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Phylogeny of Stemphylium spp. based on ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences

     United States Department of Agricultural, Agriculture Research Service, Molecular Plant Pathology Laboratory, Beltsville, Maryland 20705-2350

Peter van Berkum

     United States Department of Agriculture, Agricultural Research Service, Soybean Genomics and Improvement Laboratory, Beltsville, Maryland 20705-2350

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.

Key words: Pleospora, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The genus Stemphylium Wallr. was established in 1833 (Wallroth 1833) with S. botryosum Wallr. as the type species. There are 33 published names that represent recognizable taxa of Stemphylium (Simmons pers comm). Species of Stemphylium are dematiacious hyphomycetes with muriform, septate, usually pigmented conidia produced by a conidiophore that proliferates percurrently. The percurrently proliferating conidiophore is the principal morphological characteristic that distinguishes this genus from other genera with muriform conidia such as Ulocladium and Alternaria (Simmons 1969). In species with known teleomorphs, the sexual state is Pleospora. The association between the Stemphylium state and a Pleospora teleomorph or at least sclerotial bodies that never fully develop into an ascoma in culture has been established for 15 taxa: S. alfalfae Simmons, S. astragali Yoshii, S. bolickii Sobers & Seymour, S. botryosum, S. botryosum f. lactucum, S. drummondii Nirenberg & Plate, S. globuliferum (Vertergren) Simmons, S. gracilariae Simmons & Schatz, S. herbarum (Pers : Fries) Rabenhorst ex Cesati & DeNotaris, S. lancipes (Ellis & Everhart) Simmons, S. loti Graham, S. majusculum Simmons, S. trifolii Graham, S. trigochinicola Sutton & Pirozynski, and S. vesicarium (Wallr.) Simmons (Simmons pers comm).

Both saprotrophic and pathogenic forms of Stemphylium occur on a wide range of plants (Farr et al 1989). Many species of Stemphylium are economically important pathogens of agricultural crops. The causal agents of leaf spot in alfalfa and red clover are S. botryosum, S. globuliferum, S. herbarum, S. alfalfae, and S. vesicarium. The latter also causes purple spot in asparagus and leaf spot in onion and garlic. Gray leaf spot on tomato and potato is the result of infection by S. solani (Ellis and Gibson 1975a, Irwin 1984, Johnson and Lunden 1986, Simmons 1990, Aveling and Snyman 1993). The most widespread foliar disease of birdsfoot trefoil (Lotus corniculatus) is caused by Stemphylium loti (Seaney 1973).

Identification of Stemphylium species has relied on morphological characters such as variation in conidium, conidiophore, and ascospore morphology. However, many of these characters overlap among species, making species determinations difficult. Chaisrisook et al (1995) used RAPD data to separate isolates of five species from alfalfa into two clusters. However, studies using sequence data to reconstruct evolutionary relationships among the species of Stemphylium have not been done.

Berbee et al (1999) used ITS and gpd sequence data to estimate phylogenetic relationships in the genus Cochliobolus. In the present study we used ITS and gpd sequences to investigate phylogenetic relationships among Stemphylium species. In addition, we want to determine whether sequence divergence in these two nuclear loci is correlated with species concepts in Stemphylium based on morphological characters. We also want to determine whether species that are pathogenic to the same host are phylogenetically related.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isolates – Forty-four isolates representing 16 named species and 6 unidentified taxa of Stemphylium were included in this study. These isolates were selected to represent all the Stemphylium species that are known to be available in culture (Table I). Ex-type cultures were used when available. Isolates were prepared for long-term storage by growing them for 14 d per disks that were placed on the surface of ½ strength V-8 agar (100 mL V-8, 2 g CaCO₃, 16 g agar per L). When the filter paper was completely covered by mycelium, it was removed from the agar surface and dried for 48 h. Filter paper colonized by the fungus was then cut into small pieces and sealed in glass vials, which were stored at 10 C. Working cultures were maintained on ½ strength V-8 agar and kept at 22 C with 12/12 h light/dark cycles, and morphometric studies on these cultures were done 14 d after the plates were inoculated. At least 50 measurements of the conidia and the conidiophores were taken for each of the isolates studied (Table II).

