2014.
Type and representative specimens examined: Canada, Manitoba, from Euphorbia esula (Euphorbiaceae), 1982, K. Mortensen, culture ex-type of A. angustiovoidea CBS 195.86 = E.G.S. 36.172 = DAOM 185214. China, Hebei, from fruit of Pyrus bretschneideri (Rosaceae), 2001, R.G. Roberts, culture ex-type of A. yali-inficiens CBS 121547 = E.G.S. 50.048; Shaanxi, Hanzhong, from Platycodon grandiflorus (Campanulaceae), before Dec. 2001, T.Y. Zhang, culture ex-type of A. platycodonis CBS 121348 = E.G.S. 50.070; Shangdong, Changqing, from Broussonetia papyrifera (Moraceae), 13 Sep. 1996, T.Y. Zhang, culture ex-type of A. broussonetiae CBS 121455 = E.G.S. 50.078; Shangdong, Jinan, from Sanguisorba officinalis (Rosaceae), 19 Sep. 1996, M.X. Gao, culture ex-type of A. sanguisorbae CBS 121456 = E.G.S. 50.080. Denmark, Sjaelland, Clausdal, from Godetia sp. (Onagraceae), 27 Jul. 1942, P. Neergaard, culture ex-type of A. godetiae CBS 117.44 = E.G.S. 06.190 = VKM F-1870. Egypt, Sabet, from soil, before Jan. 1933, culture ex-type of A. soliaegyptiaca CBS 103.33 = E.G.S. 35.182 = IHEM 3319. India, from Arachis hypogaea (Fabaceae), 1 Dec. 1980, L.V. Gangawane, culture ex-epitype CBS 916.96 = CBS 110977 = CBS 115616 = E.G.S. 34.016 = IMI 254138. Israel, from Minneola tangelo (Rutaceae), before Nov. 1996, Z. Solel, culture ex-type of A. interrupta CBS 102603 = E.G.S. 45.011; Mayan Zvi, from Minneola tangelo, before Nov. 1996, Z. Solel, culture ex-type of A. dumosa CBS 102604 = E.G.S. 45.007. Japan, from fruit of Citrus unshiu (Rutaceae), 1968, K. Tubaki, culture ex-type of A. pellucida CBS 479.90 = E.G.S. 29.028; from leaf of Pyrus pyrifolia (Rosaceae), 1990, K. Nagano, culture ex-type of A. rhadina CBS 595.93. Turkey, Kuzucuoglu, from Minneola tangelo, May 1996, Y. Canihos, culture ex-type of A. turkisafria CBS 102599 = E.G.S. 44.166; Adana region, from Minneola tangelo, May 1996, Y. Canihos, culture ex-type of A. perangusta CBS 102602 = E.G.S. 44.160. UK, from Dianthus chinensis (Caryophyllaceae), 20 Feb. 1981, A.S. Taylor, representative isolate of A. tenuissima CBS 918.96 = E.G.S. 34.015 = IMI 255532. USA, from Malus sylvestris (Rosaceae), before Dec. 1924, J.W. Roberts, culture ex-type of A. mali CBS 106.24 = E.G.S. 38.029 = ATCC 13963; Arizona, Yuma, from Brassica oleracea (Brassicaceae), Apr. 1982, R.H. Morrison, culture ex-type of A. brassicinae CBS 118811 = E.G.S. 35.158; California, from fruit of Citrus sinensis (Rutaceae), before Nov. 1947, D.E. Bliss, representative isolate of A. citri CBS 102.47 = E.G.S. 02.062; California, Los Angeles, from Citrus paradisi (Rutaceae), 12 Jul. 1947, L. Davis, culture ex-type of A. citricancri CBS 119543 = E.G.S. 12.160; Colorado, from leaf of Allium sp. (Alliaceae), F.A. Weiss, culture ex-epitype of A. palandui CBS 121336 = E.G.S. 37.005 = ATCC 11680; Colorado, Fort Collins, from the root of Stanleya pinnata (Brassicaceae), 19 Jun. 2002, A. Wangeline, culture ex-type of A. seleniiphila CBS 127671 = E.G.S. 52.121; Florida, Lake Alfred, from leaf lesion of Citrus jambhiri (Rutaceae), before Jul. 1997, culture ex-type of A. limoniasperae CBS 102595 = E.G.S. 45.100; Florida, Lake Alfred, from leaf lesion of Citrus jambhiri, before Jul. 1997, culture ex-type of A. citrimacularis CBS 102596 = E.G.S. 45.090; Florida, Lake Alfred, from leaf spot of Minneola tangelo, before Feb. 1998, culture ex-type of A. citriarbusti CBS 102598 = E.G.S. 46.141; Florida, Lake Alfred, from Minneola tangelo, 19 Dec. 1980, J.O. Whiteside, culture ex-type of A. postmessia CBS 119399 = E.G.S. 39.189; Florida, Quincy, from Solanum lycopersicum (Solanaceae), June 1996, D. Chellemi, culture ex-type of A. tomaticola CBS 118814 = E.G.S. 44.048; Florida, Wauchula, from Citrus reticulata (Rutaceae), 6 Jun. 1975, J.O. Whiteside, culture