Introduction

Seiridium was introduced by Nees (1816) with the type species S. marginatum Nees occurring on rose stems in Germany. Sequence data generated to date reveal Seiridium to represent a distinct asexual coelomycetous genus in the family Amphisphaeriaceae, characterised by six-celled conidia (Nag Raj 1993; Maharachchikumbura et al. 2015). Blogiascospora and Lepteutypa have been identified as the sexual morphs of Seiridium (Shoemaker and Müller 1965). Maharachchikumbura et al. (2014) suggested that the monotypic genus Pestalotia (1839) might be a synonym of Seiridium (1816), since both genera have similar morphologies. Presently there are 39 species epithets for Seiridium in Index Fungorum (2015). Species introductions were primarily based on cultural and phenotypic characteristics, including size and morphology of conidia and presence or absence of conidial appendages (Moricca et al. 2000). However, several studies have shown that these characters are variable and inconsistent (Sutton 1980; Boesewinkel 1983; Graniti 1986; Chou 1989).

Many species of Seiridium are pathogens of various plants, and the genus is widely distributed throughout the Northern and Southern hemispheres (Tsopelas et al. 2007; Smith 2010). Seiridium canker was responsible for a world pandemic known as cypress canker affecting cypress trees and other members of the plant family Cupressaceae (Tsopelas et al. 2007). Three species of Seiridium are responsible for cypress canker (Barnes et al. 2001). Seiridium cardinale (W.W. Wagener) B. Sutton & I.A.S. Gibson is the most damaging of the three species, and was first identified on Monterey Cypress in California in 1928 (Tsopelas et al. 2007). The disease has since spread from North America to Africa, Asia, Australia, Europe, New Zealand and South America (Xenopoulos 1987; Tsopelas et al. 2007). Seiridium unicorne (Cooke & Ellis) B. Sutton and S. cupressi (Guba) Boesew. are also responsible for Seiridium canker (Graniti 1998).

In the present study, we collected a new species of Seiridium from blighted leaves of Camellia reticulata Lindl. in Yunnan Province, China, and it is introduced as Seiridium camelliae sp. nov. We also redescribe Seiridium venetum from a fresh collection of Cornus mas L. in Italy and designate it as a reference specimen.

Material and methods

Isolation and identification of fungi

Fresh specimens of Seiridium were obtained from blighted leaves of Camellia reticulata from Zixishan, Chuxiong, Yunnan Province, China, and dead twigs of Cornus mas from Italy. The samples were placed in separate plastic bags lined with tissue paper, sprayed with sterile water to create humid conditions and incubated at room temperature. The Seiridium species present on the samples were isolated using a single-spore culture technique as described by Maharachchikumbura et al. (2012). A pycnidium was cut using a razor blade, and the inner contents released in a drop of water. The water was spread on potato dextrose agar (PDA), and the plates were incubated overnight. Germinating conidia were located under a stereomicroscope and aseptically transferred to fresh PDA plates. The pure cultures were incubated at room temperature for 2–5 days and sub-cultured onto fresh PDA. Colony colour on PDA was determined with the colour charts of Rayner (1970). Microscopic preparations were made in distilled water, with 30 measurements per structure as observed under the light microscope, and photographed. All microscopic measurements were performed using the Tarosoft image framework (v. 0.9.0.7), with 30 conidial and 15 conidioma and conidiogenous cell measurements. Facesoffungi numbers are provided, as detailed in Jayasiri et al. (2015). The herbarium materials are deposited in the Mae Fah Luang University Herbarium, Chiang Rai, Thailand (MFLU) and Culture Collection, International Fungal Research & Development Centre, Chinese Academy of Forestry, Kunming, China (IFRD), and living cultures are deposited at Mae Fah Luang University Culture Collection (MFLUCC) and International Collection of Microorganisms from Plants (ICMP).

PCR and sequencing

Total genomic DNA was extracted from fresh fungal mycelia (500 mg) scraped from the margin of a colony on a PDA plate incubated at 25 °C for 7–10 days (Guo et al. 2000). The ITS and 5.8S region of the rDNA molecule was amplified using primer pairs ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) (White et al. 1990). Polymerase chain reaction (PCR) was performed with a 25-μl reaction system consisting of 19.5 μl of double-distilled water, 2.5 μl of 10× Taq buffer with MgCl2, 0.5 μl of dNTP (10 mM each), 0.5 μl of each primer (10 μM), 0.25 μl of Taq DNA polymerase (5 U/μl), and 1.0 μl of DNA template. The thermal cycling program followed Maharachchikumbura et al. (2013a). Sequencing of the PCR amplicons was conducted using the same primers as those used for the amplification reactions. The partial beta-tubulin gene (TUB) was amplified using primer pairs BT2A (5′-GGTAACCAAATCGGTGCTGCTTTC-3 ′) and BT2B (5′ ACCCTCAGTGTAGTGACCCTTGGC-3′) (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997) using the thermal cycling program followed by Maharachchikumbura et al. (2013a) (Seiridium camelliae; GenBank JQ683709, Seiridium venetum; GenBank KT438837).

