Resolving the Diplodia complex on apple and other Rosaceae hosts

Abstract

Diplodia species are known as pathogens on many woody hosts, including fruit trees, worldwide. In this study a collection of Diplodia isolates obtained mostly from apple and other Rosaceae hosts were identified based on morphological characters and DNA sequence data from ITS and EF1-α loci. The results show that the diversity of species associated with twig and branch cankers and fruit rot of apples is larger than previously recognised. Four species were identified, namely D. seriata and D. malorum (which is here reinstated for isolates with D. mutila-like conidia). Diplodia intermedia sp. nov. is closely related to D. seriata, and D. bulgarica sp. nov. is morphologically and phylogenetically distinct from all Diplodia species reported from apples.

INTRODUCTION

Species of Diplodia, like other members of the family Botryosphaeriaceae, are known to be pathogens, endophytes and saprophytes on a wide range of mainly woody hosts (Crous et al. 2006, Slippers & Wingfield 2007). Some of the more important pathogenic species include D. pinea, which causes crown wilt, dieback, cankers, shoot and tip blight, and root disease on pines (Eldridge 1961); D. mutila, the cause of black rot and canker of apples and D. seriata, which causes frog-eye leaf spot, black rot and canker of apples (Stevens 1933, Laundon 1973, Brown & Britton 1986, Brown-Rytlewski & McManus 2000); and D. corticola the cause of canker and dieback of cork and other oaks (Alves et al. 2004). There have been conflicting reports on the pathogenicity of some of the species. Thus, Larignon et al. (2001), Epstein et al. (2008) and Savocchia et al. (2007) considered D. seriata to be a primary and virulent pathogen of grapevines while Phillips (1998, 2000), van Niekerk et al. (2004) and Laveau et al. (2009) found it to be saprophytic or weakly pathogenic on this host. Also, D. seriata is regarded as an important pathogen causing canker, leaf spot and fruit rot of apple in the USA (Stevens 1933, Brown & Britton 1986, Brown-Rytlewski & McManus 2000) but as a weak secondary pathogen on the same host in England and New Zealand (Laundon 1973). These differences may be due to variations in virulence between strains, or they may be a result of the incomplete knowledge of the taxonomy of the genus, which in turn hampers accurate species recognition and identification. It is also possible that in species with a broad host range, such as D. seriata, virulence of any given isolate may vary according to the host that is being attacked.

Diplodia is a large genus with more than 1000 species currently recognised. A search of MycoBank (March 2012; www.mycobank.org) revealed 1244 names while a search of Species Fungorum (March 2012; www.speciesfungorum.org) lists 1242 names. The genus was introduced by Montagne (1834) with D. mutila as the type species. As explained by Phillips et al. (2005) the concept of Diplodia has changed over the years and has been regarded as including species with dark brown, 1-septate conidia. However, the genus is typified by D. mutila, which has hyaline, aseptate conidia that can become brown and septate with age. Phillips et al. (2005) provided an emended description of the genus. Briefly, Diplodia is circumscribed by having uni- or multilocular conidiomata lined with conidiogenous cells that form hyaline, aseptate, thick-walled conidia at their tips (Phillips et al. 2005). The conidiogenous cells proliferate internally giving rise to periclinal thickenings, or proliferate percurrently to form two or three annellations. Typically the conidia remain hyaline for a long time before they become brown and 1-septate, but in some species, such as D. pinea,D. scrobiculata and D. seriata, the conidia become coloured before discharge from the pycnidia and mostly remain aseptate (Phillips et al. 2005). No paraphyses are found in the conidiomata of Diplodia species.

Despite the relatively simple generic definition, species are less easily defined. This is largely because there are few distinguishing morphological features. For many years species in Diplodia were defined on the basis of host association, which resulted in a proliferation of species names. According to Slippers et al. (2004) host is not of primary importance in species differentiation in the Botryosphaeriaceae and thus many of the names in Diplodia are likely to be synonyms.

Since 2003 several new species have been described in Diplodia and these species were recognized mainly from DNA sequence data and minor differences in conidial morphology. For example, D. scrobiculata was differentiated from D. pinea on the basis of consistent grouping of isolates in multiple gene genealogies inferred from six protein coding genes and six microsatellite loci (de Wet et al. 2003).