ex-type of A. toxicogenica CBS 102600 = E.G.S. 39.181 = ATCC 38963; Florida, Zellwood, from Daucus carota (Apiaceae), Jan. 1984, R.H. Morrison, culture ex-type of A. daucifolii CBS 118812 = E.G.S. 37.050; Iowa, from Quercus sp. (Fagaceae), 28 Jul. 1953, A. Engelhard, culture ex-type of A. pulvinifungicola CBS 194.86 = E.G.S. 04.090 = QM 1347; Maryland, from Euphorbia esula, before Dec. 1991, culture ex-type of A. herbiphorbicola CBS 119408 = E.G.S. 40.140; Massachusetts, Hadley, from fruit of Cucumis sativus (Cucurbitaceae), 24 Sep. 1984, E.G. Simmons, representative isolate of A. caudata CBS 121544 = E.G.S. 38.022; Massachusetts, Rochester, from Cuscuta gronovii (Convolvulaceae), Aug. 1997, F. Caruso, culture ex-type isolate of A. destruens CBS 121454 = E.G.S. 46.069; New Jersey, from Vaccinium sp. (Ericaceae), Oct. 1973, R.A. Cappellini, culture ex-type of A. vaccinii CBS 118818 = E.G.S. 31.032; Wyoming, Laramie, from the root of Astragalus bisulcatus (Fabaceae), 8 Jun. 2002, A. Wangeline, culture ex-type of A. astragali CBS 127672 = E.G.S. 52.122. Unknown, from Linum usitatissimum (Linaceae), before Jul. 1934, P.K. Dey, culture ex-type of A. lini CBS 106.34 = E.G.S. 06.198 = DSM 62019 = MUCL 10030.
Notes: Both the names Torula alternata and Macrosporium tenuissimum represent sanctioned names by Fries (1832), with the basionym of tenuissimum (1818) being the older. However, the well-established name of the type species of Alternaria, A. alternata is retained above the older name A. tenuissima, as this would result in confusion among the user community, and be counterproductive. A proposal to conserve A. alternata over A. tenuissima will be compiled for submission to the Nomenclature Committee of Fungi. The isolate CBS 447.86, isolated from Malva sp. in Marocco, was stored in the CBS collection as Alternaria malvae. The original description of A. malvae was from leaf lesions of Malva crispa, from Seine-Inférieure (now called Seine-Maritime), France. Therefore A. malvae is not synonymised under A. alternata. The isolate CBS 106.34, send to the CBS by Dey in 1934 together with a reprint of his paper describing A. lini, is recognised as an ex-type isolate. Therefore A. lini is synonymised under A. alternata. The very recently described A. viniferae is synonymised based on the published gapdh and Alt a 1 sequences, which cluster within A. alternata. Because of the relative high sequence variability amongst the A. alternata isolates, no unique fixed nucleotides are assigned to A. alternata. Three formae speciales of A. alternata are currently recognised; A. alternata f. sp. mali for isolates producing the AM-toxin, f. sp. fragariae for isolates producing the AF-toxin, and f. sp. citri with two pathotypes, i.e. f. sp. citri pathotype rough lemon for isolates producing the ACR-toxin, and f. sp. citri pathotype tangerine for isolates producing the ACT-toxin.
Alternaria betae-kenyensis E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 530. 2007.
Specimen examined: Kenya, from Beta vulgaris var. cicla (Chenopodiaceae), before Jun. 2001, ex-type CBS 118810 = E.G.S. 49.159 = IMI 385709.
Unique fixed nucleotides: ITS position 464 (C); gapdh position 28 (C), 55 (A), 512 (T); rpb2 position 204 (T), 363 (T), 369 (G), 447 (G), 468 (T), 480 (A), 507 (A), 627 (G); tef1 position 213 (G), 218 (C); OPA10-2 position 63 (C), 177 (A), 199 (G), 276 (T), 309 (T), 534 (C), 567 (A), 591 (A); Alt a 1 position 55 (A), 155 (A), 311 (G), 338 (T), 359 (C), 365 (C), 379 (C), 440 (T), 473 (A); endoPG position 10 (T), 286 (T), 295 (T), 372 (G); KOG1058 position 156 (C), 522 (T), 869 (G); KOG1077 position 121 (A), 178 (C), 373 (A), 402 (C), 763 (C).
Alternaria burnsii Uppal, Patel & Kamat, Indian J. Agric. Sci. 8: 49. 1938. Fig. 3.
