Pnge 200 Homework 7-8

Keywords: Animalia, Coleoptera, Dytiscidae

Type locality.

Papua New Guinea: Madang Province, Finisterre Range, Damanti, 05°53'26.5"S; 145°57'50.6"E.

Type material studied.

Exocelinadamantiensis. Holotype: male “Stn. No. 37”, “NEW GUINEA: Madang Dist., Finisterre Mts. Damanti 3,550 ft. 2-11.x.1964.”, “M.E. Bacchus. B.M. 1965-120”, “Copelatusdamantiensis sp.n. Balke des. 1997” [red], “Holotypus” [red] (BMNH). Note: “Stn. 387” in the original description is obviously a type error. Paratypes: 2 males, 11 females “Stn. No. 37”, “NEW GUINEA: Madang Dist., Finisterre Mts. Damanti 3,550 ft. 2-11.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 16 males, 16 females “Stn. No. 38”, “NEW GUINEA: Madang Dist., Finisterre Mts. Damanti 3,550 ft. 2-11.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 3 males, 7 females, 26 exs. “Stn. No. 39”, “NEW GUINEA: Madang Dist., Finisterre Mts. Damanti 3,550 ft. 2-11.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 2 males, 1 female, 14 exs. “Stn. No. 61”, “NEW GUINEA: Madang Dist., Finisterre Mts. Budemu c. 4000 ft. 15-24.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 1 male, 1 female, 5 exs. “Stn. No. 62”, “NEW GUINEA: Madang Dist., Finisterre Mts. Budemu c. 4000 ft. 15-24.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 2 males, 1 female, 13 exs. “Stn. No. 73”, “NEW GUINEA: Madang Dist., Finisterre Mts. Budemu c. 4000 ft. 15-24.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 3 males, 11 exs. “Stn. No. 74”, “NEW GUINEA: Madang Dist., Finisterre Mts. Budemu c. 4000 ft. 15-24.x.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 6 males, 8 females “Stn. No. 78”, “NEW GUINEA: Madang Dist., Finisterre Mts. Moro.C.5550ft. 30.x.-15.xi.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 4 males, 6 females, 18 exs. “Stn. No. 82”, “NEW GUINEA: Madang Dist., Finisterre Mts. Moro.C.5550ft. 30.x.-15.xi.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 2 males, 5 exs. “Stn. No. 83”, “NEW GUINEA: Madang Dist., Finisterre Mts. Moro.C.5550ft. 30.x.-15.xi.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 2 males, 3 exs. “Stn. No. 89”, “NEW GUINEA: Madang Dist., Finisterre Mts. Moro.C.5550ft. 30.x.-15.xi.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). 5 males “Stn. No. 95”, “NEW GUINEA: Madang Dist., Nr. Sewe, c.5,300 ft. 15.xi.1964.”, “M.E. Bacchus. B.M. 1965-120” (BMNH, NHMW). All these specimens are with red paratype labels “Paratypus Copelatusdamantiensis sp.n. Balke des. 1997” [red].

Exocelinamadangensis. Paratypes: 4 males, 3 females with the same label as the holotype, except for “Paratypus Copelatusinornatus sp.n. Balke des. 1997” [red] (NHMW).

Exocelinapatepensis. Holotype: male “Stn. No. 126”, “NEW GUINEA: Morobe Dist., Lae-Bulolo Rd., Patep Ck., 28.xii.1964.”, “M.E. Bacchus. B.M. 1965-120”, “Holotypus” [red], “Copelatuspatepensis sp.n. Balke des. 1997” [red] (BMNH). Paratypes: 4 males, 1 female with the same label as the holotype, except for “Paratypus Copelatuspatepensis sp.n. Balke des. 1997” [red] (NHMW).

Exocelinarivulus. Holotype: male “IRIAN JAYA, 12.8.1992 Zentralmassiv, Borme, 140°25'E 04°24'S 900m, leg. M. Balke (8)”, “HOLOTYPUS” [red], “Copelatusrivulus Balke des. 1997” [red] (NHMW). Paratypes: 25 males, 15 females with the same label as the holotype (NHMW). 7 males, 5 females “IRIAN JAYA: Borme ca. 140°25'E 04°24'S 950m, 3.9.1993 leg. M. Balke (2)” (NHMW). 12 males, 5 females “IRIAN JAYA Zentralmassiv 140°25'E 04°24'S”, “16.8.1992 Borme, 1000m leg. Balke (15)” (NHMW). 3 males, 5 females “IRIAN JAYA, 4.9.1992 Diuremna - Nalca 139°49'E 04°24'S 1500m, leg. Balke (36)” (NHMW). 38 males, 31 females “IRIAN JAYA, 6.9.1992 Nalca 1700-1800m 139°49'E 04°24'S leg. Balke (38)” (NHMW). 3 males, 1 female “IRIAN JAYA, 7.9.1992 Kono, 1800m 139°47'E 04°21'S, leg. Balke (41)” (NHMW). 55 males, 47 females “IRIAN JAYA, 12.9.1992 Angguruk, 1400m 139°25'E 04°15'S, leg. Balke (48)” (NHMW). 5 males, 1 female “IRIAN JAYA: Angguruk, 8.10.1993 Angguruk, ca. 1350m”, “ca. 139°25'E 04°15'S leg. M. Balke (32)” (NHMW). 1 male “IRIAN JAYA: Borme Tarmlu, 1500m 6.9.1993”, “ca. 140°25'E 04°24'S, leg. M. Balke (4-6)” (NHMW). 53 males, 42 females “IRIAN JAYA: 11.9.1993 Bime – Calab Gebiet, Bime, 1400m”, “leg. M. Balke (12) ca. 140°12'E 04°20'S” (NHMW). 57 males, 20 females “IRIAN JAYA: 22.9.1993 Bime – Calab Gebiet, Bime, 1400m”, “ca.