Amongst the species with hyaline conidia, recently described new species include D. rosulata with characteristic rosulate colonies (Gure et al. 2005), and D. corticola with large conidia (Alves et al. 2004). Diplodia africana (Damm et al. 2007), D. olivarum (Lazzizera et al. 2008) and D. cupressi (Alves et al. 2006) were differentiated from D. mutila on the basis of their unique conidial morphology. All five species formed distinct clades in phylogenies based on ITS and EF1-α sequence data.

Although Slippers et al. (2004) suggested that host association may not be a suitable character for species differentiation in the Botryosphaeriaceae, many species in Diplodia do show some host preference. For example, D. pinea and D. scrobiculata occur only on conifers with rare reports on angiosperm hosts such as Prunus and Olea (Damm et al. 2007, Lazzizera et al. 2008). Diplodia rosulata has been found only on Prunus spp. (Gure et al. 2005), while D. africana initially found only on Prunus spp. (Damm et al. 2007) has meanwhile been reported as causing dieback on Juniperus phoenicea in Sardinia (Linaldeddu et al. 2011). Diplodia olivarum was considered to be restricted to Olea spp. (Lazzizera et al. 2008), and D. cupressi has been found only on Cupressus and Juniperus (Solel et al. 1987, Alves et al. 2006). Diplodia corticola has been found mainly on Quercus spp., although one isolate studied by Alves et al. (2004) was from Tsuga and another was from Cercis. More recently D. corticola has been reported from grapevines (Úrbez-Torres et al. 2010).

A collection of Diplodia isolates was obtained from stem cankers and fruit rots of mainly apple trees (Malus) and other Rosaceae hosts (Cydonia, Pyracantha, Cotoneaster) in Portugal, Bulgaria and Iran. The aim of this study was to determine the identity of the species. For this, the isolates were characterised in terms of morphology and their phylogenetic relationships to known species of Diplodia.

MATERIALS AND METHODS

Isolates

Isolations were made by spreading ascospores or conidia on the surface of Difco (Becton, Dickinson & Co, Sparks, USA) potato-dextrose agar (PDA) and incubating overnight at 25 °C. Single germinating spores were transferred to fresh plates of PDA. Isolates were cultured on half-strength PDA (1/2 PDA) or on water agar supplemented with autoclaved pine needles (Smith et al. 1996) on the agar surface. Cultures were kept on the laboratory bench at about 20–25 °C where they received diffused daylight. Growth rates were determined on PDA plates incubated in the dark at 25 °C. Representative isolates and specimens were deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, and nomenclatural data in MycoBank (Crous et al. 2004).

DNA isolation and amplification

DNA was isolated from fungal mycelium by the method of Möller et al. (1992). Procedures and protocols for DNA sequencing were as described by Alves et al. (2004). PCR reactions were carried out with Taq polymerase, nucleotides and buffers supplied by MBI Fermentas (Vilnius, Lithuania) and PCR reaction mixtures were prepared according to Alves et al. (2004), with the addition of 5 % DMSO to improve the amplification of some difficult DNA templates. All primers were synthesised by MWG Biotech AG (Elbersberg, Germany). The ITS region was amplified using the primers ITS1 and ITS4 (White et al. 1990) as described by Alves et al. (2004). The primers EF1-728F and EF1-986R (Carbone & Kohn 1999) were used to amplify part of the translation elongation factor 1-alpha (EF1-α) as described by Alves et al. (2006). The amplified PCR products were purified with the JETQUICK PCR Purification Spin Kit (GENOMED, Löhne, Germany). The PCR products were sequenced by STAB Vida Lda (Portugal).

Phylogenetic analysis

Sequences were aligned with ClustalX v. 1.83 (Thompson et al. 1997), using the following parameters: pairwise alignment parameters (gap opening = 10, gap extension = 0.1) and multiple alignment parameters (gap opening = 10, gap extension = 0.2, transition weight = 0.5, delay divergent sequences = 25 %). Alignments were checked and manual adjustments were made where necessary. Phylogenetic information contained in indels (gaps) was incorporated into the phylogenetic analyses using simple indel coding as implemented by GapCoder (Young & Healy 2003).

Phylogenetic analyses of sequence data were done using PAUP v. 4.0b10 (Swofford 2003) for Maximum-parsimony (MP) analyses, Mr Bayes v. 3.0b4 (Ronquist & Huelsenbeck 2003) for Bayesian Inference (BI) analyses and MEGA5 (Tamura et al. 2011) for Maximum-likelihood (ML) analyses. The general time-reversible model of evolution (Rodriguez et al. 1990), including estimation of invariable sites and assuming a discrete gamma distribution with six rate categories (GTR+Γ+G) was used for both ML and BI analyses. Trees were rooted to L. theobromae and visualized with TreeView (Page 1996).