Fig. 3.
Alternaria burnsii conidia and conidiophores. A–B. CBS 108.27. C–D. CBS 879.95. E–F. CBS 118816. G–H. CBS 118817. Scale bars = 10 μm.
= Alternaria tinosporae E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 508. 2007.
= Alternaria rhizophorae E.G. Simmons, CBS Biodiversity Ser. (Utrecht) 6: 510. 2007.
Specimens examined: India, from Cuminum cyminum (Apiaceae), before Dec. 1938, B.N. Uppal, culture ex-type of A. burnsii CBS 107.38; Saznakhali, from infected leaf of Rhizophora mucronata (Rhizophoraceae), 14 Mar. 1995, ex-type of A. rhizophorae CBS 118816 = E.G.S. 43.145 = IMI 368045; Punjab, from Tinospora cordifolia (Menispermaceae), before Sept. 1987, culture ex-type of A. tinosporae CBS 118817 = E.G.S. 39.14 = IMI 318433; from human sputum, Anuradha, CBS 130264. Mozambique, from stem of Gossypium sp. (Malvaceae), Aug. 1950, Quintanilha, CBS 110.50. UK, from Sorghum sp. (Poaceae), 19 Dec. 1985, M. Kalicz, CBS 879.95 = IMI 300779. Unknown, from Gomphrena globosa (Amaranthaceae), before Mar. 1927, K. Togashi, CBS 108.27.
Unique fixed nucleotides: endoPG position 196 (C), 199 (A).
Notes: Although A. burnsii only has two unique fixed nucleotides, the species can easily be distinguished from A. alternata using molecular data. The low number of unique fixed nucleotides is due to its close phylogenetic relationship to A. tomato and A. jacinthicola. Most of the nucleotide differences present between A. burnsii and the A. alternata isolates are also present in the A. tomato and / or A. jacinthicola isolates.
Alternaria eichhorniae Nag Raj & Ponnappa, Trans. Brit. Mycol. Soc. 55: 124. 1970.
Specimens examined: India, Karnataka, Bangalore, from leaf of Eichhornia crassipes (Pontederiaceae), 28 Feb. 1966, R. Charudattan, culture ex-type CBS 489.92 = ATCC 22255 = ATCC 46777 = ATCC 201659 = IMI 121518. Indonesia, from leaf of Eichhornia crassipes, before Dec. 1996, representative culture CBS 119778 = E.G.S. 45.026 = IMI 372968.
Unique fixed nucleotides: ITS position 105 (T); gapdh position 36 (G), 162 (G), 168 (T), 509 (A); rpb2 position 6 (T), 549 (G); tef1 position 12 (C), 31 (G), 223 (G); OPA10-2 position 123 (G), 366 (C), 387 (A), 582 (T), 600 (A); Alt a 1 position 67 (T), 130 (A), 298 (A), 356 (A), 397 (C); endoPG position 29 (A), 68 (C), 79 (T), 130 (A), 148 (T), 152 (A), 173 (A), 316 (G), 369 (C), 376 (C), 378 (T); KOG1058 position 16 (C), 64 (T), 254 (C), 268 (T), 269 (G), 270 (G), 278 (G), 298 (C), 536 (C), 694 (G), 711 (C); KOG1077 position 62 (T), 162 (C), 166 (C), 189 (C), 195 (C), 234 (G), 235 (C), 348 (C), 350 (C), 564 (A), 685 (A), 715 (A), 776 (T).
Alternaria gaisen Nagano ex Hara, Sakumotsu Byorigaku, Edn 4: 263. 1928.
≡ Alternaria gaisen Nagano, J. Jap. Soc. Hort. Sci. 32: 16–19. 1920. (nom. illegit., Art. 39.1).
= Alternaria kikuchiana S. Tanaka, Mem. Coll. Agric. Kyoto Univ., Phytopathol. Ser. 28: 27. 1933.
= Macrosporium nashi Miura, Flora of Manchuria and East Mongolia, Part III Cryptogams, Fungi: 513. 1928.
Specimens examined: Japan, Tottori, from Pyrus pyrifolia (Rosaceae), Jul. 1990, E.G. Simmons, representative isolate CBS 118488 = E.G.S. 90.0391; Tottori, from Pyrus pyrifolia, 11 Jul. 1990, E.G. Simmons, representative isolate CBS 632.93 = E.G.S. 90.0512. Netherlands, host unknown, Aug. 2011, S. I. R. Videira, SV01.
Unique fixed nucleotides: gapdh position 383 (C), 473 (A); rpb2 position 207 (T), 540 (G); tef1 position 241 (T); Alt a 1 position 1 (A), 13 (T), 97 (A), 339 (T), 345 (G), 413 (C); endoPG position 130 (C), 172 (A), 250 (T), 361 (T); KOG1058 position 707 (G); KOG1077 position 174 (A).