DNA sequence data

In order to investigate the phylogenetic relationships between Verticillium species, we generated DNA sequence data for 64 isolates, 317 DNA sequences were submitted to GenBank (Accessions ITS: JN187963–JN188023; ACT: JN188088–JN188151; EF: JN188216–JN188279; GPD: JN188152–JN188215; TS: JN188024–JN188087). An attempt to obtain DNA sequence data from the V. dahliae type specimen failed, as the DNA extract generated from a small part of the Dahlia sp. stem containing V. dahliae microsclerotia did not yield any PCR products (data not shown).

Single-locus analyses

To investigate whether the five single-locus datasets (ITS, ACT, GPD, EF, TS) contained similar phylogenetic information, we first analyzed each dataset individually using parsimony. For each single-locus analysis, we included only one representative of each allele. See Table 1 for descriptive statistics of the single-locus analyses. An ITS alignment, and an alignment of the combined ACT, GPD, EF and TS datasets, were submitted to TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S11756).

We did not detect any significant conflict between the most parsimonious trees from the five single-locus datasets on a 70% bootstrap support level (Figures S1, S2, S3, S4, S5), with the following exceptions. Verticillium nubilum was sister group to the clade of V. isaacii, V. klebahnii, V. tricorpus and V. zaregamsianum in the GPD tree with 89% bootstrap support (Figure S4), but in the EF and TS trees, V. nubilum was sister group to the clade of V. alfalfae, V. dahliae, V. longisporum and V. nonalfalfae with 97% support in both trees (Figures S3, S5). In the majority of trees, V. zaregamsianum was sister group to the V. isaacii, V. klebahnii and V. tricorpus clade with 100% support whereas in the EF tree (Figure S3), V. zaregamsianum was equally distantly related to all other Verticillium species. Also, the EF tree differed from the TS tree in the position of Species A1, an unknown ancestral species of the diploid hybrid V. longisporum[27]. Species A1 was a sister group to the clade of Species D1, another unknown ancestral species of V. longisporum[27] and V. dahliae in the TS tree with 76% support (Figure S5), whereas in the EF tree, Species A1 was sister to the monophyletic group of V. alfalfae, V. nonalfalfae, V. dahliae and Species D1 that were supported by 99% of the bootstrap replicates (Figure S3). In the remaining single-locus trees, the position of Species A1 was not fully resolved (Figures S1, S2, S4).

Combined analyses

With the goal of improving the phylogenetic resolution, we combined the ACT, EF, GPD and TS datasets into a single alignment for combined analysis. We did not include the ITS dataset since for V. longisporum, the ITS phylogeny does not retrace the evolution of that species [27]. The resulting combined four-locus alignment comprised 77 taxa and 2658 characters, and was submitted to TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S11756). There were a total of 32 unique multilocus haplotypes (Table 1). The Bayesian consensus tree is illustrated in Figure 1, it was congruent with the most likely tree (−ln likelihood = 12816.48) and with the 48 most parsimonious trees (Table 1) that differed at poorly supported branches within V. dahliae and the outgroup Gibellulopsis nigrescens (data not shown, but see support values in Figure 1).

Figure 1. Phylogenetic relationship of the ten Verticillium species based on the combined ACT, EF, GPD and TS dataset of 2658 characters and 77 taxa, with Gibellulopsis nigrescens as outgroup.

The Bayesian consensus tree is shown. Isolates are represented by their unique PD identifiers followed by host and geographic origin, PD identifiers in bold represent ex-type strains. Species are marked by vertical bars followed by species names, species in bold were described in this study. The two main clades recovered are indicated on the right. Numbers by the branches are Bayesian, likelihood and parsimony support values above 70 in that order, branches in bold had maximal support in all analyses. For the diploid hybrid V. longisporum, allele designations are also given following PD identifiers. Each isolate of V. longisporum has two alleles that are present in two different clades in the tree, in hypothetical Species A1, and either in hypothetical Species D1 or in V. dahliae, reflecting the hybrid origin of this species [27]. Groupings not visible in the tree but still receiving support include the clade of strains PD710 and PD743 with 71% bootstrap support, the clade of strains PD356, PD327 and PD502, with 100%, 78% and 73% Bayesian, likelihood and parsimony support, respectively; the clade of strains PD709 and PD711 and the clade of strains PD710 and PD743 with respectively 84 and 98% of the Bayesian posterior probabilities.

