Genealogical sorting index

The rooted gene genealogies resulting from each of the single gene analyses of ACT, CAL, EF1-α, ITS and TUB were submitted to the genealogical sorting index (gsi) parallel computing resource (http://www.genealogicalsorting.org/) for analysis. The gsi estimates the degree of exclusive ancestry of individuals in labelled predefined groups in a rooted tree (Cummings et al. 2008). Values range from 0 to 1 with 0 corresponding to a lack of genealogical divergence from other groups and 1 corresponding to monophyly for the predetermined clade (or species). Each isolate was assigned to a predetermined species based on Genealogical Concordance Phylogenetic Species Recognition (GCPSR) and the gsi was calculated for the best tree selected in parsimony analysis and for all trees using 10 000 permutations (Cummings et al. 2008). The assignment of each tip to groups representing the recognised species was identical for the EF1-α and combined phylogenetic trees (Fig. 2, ​,3).3). Taxa in the ITS tree that were not present in EF1-α and combined trees were not included in the calculation of gsi. The gsi and each of the probability values (P) corresponding to the species represented by more than one isolate were tabulated (Table 3). Species with one representative isolate including the outgroup were not subjected to gsi analysis. The ensemble genealogical sorting index (gsi_T) is the sum of the gsi values calculated for all individual gene trees (Table 3).

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Fig. 2

The single most parsimonious tree generated from the analysis of the EF1-α sequence alignment. MP/RAxML bootstrap values/Bayesian posterior probabilities ≥ 70 % are displayed above or below each branch. Ex-type and ex-epitype culture numbers are in bold. GenBank accessions are given for downloaded sequences and isolate codes for the newly generated sequences annotated with host and location. Isolates from Citrus are indicated in green. The tree is rooted with D. helianthi (CBS 592.81)

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Fig. 3

The single most parsimonious tree generated from the analysis of the combined ACT, CAL, EF1-α, ITS and TUB sequence alignment. MP/RAxML bootstrap values/Bayesian posterior probabilities ≥ 70 % are displayed above or below each branch. Ex-type and ex-epitype culture numbers are in bold. GenBank accessions are given for the downloaded sequences and isolate codes for the newly generated sequences annotated with host and location. Isolates from Citrus are indicated in green. Red squares indicate the epitypes designated in this study including the conserved ex-type of Diaporthe citri in Rossman et al. (2013). The tree is rooted with D. helianthi (CBS 592.81)

All the novel sequences were deposited in GenBank and the sequence alignments were submitted to TreeBASE (www.treebase.org) as S14141 (ITS), S14146 (EF1) and S14147 (combined alignment). Taxonomic novelties (MB) and typifications (MBT175959–MBT175968) were registered in MycoBank (www.mycobank.org) (Crous et al. 2004).

TAXONOMY

In this section we provide modern descriptions and illustrations of the species resolved here based on multi-gene phylogenetic analyses and morphological characters. Diaporthe citri occurs only on Citrus while D. cytosporella and D. foeniculina occur on Citrus and other woody and herbaceous hosts including high value crops. Diaporthe rudis is not known from Citrus but was previously confused with those species especially as D. medusaea and has a broad host range. Each species is described based on type and other specimens as well as ex-epitype cultures. Synonymous names of Diaporthe or Phomopsis are reviewed based on protologues, type and other specimens and cultures. When specimens with cultures from similar substrates and localities are available, epitype specimens with ex-epitype cultures are designated for both accepted and synonymous names.

Diaporthe citri (H.S. Fawc.) F.A. Wolf, J. Agric. Res. 33: 625. 1926. — Fig. 4

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Fig. 4

Diaporthe citri (ex-epitype culture AR3405 = CBS 135422). a. Sporulation on alfalfa stem in WA; b. culture on PDA (25 °C, dark, 7 d); c. pycnidial walls lined with paraphyses and conidiophores; d. section through conidiomata; e. conidiophores; f. alpha conidia; g. germinating conidia on a slide. — Scale bars: a = 500 μm; c = 20 μm; d–g = 10 μm.

Basionym. Phomopsis citri H.S. Fawc., Phytopathology 2: 109. 1912 nom. conserv. prop. non Phomopsis citri (Sacc.) Traverso & Spessa 1910.

= Diaporthe citrincola Rehm, Leafl. Philipp. Bot. 6: 2269. 1914.