Alternaria gossypina (Thüm.) J.C.F. Hopkins, Trans. Brit. Mycol. Soc. 16: 136. 1931. Fig. 4.
Fig. 4.
Alternaria gossypina conidia and conidiophores. A–B. CBS 100.23. C–D. CBS 104.32. E–F. CBS 107.36. G–H. CBS 102597. Scale bars = 10 μm.
Basionym: Macrosporium gossypinum Thüm., Herb. Mycol. Oecon.: no. 513. 1877.
= Alternaria grisea Szilv., Arch. Hydrobiol. 3: 546. 1936.
= Alternaria colombiana E.G. Simmons, Mycotaxon 70: 298. 1999.
= Alternaria tangelonis E.G. Simmons, Mycotaxon 70: 282. 1999.
Type: (Lectotype, designated in Simmons 2003) USA, South Carolina, Aiken, from stems of dead Gossypinum herbaceum, 1876, H.W. Ravenel, Macrosporium gossypinum BPI 445306.
Specimens examined: Colombia, Chinchiná, from fruit lesion of Minneola tangelo (Rutaceae), before Nov. 1996, B. L. Castro, culture ex-type of A. colombiana CBS 102601 = E.G.S. 45.017. Sumatra, Toba Heath, from soil, before Jun. 1936, A. von Szilvinyi, culture ex-type of A. grisea CBS 107.36. USA, Florida, from Minneola tangelo, before Aug. 1997, culture ex-type of A. tangelonis CBS 102597 = E.G.S. 45.114. Zimbabwe, from Gossypium sp. (Malvaceae), before Mar. 1932, J.C.F. Hopkins, culture ex-type of A. gossypina CBS 104.32. Unknown, from Malus domestica (Rosaceae), before Jun. 1923, A.S. Horne, CBS 100.23.
Unique fixed nucleotides: OPA10-2 position 172 (T); KOG1058 position 19 (A), 20 (A).
Notes: Although A. gossypina only has three unique fixed nucleotides, the species can easily be distinguished from A. alternata using molecular data. The low number of unique fixed nucleotides is due to its close phylogenetic relationship to A. longipes. Most of the nucleotide differences present between A. gossypina and the A. alternata isolates are also present in the A. longipes isolates. The isolate of A. gossypina deposited to the CBS by J.C.F. Hopkins, CBS 104.32, is recognised as ex-type culture of A. gossypina and the isolate of A. grisea deposited at the CBS by A. von Szilvinyi, CBS 107.36, is recognised as ex-type isolate of A. grisea. The isolate CBS 100.23, from Malus domestica, was deposited at the CBS as A. grossulariae. The original type description of this species, however, was from Grossularia sp., from Riga, Letland. Therefore A. grossulariae is not synonymised under A. gossypina based on this isolate pending the recollection of authentic material of the former species. By synonymising A. grisea, A. colombiana and A. tangelonis under A. gossypina, this species now has become an Alternaria species with a broad host range including host species from the Rutaceae, Malvaceae and Rosaceae.
Alternaria iridiaustralis E.G. Simmons, Alcorn & C.F. Hill, CBS Biodiversity Ser. (Utrecht) 6: 434. 2007.
Specimens examined: Australia, Queensland, Brisbane, from Iris sp. (Iridaceae), Oct. 1995, J. Alcorn, culture ex-type CBS 118486 = E.G.S. 43.014; Queensland, Brisbane, from Iris sp., Oct. 1996, J. Alcorn, CBS 118487 = E.G.S. 44.147. New Zealand, Auckland, Grey Lynn, from leaf of Iris sp., 7 Jan. 2001, C.F. Hill, CBS 118404 = E.G.S. 49.078.
Unique fixed nucleotides: ITS position 475 (A); gapdh position 33 (A), 171 (T), 174 (A), 186 (C), 218 (G), 365 (A); rpb2 position 12 (T), 489 (T), 516 (T), 591 (C); tef1 position 9 (G), 43 (T), 238 (G); OPA10-2 position 27 (G), 209 (C), 226 (A), 243 (G), 270 (C), 273 (A), 297 (C), 339 (T), 435 (A), 486 (A); Alt a 1 position 28 (T), 73 (C), 97 (G), 109 (T), 111 (G), 224 (A), 256 (T), 266 (A), 267 (G), 350 (G), 361 (A), 388 (C); endoPG position 87 (A), 93 (G), 101 (G), 210 (A), 219 (T), 338 (A), 340 (T), 374 (A); KOG1058 position 25 (C), 48 (A), 498 (C), 569 (T).
Alternaria jacinthicola Dagno & M.H. Jijakli, J. Yeast Fungal Res. 2: 102. 2011.
