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Department of Plant Pathology, Washington State University, P.O. Box 646430, Pullman, Washington 99164-6430
L.W. Timmer
Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850
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ABSTRACT |
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The causal agents of Alternaria brown spot of tangerines and tangerine hybrids, Alternaria leaf spot of rough lemon and Alternaria black rot of citrus historically have been referred to as Alternaria citri or A. alternata. Ten species of Alternaria recently were described among a set of isolates from leaf lesions on rough lemon (Citrus jambhiri) and tangelo (C. paradisi x C. reticulata), and none of these isolates was considered representative of A. alternata or A. citri. To test the hypothesis that these newly described morphological species are congruent with phylogenetic species, selected Alternaria brown spot and leaf spot isolates, citrus black rot isolates (post-harvest pathogens), isolates associated with healthy citrus tissue and reference species of Alternaria from noncitrus hosts were scored for sequence variation at five genomic regions and used to estimate phylogenies. These data included 432 bp from the 5' end of the mitochondrial ribosomal large subunit (mtLSU), 365 bp from the 5' end of the beta-tubulin gene, 464 bp of an endopolygalacturonase gene (endoPG) and 559 and 571 bp, respectively, of two anonymous genomic regions (OPA1–3 and OPA2–1). The mtLSU and beta-tubulin phylogenies clearly differentiated A. limicola, a large-spored species causing leaf spot of Mexican lime, from the small-spored isolates associated with citrus but were insufficiently variable to resolve evolutionary relationships among the small-spored isolates from citrus and other hosts. Sequence analysis of translation elongation factor alpha, calmodulin, actin, chitin synthase and 1, 3, 8-trihydroxynaphthalene reductase genes similarly failed to uncover significant variation among the small-spored isolates. Phylogenies estimated independently from endoPG, OPA1–3 and OPA2–1 data were congruent, and analysis of the combined data from these regions revealed nine clades, eight of which contained small-spored, citrus-associated isolates. Lineages inferred from analysis of the combined dataset were in general agreement with described morphospecies, however, three clades contained more than one morphological species and one morphospecies (A. citrimacularis) was polyphyletic. Citrus black rot isolates also were found to be members of more than a single lineage. The number of morphospecies associated with citrus exceeded that which could be supported under a phylogenetic species concept, and isolates in only five of nine phylogenetic lineages consistently were correlated with a specific host, disease or ecological niche on citrus. We advocate collapsing all small-spored, citrus-associated isolates of Alternaria into a single phylogenetic species, A. alternata.
Key words: anonymous region, beta-tubulin, black rot, endopolygalacturonase, mitochondrial LSU rDNA, phylogeny, phytopathogen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Otani et al 1995) that are demonstrated pathogenicity factors in disease (Hatta et al 2002, Johnson et al 2000, 2001, Tanaka et al 1999). The four described Alternaria diseases of citrus include: (i) Alternaria brown spot of mandarins, tangerines and various tangerine hybrids; (ii) Alternaria leaf spot of rough lemon (C. jambhiri Lush.); (iii) black rot of citrus, a post harvest disease; and (iv) citrus leaf spot (mancha foliar de los citricos) of Mexican (Key) lime (C. aurantiifolia [Christm.] Swingle).
Alternaria brown spot of mandarin and tangerines, caused by A. citri Ellis & Pierce, first was described on emperor mandarin in Australia in 1903 (Cobb 1903). Alternaria leaf spot of rough lemon, also attributed to A. citri, originally was described from South Africa in 1929 (Doidge 1929) and Florida in 1937 (Ruehle 1937). The former pathogen has been referred to as A. alternata (Fr. : Fr.) Keissl. "tangerine pathotype" and the latter as A. alternata "rough lemon pathotype" because each form is host-specific and produces a chemically distinct host-specific toxin (Pegg 1966, Kohmoto et al 1979, Whiteside 1976). Black rot of citrus also has been attributed to A. citri, and symptoms include a stem-end decay of mature fruit in storage, which can occur on all commercial citrus cultivars (Bliss and Fawcett 1944). Mancha foliar de los citricos is caused by A. limicola Simmons & Palm (Palm and Civerolo 1994, Simmons 1990) and is a weak pathogen of other species of Citrus L.