Phylogenetic analysis

Sequences were optimized manually to allow maximum alignment and maximum sequence similarity, as detailed in Maharachchikumbura et al. (2012) (Table 1). Phylogenetic analyses of the sequence data consisted of Bayesian inference (BI) and maximum likelihood (ML) using MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) and raxmlGUI v. 1.3 (Silvestro and Michalak 2011). For Bayesian inference, a suitable model was selected using models of nucleotide substitution for gene, as determined using MrModeltest (Nylander 2004). The gamma model was selected for ITS and was incorporated into the analysis. The analyses of four Markov chain Monte Carlo (MCMC) chains were run from random trees for 100,000,000 generations and sampled every 1,000th generation. The temperature value was lowered to 0.15, burn-in was set to 0.25, and the run was automatically stopped as soon as the average standard deviation of split frequencies reached a value below 0.01. For maximum likelihood analyses, the tree search was conducted with 1000 separate runs, using the default algorithm of the program from a random starting tree for each run. The final tree was selected among suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA substitution model. The resulting trees were printed with FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/), and the layout was created in Adobe Illustrator CS v. 6. Sequences generated in this study were deposited at GenBank.

Table 1 Isolates used in this study and their ITS GenBank accession numbers
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Results

Phylogenetic analysis

The alignment comprised 22 strains of Seiridium, 16 isolates of other genera belong to the Amphisphaeriaceae and Hypoxylon fragiforme, and Melogramma campylosporum as the outgroup taxa. The manually adjusted dataset comprised 598 characters including gaps. Dirichlet base frequencies and the HKY+G model with the gamma-distributed rate model were recommended by MrModeltest analysis and used in the Bayesian analysis. The Bayesian analysis resulted in a tree with the same topology and clades as the ML trees. The novel taxon Seiridium camelliae (MFLUCC 12–0647) clustered in Seiridium, but separated from other species in the genus. The reference specimen of S. venetum nested with Seiridium ceratosporum (strain 143331 and J21) and Seiridium sp. (strain 51). The S. banksiae clusters somewhat apart from other species of Seiridium, as sister taxon to Monochaetia kansensis.

Taxonomy

Seiridium camelliae Maharach. & K.D. Hyde, sp. nov.

Index Fungorum number: IF551385; Facesoffungi number: FoF584 (Figs. 1, 2 and 3)

Fig. 1
figure 1

Consensus phylogram (50 %) majority rule resulting from a Bayesian analysis of the ITS sequence alignment of Seiridium and other genera in family Amphisphaeriaceae. Bayesian posterior probabilities (PP) above 90 % and RAxML bootstrap support values (ML) above 50 % are given at the nodes (PP/ML). The GenBank number followed by the species name. The scale bar represents the expected number of changes per site. The tree is rooted to Hypoxylon fragiforme and Melogramma campylosporum

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Fig. 2
figure 2

Seiridium camelliae (IFRD OP016). a–b. Leaf spot on Camellia reticulata c. Conidiomata d–e. Conidia. Scale bars: d–e = 10 μm

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Fig. 3
figure 3

Seiridium camelliae (IFRD OP016). a Conidiogenous cells. Scale bars: a = 10 μm

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Holotype: IFRD OP016.

Etymology: Named after the host genus, Camellia, from which the species was isolated.

Description:

Forming leaf blight on leaves of Camellia reticulata (Fig. 2). Sexual morph: Undetermined. Asexual morph: Conidiomata pycnidial, globose, solitary, embedded or semi-immersed, dark brown to black, up to 400 μm diam.; exuding globose, brown to black conidial masses. Conidiophores reduced to conidiogenous cells or, more often, branched at the base and with 1–3 septa, hyaline, smooth-walled, up to 20 μm long. Conidiogenous cells discrete, cylindrical to subcylindrical, with up to 3 proliferations, hyaline, smooth-walled. Conidia 18–28 × 6–8.5 (mean = 23 × 7.5) μm, fusiform to ellipsoid, wall constricted at septa, straight or somewhat curved, basal cell obconic with a truncate base, hyaline, smooth-walled, 2–3.5 (mean = 2.8) μm long; 4 median cells short-cylindric, unequal, brown, periclinal wall and septa darker than the central cell area, together 10–18 (mean = 13) μm long; second cell from base (mean = 4.3) 4–5 μm long; third cell 3–4.5 (mean = 4) μm long; fourth cell 2.5–4 (mean = 3) μm long; fifth cell 3–4.5 (mean = 3.5) μm long; apical cell conical, hyaline, smooth-walled, 2–4 (mean = 3) μm long; apical appendages tubular, corniform, unbranched, single, 1–4 (mean = 2) μm long; basal appendage corniform, unbranched, single, centric, 1–3 μm long.