= Alternaria capsicicola A. Nasehi, J. Kadir & F. Abed-Ashtiani, Mycol. Progr. 13: 1044. 2014. (nom. inval., Art. 8.1, Melbourne Code).
Specimens examined: Mali, from leaf of Eichhornia crassipes (Pontederiaceae), 2006, K. Dagno, culture ex-type CBS 133751 = MUCL 53159. Mauritius, from leaf spot of Arachis hypogaea (Fabaceae), 2 Sep. 1959, S. Felix, CBS 878.95 = IMI 77934b. Unknown, from imported fruit of Cucumis melo (Cucurbitaceae) bought in Dutch supermarket, Feb. 2013, U. Damm, UD03.
Unique fixed nucleotides: gapdh position 479 (A); rpb2 position 6 (T), 549 (G); OPA10-2 position 159 (C); Alt a 1 position 295 (C), 353 (C), 364 (G); endoPG position 19 (T).
Notes: Although A. jacinthicola only has a few unique fixed nucleotides, the species can easily be distinguished from A. alternata using molecular data. The low number of unique fixed nucleotides is due to its close phylogenetic relationship to A. tomato and A. burnsii. Most of the nucleotide differences present between A. jacinthicola and the A. alternata isolates are also present in the A. tomato and / or A. burnsii isolates. By including two other isolates with A. jacinthicola, it has become an Alternaria species with a broad host range including species from the Pontederiaceae, Cucurbitaceae and Fabaceae. The recently described A. capsicicola (Nasehi et al. 2014) is synonymised under A. jacinthicola based on its Alt a 1 (KJ508068, KJ508069) and gapdh (KJ508064, KJ508065) sequences which are 100 % identical to A. jacinthicola. The name A. capsicicola is invalid, as two accessions were designated as holotype specimens.
Alternaria longipes (Ellis & Everh.) E.W. Mason, Mycol. Pap. 2: 19. 1928.
Basionym: Macrosporium longipes Ellis & Everh., J. Mycol. 7: 134. 1892.
= Alternaria brassicae var. tabaci Preissecker, Fachliche Mitt. Österr. Tabakregie 16: 4. 1916.
Specimens examined: USA, North Carolina, from Nicotiana tabacum (Solanaceae), 1967, E.G. Simmons, CBS 917.96; North Carolina, from Nicotiana tabacum, before Nov. 1971, representative isolate CBS 540.94 = E.G.S. 30.033 = QM 9589; North Carolina, Colombus County, from Nicotiana tabacum, Aug. 1963, E.G. Simmons, CBS 539.94 = QM 8438; North Carolina, from Nicotiana tabacum, before Nov. 1971, representative isolate CBS 121332 = E.G.S. 30.048; North Carolina, from Nicotiana tabacum, before Nov. 1971, representative isolate CBS 121333 = E.G.S. 30.051. Unknown, from leaf spot of Nicotiana tabacum, before Oct. 1935, W.B. Tisdale, CBS 113.35.
Unique fixed nucleotides: SSU position 654 (G); ITS position 491 (C); gapdh position 144 (G); OPA10-2 position 51 (T), 85 (G); KOG1058 position 848 (C).
Notes: Although A. longipes only has a few unique fixed nucleotides, the species can easily be distinguished from A. alternata using molecular data. The low number of unique fixed nucleotides is due to its close phylogenetic relationship to A. gossypina. Most of the nucleotide differences present between A. longipes and the A. alternata isolates are also present in the A. gossypina isolates.
Alternaria tomato (Cooke) L.R. Jones, Bull. Torrey Bot. Club 23: 353. 1896.
Basionym: Macrosporium tomato Cooke, Grevillea 12: 32. 1883.
Specimens examined: Unknown, from Solanum lycopersicum (Solanaceae), before Apr. 1930, A.A. Bailey, CBS 103.30; from Solanum lycopersicum, before Mar. 1935, G.F. Weber, CBS 114.35.
Unique fixed nucleotides: gapdh position 356 (T); rpb2 position 21 (T), 252 (C), 567 (C); tef1 position 36 (T); Alt a 1 position 187 (G); KOG1058 position 60 (A), 183 (A); KOG1077 position 588 (T).
Notes: Although A. tomato only has a few unique fixed nucleotides, the species can easily be distinguished from A. alternata using molecular data. The low number of unique fixed nucleotides is due to its close phylogenetic relationship to A. burnsii and A. jacinthicola. Most of the nucleotide differences present between A. tomato and the A. alternata isolates are also present in the A. burnsii and / or A. jacinthicola isolates.
Alternaria arborescens species complex (Fig. 5).
Fig. 5.
Alternaria arborescens species complex conidia and conidiophores. A–B. A. geophila CBS 101.13. C–D. A. arborescens CBS 102605. E–F. A. cerealis CBS 119544. G–H. A. senecionicola CBS 119545. Scale bars = 10 μm.