= Phomopsis caribaea W.T. Horne, Phytopathology 12: 417. 1922.

Perithecia on decaying twigs, black, globose to conical, 130–200 μm diam, scattered, solitary or in groups, immersed deep in bark with tapering perithecial necks 190–700 μm long. Asci unitunicate, 8-spored, sessile, elongate to clavate, (37.3–)40.5–50.5(–55) × (9–)10.5–12(–12.2) μm. Ascospores hyaline, 2-celled, often 4-guttulate, with larger guttules at centre and smaller one at ends, elongated to elliptical, (12–)12.4–14(–14.2) × 3.2–3.6(–3.8) μm (av. ± SD = 13.2 ± 0.8 × 3.3 ± 0.2, n = 30). Pycnidia on alfalfa twigs on WA: globose, 200–250 μm diam, later conical, embedded in tissue, erumpent at maturity, up to 450 μm diam, 65–100 μm high, with an elongated black neck, often with a yellowish, spiral conidial cirrus extruding from ostiole; walls parenchymatous, consisting of 3–4 layers of medium brown textura angularis. Conidiophores hyaline, smooth, unbranched, ampulliform, straight to sinuous, 10–15 × 1–2 μm. Conidiogenous cells phialidic, cylindrical, terminal, slight tapering towards apex, 0.5–1 μm diam. Paraphyses abundant among conidiophores, 20–40 × 1–2 μm. Alpha conidia aseptate, hyaline, smooth, ovate to ellipsoidal, mono- to biguttulate, rarely 3-guttulate, base subtruncate, (7.6–)8–9(–10.2) × 3–4.2 μm (av. ± SD = 8.5 ± 0.8 × 3.7 ± 0.2 , n = 30). Beta and gamma conidia not observed on alfalfa twigs or in culture.

Culture characteristics — In dark at 25 °C for 1 wk, colonies on PDA slow growing, 4.2 ± 0.2 mm/day (n = 8), white, fluffy aerial mycelium, reverse centre greenish yellow pigmentation developing in centre.

Host range — Causing melanose and stem end rot disease, associated with dying or dead twigs of Citrus spp. and closely related hosts including C. aurantiifolia, C. aurantium, C. maxima (= C. grandis), C. nobilis, C. paradisi, C. reticulata, C. sinensis, C. sudachi, C. unshiu, × Citrofortunella microcarpa (= × C. mitis), Fortunella japonica (Kobayashi 2007), F. margarita, Poncirus trifoliata.

Distribution — Probably throughout Citrus-growing regions of the world. Reported from Brazil, China, Cuba, Japan, Korea, New Zealand, The Philippines, Puerto Rico and the United States (Florida, Texas).

Type specimens examined. USA, Florida, Lake Alfred, Ana, on twigs of Citrus sp, 26 Apr. 2000, L.W. Timmer, dried specimen from culture sporulating on alfalfa stem (type of Phomopsis citri proposed for conservation in Rossman et al. (2013) (BPI 892456, ex-type culture AR 3405 = CBS 135422). – PHILIPPINES, Los Baños, on dead twigs of Citrus nobilis, Oct. 1913, coll. M.B. Raimundo, comm. C.F. Baker, no. 1875 (holotype of Diaporthe citrincola S-F52860). – CUBA, Isle of Pines, on Citrus paradisi, 30 Oct. 1917, Fredrick Maskew, intercepted in San Francisco, derived culture sporulating on Citrus twig (lectotype of Phomopsis caribaea designated here BPI 358328, isolectotype NY01097305; MBT175959).