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We analyzed the four single-locus datasets jointly despite topological conflicts between them (Figures S2, S3, S4, S5). To evaluate whether single-locus datasets should be concatenated for combined analyses, a conditional combinability approach is often used which states that datasets should not be combined if there are significant differences between them [28], [29]. There is no agreement how much the single-locus datasets are allowed to differ, but topological differences supported by 70–90% of the bootstrap replicates have been used as cutoffs [28], [30]. In our case, there were topological differences supported by up to 100% of the parsimony bootstrap replicates between the single-locus datasets, involving the positions of V. nubilum, Species A1 and V. zaregamsianum. However, we found that the four-locus phylogeny comprised 35 groups with >70% support, higher than any of the single-locus trees (Table 1). Also, for V. nubilum, Species A1 and V. zaregamsianum, the combined analyses resulted for each species in the topology that had strongest overall support from the single-locus phylogenies. But the phylogenetic affinities of V. nubilum and Species A1 remain uncertain (Figure 1), and more data is needed to conclusively determine the closest relatives of these two species in Verticillium. In the combined analyses and in all single-locus datasets but the EF dataset, V. zaregamsianum formed a well-supported clade with V. isaacii, V. klebahnii and V. tricorpus (Figure 1). One possibility that could explain this divergence is origin by horizontal transfer of the EF gene in V. zaregamsianum. In conclusion, combined analysis of the single-locus datasets generated a phylogeny with higher overall support than any of the single-locus phylogenies, but did not conclusively settle the phylogenetic positions of V. nubilum and Species A1.

Phylogenetic groups obtained

We were able to infer a robust phylogenetic tree of Verticillium. The majority of the branches received maximal support, species were distinct and well defined, and the relationships between species were generally well resolved. As expected, branches with lower support were mainly present within species [31].

We recognized ten different species based on a phylogenetic species concept [31]. Except for the diploid hybrid V. longisporum[27], species were defined as terminal or subterminal clades receiving maximal support in the phylogenetic analyses based on the combined four-locus dataset (Figure 1). Each of the nine species level clades contained a single ex-type strain representing herbarium material, and thus linking each clade to one of the following nine species names (Figure 1). Verticillium albo-atrum, V. alfalfae, V. dahliae, V. isaacii, V. klebahnii, V. nonalflalfae, V. nubilum, V. tricorpus and V. zaregamsianum. Alleles of the diploid hybrid V. longisporum were present in the three different clades Species A1, Species D1 and V. dahliae (Figure 1), reflecting the hybrid origin of V. longisporum[27]. Verticillium longisporum evolved at least three different times from four different parental lineages in Species A1, Species D1 and V. dahliae[27]. Species A1 and Species D1 were not linked to any type material and could not be officially described, since Species A1 and Species D1 have never been found [27].

The evolutionary relationships among the Verticillium species was overall well resolved, the species fell into two major clades reflecting morphological similarity. The major clades were Clade Flavexudans containing species producing yellow-pigmented hyphae including V. albo-atrum, V. isaacii, V. klebahnii, V. tricorpus and V. zaregamsianum, and Clade Flavnonexudans with species devoid of yellow-pigmented hyphae, including V. alfalfae, V. dahliae, V. nonalfalfae, and V. longisporum (Figure 1). The exception was V. nubilum whose placement in Clade Flavnonexudans agreed with morphological data, but was only supported in the parsimony analyses (Figure 1). The other exception was the position of the V. longisporum ancestor Species A1 whose placement in the Bayesian consensus tree (Figure 1) contradicted phylogenetic analyses by Inderbitzin et al. [27] who used a different dataset.

Mating type distribution in V. alfalfae and V. nonalfalfae

All seven V. alfalfae and nine V. nonalfalfae isolates were screened for presence of MAT1-1 and MAT1-2 idiomorphs which are the two mating compatibility alleles in ascomycetes [32]. All V. alfalfae isolates showed the MAT1-1 specific PCR band whereas all V. nonalfalfae isolates lacked that band. All V. alfalfae isolates lacked the MAT1-2 specific band, whereas the MAT1-2 specific band was present in all V. nonalfalfae isolates (Figure 2). Thus, all V. alfalfae isolates likely have MAT1-1 idiomorphs whereas V. nonalfalfae isolates have MAT1-2 idiomorphs.

Figure 2. PCR gels documenting the results of the mating type PCR screens of Verticillium alfalfae and V. nonalfalfae using MAT1-1 and MAT1-2 specific primers.

Lanes 1–19: MAT1-1 specific PCR assay using primer set Alf/MAT11r. Lanes 1, 19: 2-log DNA ladders. Lane 18: Negative control. Lanes 2–17: V. alfalfae isolates followed by V. nonalfalfae isolates, in the same order as listed in Table S1. Lanes 20–38: MAT1-2 specific PCR assay using primer set HMG21f/MAT21r. Lanes 20, 38: 2-log DNA ladders. Lane 37: Negative control. Lanes 21–36: V. alfalfae isolates followed by V. nonalfalfae isolates, in the same order as listed in Table S1.

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Taxonomy

The genus Verticillium sensu stricto corresponds to a monophyletic group of taxa comprising V. dahliae that has been conserved as the type of Verticillium[15], [16]. We recognize ten species in Verticillium sensu stricto that are listed below in alphabetic order. The information provided for each species was obtained from morphological examination of cultures and herbarium specimens (Figure 3), literature surveys and phylogenetic analyses (Figure 1).

Figure 3. Type material examined in this study.