Alternaria arborescens E.G. Simmons, Mycotaxon 70: 356. 1999.
Alternaria cerealis E.G. Simmons & C.F. Hill, CBS Biodiversity Ser. (Utrecht) 6: 600. 2007.
Alternaria geophila Dasz., Bull. Soc. Bot. Genève, 2 Sér. 4: 294. 1912.
Alternaria senecionicola E.G. Simmons & C.F. Hill, CBS Biodiversity Ser. (Utrecht) 6: 658. 2007.
Type specimens examined: New Zealand, Auckland, Grey Lynn, from blighted Senecio skirrhodon (Compositae), Jul. 2000, C.F. Hill, culture ex-type of A. senecionicola CBS 119545 = E.G.S. 48.130; Auckland, from Avena sativa (Gramineae), Nov. 1995, C.F. Hill, culture ex-type of A. cerealis CBS 119544 = E.G.S. 43.072. Switzerland, from peat soil, before 1913, W. Daszewska, culture ex-type of A. geophila CBS 101.13. USA, California, from Solanum lycopersicum (Solanaceae), 23 Apr. 1990, D. Gilchrist, culture ex-type of A. arborescens CBS 102605 = E.G.S. 39.128.
Unique fixed nucleotides: rpb2 position 18 (A), 385 (T); tef1 position 42 (T), 44 (A), 111 (G); OPA10-2 position 330 (G), 504 (C); Alt a 1 position 333 (T); endoPG position 349 (C); KOG1058 position 625 (C); KOG1077 position 207 (A), 276 (−), 429 (G), 651 (T).
Notes: Although A. geophila is the oldest name in this species complex, the well-known name A. arborescens is retained above the relatively unknown name A. geophila for the species complex. The morphospecies present in this complex could not be resolved with the set of partial gene sequences used in this study and a more detailed study, possibly using whole-genome sequences of additional isolates from this species complex, is needed. Should this species complex be resolved and A. geophila and A. arborescens have to be synonymised, priority of the name A. arborescens over A. geophila is strongly suggested. The isolate CBS 126.60 was deposited in the CBS collection as A. maritima; however, the type material of A. maritima is unknown, and therefore A. maritima is not included within the AASC pending the recollection of suitable material of A. maritima.
Discussion
The aim of the present study was to employ genome comparisons and molecular phylogenies to clarify the species present in Alternaria sect. Alternaria. The Alternaria genomes generated in this study ranged in size from 32.0–39.1 Mb (Table 2), which can only be partly explained by differences in repeat content between the genomes. The isolates with the highest repeat content, A. avenicola (∼12 % repeats) and A. alternantherae (∼16 % repeats), have a relatively large genome size (39.1 and 35.0 Mb), but A. infectoria with a genome size of 36.5 Mb contains only ∼5 % of repeats (Table 2). The percentage of repeats within sect. Alternaria is relatively low, 1.4–2.7 %, with the highest percentage of repeats in the A. arborescens genome. The isolates which are now named A. alternata, only ranged from 1.4–1.7 %. The genome assembly shows a high similarity between the isolates within sect. Alternaria; 96.7–98.2 % genome identity within sect. Alternaria, compared to 85.1–89.3 % genome identity between isolates from other sections with the reference genome of A. alternata (CBS 916.96). This is confirmed by the percentage of SNPs found in the whole-genome and transcriptome reads; 1.4–2.8 % and 0.8–1.8 % SNPs in respectively the whole-genome and transcriptome reads between isolates from sect. Alternaria, compared to 8.0–10.3 % and 6.1–8.5 % SNPs found in isolates from different sections with the A. alternata reference genome. The phylogenetic species boundaries proposed here for sect. Alternaria are corroborated by the percentage of SNPs found in both the genome and transcriptome studies. The morphospecies now synonymised under A. alternata show 1.4–1.5 % SNPs in their whole-genome reads compared to 2.8 % in A. gaisen and ≤1 % of SNPs in their transcriptome reads compared to the reference isolate, while the species retained as separate, A. gaisen and A. arborescens, both show 1.8 % of SNPs in the transcriptome reads.