Culture characteristics: Colonies on PDA reaching 20–30 mm diam. after 30 d at 25 °C, entire at the edge, whitish, with sparse aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in color. Cultures sporulated poorly on PDA.

Material examined: China, Yunnan Province, Chuxiong, Zixishan, on leaves of Camellia reticulata Lindl. (Theaceae), Aug 2010, Y.M. Zhang (IFRD OP016, holotype); ex-type living culture MFLUCC 12–0647, ICMP.

Notes: Seiridium camelliae (Figs. 2, 3) is presently confined to Camellia reticulata in China, and forms a separate lineage in the DNA phylogeny as sister to a species assemblage including S. phylicae and Seiridium spp. (CHTAM66 and CHTAM69). Seiridium camelliae is distinct from S. phylicae (conidia 23–35 × 9–11 μm; Crous et al. 2012) in having smaller conidia. Seiridium eriobotryae was recorded from leaves of Eriobotrya japonica in Guangxi Province, China (Chen et al. 2002). However, conidia of S. eriobotryae (24–33 μm) are longer than those of S. venetum. A Mega BLAST search of the NCBI GenBank nucleotide sequence database using the partial β-tubulin (TUB) sequence of S. camelliae retrieves as closest hits S. cupressi [GenBank DQ926979; identities = 306/329 (93 %), gaps = 2/329 (0 %)] and S. phylicae [GenBank KC005819; identities = 4361/400 (90 %), gaps = 8/400 (2 %)], amongst others.

Seiridium venetum (Sacc.) Nag Raj, Mycotaxon 35(2): 293. 1989. Fig. 4.

Fig. 4
figure 4

Seiridium venetum (MFLUCC 14–0265). a Conidiomata on Cornus mas b Conidiomata sporulating on PDA. c–e Conidiogenous cells. f–h Conidia. Scale bars: c–h = 10 μm

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Basionym: Pestalotia veneta Sacc., Michelia 1(1): 92. 1877.

≡Monochaetia veneta (Sacc.) Sacc. & D. Sacc., Syll. fung. (Abellini) 18: 485. 1906.

≡Pestalotia corni Allesch., Bot. Zbl. 42: 106. 1890.

≡Seiridium corni (Allesch.) B. Sutton, Can. J. Bot. 47(12): 2091. 1969.

FacesofFungi number: FoF585

Description:

Associated with dead twigs of Cornus mas (Fig. 4). Sexual morph: Undetermined. Asexual morph: Conidiomata 200–400 μm diam., pycnidial, globose to clavate, solitary or aggregated, semi-immersed, dark brown, exuding globose, dark brown, glistening conidial masses. Conidiophores 60–100 μm long, lining the cavity of conidioma, septate, irregularly branched, colorless, smooth-walled, invested in mucus. Conidiogenous cells 10–20 × 1–2.5 (mean = 13 × 1.5) μm, discrete, cylindrical, with minute apical periclinal thickenings, colorless, smooth, proliferating 2–5 times percurrently. Conidia 20–30 × 6.5–8.5 (mean = 24.5 × 7.6) μm, fusiform to ellipsoid, straight or somewhat curved, basal cell obconic with a truncate base, hyaline, smooth-walled 3–4 (mean = 3.4) μm long; 4 median cells doliiform, unequal, brown, thick-walled, together 13–22 (mean = 18) μm long; second cell from base 4–5.5 (mean = 5) μm long; third cell 3–5 (mean = 4) μm long; fourth cell 2.5–5 (mean = 3.5) μm long; fifth cell 3–5 (mean = 5.5) μm long; apical cell, conical, hyaline, smooth-walled, 2–4 (mean = 3) μm long; apical appendages tubular, attenuated, flexuous single, unbranched or once or twice branched, 10–35 (mean = 20) μm long; basal appendage single, unbranched or twice branched, centric, 2–5 μm long.