PCR amplifications – Primers NS1 and ITS4 (White et al 1990) were used to amplify the ITS region and small subunit (SSU) rRNA gene and part of the large subunit (LSU) rRNA gene (Câmara et al 2000). We designed primers to amplify and to sequence a partial region of the gpd gene based on the sequence of Cochliobolus heterostrophus, GenBank accession X63516. The forward primer, gpd f 5'-GCA CCG ACC ACA AAA ATC-3' was located at bases 574-- 591 and the reverse primer, gpd r 5'-GGG CCG TCA ACG ACC TTC-3', was located at bases 1499–1482. A PCR optimizer kit (Invitrogen, Carlsbad, California) was used to optimize the PCR reaction mixture for each locus. The following reaction conditions were used for both the ITS and the gpd regions: 10 µL of 5x buffer C (60 mM tris-HCl, 15 mM (NH₄)₂SO₄, 2.5 mM MgCl₂ at a final pH of 8.5), 1.25 µL each of 10 mM dATP, dCTP, dGTP, dTTP, 5 0 pmol of the primers ITS4 and NS1 (ITS) or gpd f and gpd r (gpd region), 2 µL Perkin Elmer Taq polymerase, and 22 µL of sterile water. Amplifications were performed with an ERICOMP Delta Cycler II^TM system using the following program parameters: 35 cycles of 94 C for 30 s, 57 C for 1 min, 72 C for 1.5 min, and a final extension at 72 C for 3 min. The presence of the PCR products was verified by UV illumination of horizontal agarose (0.7% w/v) gels after electrophoresis in the presence of EtBr (0.08 µg/mL).

DNA sequencing – The PCR products were purified using QIAquick spin columns (Qiagen Inc., Chatsworth, California) and both strands were sequenced with an ABI 377 automated DNA sequencer (Applied Biosystems Inc., Foster City, California) using a Taq Dye-Deoxy Terminator Cycle Sequencing Kit (ABI, Foster City, California). The ITS region was sequenced using primers ITS3, ITS4, ITS5 (White et al 1990) and ITS2c (5'-CAGTAAACATGGAAGTTCGA-3'), a new primer designed to provide overlap with the sequence derived from primer ITS3. The gpd region was sequenced with forward primers gpd ef, 5'-CGG CTT CGG TCG CAT G-3' (790–805), and gpd if, 5'-CAC GGC CAG TTC AAG-3' (1084–1098), and reverse primers gpd er, 5'-GCC AAG CAG TTG GTT GTG-3' (1400–1383), and gpd ir, 5'-GGC GGG GTC CTT CTC C-3' (1176–1161). Sequence data were edited and assembled with Factura and Autoassembler (Applied Biosystems Inc., Foster City, California) on a Macintosh computer.

Analysis of sequence data – Sequences were aligned by using the PILEUP program in the Wisconsin package of the Genetics Computer Group (Madison, Wisconsin). ITS sequences of strains identified as S. botryosum (AF229481), S. callistephi Baker & Davis (AF 229482), S. solani Weber (AF 203451) and S. vesicarium (AF229484), obtained from GenBank, were included in the analysis in addition to the 37 isolates of Stemphylium sequenced in this study. The gpd sequences of Stemphylium available from GenBank (S. botryosum and S. vesicarium) were identical to sequences of the same species obtained in this work and were not used in the analysis. Alignment parameters were empirically adjusted to a gap penalty of one and gap extension penalty of zero. Alignments were manually inspected for ambiguities and adjustments were made where necessary by using GeneDoc 2.5 (Nicholas and Nicholas 1997). The sequences obtained in this study were deposited in GenBank (Table I) and the alignment of the sequences was deposited in TreeBase (Study accession number = S687; matrix accession number = M1079).