To be able to determine whether an isolate should be referred to as forma specialis or pathotype, the species boundaries should first be firmly established. From the seven described pathotypes of A. alternata (Akimitsu et al. 2014), two are now recognised as separate phylogenetic species in sect. Alternaria, namely A. gaisen and A. longipes, and one belongs to the A. arborescens species complex (AASC). The terms forma specialis (e.g. Neergaard, 1945, Joly, 1964, Grogan et al., 1975, Yoon et al., 1989, Vakalounakis, 1989) and pathotype (Nishimura & Kohmoto 1983) have both been used to specify the host affinity of strains of A. alternata. This affinity to a specific host is in most cases caused by the ability to produce a unique host-specific toxin (HST), which is needed for infection of the specific host. We propose here to standardise the taxonomic terms used according to Rotem's approach (1994). He favoured the use of the trinomial system in which the third epithet, the forma specialis, defines the affinity to a specific host in accordance with the produced toxin. When different toxins are produced on the same host, but these toxins affect different host species, like for instance on Citrus where the ACT- and / or ACR-toxin can be produced by the same f. sp., which affect tangerine and / or rough lemon, respectively (Masanuka et al. 2005), the term pathotype will be used. The four previously described pathotypes which still reside in A. alternata (Akimitsu et al. 2014), will therefore be named A. alternata f. sp. mali for isolates producing the AM-toxin, f. sp. fragariae for isolates producing the AF-toxin, f. sp. citri pathotype rough lemon for isolates producing the ACR-toxin, and f. sp. citri pathotype tangerine for isolates producing the ACT-toxin. All A. alternata isolates which are not confined to specific hosts and / or toxins should retain only the binomial name until such specificity is found. Multiple studies showed that HST gene clusters are located on small conditionally dispensable (CD) chromosomes (Tanaka and Tsuge, 2000, Hatta et al., 2002, Akamatsu, 2004, Harimoto et al., 2007, Harimoto et al., 2008, Hu et al., 2012) which can be lost (Johnson et al. 2001) or gained (Salamiah et al., 2001, Masanuka et al., 2005, Akagi et al., 2009), making an isolate either non-pathogenic or pathogenic to the specific host affected by the HST. With the species boundaries set in this study, this loss or gain of a specific gene cluster will not change the binomial part of the species name of an isolate.
Stewart et al. (2013a) have suggested that sequence data derived from SCARs would provide sufficient resolution to address lower level phylogenetic hypotheses in Alternaria. The authors developed SCARs from randomly amplified and cloned RAPD-PCR amplicons of which six of the 19 tested on small-spored Alternaria isolates were highly polymorphic. One of them was too variable which made it difficult to align and amplify this region; the remaining five were all more variable then ITS, gapdh and tef1, but only one (OPA10-2) showed a higher variability than endoPG. The other four were equally variable as or slightly more variable than endoPG. Both endoPG and OPA10-2 are used in the multi-gene phylogeny presented here, but could only distinguish 11 species of the 52 morphospecies previously described. Also, the molecular phylogenies obtained from the relative low conservative genes based on genome sequencing, KOG1058 and KOG1077, could not provide sufficient resolution to distinguish the known morphospecies. The incongruencies between the single-gene phylogenies, together with the high similarity found in the sequenced genomes of sect. Alternaria and the low SNP count derived by the genomic and transcriptomic data between isolates of sect. Alternaria led to the conclusion to synonymise 35 Alternaria morphospecies under A. alternata. As mentioned above, the detection of host-specific toxins could eventually give rise to several new formae speciales of A. alternata.
In a later study the same authors (Stewart et al. 2014) estimated the evolutionary histories of four nuclear loci on a worldwide sample of A. alternata isolates, causing citrus brown spot, using the coalescent theory. Next to the phylogenetic species concepts for estimating the species boundaries, two approaches were used that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common. The coalescent analyses revealed that the phylogenetic lineages are strongly influenced by incomplete lineage sorting and recombination. Also a study of the mating system of A. alternata isolates causing citrus brown spot found signatures of recombination (Stewart et al. 2013b). Andrew et al. (2009) already hypothesised that recombination and incomplete lineage sorting could explain the significant incongruence they found among gene genealogies in a four-gene species phylogeny on small-spored Alternaria, and the several putative recombination events that were identified within two non-coding regions. In agreement with our findings, little support was found for most of the morphospecies, when using these quantitative species recognition approaches.
Most of the synonymised morphospecies (10 / 35 species) under A. alternata were described in 2007 (Simmons), and are only based on a single isolate that was collected long before the year of description (A. brassicinae, A. citricancri, A. herbiphorbicola, A. pulvinifungicola, A. postmessia, A. soliaegyptiaca, A. vaccinii). As far as known, no new isolates of these species were reported in literature after their original description. Studies on the presence of host-specific toxins in these isolates could show if they should become a new f. sp. of A. alternata. Nine of the synonymised morphospecies are described in a paper on the classification of citrus pathogens (Simmons 1999). The validity of all these small-spored species described from citrus was already questioned by a molecular study performed in later years (Peever et al. 2004). The authors already advocated that all small-spored citrus-associated isolates of Alternaria should collapse into a single phylogenetic species, A. alternata. Also the validity of the name A. mali, the causal agent of Alternaria blotch of apple, which occurs on the European quarantine lists, was questioned in recent years (Rotondo et al., 2012, Harteveld et al., 2013). The authors describe the association of multiple Alternaria species-groups with leaf blotch and fruit spot diseases of apple in Italy and Australia respectively, and could not separate the A. mali reference isolate from ‘A. tenuissima’ isolates with molecular data. Based on the approach described in the present study, the only way to distinguish A. alternata f. sp. mali, which is of high importance as quarantine organism, is to detect the AM-toxin that gives the name to these isolates (Johnson et al. 2000).
