Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species

Abstract

Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen. A morphology-based key is provided for identification to species or species groups.

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 . 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.

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.

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.

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.

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.

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.

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.

Figure 8. Morphological features of Verticillium klebahnii.

8a. Colony of strain PD659 after 10 days on PDA, frontal view. 8b. Colony of strain PD659 after 10 days on PDA, reverse view. 8c. Conidiophore of strain PD659 after 24 days on WA-p. 8d. Conidia of strain PD401 after 35 days on WA-p. 8e. Resting mycelium of strain PD657 after 32 days on WA-p. 8f. Chlamydospores of strain PD657 after 32 days on WA-p. 8g. Microsclerotia of strain PD401 after 32 days on WA-p. 8h. Hyphal cells of strain PD401 with yellow pigment and yellow crystals after 20 days on PDA. Scale bar: 8a, 8b = 2 cm; 8c = 50 µm; 8d–8h = 20 µm. Imaging method: 8a, 8b = DS; 8c, 8e–8h = BF; 8d = DIC.

Figure 9. Select morphological features of Verticillium longisporum.

9a. Colony of strain PD356 after 10 days on PDA, frontal view. 9b. Colony of strain PD356 after 10 days on PDA, frontal view. 9c. Conidia of strain PD348 after 35 days on PDA. 9d. Elongate microsclerotium of strain PD356 after 35 days on PDA. 9e. Rounded microsclerotium of strain PD356 after 35 days on PDA. 9f. Short brown-pigmented hyphae attached to microsclerotium in strain PD348 after 35 days on PDA 9g. Elongate microsclerotium from V. longisporum holotype specimen CBS H-19247. Scale bar: 9a, 9b = 1 cm; 9c–9g = 20 µm; Imaging method: 9a, 9b, = DS; 9c = DIC; 9d–9g = BF.

Figure 10. Morphological features of Verticillium nonalfalfae.

10a. Colony of strain PD592 after 14 days on PDA, frontal view. 10b. Colony of strain PD592 after 14 days on PDA, reverse view. 10c. Branched conidiophore of strain PD616 after 13 days on WA-p. 10d. Conidiophore of strain PD616 after 13 days on WA-p. 10e. Phialide of apical whorl of strain PD616 after 13 days on WA-p. 10f. Conidia of strain PD808 after 31 days on WA-p. 10g. Resting mycelium of strain PD811 after 18 days on WA-p. 10h. Intertwined hyphae of resting mycelium in strain PD810 in the lumen of a thick-walled plant cell after 18 days on WA-p. 10i. Torulose hyphal cells of resting mycelium in strain PD592 after 18 days on WA-p. Scale bar: 10a, 10b = 2 cm; 10c, 10e–10i = 20 µm; 10d = 50 µm. Imaging method: 10a, 10b = DS; 10c, 10e, 10f = DIC; 10d, 10g–10i = BF.

Figure 11. Morphological features of Verticillium nubilum.

11. Colony of strain P742 after 13 days on PDA, frontal view. 1b. Colony of strain PD742 after 13 days on PDA, reverse view. 11c. Conidiophore of strain PD621 after 17 days on WA-p. 11d. Apical phialide of strain PD621 after 17 days on WA-p. 11e. Conidia of strain PD621 after 17 days on WA-p. 11f. Solitary chlamydospore of strain PD742 after 17 days on WA-p. 11g. Linear chain of chlamydospores of strain PD742 after 17 days on WA-p. 11h. Angular chain of chlamydospores of strain PD621 after 25 days on PDA. 11i. Brown-pigmented hyphae of strain PD621 after 25 days on PDA. Scale bar: 11a, 11b = 1 cm; 11c–11i = 20 µm; Imaging method: 11a, 11b, = DS; 11c = PC; 11d, 11e = DIC; 11f–11i = BF.

Figure 12. Select morphological features of Verticillium tricorpus.

12a. Colony of strain P685 after 10 days on PDA, frontal view. 12b. Colony of strain PD685 after 10 days on PDA, reverse view. 12c. Conidia of strain PD685 after 38 days on PDA. 12d. Resting mycelium of strain PD685 after 38 days on PDA. 12e. Chain of chlamydospores and microsclerotium of strain PD685 after 38 days on PDA. 12f. Microsclerotium of strain PD685 after 38 days on PDA. 12g. Microsclerotium of lectotype specimen IMI 51602. 12h. Yellow-pigmented hypha of strain PD685 after 38 days on PDA. Scale bar: 12a, 12b = 1 cm; 12c–12h = 20 µm; Imaging method: 12a, 12b = DS; 12c = DIC; 12d–12h = BF.

Figure 13. Morphological features of Verticillium zaregamsianum.

13a. Colony of strain PD736 after 10 days on PDA, frontal view. 13b. Colony of strain PD736 after 10 days on PDA, reverse view. 13c. Conidiophore of strain PD736 after 32 days on WA-p. 13d. Solitary phialide of strain PD733 after 31 days on WA-p. 13e. Conidia of strain PD736 after 44 days on PDA; Inset: Brown, septate and constricted conidium of strain PD733 after 44 days on WA-p. 13f–13i. Microsclerotia of strain PD586 after 31 days on WA-p. 13f. Microsclerotium initial resembling chlamydospores. 13g. Microsclerotium initial with cells that originated by lateral cell divisions. 13h. Small microsclerotium. 13i. Microsclerotium. 13j. Hypha of resting mycelium in strain PD586 after 31 days on WA-p. 13k. Hyphal cells of strain PD733 containing yellow pigment after 44 days on PDA. 13l. Hyphal cells of strain PD736 encrusted by yellow crystals after 10 days on PDA. Scale bar: 13a, 13b = 2 cm; 13c = 50 µm; 13d–13l = 20 µm. Imaging method: 13a, 13b = DS; 13c, 13f–13l = BF; 13d, 13e = DIC.

Figure 14. Key for the identification of Verticillium species from PDA medium using morphological characters.