The taxonomic status of the fungi that cause Alternaria brown spot and Alternaria leaf spot is unclear. These pathogens originally were identified as A. citri based on morphological similarities to isolates causing black rot (Doidge 1929, Kiely 1964, Pegg 1966, Ruehle 1937) and later were considered to represent a distinct strain based on their ability to infect leaves and young fruit and produce host-specific toxins (Kiely 1964, Whiteside 1976). Nishimura and Kohmoto (1983) treated these pathogens as A. alternata based on a published description of the morphology and size of the conidia of this species. The fungi causing Alternaria brown spot also have been called A. alternata pv. citri (Solel 1991) to differentiate them from saprophytic isolates of A. alternata. The taxonomic status of isolates causing black rot similarly is uncertain. Isolates causing black rot are considered biologically and taxonomically distinct from the brown spot pathogens because they are unable to cause disease on leaves or young fruit and do not produce host-specific toxins (Kiely 1964, Pegg 1966, Ruehle 1937). However, they are also small-spored and morphologically similar to the brown spot pathogens and it is not clear if they represent a distinct taxon.
Phylogenetic analyses of species of Alternaria based on DNA sequence data from several regions of the genome have revealed that small-spored species such as A. alternata, A. longipes (Ellis & Everh.) Mason and A. tenuissima (Nees : Fr.) Wiltshire are readily distinguished from large-spored species such as A. solani Sorauer (Kusaba and Tsuge 1995, McKay et al 1999, Pryor and Gilbertson 2000, Pryor and Michailides 2002). In contrast, differentiation of the small-spored species has been difficult due to lack of variation in nuclear ribosomal internal transcribed spacer (ITS) and beta-tubulin sequences, two genomic regions typically used in fungal systematics. Analysis of nuclear rDNA and mitochondrial RFLP also failed to differentiate several small-spored species including A. alternata, A. citri, A. gaisen Nagano, A. longipes and A. mali Roberts (Kusaba and Tsuge 1994, 1997). Analysis of ITS sequences revealed that small-spored, toxin-producing taxa such as A. alternata tangerine pathotype, A. alternata rough lemon pathotype, A. alternata strawberry pathotype, A. alternata tomato pathotype, A. gaisen and A. mali could not be differentiated from each other or from several saprophytic isolates of A. alternata (Kusaba and Tsuge 1995). Pryor and Gilbertson (2000), Pryor and Michailides (2002), Kang et al (2002) and McKay et al (1999) also failed to differentiate small-spored Alternaria species using ITS sequence data.
Recent research has attempted to clarify the systematics of the species of Alternaria associated with Alternaria brown spot and leaf spot of citrus (Simmons 1999a). One hundred thirty-five isolates from a worldwide collection, including isolates from leaf lesions on rough lemon, tangerine and tangerine x grapefruit hybrids, were examined (Simmons 1999a). Ten morphological species of Alternaria were described from this material, none of which was considered representative of A. alternata or A. citri. Because this research was based primarily on isolates from the culture collections of T.L. Peever and L.W. Timmer (Peever et al 1999, 2002), it represented an ideal opportunity to test the hypothesis that Alternaria species defined under a morphological species concept were concordant with taxa defined under a phylogenetic species concept using sequence data from multiple regions of the genome. To test this hypothesis, we used sequence data from the mitochondrial ribosomal large ribosomal subunit (mtLSU), the 5' end of the beta-tubulin gene, an endopolygalacturonase gene (endoPG) and two anonymous regions of the genome (OPA1–3, OPA2–1) that together provided the necessary variation to resolve a phylogeny among all known citrus-associated isolates of Alternaria. A secondary objective was to test the hypothesis that isolates associated with citrus black rot are monophyletic and phylogenetically distinct from citrus brown spot isolates. A preliminary report of this research has been published (Su et al 2001).