3a. Holotype specimen of Verticillium dahliae from HBG comprising a stem of Dahlia sp. cv. Geiselher infected with V. dahliae. 3b. Holotype specimen of V. longisporum (CBS H-19247) consisting of a dried agar culture (bottom) mounted in a cardboard sleeve (top). The number ‘19247’ was written on an envelope the specimen was placed in (not shown). 3c. Lectotype specimen of V. tricorpus (K(M) 172015, IMI 51602) comprising a dried agar culture (bottom) mounted in a cardboard sleeve (top). Scale bar: 3a = 8 cm; 3b, 3c = 3 cm.

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Verticillium albo-atrum Reinke & Berthold, Untersuchungen aus dem Botanischen Laboratorium der Universität Göttingen 1:75 (1879) Figure 4

Figure 4. Morphological features of Verticillium albo-atrum.

4a. Colony of strain PD747 after 10 days on PDA, frontal view. 4b. Colony of strain PD747 after 10 days on PDA, reverse view. 4c. Conidiophore of strain PD748 after 29 days on WA-p. 4d. Branched conidiophore of strain PD670 after 29 days on WA-p. 4e. Phialide of strain PD670 after 29 days on PDA. 4f. Conidia of strain PD670 after 29 days on PDA; Insets: Pigmented, septate and constricted conidium of strain PD670 after 29 days on PDA, budding conidium and conidium germinating by formation of a phialide, both of strain PD748 after 29 days on WA-p. 4g. Resting mycelium of strain PD747 after 33 days on WA-p. 4h. Aggregated hyphae of resting mycelium in strain PD670 after 28 on WA-p. 4i. Microsclerotium of strain PD670 after 47 days on PLYA. 4j. Microsclerotium of strain PD670 after 28 on WA-p. 4k. Microsclerotium of strain PD747 formed in the lumen of a thick-walled plant cell after 32 days on WA-p. 4l. Hypha of strain PD747 containing yellow pigment after 10 days on PDA. Scale bar: 4a, 4b = 2 cm; 4c, 4d = 50 µm; 4e–4h, 4j–4l = 20 µm; 4i = 100 µm. Imaging method: 4a, 4b = DS; 4c, 4d, 4g–4l = BF; 4e, 4f = DIC.

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MycoBank: MB199278 (as V. alboatrum)

Description.

Colonies on PDA after two weeks 4.5–5.5 cm diam, white at first, later turning yellow to orange due to the formation of yellow-pigmented hyphae, then darkening due to formation of resting mycelium immersed in the agar medium (Figures 4a, 4b). Aerial mycelium generally abundant, floccose to pruinose, hyphae smooth-walled, (1–) 1.5–4 µm wide. Conidiophores erect or slanted, generally determinate (Figure 4c), branched or unbranched (Figure 4d), formed disjointedly throughout the colonies, hyaline, base brown-pigmented at times, 80–480 µm in length, 3–6 µm wide, narrowing towards the apex to 2–2.5 µm, transversely septate, septa spaced more narrowly towards the apex. Conidiogenous cells are phialides, arranged in 1–4 (–6) whorls along conidiophores (Figures 4c, 4d), arising below transverse septum (Figure 4e). Whorls spaced 20–140 µm apart, closer towards the apex, consisting of (1–) 2–4 (–6) phialides (Figures 4c, 4d, 4e). Apical whorls consisting of one apical and one to several lateral phialides (Figures 4c, 4d, 4e). Phialides subulate, tapering from 1.5–3 µm at the base to 1–1.5 µm at the tip, terminal phialides 40–80 µm long, lateral phialides 20–50 µm long (Figure 4e). Conidia hyaline, smooth-walled, cylindrical with rounded apices to oval (Figure 4f), tapering at times, (3.0–) 6.0 µm±1.5 µm (–10.5)×(2.0–) 3.0 µm±0.5 µm (–6.0) (l/w = (1.1–) 2.0±0.4 (–3.0), n = 86), accumulating at the tip of the phialides (Figures 4c, 4d). After 4 wks, a small number of conidia (generally <1%) with central septum, constricted at the septum at times (Figure 4f). Budding conidia and conidia germinating by formation of a phialide observed (Figure 4f). Resting mycelium present, consisting of brown-pigmented hyphae, up to 7 µm wide, thick-walled, straight or curved, solitary or aggregated, up to 25 µm wide (Figures 4g, 4h). Microsclerotia present, composed of tightly interwoven, torulose brown-pigmented hyphae, rounded or variously shaped, up to 230 µm diam and consisting of rounded to elongate cells, up to 10 µm diam (Figures 4i, 4j, 4k). Yellow-pigmented hyphal cells present at times, up to 5 µm wide (Figure 4l).

Types.

Holotype: Missing, not found at GOET, B, M; Lectotype (designated herein): Illustrations from protolog: Figures of Plate (‘Tafel’) 8 and Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 of Plate 9 in Reinke and Berthold [21], available online at http://books.google.com/books?id=iWgVAAAAYAAJ&dq=Die%20Kr%C3%A4uselkrankheit%20der%20Kartoffel&pg=PA107#v=onepage&q=Die%20Kr%C3%A4uselkrankheit%20der%20Kartoffel&f=false (accessed on October 5, 2011); Epitype (designated herein): Dried culture of Verticillium albo-atrum strain PD747 (Canada: Prince Edward Island; potato field soil) deposited at UC (UC 1953892), an ex-epitype culture at CBS (CBS 130340) and NRRL (NRRL 54797).