The isolates constituting the AASC show some internal molecular and morphological variation, but can only clearly be separated from the A. alternata cluster based on molecular data. Both A. cerealis and A. senecionicola were marked by Simmons (2007) as having an arborescent-like sporulation pattern, but not all isolates from the AASC display this typical arborescent-like sporulation pattern (Fig. 5). This is illustrated by the fact that 12 out of the 28 isolates, which cluster in the AASC, were stored in the CBS collection as either A. alternata or A. tenuissima (Table 1). Because of the inconsistencies in morphology and molecular data in the AASC, more research is needed before conclusions can be drawn on the phylogenetic species present in this complex. Next to the known pathogenicity of A. arborescens on tomato, caused by the production of the AL-toxin, studies on Alternaria spp. show that isolates from the AASC can also cause diseases on apple (Rotondo et al., 2012, Harteveld et al., 2013, Harteveld et al., 2014) and can act as postharvest pathogens on apple and citrus (Kang et al., 2002, Serdani et al., 2002). The presence of multiple human isolates in the AASC stresses the importance of additional research on this species complex. To our knowledge, A. arborescens was not previously recognised as being of medical importance. One recent publication (Hu et al. 2014) does describe A. arborescens as the causative agent of a cutaneous Alternariosis in a healthy person, but the identification was based on ITS alone, a locus which cannot distinguish A. arborescens from multiple other species now recognised in sect. Alternaria (Table 4). In the end it might well be that A. arborescens needs the same treatment as A. alternata, and that it will be divided into different formae speciales based on the specific host they infect, and the toxin gene cluster they exploit.
The need for this research is stressed by examining recent publications on Alternaria spp. from sect. Alternaria. Two Alternaria species that were both argued as new based on phylogenetic data, and which were published during the writing of this manuscript, are both placed in synonymy under an older species name in this study. Based on molecular comparisons, Alternaria capsicicola (Nasehi et al. 2014) is synonymised under A. jacinthicola, and A. viniferae (Tao et al. 2014) is synonymised under A. alternata. Furthermore, the recent descriptions based on ITS alone of A. arborescens as the cause of cutaneous Alternariosis in a healthy person (Hu et al. 2014) and of A. longipes as the cause of a severe leaf spot disease on potato (Shoaib et al. 2014) need to be re-investigated by employing a more robust molecular dataset. As already mentioned above, A. arborescens cannot be separated from A. alternata based on the ITS region alone, and the 1 unique fixed nucleotide in the ITS sequence which separates A. longipes from A. alternata is not present in the ITS sequence from the isolate causing the leaf spot in potato. These are most likely not the only examples of species of Alternaria sect. Alternaria treated in recently published manuscripts that need to be confirmed by, or subjected to, a multilocus sequence analysis in light of the present study. The research presented here will hopefully make the correct identification of species in sect. Alternaria easier for other researchers confronted with these species.
Conclusions
Based on genome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of 11 phylogenetic species and one species complex. Thirty-five morphospecies, which cannot reliably be distinguished based on the multi-gene phylogeny, are synonymised under A. alternata. When a specific HST-gene cluster is demonstrated in an A. alternata isolate, this isolate will be named as a f. sp. of A. alternata. Currently three formae speciales of A. alternata are recognised, of which f. sp. citri consists of two pathotypes, according to the host species the HST acts upon. The AASC can be distinguished from all species now recognised within sect. Alternaria, but the inconsistencies in morphology and molecular data makes further research necessary. By providing guidelines for the naming and identification of phylogenetic species in Alternaria sect. Alternaria, a stable and consistent taxonomic treatment of this section can hopefully be accomplished for the future. The provided unique fixed nucleotides will help plant pathologists and medical mycologists to choose which genes to sequence for quick and accurate identification of their species of interest.
Acknowledgements
The authors would like to acknowledge E.G. Simmons (1920–2013) for his monumental taxonomic revision of Alternaria over the past few decades, and especially for making his strains available to facilitate this study. The research was supported by the Dutch Ministry of Education, Culture and Science through an endowment of the FES programme “Making the tree of life work”. Research in the laboratory of BPHJT is supported by the Research Council for Earth and Life Sciences (ALW) of the Netherlands Organisation for Scientific Research (NWO). Ion Torrent sequencing at the CBS-KNAW was financially supported by the European Community Research Infrastructures program under FP7 call ‘Synthesis of Systematic Resources’, grant number 226506-CP-CSA-Infra.
Footnotes
Peer review under responsibility of CBS-KNAW Fungal Biodiversity Centre.
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