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MATERIALS AND METHODS |
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, 2002), and examined by Simmons (1999a); (ii) isolates causing leaf spot of Mexican lime (Simmons 1990, Palm and Civerolo 1994); (iii) isolates from healthy citrus tissue; and (iv) isolates sampled from mature citrus fruit with symptoms of black rot. When available, ex-type strains or strains considered representative of each described morphospecies were used (Table I). All isolates were stored on sterile filter paper as previously described (Peever et al 1999). For production of mycelium, isolates were grown 4 d in 2-YEG medium (10 g dextrose, 2 g yeast extract per L) on a rotary shaker, and mycelium was collected and lyophilized as previously described (Peever et al 1999). Genomic DNA was extracted from 50 µg lyophilized mycelium as described by Peever et al (1999) with modifications. One phenol/chloroform/isoamyl alcohol (25:24:1) extraction and one chloroform/isoamyl alcohol (24:1) extraction were used. DNA concentrations were estimated visually in 0.7% agarose gels containing 5 µg/mL ethidium bromide by comparing band intensity with known quantities of 
DNA/Hind III markers.
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) were used to amplify 1700 bp of the mtLSU from A. alternata (EGS 34–016), A. gaisen (EGS 90–0512), A. tangelonis (SH-MIL-4s), A. limicola (Colima A90), A. longipes (isolate EGS 30–033) and A. solani (49ss) (Table I). This 1700 bp amplicon corresponded to nucleotide positions 587–2436 of the coding region of the Penicillium chrysogenum Thom mtLSU rDNA (GenBank accession D13859). Twenty five µL PCR contained 1x reaction buffer, 0.2 µM of each primer, 200 µM dNTP, 2.5 mM MgCl2, 25 ng of DNA and 1 unit of Taq polymerase. PCR was carried out in a Hybaid Omn-E thermocycler (Hybaid, Ashford, Middlesex, U.K.), and cycling conditions consisted of 95 C for 1 min followed by 35 cycles of 95 C for 1 min, 60 C for 1 min and 72 C for 1 min. Amplicons from all six isolates were cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, California) and used to chemically transform competent TOP 10 Escherichia coli cells (Invitrogen) following the manufacturer's directions. Transformed cells were grown overnight in Luria-Bertani broth amended with 100 µg/mL ampicillin, and plasmid DNA was extracted using QIAprep Spin Mini Prep columns (Qiagen, Valencia, California) or BIO 101 RPM spin columns (Q-BIOgene, Carlsbad, California). Concentrations of plasmid DNA were determined using a fluorometer (Dynatech Laboratories, Chantilly, Virginia). Inserts were sequenced using T3 and T7 primers with each sequence reaction containing 40–90 ng DNA, 320 nM primer, 4 µL BigDye Terminator Cycle Sequencing Ready Reaction Mix (Applied Biosystems, Foster City, California) and sterile distilled water in 10 µL total volumes. Cycle sequence reactions were performed in a Hybaid Omn-E thermal cycler and cycling conditions consisted of 25 cycles of 96 C for 15 s, 50 C for 15 s, and 60 C for 4 min. Products were purified using Centriflex Gel Filtration Cartridges (Edge BioSystems, Gaithersburg, Maryland), dried in a rotary evaporator and sequenced on a PE Biosystems Model 377 Automated DNA Sequencer (Applera Corp., Norwalk, Connecticut). All sequencing was performed in the Laboratory for Biotechnology and Bioanalysis, School of Molecular Biosciences, Washington State University. Based on alignment of mtLSU sequences from the six species of Alternaria, a new reverse primer ML-R1 (5'-GCCCTTCCGAGAGCAAATAC-3') was designed using Primer3 software to amplify a product of convenient size for direct sequencing each strand in a single sequencing reaction. Primers ML1 and ML-R1 amplified a 460 bp fragment from all tested isolates using the same PCR conditions as described above. This amplicon corresponded to nucleotide positions 611–1081 of the P. chrysogenum mtLSU rDNA. Amplicons were purified through QiaQuick PCR purification columns (Qiagen) and direct sequenced on both strands using the same conditions described above with primers ML-1 and ML-R1. Cycle sequence reactions and automated sequencing were carried out as described above. MtLSU sequences have been deposited in GenBank (accession numbers AY293857–64).