Additional materials examined. BRAZIL, Escola Agr., Vicosa, Minas Gerais, on peel of Citrus sp., 17 May 1932, P.H. Rolfs (BPI 615855); intercepted New York #pi 7163, on fruit of Citrus sinensis, 22 June 1924, A.C. Hill, det. A.J. Bruman (BPI 358408). – JAPAN, Yokohama, intercepted Seattle Washington #pi 4780, on fruit of Citrus sinensis, 14 Jan. 1940, A.G. Webb, det. J.A. Stevenson (BPI 358405). – MEXICO, intercepted Brownsville Texas #692229, on leaves of Citrus sp., 30 Jan. 1930, Mueller, det. D.J. Smith, A.E. Jenkins, J.A. Watson (BPI 615856); intercepted Laredo Texas #50818, on leaves of Citrus sp., 23 Jan. 1951, Trotter, det. A.H. Lewis, J.A. Watson (BPI 615857). – PUERTO RICO, Bayamon, on leaves of Citrus grandis, 22 Aug. 1933, C.G. Anderson (BPI 358392). – USA, Florida, Orlando, on dead stems of Citrus aurantifolia, July 1925, F.A. Wolf (BPI 615860); Florida, Orlando, on dead stems of Citrus sp., Jan. 1926, F.A. Wolf (BPI 615959); Florida, Orlando, on leaves of Citrus sinensis, Mar. 1922, J.R. Winston (BPI358409); Florida, Eustis, on leaves of Citrus grandis, 8 Jan. 1932, H.S. Fawcett (BPI 358391); Florida, St. Nicholas, on stem of Citrus sinensis, 28 Nov. 1895, det. F. Albert, W.W. Diehl (BPI 358404); Florida, Fort Myers, on stems of Citrus sinensis, 16 Feb. 1924, J.A. Stevenson (BPI 358407); Florida, Gainesville, Florida Agricultural Experiment Station, on stems of Citrus sinensis, 16 Mar. 1910, H.S. Fawcett (BPI 358406); Florida, Winter Park, on stems of Citrus grandis, 21 Feb. 1923, C.L. Shear (BPI 615868); Florida, Winter Park, on stems of Citrus grandis, 20 Jan. 1925, H.E. Stevens, det. C.L. Shear (BPI 615869); Florida, on dead stem of Citrus sp., 1913, J.G. Grossenbacker, det. C.L. Shear (BPI 615858); Florida, on dead stem of Citrus sp., 8 July 1929, F.A. Wolf, det. C.L. Shear (BPI 615854); Florida, on leaves of Citrus grandis, 6 Jan. 1932, H.S. Fawcett (BPI 358393).

Notes — The name D. citri is based on Phomopsis citri H.S. Fawc. 1912, a later homonym of Phomopsis citri (Sacc.) Traverso & Spessa 1910. A conservation proposal has been published to continue the use of the widely used name for the species associated with melanose or stem end rot of Citrus as D. citri (Rossman et al. 2013). Diaporthe citri based on the basionym Phomopsis citri H.S. Fawc. has priority over the later synonyms D. citrincola and P. caribaea. This is also in accordance with the change to unit nomenclature with the older genus Diaporthe serving as the correct name for all species in Diaporthe-Phomopsis (McNeill et al. 2012). No type specimen for P. citri could be located at BPI or FLAS, leaving only an illustration (Fawcett 1912) as a potential, but unsatisfactory, iconotype, thus P. citri is proposed for conservation with a new type specimen (Rossman et al. 2013). The type specimens of D. citrincola and P. caribaea were examined and contributed to the conclusions that these names are synonyms of D. citri. A lectotype of P. caribaea is designated.

The fruiting structures of D. citri are found on dead twigs, stems and fruits of Citrus affected by melanose and stem end rot (Wolf 1926, Fawcett 1932, Whiteside & Timmer 2000b). The fungus generally propagates itself on dead twigs of Citrus. A few days after infecting leaf tissue or fruit, the melanose symptoms appear as small, brown, discrete or confluent, sunken spots. A few epidermal cell layers on infected tissue are killed and become impregnated with reddish brown gum that later become raised black pustules (Timmer 2000). Although pustules on leaves are initially surrounded by yellow halos, they recover and become green again and corky pustules are often the only symptoms (Bach & Wolf 1928, Nelson 2008). Fungal structures such as pycnidia or perithecia are never visible in these melanose lesions, therefore, the fungus cannot be observed in the infected leaves or fruit. When the fruiting structures are present on dead twigs or bark of the stems, the pycnidia or ascomata are abundant deep in the tissue.

Diaporthe cytosporella (Penz. & Sacc.) Udayanga & Castl., comb. nov. — MycoBank MB803986; Fig. 5, ​,66

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Fig. 5

Diaporthe cytosporella (AR5149). a. Sporulating pycnidia on alfalfa stem; b. culture on PDA (25 °C, dark, 7 d); c. concentric pycnidial in rings on culture; d. pycnidia on culture; e. conidiophores; f. alpha conidia. — Scale bars: a, c = 2 000 μm; d = 3 000 μm; e, f = 20 μm.