Specimens examined.

The description was based on Verticillium albo-atrum strains PD670 (USA: WI; Irish potato), PD693 (UK; Irish potato), PD746 (Canada: New Brunswick; potato field soil), PD747 (Canada: Prince Edward Island; potato field soil) and PD748 (Canada: Prince Edward Island; potato field soil)(Table S1).

Distribution and host range.

Currently known from Canada, Germany, UK and USA (WI). Substrates include Irish potato and soil collected from Irish potato fields.

Commentary.

Verticillium albo-atrum was described by Reinke and Berthold in 1879 from diseased potato plants collected near Göttingen, Germany [21]. The protolog of V. albo-atrum contains detailed descriptions and drawings, but no reference is made to type material. We inquired at the herbaria of Göttingen (GOET), Berlin (B) and München (M), none of which has any V. albo-atrum type material or any other V. albo-atrum material deposited by Reinke and Berthold. According to Klebahn [33], original cultures of V. albo-atrum are no longer available. We did not find any V. albo-atrum cultures by Reinke and Berthold in any of the major culture collections (CBS, IMI, DSMZ, ATCC). Thus, in absence of any original fungal material, we designated the illustrations from the V. albo-atrum protolog in Plate 8 (Figures 1, 2, 3, 4, 5, 6, 7, 8) and Plate 9 (Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) in Reinke and Berthold [21] as the lectotype for V. albo-atrum. According to The International Code of Botanical Nomenclature (ICBN) [34] this is permissible when the holotype, all cited or uncited original specimens and all original cultures are missing (Art. 8.4, Art. 9.2, Art. 9.10, Art. 37.4). To serve as an interpretive type, we designated a V. albo-atrum epitype with an ex-epitype culture for molecular studies. Designation of an epitype is permissible according to ICBN when serving the precise application of a name (Article 9.7).

The original description of V. albo-atrum by Reinke and Berthold [21] was based on observations from decaying potato stems, and is congruent with our observations from the V. albo-atrum isolates examined in this study. The exception was the presence of yellow pigment associated with hyphal cells (Figure 4l) not seen by Reinke and Berthold [21]. However, Klebahn [35] reported that V. albo-atrum mycelium on Salep Agar medium was white with a yellow tinge (p. 64), whereas V. dahliae mycelium was described as white (p. 65).

In addition to resting mycelium, Verticillium albo-atrum also forms microsclerotia (Figures 4i, 4j, 4k). Microsclerotia are very ‘small, firm, frequently rounded masses of hyphae with or without the addition of host tissue or soil.’ [36]. The V. albo-atrum microsclerotia were described and illustrated in the protolog on pages 74 and 75, and in Figures 1 and 2 of Plate 9 [21], a translation from the German original is provided by Isaac [22]. The microsclerotia consist of aggregations of brown-pigmented, thick-walled hyphae, no lateral cell divisions are involved in their formation [21]. This is opposed to the microsclerotia of V. dahliae where an increase in width is achieved by the lateral divisions of hyphal cells as described by Klebahn [35] on pages 56 and 57, and illustrated in Klebahn's [35]Figure 8. Microsclerotia were only observed on WA-p and PLYA media, they were absent from strains cultured on PDA. Verticillium albo-atrum has frequently been confused with V. alfalfae and V. nonalfalfae that form resting mycelium but no microsclerotia.

The name ‘V. albo-atrum’ is correct with or without hyphen (Art. 23.1), the hyphenated form is more commonly encountered in the literature.

Verticillium alfalfae Inderb., H. W. Platt, R. M. Bostock, R. M. Davis & K. V. Subbarao, sp. nov.Figure 5

Figure 5. Morphological features of Verticillium alfalfae.

5a. Colony of strain PD682 after 24 days on PDA, frontal view. 5b. Colony of strain PD682 after 24 days on PDA, reverse view. 5c. Conidiophore of strain PD682 after 31 days on WA-p. 5d. Phialide of strain PD489 after 30 days on WA-p. 5e. Conidia of strain PD682 after 30 days on WA-p. 5f. Resting mycelium of strain PD489 after 30 days on WA-p. 5g. Aggregated hyphae of resting mycelium in strain PD682 after 73 days on PDA. 5h. Resting mycelium of strain PD683 in the lumen of a thick-walled plant cell after 32 days on WA-p. Scale bar: 5a, 5b = 2 cm; 5c = 50 µm; 5d–5h = 20 µm. Imaging method: 5a, 5b = DS; 5c, 5f–5h = BF; 5d, 5e = DIC.

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MycoBank: MB563552

Etymology: Medicago sativa (‘alfalfa’), the only currently known host of this species.

Latin diagnosis.