Cloning and sequencing of the beta-tubulin gene –
One thousand one hundred twenty-four bp of the beta-tubulin gene was amplified from A. alternata (EGS 34–016), A. limicola (Colima A90), A. solani (49ss) and A. tangelonis (SH-MIL-4s) (Table I) using primers T1 (O' Donnell and Cigelnik 1997) and beta-tub-2 (5'-ATCATGTTCTTGGGGTCGAA-3'). Primer beta-tub-2 was designed to prime at nucleotide positions 806–787 relative to a partial A. alternata beta-tubulin sequence (GenBank accession Y17073) and nucleotide positions 1641–1661 in exon 6 of the Venturia inaequalis Aderhold beta-tubulin gene (GenBank accession M97951). These 1100 bp amplicons corresponded to nucleotide positions 447–1662 of the V. inaequalis beta-tubulin gene and included 
900 bp of putative exon sequence and 200 bp of putative intron sequence. Amplification of partial beta-tubulin sequences was carried out as described above, and amplicons were cloned and sequenced as described above. Sequences were aligned to design degenerate primers beta-3 (5'-GAGATTGYAAGTATCGCCTGSM-3') and beta-4 (5'-GCACGAACTTGTTGTTGGAS-3') that amplified a 400 bp fragment corresponding to nucleotide positions 456–943 of the V. inaequalis beta-tubulin gene. This amplicon included 300 bp of putative exon sequence and 100 bp of putative intron sequence. Amplicons were purified and direct sequenced with primers beta-3 and beta-4 as described above. Cycle sequence reactions and sequencing were carried out as described above. Beta-tubulin sequences have been deposited in GenBank (accession numbers AY293867–82).
Sequencing of additional gene regions –
Primers PG3 and PG2 were used to amplify a portion of an endopolygalcturonase gene (endoPG, GenBank accession ABO47682) characterized from the rough lemon pathotype of A. alternata (Isshiki et al 2001). These primers amplified a 500 bp amplicon consisting entirely of exon sequence. Amplifications were carried out as described for the mtLSU and beta-tubulin gene regions. Cycling conditions consisted of 95 C for 2 min followed by 35 cycles of 95 C for 1 min, 50 C for 1 min, and 72 C for 1 min. These cycles were followed by a final 5 min elongation cycle at 72 C. Amplicons were purified and sequenced as described above. EndoPG sequences have been deposited in GenBank (accession numbers AY295020–33). Five additional regions of the genome were amplified from selected isolates with variable endoPG sequences. These included actin (ACT), calmodulin (CAL), chitin synthase (CHS), translation elongation factor alpha (EF-1
) and 1, 3, 8-trihydroxynaphthalene (THN) reducatase. Primers EF1–728F and EF1–986R, CAL-228F and CAL-737R, ACT-512F and ACT-783R, CHS-79F and CHS-354R (Carbone and Kohn 1999) were used to amplify a 300 bp fragment of EF-1 including 230 bp of intron sequence, a 550 bp fragment of CAL including 350 bp of intron sequence, a 240 bp fragment of ACT including 130 bp of intron sequence and a 270 bp fragment of CHS including no intron sequences, respectively. Amplification of EF-1 was carried out as described for mtLSU, beta-tubulin and endoPG. Cycling conditions consisted of 95 C for 2 min followed by 35 cycles of 95 C for 1 min, 60 C for 1 min, and 72 C for 1 min. These cycles were followed by a final 5 min elongation cycle at 72 C. Amplifications of CAL, ACT and CHS were carried out in a PE Applied Biosystems Gene Amp 9700 thermocycler (Applied Biosystems, Norwalk, Connecticut), and reagents and cycling conditions were similar to those used for EF-1 except that a 55 C annealing temperature was used. Primers melanin-3 (5'-TCAATCGAGCAGACATGGAG-3') and melanin-4 (5'-CAACGCAGTTGACGGTGAT-3') were designed to amplify a 660 bp fragment corresponding to nucleotide positions 903–1570 of the A. alternata THN reductase gene, BRM2 (GenBank accession AB015743) involved in melanin biosynthesis. The amplified sequence included 570 bp of exon sequence and 90 bp of intron sequence. PCR were carried out in a Hybaid Omn-E thermocycler as described above. Cycling conditions consisted of 95 C for 2 min followed by 35 cycles of 95 C for 1 min, 62 C for 1 min, and 72 C for 1 min. These cycles were followed by a final 5 min elongation cycle at 72 C. Amplicons were purified and sequenced as described above.