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Fig. 6

Holotype specimen of Diaporthe cytosporella (BPI 798526). a. Pycnidia-bearing bark of Citrus sp.; b. branched stroma and sporulating pycnidia; c. section through pycnidia with pycnidial wall and conidiophores; d. alpha conidia. — Scale bars: a = 1 000 μm; b = 50 μm; c, d = 15 μm.

Basionym. Phoma cytosporella Penz. & Sacc., Fung. Agron.: 361. 1887.

≡ Phomopsis cytosporella (Penz. & Sacc.) H.S. Fawc. & H.A. Lee, Citrus diseases and their control, Ed. 1: 407. 1926.

Perithecia unknown. Pycnidia on alfalfa twigs on WA: globose, 150–200 μm diam, mostly embedded in tissue and erumpent at maturity, up to 450 μm diam, 65–100 μm high, with an elongated black neck, often with a yellowish conidial cirrus extruding from ostiole; walls parenchymatous consisting of 3–4 layers of medium brown textura angularis. Paraphyses lacking. Conidiophores 7–18 × 1–2 μm, hyaline, smooth, branched or unbranched, ampulliform, cylindrical, wider at base, occurring in dense clusters. Conidiogenous cells phialidic, cylindrical, terminal, with slight tapering towards apex, 0.5–1 μm diam. Alpha conidia (6.9–)8–9(–12.6) × (2.3–)2.6–3.2 μm (av. ± SD = 8.8 ± 0.9 × 3.0 ± 0.3, n = 30), aseptate, hyaline, smooth, ovate to ellipsoidal, biguttulate or multi-guttulate, base subtruncate, occasionally larger alpha conidia present in culture and on alfalfa stems. Beta and gamma conidia not observed on alfalfa twigs or in culture.

Culture characteristics — In dark at 25 °C for 1 wk, colonies on PDA relatively slow growing, 4.0 ± 0.2 mm/day (n = 8). On PDA white, fluffy aerial mycelium, reverse with ash colour pigmentation developing in centre. In a 2-wk-old culture, clusters of black, branched stromata occurring in concentric rings with sporulating pycnidia.

Host range — Citrus limon, C. sinensis and Vitis vinifera.

Distribution — Europe (Spain, Italy), United States (California).

Type specimens examined. ITALY, Rome, Modena, on Citrus limonia, Jan. 1886 (holotype of Phoma cytosporella BPI 798526). – SPAIN, on Citrus limon, M.E. Palm, dried culture (epitype of Phoma cytosporella designated here: BPI 892459, living culture FAU461 = CBS 137020; MBT 175960).

Additional material examined. USA, California, on twigs of Citrus sinensis, 4 Oct. 2011, A. Eskalen UCR1751, dried culture with pycnidia sporulating on alfalfa stems (BPI 892457, living culture AR5149); ibid., UCR1750, dried culture with pycnidia sporulating on alfalfa stems (BPI 892458, living culture AR5148).

Notes — Diaporthe cytosporella is phylogenetically closely related to D. foeniculina but clearly distinguished based on ITS and EF1-α sequences (Fig. 1, ​,2).2). The species was first described from Italy and later synonymised under D. citri. Although in this study this species is primarily recognized using isolates from Citrus limon in Europe (Spain) and the United States (California), two ITS sequences (FJ94470, AY745085) from GenBank are 100 % identical suggesting that this species may also occur on Vitis and other host species in California (retrieved on 1 Feb. 2013). At maturity cultures of D. cytosporella (AR5148 and AR5149) on PDA produce distinctive black, branched stromata. Perithecia were not observed in culture. Morphological characters were highly similar among the type specimen (Fig. 6) and the isolates used in epitype and genetically similar additional materials examined.

Diaporthe foeniculina (Sacc.) Udayanga & Castl., comb. nov. — MycoBank MB803929; Fig. 7

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Fig. 7

Diaporthe foeniculina (ex-epitype culture DP0391 = CBS 111553). a. Sporulation on alfalfa stem in WA; b. culture on PDA (25 °C, dark, 7 d); c. conidiophores; d. alpha conidia; e. beta conidia. — Scale bars: a = 2 000 μm; all others = 10 μm.

Basionym. Phoma foeniculina Sacc., Michelia 2: 95. 1880.