Culture characteristics: Colonies on PDA reaching 30–40 mm diam. after 7 days at 25 °C, entire at the edge, whitish, with dense aerial mycelium on the surface, and black, gregarious conidiomata; reverse similar in color.

Material examined: Italy, Selva, on Cornus sanguinea, 1873 (PAD, holotype); Italy, Trento Province. Val di Sole, Croviana, from dead twigs of Cornus mas, 29 July 2013, E. Camporesi IT 1393 (MFLU 15–0396, reference material of Seiridium venetum designated here); culture MFLUCC 14–0265, ICMP.

Notes: This species was originally described from dry twigs of Cornus sanguinea in Conegliano, Italy. Nag Raj (1993) noted S. venetum as being distributed on Cornus alba, C. sanguinea, C. stolonifera and Cornus sp. in Canada, Germany and Italy. In Seiridium venetum, the apical appendage is single and may branch at some distance from the point of insertion on the apical cell. Sutton (1969) revised and preferred to regard Pestalotia as a monotypic genus, and many species have been transferred to other genera. In his revision of Pestalotia, Sutton (1969) transferred P. corni to Seiridium. Nag Raj (1993) introduced a new combination of Seiridium venetum, which was originally described by Saccardo (1877) as Pestalotia veneta. Furthermore, Seiridium corni was synonymized under S. venetum. The first alternative for supplementing poor type material is by means of epitypification (Hyde and Zhang 2008). The holotype of Seiridium venetum (at herbarium PAD) has the same host, morphology and country of origin as our fresh collection. However, “selection of an epitype is not always justifiable under the current provisions of the ICN, and cannot be undertaken simply because no sequence data is obtainable from the name-bearing type” (Ariyawansa et al. 2014). Therefore, in this paper we designate our fresh collection as a reference specimen. In the ITS analysis, S. venetum received low support, with branches resulting in a polytomy with S. ceratosporum (strain J21 and 143331) and Seiridium sp. (strain 51). However, S. venetum is distinct from S. ceratosporum (conidia 29–35 × 10–12 μm; Nag Raj 1993) by its smaller conidia and presence of longer branched or unbranched apical conidial appendage. Based on a Mega BLAST search of NCBI GenBank nucleotide database, the closest hits using the TUB gene sequence are S. cupressi [GenBank DQ926979; identities = 318/346 (92 %), gaps = 1/346 (0 %)] and Seiridium cardinale [(DQ926973; identities = 329/362 (91 %), gaps = 4/362 (1 %)].

Discussion

In this study, one new species, Seiridium camelliae (from blighted leaves of Camellia reticulata in China), and a fresh collection of Seiridium venetum were characterized in terms of morphology and DNA sequence comparison. Both S. camelliae and S. venetum have distinct morphological characters. Species in the genus Seiridium can cause disease in many economically important plants (Tsopelas et al. 2007), and rapid spreading of several species of Seiridium has now been reported in various areas (Smith 2010). Therefore, swift identification of the causative agent is essential for preventing spread and infection.

Sutton (1980) and Nag Raj (1993) preferred to adopt a narrow concept for Seiridium to include five-septate conidial forms, and the type species of Seiridium, S. marginatum, characterised by five-septate conidia with long attenuated, flexuous apical and basal appendages. However, sequence data generated to date have revealed Seiridium to represent a distinct genus, characterised by six-celled conidia (Maharachchikumbura et al. 2014). Seiridium banksiae, which was isolated on leaves of Banksia marginata in Australia, grouped separately from other species in the genus. This species is characterised by four-celled conidia. Therefore, it is possible that S. banksiae is not congeneric with Seiridium (Crous et al. 2011). Our analysis also did not confirm the monophyly of the genus. However, to date, no published studies have included sequences of Seiridium marginatum, the type species of the genus, and thus the taxonomic placement is still not completely resolved.

Although the ITS is considered the universal barcode for fungi (Schoch et al. 2012; Nilsson et al. 2014) and has a high resolving power within some fungal lineages (Bridge et al. 2005; Schoch et al. 2012), ITS in the present study did not reveal high variation among species, and thus discrimination between taxa was not achievable. However, certain taxa can be better resolved using protein-coding genes (Liu et al. 1999; Liu and Hall 2004; Maharachchikumbura et al. 2013b; Hyde et al. 2014). Barnes et al. (2001) used a TUB gene fragment to resolve Seiridium spp. associated with cypress canker. This region has also been shown to resolve species in other related genera in groups such as Discosia (Tanaka et al. 2011), Seimatosporium (Tanaka et al. 2011) and Seiridium (Barnes et al. 2001).