Sequencing of anonymous regions –
Two anonymous regions of the Alternaria genome were amplified using RAPD-PCR with commercially available random decamer primers. Polymorphic amplicons were cloned and sequenced to design specific primers that would amplify regions of the genome useful for phylogenetic analysis among the small-spored citrus-associated species of Alternaria. Four isolates of Alternaria from citrus (SH-MIL-1s, 4s, 5s, 37s) previously shown to have divergent RAPD haplotypes (Peever et al 1999) were used to screen for polymorphisms with primers OPA1 and OPA2 ("Kit OPA", Operon). Twenty five µL PCR reactions contained 1x reaction/loading buffer, 0.2 µM of either primer, 200 µM dNTP, 2.0 mM MgCl2, 25 ng of DNA and 1 unit of Taq polymerase. PCR was carried out in a Hybaid Omn-E thermocycler (Hybaid) with cycling conditions of 97 C for 1 min followed by 35 cycles of 96 C for 15 s, 45 C for 15 s, and 72 C for 15 s. A polymorphic 922 bp amplicon produced by primer OPA1 from isolate SH-MIL-37s and a polymorphic 614 bp amplicon produced by primer OPA2 from isolate SH-MIL-4s were purified from agarose gels using QiaQuick Gel Extraction columns (Qiagen) and cloned into pCR4-TOPO (Invitrogen) as described above. Plasmid DNA was prepared as described above, and insert size was verified by EcoR1 digestion and electrophoresis. Plasmid inserts were sequenced on both strands using T3 and T7 primers as described above. Primers were designed to the ends of the inserts using Primer 3 so that the new primers included the decamer priming site. Primers OPA1–3-L (5'-CAGGCCCTTCCAATCCAT-3') and OPA1–3-R (5'-AGGCCCTTCAAGCTCTCTTC-3') or OPA2–1-L (5'-TGCCGAGCTGTCAGATAATTG-3') and OPA2–1-R (5'-GCCGAGCTGGTGGAGAGAGT-3') were used to amplify a 900 bp or 600 bp fragment, respectively, from selected small-spored species of Alternaria with different endoPG haplotypes (approximately two isolates representing each endoPG clade) to facilitate comparison among all three datasets and minimize sequencing effort. BLAST searches revealed no significant matches to these sequences in the databases. Ten µL PCR reactions contained 1x reaction/loading buffer, 0.3 µM of each primer, 200 µM dNTP, 2 mM MgCl2, 10 ng DNA, 0.8 M betaine and 0.5 units Taq polymerase. PCR was carried out in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems), and cycling conditions consisted of 94 C for 1 min followed by 35 cycles of 94 C for 20 s, 56 C for 20 s and 72 C for 40 s followed by a final extension at 72 C for 7 min. Amplicons were purified and sequenced as described above. OPA1–3 and OPA 2–1 sequences have been deposited in GenBank (accession numbers AY295034–53 and AY295054–72, respectively).
DNA sequence alignment and phylogenetic analysis –
MtLSU and beta-tubulin sequences were aligned using ClustalX 1.8 (Thompson et al 1997) and a rooted, maximum likelihood phylogeny was estimated for each gene using the DNAML program in PHYLIP (Felsenstein 1993) with A. infectoria as the outgroup. Previous phylogenetic studies of Alternaria have revealed that A. infectoria consistently forms the basal clade of the genus (McKay et al 1999, Pryor and Gilbertson 2000, Pryor and Michailides 2001). Models of sequence evolution were tested and model parameter estimates obtained for each alignment using MODELTEST 3.06 (Posada and Crandall 1998) as implemented in PAUP* 4.0b10 (Swofford 2002). For the mtLSU data, MODELTEST selected the F81 + G model with unequal base frequencies, a gamma shape parameter of 0.0083 and a transition:transversion ratio of 1.0. For the beta-tubulin data, MODELTEST selected the HKY + G model with unequal base frequencies, a gamma shape parameter of 0.462, and a transition:transversion ratio of 1.571. Statistical support for phylogram topologies was estimated using 100 bootstrapped datasets generated in the SEQBOOT program of PHYLIP. One hundred phylograms were estimated using DNAML with transition:transversion ratios, gamma shape parameters, base frequencies estimated using MODELTEST and random input order of taxa with three jumbling steps. Insertions and deletions in the alignments are ignored by DNAML. A majority-rule consensus tree was produced by the program CONSENSE and the consensus phylograms were visualized in TREEVIEW (Page 1996). Maximum-likelihood branch lengths for the consensus trees were estimated by removing the branch lengths from the consensus tree and using this tree as a user tree in DNAML. Clades with bootstrap values greater than 80% were considered significantly supported. EndoPG, OPA1–3 and OPA2–1 sequences were aligned using Clustal-X and rooted, maximum-likelihood phylogenies were estimated independently for each sequence using DNAML with Alternaria gaisen as outgroup. Alternaria gaisen has been demonstrated to be phylogenetically distinct from the small-spored citrus isolates and forms the basal clade in phylogenetic analyses of the small-spored species of Alternaria (Peever et al 2002). For the endoPG data, MODELTEST selected the K80 model with equal base frequencies, equal rates among sites and a transition:transversion ratio of 3.31. For the OPA1–3 data, MODELTEST selected the K80 + G model with equal base frequencies, a gamma shape parameter of 0.329, and a transition:transversion ratio of 2.08. For the OPA2–1 data, MODELTEST selected the K80 model with equal base frequencies, and a transition:transversion ratio of 4.686. For the combined endoPG, OPA1–3 and OPA2–1 data, MODELTEST selected the K80 + G model with equal base frequencies, a gamma shape parameter of 0.013, and a transition:transversion ratio of 2.589. These parameter estimates were used in DNAML for phylogenetic analyses. Topologies of the resulting phylograms were compared using incongruence length difference (ILD) tests (Farris et al 1994) to determine the suitability of combining the endoPG, OPA1–3 and OPA2–1 data. ILD tests were implemented in PAUP* (referred to as "partition homogeneity tests" in PAUP*) with invariant characters removed and 500 randomized partitions. Tested data partitions included: (i) endoPG, OPA1 and OPA2; (ii) endoPG and OPA1; (iii) endoPG and OPA2; and (iv) OPA1 and OPA2. Data partitions were considered significantly different at P < 0.01 (Sullivan 1996). Sequence alignments have been deposited in TreeBASE (study accession number S891, data matrices accession numbers M1461–64).
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RESULTS |
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and THN –
Alignment of sequences from five additional regions of the Alternaria genome revealed little polymorphism among selected small-spored Alternaria isolates. Isolates selected from clades 3, 4, 5, 6, 7, 8 and 9 of the combined analysis were invariant for ACT and THN (results not shown). Alignment of EF-1 from nine small-spored isolates representing different clades in the combined analysis revealed four polymorphic sites, one of which separated A. gaisen from all other isolates and three sites that separated A. tangelonis (EV-MIL-2s) from all other isolates. The same set of isolates revealed three polymorphic CAL sites and 2 polymorphic CHS sites. |
DISCUSSION |
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) and are easily distinguished morphologically from the small-spored citrus isolates, which generally are 30–50 µm in length (Simmons 1999). MtLSU and beta-tubulin sequence data indicated that A. limicola is most closely related to other large-spored species such as A. solani and A. linicola. Symptoms of mancha foliar de los citricos on Mexican lime include the production of water-soaked pustules that more closely resemble a bacterial disease; the disease originally was mistaken for citrus canker (Palm and Civerolo 1994). These symptoms are very different from those associated with Alternaria brown spot and leaf spot, and A. limicola has never been associated with black rot of citrus. The second well-supported clade inferred from mtLSU and beta-tubulin sequences included small-spored species of Alternaria associated with Alternaria brown spot of tangerines and hybrids, Alternaria leaf spot of rough lemon and citrus black rot. These gene regions were insufficiently variable to estimate a phylogeny among the small-spored isolates as were EF-1, CAL, CHS and THN sequences. This result is similar to that reported previously for phylogenetic analyses of beta-tubulin (McKay et al 1999) and ITS sequences (Kusaba and Tsuge 1995, McKay et al 1999, Pryor and Gilbertson 2000, Pryor and Michailides 2002).