≡ Phomopsis foeniculina (Sacc.) Sousa da Câmara, Agron. Lusit. 9: 104. 1947.

= Diaporthe theicola Curzi, Atti Ist. Bot. Lab. Crittog. Univ. Pavia 3 Sér. 3: 60. 1927.

= Phomopsis theicola Curzi, Atti Ist. Bot. Lab. Crittog. Univ. Pavia 3 Sér. 3: 64. 1927.

= Phomopsis californica H.S. Fawc., Phytopathology 12: 419. 1922.

= Diaporthe neotheicola A.J.L. Phillips & J.M. Santos, Fung. Diversity 34: 120. 2009.

= Diaporthe rhusicola Crous, Persoonia 26: 135. 2011.

Perithecia on decaying twigs black, globose to subglobose (200–)360 × 200 μm, scattered, solitary or in groups, with tapering perithecial necks barely protruding through epidermis. Asci unitunicate, 8-spored, sessile, cylindrical to clavate, (40–)50.5–60.5(–65) × 8–10(–12.2) μm. Ascospores hyaline, 2-celled, often with four guttules, larger guttules near centre and smaller ones at ends, elongated to elliptical, (9.0–)12.4–14(–15.2) × (3.2–)3.4–3.6(–5.2) μm (av. ± SD = 13.2 ± 0.8 × 3.5 ± 0.1, n = 30). Pycnidia on alfalfa twigs on WA: globose to subglobose 400–700 μm diam, erumpent at maturity, (300–)500–800(–930) μm high, with an elongated, black neck, mostly embedded in tissue, often with a yellowish, drop-like conidial cirrus extruding from ostiole; walls parenchymatous, consisting of 2–3 layers of medium brown textura angularis. Paraphyses lacking. Conidiophores hyaline, smooth, unbranched, cylindrical, straight to sinuous, 9–15(–18) × 1–2 μm. Conidiogenous cells phialidic, cylindrical, terminal, with slight taper towards apex, 0.5–1 μm diam. Alpha conidia aseptate, hyaline, smooth, ellipsoidal or fusiform, with none, two or many guttules, rarely with subtruncate base, (7.5–)8.5–9(–9.2) × (2–)2.3–2.5(–2.7) μm (av. ± SD = 8.8 ± 0.3 × 2.4 ± 0.1 μm, n = 30). Beta conidia hyaline, aseptate, eguttulate, hamate or slightly curved, abundant, base subtruncate, acute apex, (20–)22–28(–29) × (1.1–)1.4–1.6(–2) μm (av. ± SD = 25.1 ± 3.3 × 1.5 ± 0.1 μm, n = 30).

Culture characteristics — In dark at 25 °C for 1 wk, colonies on PDA slow growing, 5.2 ± 0.2 mm/day (n = 8), white, sparse aerial mycelium, greenish yellow pigmentation developing in reverse centre.

Host range — Acacia, Acer, Actinidia deliciosa, Aspalathus linearis, Bougainvillea spectabilis, Camellia sinensis, Castanea, Citrus limon, C. limonia, Crataegus, Diospyros, Foeniculum vulgare, Fuchsia, Hydrangea, Juglans, Malus, Olea, Prunus, Pyrus, Quercus, Rhus, Ribes, Vitis vinifera and Wisteria sinensis. In addition to the hosts on the specimens listed below, these hosts are represented in Fig. 1 based on the ITS phylogeny and Gomes et al. (2013) as D. foeniculacea.

Distribution — Argentina, Australia, Europe (Greece, Portugal, Spain, Italy), New Zealand, South Africa and USA (California).