Verticillio nonalfalfae morphologia simile, sed characteribus sequentiarum nucleidearum distinguendum. Actin positione 21 (T), 72 (G), 78 (T), 459 (A), 462 (A); Elongation factor 1-alpha positione 149 (G), 157 (G), 175 (G), 225 (A), 265 (T), 266 (A), 271 (A), 280 (C), 304 (T), 346 (C), 428 (T), 429 (T), 441 (G), 465 (T), 469 (C), 474 (T), 591 (C), 596 (T), 600 (C), 624 (T); Glyceraldehyde-3-phosphate dehydrogenase positione 173 (A), 324 (C); Tryptophan synthase positione 87 (A), 161 (T), 169 (C), 246 (T), 273 (T), 315 (T), 583 (T), 601 (C).

Description.

Colonies on PDA after two weeks 3.5–4.5 cm diam, white at first (Figures 5a, 5b), later darkening due to the formation of resting mycelium immersed in the agar. Aerial mycelium generally abundant, floccose to pruinose, hyphae smooth-walled, 2–3 µm wide. Conidiophores erect or slanted, generally determinate (Figure 5c), branched or unbranched, formed disjointedly throughout the colonies, hyaline, base brown-pigmented at times, enlarged to up to 11 µm at times, 70–570 µm in length, 4.5–6.5 µm wide, narrowing towards the apex to 2–2.5 µm, transversely septate, septa spaced more narrowly towards the apex (Figure 5c). Conidiogenous cells are phialides (Figure 5d), arranged in 1–4 (–6) whorls along conidiophores (Figure 5c). Whorls spaced 30–130 µm apart, closer towards the apex, consisting of (1–) 2–5 (–6) phialides, arising below transverse septum (Figures 5c, 5d). Apical whorls consisting of one apical and one to several lateral phialides (Figures 5c, 5d). Phialides subulate, tapering from 2–3 µm at the base to 1–2.5 µm at the tip, terminal phialides 40–60 µm long, lateral phialides 20–40 µm long (Figure 5d). Conidia hyaline, smooth-walled, cylindrical with rounded apices to oval (Figure 5e), allantoid at times, (4.5–) 6.0 µm±1.0 µm (–11.0)×(2.5–) 3.0 µm±0.5 µm (–4.0) (l/w = (1.4–) 1.9±0.3 (–2.9), n = 68), accumulating at the tip of the phialides (Figure 5c). Resting mycelium present (Figures 5f, 5g, 5h), consisting of brown-pigmented hyphae, up to 9 µm wide, thick-walled (Figures 5f, 5g), straight or curved, solitary or aggregated (Figure 5g), torulose at times.

Types.

Holotype: Dried culture of V. alfalfae strain PD489 (USA; alfalfa) deposited at UC (UC 1953895), an ex-holotype culture at CBS (CBS 130603) and NRRL (NRRL 54790).

Specimens examined.

The description was based on V. alfalfae strains PD338 (USA: PA; alfalfa), PD353 (USA: PA; alfalfa), PD489 (USA; alfalfa), PD620 (Canada; alfalfa), PD681 (USA; alfalfa), PD682 and PD683 (Japan: Hokkaidou; alfalfa) (Table S1).

Distribution and host range.

Currently known from Canada, Japan and the USA (PA), only from alfalfa.

Commentary.

Verticillium alfalfae is morphologically indistinguishable from V. nonalfalfae.

Verticillium dahliae Kleb., Mycologisches Centralblatt 3: 66 (1913) Figures 3a, 6

MycoBank: MB196942

Description.

Colonies on PDA after two weeks 4–6 cm diam, white at first, later darkening due to the formation of microsclerotia (Figures 6a, 6b). Aerial mycelium generally abundant, floccose, at times sparse and pruinose, or appressed to the agar and appearing water-soaked. Aerial hyphae smooth-walled, (1.5–) 2–4 µm wide, at times containing inflated cells up to 9 µm wide (Figure 6c). Conidiophores erect or slanted, generally determinate (Figure 6d), branched or unbranched (Figure 6e), formed disjointedly throughout the colonies, hyaline, 80–800 µm in length, 3–4 µm wide, narrowing towards the apex, transversely septate, septa spaced more narrowly towards the apex. Conidiogenous cells are phialides (Figure 6f), arranged in (1–) 2–3 (–10) whorls along conidiophores (Figures 6d, 6e), arising below transverse septum (Figure 6f). Whorls spaced 50–100 µm apart, closer towards the apex, consisting of (1–) 2–4 (–6) phialides (Figures 6d, 6e). Apical whorls consisting of one apical and one to several lateral phialides (Figure 6d). At times solitary phialides are formed laterally from vegetative hyphae (Figure 6g). Phialides subulate, tapering from 2–3 µm at the base to 1–2 µm at the tip, terminal phialides 40–60 µm long, lateral phialides 25–50 µm long (Figures 6d, 6e, 6f, 6g). Conidia hyaline, smooth-walled, non-septate, cylindrical with rounded apices to oval (Figure 6h), allantoid or tapering at times, (3.5–) 6.5 µm±1.5 µm (–13.5)×(2.0–) 3.0 µm±0.5 µm (–4.5) (l/w = (1.4–) 2.2±0.3 (–3.4), n = 80), accumulating at the tip of the phialides (Figures 6d, 6e). Microsclerotia immersed in agar, regularly or irregularly distributed throughout the colonies, composed of rounded, brown-pigmented cells up to 13 µm diam, solitary microsclerotia rounded to elongate or irregular in shape, 25–100 µm diam, aggregates of microsclerotia up to 200 µm diam (Figures 6i, 6j, 6k). At times short, brown-pigmented hyphae attached to microsclerotia present (Figure 6k).