Maximum parsimony trees were inferred by using the heuristic search option with the random sequence addition and branch swapping with tree bisection-reconnection options in PAUP 4.0b2 (Swofford 1999). PAUP 4.0b2 (Swofford 1999) was also used to generate Jukes Cantor distances among the sequences to produce a Neighbor-joining tree. Based on results reported by Berbee et al (1999), representatives of four genera within the Pleosporales (Alternaria, Pyrenophora, Setosphaeria, and Cochliobolus) were chosen as outgroups. The Shimodaira-Hasegawa (Shimodaira and Hasegawa 1999) test was used to identify the tree with a likelihood score closest to zero when more than one most parsimonious tree was obtained for the purpose of showing the most likely phylogenetic relationships. This test also was used to determine whether other hypothetical trees, constrained for monophyly of the Stemphylium spp. pathogenic to alfalfa, would be less likely than the most parsimonious trees. Relative support for the phylogram branches was estimated with 1000 bootstrap replications of the data sets with random addition input order of sequences (10 replicates) during each heuristic search. Molecular characters were unordered and given equal weight during analysis and all were included in the analysis after removing indels. Partition homogeneity analysis (Farris et al 1995, Huelsenbeck et al 1996) was used to determine if the two data sets (ITS and gpd) could be combined, and a combined analysis was run using the parameters described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Parsimony analysis of the ITS region generated more than 1000 most parsimonious trees (tree length of 279 steps, CI = 0.8029, RI = 0.8276, and RC = 0.6644). Sequences of the ITS1, ITS2, and 5.8S rDNA of the Stemphylium species ranged from 499 to 507 bp. From a total of 566 evaluated characters, 405 were constant and 97 were parsimony-informative. Analysis of the gpd region generated 1 most parsimonious tree (length of 469 steps, CI = 0.6716, RI = 0.7722, and RC = 0.5186). Of 605 characters evaluated, 391 were constant and 139 were parsimony-informative. Sequences of the gpd gene of the Stemphylium species ranged from 556 to 562 bp. Two introns and three exons were present in the gpd fragment. The introns accounted for 172 nucleotides, of which 75 were variable, while the three exons accounted for 385 nucleotides, of which 94 were variable. Among the species of Stemphylium the numbers of variable sites were 71 and 49 for the introns and exons, respectively.

The species of Stemphylium formed a well-supported monophyletic group in parsimony trees constructed from aligned sequences of the ITS region or gpd gene (Figs. 1, 2) with bootstrap values of 98% and 77%, respectively. The partition homogeneity test for incongruence between the two loci was not significant and we concluded that the ITS and the gpd data sets could be combined (P = 0.45). The combined data set generated 6 most parsimonious trees (tree length of 748 steps, CI = 0.7139, RI = 0.7827, and RC = 0.5588). Of 1226 characters evaluated, 846 were constant and 246 were parsimony-informative. Analysis of the two data sets individually led to trees with different levels of resolution among species but their topologies were similar. Within the Stemphylium species groupings were resolved in t he tree constructed from the ITS region, including the 5.8S rRNA gene, but only three of the groups were well supported (Fig. 1). The groups with S. loti and S. sarciniforme (Cav.) Wilts., with S. botryosum and S. globuliferum, and with S. xanthosomatis Huguenin and S. lycopersici (Enjoji) Yamamoto were well supported (bootstrap >78%).

View larger version (40K): [in this window] [in a new window]    FIG. 1. One of more than 100 equally parsimonious trees resulting from an analysis of the ITS1–5.8S-ITS2 sequences from Stemphylium species (tree length of 279 steps, CI = 0.8029, RI = 0.8276, and RC = 0.6644). Parsimony analysis of the ITS region generated more than 1000 most parsimonious trees. Bootstrap values (>50%) from 1000 replicates were included at the nodes

View larger version (40K): [in this window] [in a new window]    FIG. 2. One single most parsimonious tree resulting from an analysis of the glyceraldehyde-3-phosphate dehydrogenase sequences from Stemphylium species (tree length of 469 steps, CI = 0.6716, RI = 0.7722, and RC = 0.5186). Bootstrap values (>50%) from 1000 replicates were included at the nodes

Stemphylium solani and S. callistephi grouped together with a bootstrap value of 92% (Fig. 2, group D). Stemphylium sarciniforme, S. loti, S. trifolii, S. trigochinicola, and the isolate EGS 42-138 with morphological characteristics resembling S. globuliferum (Fig. 2, group E) also were grouped together (bootstrap 89%). The tree generated from the gpd data alone and the combined data set (ITS + gpd) had similar topologies but the combined tree had better bootstrap support for some of the internal branches (Fig. 3).