Type specimens examined. FRANCE, on stems of Foeniculum ‘arvensis’, Brunaud, cited in Phillips (2003) with illustration (holotype of Phoma foeniculina PAD 281 – unavailable, not examined). – PORTUGAL, Madeira, Serra da Agua, at base of 2-yr-old stem of Foeniculum vulgare, Aug. 2001, A.J.L. Phillips (epitype of Phoma foeniculina designated here LISE 94791, ex-epitype culture from single ascospores CBS 111553 = DP0391; MBT175961). – USA, California, Santa Barbara County, on dead outer bark and decaying fruit of Citrus limonia, 3 Mar. 1922, H.S. Fawcett (holotype of Phomopsis californica BPI0358313); California, San Diego, on branch of Citrus limonia, 16 Nov. 2012, Akif Eskalen (epitype of Phomopsis californica designated here BPI 892460, ex-epitype culture AR5142 = CBS135430; MBT 175962). – ITALY, on Camellia sinensis, Curzi, dried culture specimen (epitype of Diaporthe theicola designated here BPI 892462, ex-epitype culture CBS 187.27, same as ex-isotype culture of Phomopsis theicola; MBT175963); Illustration in Atti dell’Istituto Botanica della Universita e Laboratoria Crittogamico di Pavia 3 Sér. 3: 60 (1926) [1927] (lectotype of Phomopsis theicola designated here; MBT175964). – PORTUGAL, Évora, on Foeniculum vulgare, Nov. 2007, A.J.L. Phillips (holotype of Diaporthe neotheicola CBS-H 20131, ex-holotype culture (Di-C004/5 = CBS 123208).

Additional specimens examined. PORTUGAL, Madeira, Serra da Agua, at base of 2-yr-old stem of Foeniculum vulgarae, Aug. 2001, A.J.L. Phillips (LISE 94792 as Diaporthe foeniculacea, culture from single ascospores DP0392 = CBS 111554. – SPAIN, on fruit of Citrus limon, intercepted at Elizabeth, New Jersey, 20 Mar. 1987, C. Markham 001514, M.E. Palm (BPI 1107900, living culture MEP1289); ibid. (BPI 747926); ibid. (BPI 747927); on peel of Citrus limonia, intercepted New York #87452, 13 Nov. 1940, Hodson, E.A. Jenkins (BPI 615878); ibid., on fruit of Citrus limon, M.E. Palm (BPI 892461, culture FAU460 = CBS).

Notes — Diaporthe foeniculina is known to occur on Citrus and many other woody plants hosts in temperate and tropical regions. This species causes a stem end rot of lemandarin (Citrus limonia) in Europe and the United States (California) and was observed as a saprobe on branches of this host. As D. neotheicola, this species has been reported to cause diseases of temperate and tropical fruits from Australia, Europe and South Africa. Our results indicate that isolates from Citrus in Spain are conspecific with the type isolate of the recently described D. neotheicola from Foeniculum as well as isolates from other hosts now considered to be D. foeniculina. We reviewed the possible synonyms of this species based on available molecular data, living cultures and type specimens. The specimen deposited in LISE 94792 corresponding to the living culture CBS 111554 was selected as the epitype specimen for Phoma foeniculina, now recognised as D. foeniculina. Molecular data derived from the epitype of Phoma foeniculina, now D. foeniculina, and additional isolates show that this taxon is conspecific with the ex-type isolates of D. neotheicola and P. theicola (Phillips 2003, Santos & Phillips 2009, Gomes et al. 2013) as well as isolates from Citrus in Spain. The name D. neotheicola has been widely used for this taxon (Santos & Phillips 2009, Udayanga et al. 2012a, Thomidis et al. 2013).

Phomopsis foeniculina (syn. Phoma foeniculina) was considered a synonym of D. foeniculacea (syn. Sphaeria foeniculacea) by Phillips (2003). We examined the type specimen of Sphaeria foeniculacea and agree with von Arx & Müller (1954) who recognised this species as Guignardia foeniculacea (Mont.) Arx & E. Müll. (as G. foeniculata). Gomes et al. (2013) used the name D. foeniculacea to refer to this species based on Phillips (2003). However, observation of type specimens of Sphaeria foeniculacea confirmed that this name cannot be applied to a species of Diaporthe. This is further explained under the excluded species.

Although type specimens of Phomopsis theicola and D. theicola could not be located, an ex-type culture of P. theicola exists as mentioned by Santos & Phillips (2009). They stated that P. theicola was not the same as D. theicola based on the illustrations in the protologues of these taxa and described the name D. neotheicola for the sexual state of P. theicola, with an ex-holotype culture from Foeniculum. However, measurements of asci and ascospores in Curzi’s (1927) original description of D. theicola are consistent with those of D. foeniculina specimens examined in this study. We agree with the opinion of Curzi (1927) that D. theicola and P. theicola are known sexual and asexual states of the same fungus, simultaneously described from the same specimen and therefore here we epitypify the name D. theicola with Curzi’s ex-type culture of P. theicola.