Figure 6. Morphological features of Verticillium dahliae strain PD322 (ex-epitype) unless otherwise noted.

6a. Colony after 14 days on PDA, frontal view. 6b. Colony after 14 days on PDA, reverse view. 6c. Inflated cells present in mycelium after 28 days on PDA. 6d. Conidiophore after 15 days on WA-p. 6e. Branched conidiophore after 12 days on WA-p. 6f. Whorl phialide after 25 days on WA-p. 6g. Solitary phialide after 14 days on PDA. 6h. Conidia after 9 days on PDA. 6i. Microsclerotia after 12 days on WA-p. 6j. Microsclerotia of the V. dahliae holotype material from stem of Dahlia sp. 6k. Short brown-pigmented hypha composed of torulose cells attached to microsclerotium after 49 days on PDA. Scale bar: 6a, 6b = 2 cm; 6c, 6f–6k = 20 µm; 6d, 6e = 50 µm. Imaging method: 6a, 6b = DS; 6c, 6f–6h = DIC; 6d, 6e, 6i, 6k = BF; 6j = PC.

https://doi.org/10.1371/journal.pone.0028341.g006

Types.

Holotype: Specimen V. dahliae (Germany; Dahlia sp.) at HBG (Figures 3a, 6j) [16]; Epitype (designated herein): Dried culture of Verticillium dahliae strain PD322 (USA: CA, lettuce) deposited at UC (UC 1953893), an ex-epitype culture at CBS (CBS 130341) and NRRL (NRRL 54785).

Specimens examined.

The description was based on Verticillium dahliae strains PD322 (USA: CA; lettuce), PD327 (USA: CA; bell pepper) and PD502 (USA: WI; maple) (Table S1). The V. dahliae holotype specimen was also examined (Figures 3a, 6j).

Distribution and host range.

Currently known from Brazil, Canada, Denmark, France, Germany, Iran, Israel, Italy, Japan, Netherlands, Russia, Spain, Sweden, UK, Ukraine, and USA (CA, ID, IL, OR, TX, WA, WI) [27]. Substrates include Anaheim pepper, annual sunflower, apricot, ash, bell pepper, cabbage, celandine, chili pepper, common flax, eggplant, European smoketree, garden tomato, globe artichoke, horseradish, hybrid strawberry, Icelandic poppy, Irish potato, jalapeno, Japanese maple, lettuce, maple, olive, opium poppy, paprika, pepper, peppermint, pistachio nut, purple coneflower, rape, scentless false mayweed, spinach, stock, sweet almond, udo, upland cotton, and watermelon [27] that represent fourteen different plant families (Aceraceae, Amaranthaceae, Anacardiaceae, Araliaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Linaceae, Malvaceae, Oleaceae, Papaveraceae, Rosaceae, Solanaceae).

Commentary.

Verticillium dahliae is the type of Verticillium and was described by Klebahn [35] from Dahlia sp. cv. Geiselher in Germany (Figure 3a). Verticillium dahliae is not the oldest species of the genus, but it has the largest impact as a pathogen, is common and genetically relatively homogenous, and has thus been conserved as the type of the genus [16], [34]. Since a viable ex-holotype culture is no longer available [33], and DNA extraction attempts from the holotype specimen failed, we designated a V. dahliae epitype with an ex-epitype culture that serves as an interpretive type for molecular studies.

The original description of V. dahliae by Klebahn [35] was based on material from Dahlia sp., and from cultures on Salep Agar medium which is a mixture of polysaccharides contained in orchid tubers [37]. The composition of Klebahn's medium is unknown, but as a reference, Noël [38] isolated fungal symbionts of orchids using a clear, weak decoction of salep containing 2% agar. We examined the V. dahliae holotype material which contains an approximately 50 cm long stalk of Dahlia sp. ‘Sorte Geiselher’ and several leaves (Figure 3a). The microsclerotia present on the stem (Figure 6j) were similar to the microsclerotia formed in culture (Figure 6i). No conidiophores were observed, these are difficult to detect on Dahlia sp. [35], but are illustrated as part of the protolog [35].

The description of V. dahliae based on V. dahliae strains PD322, PD327 and PD502 agreed with the original description by Klebahn [35] except that we failed to detect strands of erect, hyphal aggregates containing conidia and microsclerotia. Klebahn [35] reported the presence of a slightly wider cell (foot cell) at the base of conidiophores. Since foot cells were absent in culture and we did not inoculate live plants, we were unable to confirm the presence of foot cells in V. dahliae. The dimensions provided by Klebahn [35] for microsclerotia, conidiophores, conidiogenous cells and conidia were at the lower end of the range of dimensions that we observed. Our dimensions were similar to reports in the literature for conidia [22] and microsclerotia [22], [39], [40], [41]. Short brown-pigmented hyphae attached to microsclerotia were illustrated by Klebahn [35], the ones that we observed resemble immature microsclerotia as illustrated by Klebahn [35] and Isaac [22]. Verticillium dahliae resembles V. longisporum but has smaller conidia.