Maximum-parsimony analyses were performed using the heuristic search option with 1000 random taxa addition and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight and gaps were treated as missing data. Maxtrees were set to 500, branches of zero length were collapsed, and all multiple equally parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated from 1 000 bootstrap replications (Hillis & Bull 1993). Other measures used were consistency index (CI), retention index (RI) and homoplasy index (HI).

Bayesian analyses employing a Markov Chain Monte Carlo method were performed. Four MCMC chains were run simultaneously, starting from random trees for 1000000 generations. Trees were sampled every 100th generation for a total of 10000 trees. The first 1000 trees were discarded as the burn-in phase of each analysis. Posterior probabilities (Rannala & Yang 1996) were determined from a majority-rule consensus tree generated with the remaining 9000 trees. This analysis was repeated three times starting from different random trees to ensure trees from the same tree space were sampled during each analysis.

ML analyses were performed on a starting tree automatically generated by the software. Nearest-Neigbor-Interchange (NNI) was used as the heuristic method for tree inference and 1000 bootstrap replicates were performed. Bootstrapping analysis was computed using ML with an estimated proportion of invariant sites and empirical base frequencies with the indicated outgroup sequences.

A comparison of highly supported clades (bootstrap support values ≥ 70 %) among trees generated from MP analyses of individual datasets was performed in order to detect conflict between individual phylogenies (Alves et al. 2008).

RESULTS

DNA phylogeny

Approximately 550 and 300 bases were determined for the ITS and EF1-α genes, respectively. New sequences were deposited in GenBank (Table 1) and the alignment in TreeBase (submission ID 12819). No major conflicts were detected between single gene phylogenies indicating that the genes could be combined. After alignment the combined ITS and EF1-α dataset consisted of 946 characters (including alignment gaps) for 74 ingroup taxa and 2 outgroup taxa. Of the 946 characters, 735 were constant and 18 were variable and parsimony-uninformative. Maximum parsimony analysis of the remaining 193 parsimony-informative characters resulted in 500 most parsimonious trees of 312 steps (CI = 0.753, RI = 0.955, HI = 0.247) and one is shown in Fig. 1. ML and BI analyses retrieved phylogenetic trees whose topologies were identical to the MP tree presented.

Table 1.

Isolates of Diplodia species considered in this study.