Diaporthe rudis (Fr.) Nitschke, Pyrenomycetes Germanici 2: 282. 1870. — Fig. 8

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Fig. 8

Diaporthe rudis. a, b. Ectostroma and perithecia on Laburnum anagyroides; c. perithecia in transverse section; d. perithecial wall in longitudinal section; e, f. perithecia in longitudinal section; g–j. asci; k. ascospores; l. conidiophores developing on alfalfa stem in culture; m. alpha and beta conidia developing on alfalfa stem in culture (a–k. Epitype specimen BPI 748231; l–n. ex-epitype culture AR3422 = CBS 109292). — Scale bars: a = 2 000 μm; b, c = 1 000 μm; d = 50 μm; e, f = 100 μm; g–k = 25 μm; l–n = 15 μm.

Basionym. Sphaeria rudis Fr., Elench. Fung. (Griefswald) 2: 98. 1828.

≡ Rabenhorstia rudis (Fr.) Fr., Summa Veg. Scand., Section Post. (Stockholm): 410. 1849.

≡ Aglaospora rudis (Fr.) Tul. & C. Tul., Select. Fung. Carpol. (Paris) 2: 165. 1863.

= Phoma rudis Sacc., Michelia 1: 257. 1878.

≡Phomopsis rudis (Sacc.) Höhn., Sitzungsber. Kaiserl. Akad. Wiss., Math.-Naturwiss. Cl., Abt. 1, 115: 680. 1906.

= Diaporthe faginea Sacc., Syll. Fung. 1: 619. 1882.

= Diaporthe macrostoma Nitschke, Pyrenomycetes Germanici 2: 284. 1870.

= Diaporthe medusaea Nitschke, Pyrenomycetes Germanici 2: 251. 1870.

= Diaporthe viticola Nitschke, Pyrenomycetes Germanici 2: 264. 1870.

=Diaporthe silvestris Sacc. & Berl., Atti Rev. Instit. Venet. Ser. II, 6: 737. 1885.

Perithecia black, clustered, globose, 300–350 μm, with tapering perithecial necks, 200–700 μm long. Asci unitunicate, sessile, elongate to clavate, (50.3–)53.5–58.5(–59.6) × (8.9–)10.6–12(–12.3) μm. Ascospores hyaline, 2-celled, often tetra-guttulate, with larger guttules at centre and smaller at ends, elongated to clavate, (11.6–)12–14.2(–15) × (2.8–)3.5–3.7(–3.8) μm (av. ± SD = 13.2 ± 1.1 × 3.6 ± 0.1, n = 30). Pycnidia on alfalfa twigs on WA: globose 200–250 μm diam, erumpent at maturity, up to 400–500 μm diam; walls 60–150 μm diam, parenchymatous, consisting of 3–4 layers of medium brown textura angularis. Conidiophores cylindrical, hyaline, smooth, branched, ampulliform, straight to sinuous, 20–45 × 2–2.4 μm. Conidiogenous cells phialidic, cylindrical, terminal, with slight tapering towards apex, 0.5–1 μm diam. Paraphyses abundant among conidiophores 20–40 × 1–2 μm. Alpha conidia aseptate, hyaline, smooth, ovate to ellipsoidal, biguttulate, base subtruncate (6.3–)7–8(8.7) × 2–2.5 μm (av. ± SD = 7.5 ± 0.4 × 2.2 ± 0.2, n = 30). Beta conidia aseptate, hyaline, smooth, fusiform or hooked, base subtruncate, 27–31(–35.2) × (3–)3.4–3.8(–4.2) μm (av. ± SD = 29.5 ± 2 ×3.6 ± 2, n = 30). Gamma conidia aseptate, hyaline, smooth, fusiform, mostly biguttulate, base subtruncate (10–)14–15 × 1–2 μm (av. ± SD = 14.4 ± 0.2 × 1.7 ± 0.24, n = 30).

Culture characteristics — In dark at 25 °C for 1 wk, colonies on PDA relatively slow growing, 4.2 mm/day, white, fluffy aerial mycelium, reverse with yellow pigmentation developing in centre.

Host range — Acer, Asphodelus albus, Aucuba japonica, Brugmansia, Castanea, Corylus, Dipsacus fullonum, Epilobium, Eucalyptus, Fagus, Fraxinus, Holcus, Hydrangea, Ileostylis, Laburnum, Lupinus, Malus, Protea, Pyrus, Rosa, Sambucus, Salix, Vaccinium and Vitis vinifera. In addition to the hosts on the specimens listed below, these hosts are represented in Fig. 1 based on ITS phylogeny and Gomes et al. (2013) as D. viticola.