Verticillium isaacii Inderb., R. M. Bostock, R. M. Davis & K. V. Subbarao, sp. nov.Figure 7

Figure 7. Morphological features of Verticillium isaacii.

7a. Colony of strain PD619 after 10 days on PDA, frontal view. 7b. Colony of strain PD619 after 10 days on PDA, reverse view. 7c. Conidiophore of strain PD618 after 21 days on WA-p. 7d. Phialides of strain PD660 as part of an apical whorl after 21 days on WA-p. 7e. Conidia of strain PD611 after 22 days on WA-p; Insets: One-septate, constricted conidium and two septate conidium of strain PD660 after 21 days on WA-p. 7f. Hypha of resting mycelium and yellow crystal of strain PD752 after 35 days on PDA. 7g. Chlamydospores of strain PD752 after 35 days on PDA. 7h. Microsclerotium of strain PD752 after 35 days on PDA. 7i. Hyphal cell of strain PD660 encrusted with yellow pigment after 20 days on PDA. Scale bar: 7a, 7b = 2 cm; 37c = 50 µm; 7d–7i = 20 µm. Imaging method: 7a, 7b = DS; 7c, 7f–7i = BF; 7d, 7e = DIC.

https://doi.org/10.1371/journal.pone.0028341.g007

MycoBank: MB563553

Etymology: Named after Ivor Isaac (1914–1978), in recognition of significant contributions to Verticillium taxonomy.

Latin diagnosis.

Verticillio tricorpus morphologia simile, sed characteribus sequentiarum nucleidearum distinguendum. Actin positione 79 (T), 115 (T), 292 (T), 380 (T), 410 (T), 432 (A); Elongation factor 1-alpha positione 142 (C), 162 (A), 166 (T), 185 (A), 190 (T), 230 (A), 235 (G), 248 (A), 260 (A), 331 (A), 363 (T), 366 (G); Glyceraldehyde-3-phosphate dehydrogenase positione 153 (C), 278 (C); Tryptophan synthase positione 133 (A), 143 (A), 383 (G).

Description.

Colonies on PDA after two weeks 2.5–6 cm diam, white at first, later yellow, reverse orange to yellow, then darkening due to the formation of resting mycelium, chlamydospores and microsclerotia (Figures 7a, 7b). Aerial mycelium generally abundant, floccose, hyphae smooth-walled, 1–3.5 µm wide. Conidiophores erect or slanted (Figure 7c), generally determinate, branched or unbranched, formed disjointedly throughout the colonies, hyaline, verruculose surface ornamentation present at times, 105–690 µm in length, 3–6 µm wide, narrowing towards the apex to 2–2.5 µm, transversely septate, septa spaced more narrowly towards the apex. Conidiogenous cells are phialides (Figure 7d), arranged in (1–) 2–4 (–6) whorls along conidiophores (Figure 7c), arising below transverse septum. Whorls spaced 25–60 µm apart, closer towards the apex, consisting of (1–) 3–5 (–6) phialides (Figure 7c). Apical whorls consisting of one apical and one to several lateral phialides (Figures 7c, 7d). Phialides subulate, tapering from 2–3.5 µm at the base to 1–1.5 µm at the tip, terminal phialides 30–65 µm long, lateral phialides 20–40 µm long (Figure 7d). Conidia hyaline, smooth-walled, cylindrical with rounded apices to oval (Figure 7e), tapering at times, (3.5–) 6.0 µm±1.5 µm (–14.5)×(1.5–) 3.0 µm±0.5 µm (–5.0) (l/w = (1.4–) 1.9±0.3 (–3.5), n = 73), accumulating at the tip of the phialides (Figure 7c). Conidia rarely one- or two-septate, constricted at the septum at times (Figure 7e). Resting mycelium, chlamydospores and microsclerotia present. Resting mycelium consisting of brown-pigmented hyphae, up to 5 µm wide (Figure 7f), chlamydospores solitary or in chains, up to 12 µm wide (Figure 7g), microsclerotia rounded or variously shaped, up to 70 µm diam and consisting of rounded to elongate cells, up to 10 µm wide (Figure 7h). Yellow-pigmented hyphal cells present, up to 5.5 µm wide, containing globules of yellow pigment, at times pigment accumulating as crystals outside the cells, up to 21 µm diam (Figures 7f, 7i).

Types.

Holotype: Dried culture of V. isaacii strain PD660 (USA: CA; lettuce) deposited at UC (UC 1953896), an ex-holotype culture at CBS (CBS 130343) and NRRL (NRRL 54792).

Specimens examined.

The description was based on V. isaacii strains PD341, PD343, PD367, PD437, PD610, PD611, PD612, PD613, and PD660 (USA: CA; lettuce), PD618 (UK; garden tomato), PD619 (Canada; soil), PD661 (USA: WA; lettuce), PD752 and PD753 (USA, WA; spinach) (Table S1).

Distribution and host range.

Currently known from Canada, UK and USA (CA, WA). Substrates include garden tomato, globe artichoke, hairy nightshade, lettuce, spinach and soil.

Commentary.

Verticillium isaacii is morphologically indistinguishable from V. klebahnii and V. tricorpus.

Verticillium klebahnii Inderb., R. M. Bostock, R. M. Davis & K. V. Subbarao, sp. nov.Figure 8

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