View larger version (49K): [in this window] [in a new window]    FIG. 3. Best (-ln L) of six equally most parsimonious trees resulting from an analysis of the combined data set (glyceraldehyde-3-phosphate dehydrogenase + ITS1–5.8s-ITS2) from Stemphylium species (tree length of 748 steps, CI = 0.7139, RI = 0.7827, and RC = 0.5588). Bootstrap values (>50%) from 1000 replicates were included at the nodes

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Phylogenetic analysis of the Stemphylium species and isolates generally confirmed the previously described classifications based on morphological characters. From both the ITS and gpd data sets we concluded that there was strong support for the monophyly of the genus Stemphylium. Species with known sexual states and those for which only the asexual state is known clustered together with their predicted sexual relatives. Analysis of the gpd fragment was more useful for establishing well-supported relationships among the species and isolates of Stemphylium due to its greater resolving power.

All the species of Stemphylium with no known sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Each of the Pleospora spp. is associated with one Stemphylium anamorph. A similar relationship among sexually and asexually reproducing species of Curvularia and Bipolaris has been reported (Berbee et al 1999). If sexuality is ancestral to asexuality, this pattern may indicate that the ability to reproduce sexually was lost more than one time during the evolutionary history of this genus. However, we wish to emphasize that characterization of a fungus' ability to reproduce sexually is often determined in pure culture and may not necessarily represent the character outside the laboratory. Therefore, it is possible that some or all of the Stemphylium species or isolates do reproduce sexually in nature or under more ideal conditions in culture. Therefore, whether or not a specific culture is truly asexual is unknown.

From analysis of the two loci we placed the alfalfa pathogens into two distinct clusters (groups B and C) that were similar to the groups identified using RAPD analysis (Chaisrisook et al 1995). One group included S. botryosum and S. globuliferum while the other included S. alfalfae, S. herbarum and S. vesicarium.

Stemphylium botryosum and two isolates with a morphology similar to S. globuliferum grouped together (Fig. 1; group B in Figs. 2, 3). The morphology of these two isolates and the description of S. globuliferum (Simmons 1969) are similar to that of isolate EGS 42-138 originating from Malus sylvestris. However, isolate EGS 42-138 was placed quite distant from the pathogens of Medicago sp. in our phylogenetic analysis. This result may imply that morphological characterization of species and isolates of Stemphylium may not necessarily indicate their phylogenetic placement as reconstructed from ITS and gpd sequence divergence. The morphology of these two isolates and EGS 42-138 differs from the type description of S. globuliferum in conidia and conidiophore ornamentation and color intensity. Therefore, these two isolates and EGS 42-138 may represent more than one species.

The species S. botryosum and S. globuliferum are distinguished by small differences in conidial and ascospore size, intensity and type of conidial ornamentation, the presence or absence of ornamentation on the tip of the conidiophore, and the pattern of septum development in both the conidia and the ascospores (Simmons 1969, 1985). However, they share a number of morphological characters that include conidial shape, a conidial length-width ratio (L/W) of 1–1.5, thick ascomatal walls, host range, and slow ascomatal development. Unfortunately a representative culture of S. globuliferum was not available in our study for phylogenetic placement and for morphological analysis.

The Stemphylium spp. from New Zealand (EGS 48-077; EGS 48-079), S. astragali, S. herbarum, S. vesicarium, S. alfalfae, S. majunsculum, and S. gracilariae are pathogens of legumes that share the similar morphological characteristics of having conidia that are constricted in more than one transverse septum, where the conidial wall is verrucose or heavily punctate, and that develop the sexual state fairly rapidly (Simmons 1969, 1985, 1989). These species grouped together in our molecular systematic analysis (group C, Figs. 2, 3).