Distribution — Australia, Canada, Chile, Europe (Austria, Germany, Italy, Latvia, Portugal, Spain, Sweden, Switzerland), New Zealand and South Africa.

Type specimens examined. FRANCE, on a dead branch of Laburnum anagyroides (as Cytisus laburnum), ex herb. Guépin no. 163 (holotype of Sphaeria rudis UPS F-004948). – AUSTRIA, Vienna, 19. 7763/2, Reisenbergbach-Weg, on stem of Laburnum anagyroides, 8 Apr. 2000, W. Jaklitsch (epitype of Sphaeria rudis designated here BPI 748231, ex-epitype culture AR 3422 = CBS 109292; MBT175965). – GERMANY, Nordrhein-Westfalen, Landkreis Unna, Cappenberg, Schloßgarten zu Cappenberg, on twigs of Laburnum anagyroides (syn. Cytisus laburnum), 18 Aug. 1866, T. Nitschke (holotype of Diaporthe medusaea B 70 0009168). – AUSTRIA, Vienna, 19. 7763/2, Reisenbergbach-Weg, on stem of Laburnum anagyroides, 8 Apr. 2000, W. Jaklitsch (epitype of Diaporthe medusaea designated here BPI 748231, ex epitype culture AR3422 = CBS 109292; MBT175966). – GERMANY, Nordrhein-Westfalen, Munsterland, Munster Botanischer Garten, on thin branch of Fagus sylvatica, 18 May 1866, T. Nitschke (holotype of Diaporthe macrostoma B 70 0009167). – GERMANY, Westfalen, Munster, bei der Wienburg, on Vitis vinifera, Feb. 1866, Nitschke (holotype of Diaporthe viticola B: not seen), ibid. (isotype BPI 797316). – PORTUGAL, Santo Tirso, Burgaes, on Vitis vinifera,16 Feb. 1998, A.J.L. Phillips (epitype of D. viticola designated in van Niekerk et al. (2005) CBS-H 7950 not seen, ex-epitype culture STE-U 5683 = CBS 113201). – ITALY, “In sarmentis Vitis viniferae silvestris, Cervarese” (holotype of Diaporthe silvestris: PAD 228 not seen). The synonymy of this name is based on van Niekerk et al. (2005) in which the holotype specimen was observed and considered to be D. viticola.

Additional specimens examined. AUSTRIA, Vienna, stem of Rosa canina, 13 May 2001, W. Jaklitsch (BPI 840948, living culture AR3654); Vienna, stem of Acer pseudoplatanus, 31 Mar. 2001, W. Jaklitsch (BPI840940, living culture AR3634). – GERMANY, urban residential area, container plant, dead stem of Brugmansia sp., 31 Oct. 2012, R. Schumacher (BPI 892463, living culture DA243 = CBS135435). – ITALY, on dead stem of Acer opalus, 2 May 2012, E. Camporesi ER285 (BPI 892464, living culture ER285A = CBS 135437); ibid., (ER 286, BPI 892465, living culture ER286D).

Notes — The name D. rudis is based on the oldest epithet of the many synonyms for this species including D. medusaea. Diaporthe medusaea, originally described from Laburnum anagyroides in Germany, has been used as the name for the fungus causing melanose and stem end rot of Citrus in North America. We observed holotype material as well as isolates on the same host from Austria in order to recognise the similarities or differences as discussed herein. Wehmeyer (1933) listed a number of synonyms for D. medusaea including D. citri, D. citrincola, D. faginea, D. rudis and D. viticola. Diaporthe citrincola is here recognised as a synonym of D. citri. Diaporthe faginea was established as a legitimate name for Sphaeria faginea Curr., Trans. Linn. Soc. London 22: 281. 1859 nom. illeg. non S. faginea Pers. 1794. No specimen as D. faginea exists in PAD. Based on an ITS sequence (EF155490) of the isolate from Fagus in Germany and a morphological comparison of the protologue, this name is accepted as a synonym of D. rudis. Although D. viticola was recognised as a distinct taxon and characterised and epitypified using a specimen on Vitis by van Niekerk et al. (2005), it is here determined to be a synonym of D. rudis.