MtLSU sequence data were sufficiently variable to differentiate the large-spored species of Alternaria (A. solani, A. linicola and A. limicola) from the small-spored species (A. alternata and A. tenuissima). However, analysis of this gene region did not allow us to differentiate A. brassicicola or A. infectoria from any of the small-spored citrus isolates or reference isolates such as A. alternata and A. tenuissima. The mtLSU appears to be considerably less variable among species of Alternaria than the mtSSU (Pryor and Gilbertson 2000), a gene region used to differentiate A. alternata, A. brassicicola and A. infectoria. Sequence data from the 5' end of the beta-tubulin gene revealed four clades in the current study and appears to be slightly more variable than either ITS, mtSSU, or exon 6 of the beta-tubulin gene (Kusaba and Tsuge 1995, Pryor and Gilbertson 2000, Pryor and Michailides 2002, McKay et al 1999). This portion of the beta-tubulin gene has been successful in delimiting phylogenetic species of Cylindrocladium Morgan (Crous et al 1999), Fusarium Link : Fr. (O'Donnell et al 1998, 2000) and basidiomycetes (Thon and Royse 1999).
Phylogenetic analysis of the combined endoPG, OPA1–3 and OPA2–1 data revealed substantially more variation than the beta-tubulin phylogeny for the small-spored isolates from citrus as well as several reference species. The analysis revealed nine clades that generally were congruent with described morphospecies (Fig. 4). These nine clades could be interpreted as defining nine species under a phylogenetic species concept with genealogical concordance (Taylor et al 2000). Other regions of the genome, including EF-1, CAL, ACT and CHS, have been useful in estimating phylogenies at the inter- and intraspecific levels in other fungi (Carbone and Kohn 1999, 2001,Crous et al 1999, O'Donnell et al 1998, 2000), but had little variation among the small-spored Alternaria isolates associated with citrus. We consider this further evidence of the close evolutionary relationship among these fungi. EndoPG has proven useful for the elucidation of phylogeographical patterns among Alternaria brown spot and leaf spot isolates (Peever et al 2002), and several isolates from that study were included here. EndoPG also might prove useful for phylogenetic or phylogeographic studies of other closely related fungi. To our knowledge, this is the first reported use of this gene for molecular systematics. The results of Peever et al (2002) revealed three endoPG clades among a worldwide sample of citrus brown spot isolates that correspond to clades 2, 4 and 6 of the combined analysis in the present study. The anonymous regions that were generated from RAPD for this study also appear to be very useful for phylogenetic studies of closely related Alternaria species.
In almost all cases where more than one isolate of a morphospecies was analyzed, both isolates clustered together in the same clade. However, the number of morphospecies exceeded that which could be supported phylogenetically and at least one morphospecies appeared to be polyphyletic (A. citrimacularis). Only two morphospecies, A. limoniasperae and A. toxicogenica, were associated uniquely with a single clade in the combined analysis. The eight remaining morphospecies were not associated uniquely with a specific clade, host, disease or ecological niche. The small-spored citrus isolates examined in this study were sampled from different ecological niches (leaf spots, fruit rots, healthy tissue) and have been shown to differ in host specificity and virulence (Peever et al 1999, 2002). However, these ecological differences did not map uniquely to specific clades defined through phylogenetic analysis of the combined endoPG, OPA1 and OPA2 data. Among the brown spot isolates, there were more morphospecies than could be supported by the phylogenetic analysis. This pattern contrasts with that revealed in many phylogenetic studies of morphologically well-characterized fungi. In these cases, morphospecies have been found to be polytypic and new phylogenetic species are typically uncovered (Geiser et al 1998, Giraud et al 1997, O'Donnell et al 1998). One definition of a fungal species consists of monophyletic groups that have unique, diagnosable phenotypic characters (Harrington and Rizzo 1999), and we advocate this species concept for the small-spored Alternaria isolates associated with citrus. The lack of strict correlation between each phylogenetic lineage and unique phenotypic, ecological or biological characters among the small-spored citrus-associated Alternaria and the occurrence of multiple morphospecies in several clades calls into question the practical utility of both the morphospecies and species defined by strictly phylogenetic criteria. Until such time as unique phenotypic, biological or ecological differences can be consistently and uniquely associated with isolates in a given clade, we advocate collapsing the small-spored species of Alternaria from citrus into a single phylogenetic species, A. alternata.