Within group C, we observed a well-supported sub-group (Figs. 2, 3) of Stemphylium species that are pathogenic to Medicago spp. (S. herbarum, S. vesicarium, S. alfalfae). Only small differences in the patterns and in the septation of conidia and ascospores have been used to distinguish among these three species (Simmons 1969, 1985, 1989). However, they share other characteristics such as conidial shape (oblong to broadly oval, especially in culture) with L/W ratio close to 2.0 (1.9, 1.7, 2.5, respectively), and thin-walled, fast-maturing ascomata in culture. Also, these three species had identical sequences for the gpd region and only a single nucleotide difference was observed in the ITS region (Table III). Evidence for separating S. vesicarium, S. herbarum and S. alfalfae into separate species is based on morphological data (Simmons 1969, 1985, 1989), which is not supported by our phylogenetic analysis.

Stemphylium trifolii, S. loti, S. sarciniforme, S. triglochinicola, and one isolate with morphology similar to S. globuliferum (EGS 42-138) were placed in a single group in reconstructions using both loci and the combined data set. This group was well supported in analyses of the gpd and the combined data set (Figs. 2, 3). Most of the isolates that share this group have smooth-walled conidia, are pathogens of legumes (Stemphylium trifolii, S. loti, S. sarciniforme), and have an unknown sexual state. Only S. triglochinicola, isolated from the salt marsh arrow grass Triglochin maritime produces the sexual state abundantly in culture (Simmons 1969).

There was one well- supported subgroup (S. sarciniforme and S. loti) within group E (Figs. 1, 3). This group contains pathogens of the legumes Trifolium and Lotus, respectively, which share similar morphological characteristics that include conidial shape, lack of wall ornamentation and conidiophore size (Table II). However, fully developed S. loti conidia are larger with more frequent septations than those of S. sarciniforme. Stemphylium loti also produces stromatic bodies in culture but these never completely mature into a teleomorph. Differences in the production of stromatic bodies by S. loti and host range specificity (Graham 1953) are characteristics used to distinguish these two species, and is in agreement with our phylogenetic analysis.

Stemphylium lycopersici and S. xanthosomatis (group A) share such characters as long conidiophores and conidia with pointed apices, and 1–3 major transverse septa (Yamamoto 1960, Huguenin 1965). The two had nearly identical sequences for both genes; the ITS sequences were identical and there was only one nucleotide difference between their gpd sequences. Stemphylium lycopersici has a worldwide distribution and infects a broad range of hosts including Lycopersicon, Allium, Carthamus, Gladiolus and others (Ellis and Gibson 1975b). Differences between the two are based primarily on sizes of conidia and conidiophores (Yamamoto 1960, Huguenin 1965) with measurements for morphological variation made using fungal-infected plant tissues. From our measurements, using defined media for growing each of the two fungal isolates, we concluded that morphologically they are very similar (Table II). Since both of these fungal isolates appear to be very similar in morphology and in phylogenetic placement, the differences in morphology may in fact represent intra-specific variation. Clearly additional information is required with a larger number of isolates under standard growing conditions for the morphometric measurements to help to clarify whether morphological and phylogenetic species concepts (Taylor 2000) are congruent in this case.

The phylogenetic relationships based on the sequence data of the gpd locus presented here agree with the morphological species concepts of most Stemphylium species included in this study

We observed that some phenotypic characters currently used in the taxonomy of this genus may be more useful than certain genotypic characters. For instance, morphological variation and our sequencing results of the gpd locus separated Stemphylium species that had identical ITS sequences. Since different morphologically described species had identical ITS region sequences it is unclear whether they actually represent separate taxa, even though they could be separated genetically by using the gpd locus. Therefore, it is possible that our observation with Stemphylium is another example in which the ITS region is insufficiently informative to separate morphologically described species.

	ACKNOWLEDGMENTS

 
The authors extend sincere thanks to Patrick Elia for technical support in the sequencing analysis. We sincerely appreciate the assistance of Lisa Castlebury and Steve Rehner in reviewing this manuscript. We also thank Dr. Emory Simmons for providing most of the isolates used in this study and for helpful comments on the taxonomy of the Stemphylium spp.

	FOOTNOTES

 
¹ Corresponding author, Email: oneilln{at}ba.ars.usda.gov

Accepted for publication December 7, 2001.

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