Isolates sampled from citrus fruit with symptoms of black rot were polyphyletic. It is not known if the seven black rot isolates studied here fit the morphological description of A. citri (Simmons 1990) but our data clearly indicate that black rot can be caused by several phylogenetically distinct small-spored Alternaria isolates. Black rot isolates and brown spot isolates sampled from different citrus hosts in several parts of the world had identical beta-tubulin sequences, and the former were distributed throughout the combined analysis in three of nine clades with the brown spot isolates. This might indicate that phylogenetically distinct isolates associated with citrus (including brown spot isolates) can induce citrus black rot. A similar result was obtained by Kang et al (2002), who found that phylogenetically diverse small-spored Alternaria isolates were associated with black rot of citrus in South Africa. In the present study, one of the black rot isolates (Acitri-1) was found in a clade with A. arborescens, a host specific toxin-producing tomato pathogen. We speculate that most or all small-spored species of Alternaria associated with citrus or other hosts can cause black rot. The morphological similarity of citrus black rot isolates and brown spot isolates in culture has been observed previously, and brown spot isolates were initially described as a distinct strain of A. citri (Doidge 1929, Kiely 1964, Pegg 1966, Ruehle 1937, Whiteside 1976). Based on our data, there appears to be no basis for considering black rot isolates as a distinct taxon and we also advocate the use of A. alternata for these isolates.
Alternaria brown spot and leaf spot pathogens were found in two of five clades in the endoPG phylogeny and five of nine clades in the combined endoPG, OPA1–3 and OPA2–1 phylogeny. Rooting the endoPG phylogeny by A. limicola (data not shown) revealed that A. colombiana, A. gaisen, A. longipes and A. tangelonis occupy a basal position in this phylogeny. Citrus morphospecies in this basal clade (A. colombiana, A. tangelonis) are pathogenic on tangerine and tangerine hybrids and produce host-specific ACT-toxin (Masunaka et al 2000). Alternaria gaisen, which causes black spot of Japanese pear, is known to produce host-specific AK-toxins (Nishimura and Kohmoto 1983, Simmons and Roberts 1993, Tanaka et al 1999) that are structurally similar to ACT-toxins produced by citrus brown spot isolates (Otani et al 1995). Homologs of genes controlling AK-toxin production have been identified in brown spot isolates, including isolates in this basal clade (Masunaka et al 2000). The occurrence of toxin-producing citrus brown spot isolates in several clades derived from the A. gaisen clade indicates that toxin production may be an ancestral character in this group of organisms. In addition, it appears that rough lemon leaf spot pathogens (A. limoniasperae) also have evolved from an ACT-toxin producing ancestor, even though they produce an unrelated toxin (ACRL-toxin) and are specific to rough lemon (Peever et al 1999). A. arborescens, A. longipes, and A. mali similarly appear to have evolved from an AK-toxin-producing ancestor yet produce host-specific toxins that are unrelated to AK- and ACT-toxin (Nishimura and Kohmoto 1983, Otani et al 1995). The recent availability of sequence data from the entire AK- and AF-toxin gene clusters (Hatta et al 2002, Tanaka et al 1999, 2000) and knowledge of their chromosomal location (Hatta et al 2002, Johnson et al 2001) will allow tests of specific hypotheses about the evolution of these toxin genes, the evolution of host specificity, correlations between toxin production and virulence and the molecular mechanisms conferring loss of toxin production in specific phylogenetic lineages of these fungi.
The basal position of A. gaisen in the combined phylogeny also suggests that citrus black rot isolates have evolved from a toxin-producing ancestor. Black rot isolates are nonpathogenic on leaves and immature fruit (Kiely 1964, Ruehle 1937, Masunaka et al 2000), and the few isolates that have been tested do not produce host-specific toxins (Pegg 1966, Masunaka et al 2000). Although the black rot isolates employed in the present study have not been tested for ACT-toxin production, they were found in three of nine clades with known toxin-producing strains. It is possible that black rot isolates have lost the ability to produce toxins but have retained some or all of the genes controlling their synthesis. On the other hand, toxin sequences might have been lost due to the loss of entire chromosomes carrying a cluster of toxin gene sequences (Hatta et al 2002, Johnson et al 2001). Our working hypothesis is that all small-spored isolates of Alternaria associated with citrus are potential black rot pathogens, and this hypothesis is being tested using host inoculations and a larger sample of isolates from diverse geographic regions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication June 2, 2003.
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