					GenBank Numbers2
Species	Accession number1	Host	Location	Collector	ITS	EF1-α
“Botryosphaeria” tsugae	CBS 418.643	Tsuga heterophylla	British Columbia, Lake Cowichan, Canada	A. Funk	DQ458888	DQ458873
Diplodia corticola	CBS 112547	Quercus ilex	La Rozuela, Córdoba	M.E. Sánchez	AY259110	DQ458872
	CBS 112549	Quercus suber	Requeixo, Aveiro, Portugal	A. Alves	AY259100	AY573227
Diplodia africana	CBS 120835	Prunus persica	Paarl, Western Cape, South Africa	U. Damm	EF445343	EF445382
	CBS 121104	Prunus persica	Paarl, Western Cape, South Africa	U. Damm	EF445344	EF445383
Diplodia bulgarica	CBS 124135	Malus sylvestris	Plovdiv, Bulgaria	S. Bobev	GQ923852	GQ923820
	CBS 124254	Malus sylvestris	Plovdiv, Bulgaria	S. Bobev	GQ923853	GQ923821
	CBS 124136	Malus sylvestris	Plovdiv, Bulgaria	S. Bobev	GQ923854	GQ923822
	IRAN1530C	Malus domestica	Gahvareh village, Kermanshah, Iran	J. Abdollahzadeh	JX152582	JX152578
	IRAN1532C	Malus domestica	Gahvareh village, Kermanshah, Iran	J. Abdollahzadeh	JX152583	JX152579
	IRAN1548C	Malus domestica	Gahvareh village, Kermanshah, Iran	J. Abdollahzadeh	JX152584	JX152580
Diplodia cupressi	CBS 168.87	Cupressus sempervirens	Bet Dagan, Israel	Z. Solel	DQ458893	DQ458878
	CBS 261.85	Cupressus sempervirens	Bet Dagan, Israel	Z. Solel	DQ458894	DQ458879
Diplodia intermedia	CAA 147	Malus domestica (fruit rot)	Aveiro, Portugal	A. Alves	GQ923857	GQ923825
	CBS 112556	Malus sylvestris (canker)	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	AY259096	GQ923851
	CBS 124134	Cydonia sp. (fruit rot)	Torres Vedras, Portugal	S. Santos	HM036528	GQ923851
	CBS 124462	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923858	GQ923826
	IRAN1559C	Unknown woody plant	Rezvanshahr, Rasht, Gilan, Iran	J. Abdollahzadeh	JX152585	JX152581
Diplodia sp.	CAP 330	Pyracantha coccinea	Plovdiv, Bulgaria	S. Bobev	GQ923881	GQ923849
Diplodia malorum	CAP 265	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923859	GQ923827
	CAP 266	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923860	GQ923828
	CAP 267	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923861	GQ923829
	CAP 268	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923862	GQ923830
	CAP 269	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923863	GQ923831
	CAP 270	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923864	GQ923832
	CBS 124130	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923865	GQ923833
	CAP 272	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923866	GQ923834
	CBS 124253	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923867	GQ923835
	CAP 275	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923868	GQ923836
	CAP 277	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923869	GQ923837
	CAP 278	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923870	GQ923838
	CAP 340	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923871	GQ923839
	CAP 341	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	GQ923872	GQ923840
	CBS 112554	Malus sylvestris	Monte da Caparica, Setúbal, Portugal	A.J.L. Phillips	AY259095	DQ458870
Diplodia mutila	CBS 112553	Vitis vinifera	Montemor-o-Novo, Portugal	A.J.L. Phillips	AY259093	AY573219
	CBS 230.30	Phoenix dactylifera	California, USA	L.L. Huillier	DQ458886	DQ458869
Diplodia olivarum	CAP 301	Ceratonia siliqua	Sicily, Italy	A. Sidoti	GQ923873	GQ923841
	CAP 222	Olea europaea	Cutrofiano, Lecce, Puglia, Italy	S. Frisullo	EU392295	EU392272
	CAP 224	Olea europaea	Salice Salentino, Lecce, Puglia, Italy	S. Frisullo	EU392296	EU392273
	CAP 225	Olea europaea	Campi Salentino, Lecce, Puglia, Italy	S. Frisullo	EU392297	EU392274
	CBS 121887	Olea europaea	Italy, Puglia, Lecce, Bosco Belvedere, Scorrano	S. Frisullo	EU392302	EU392279
	CAP 257	Olea europaea	Montesano Salentino, Lecce, Puglia, Italy	S. Frisullo	GQ923874	GQ923482
Diplodia pinea	CAP 166	Olea europaea	Scanzano, Matera, Basilicata, Italy	S. Frisullo	EU392284	EU392261
	CAP 168	Olea europaea	Scorano, Lecce, Puglia, Italy	S. Frisullo	EU392285	EU392262
	CAP 169	Olea europaea	Cutrofiano, Lecce, Puglia, Italy	S. Frisullo	EU392286	EU392263
	CAP 339	Pinus sp.	Belgium	S. Bobev	GQ923875	GQ923843
	CBS 393.84	Pinus nigra	Putten, Netherlands	H.A. van der Aa	DQ458895	DQ458880
	CBS 109727	Pinus radiata	Stellenbosch, South Africa	W.J. Swart	DQ458897	DQ458882
	CBS 109725	Pinus patula	Habinsaran, Indonesia	M.J. Wingfield	DQ458896	DQ458881
	CBS 109943	Pinus patula	Indonesia	M.J. Wingfield	DQ458898	DQ458883
Diplodia rosulata	CBS 116472	Prunus africana	Gambo, Ethiopia	A. Gure	EU430266	EU430268
	CBS 116470	Prunus africana	Gambo, Ethiopia	A. Gure	EU430265	EU430267
Diplodia scrobiculata	CMW 189	Pinus banksiana	USA	M.A. Palmer	AY253292	AY624253
	CBS 109944	Pinus greggii	Mexico	M.J. Wingfield	DQ458899	DQ458884
	CBS 113423	Pinus greggii	Mexico	M.J. Wingfield	DQ458900	DQ458885
	CAP 163	Olea europaea	Supersano, Lecce, Puglia, Italy	S. Frisullo	EU392283	EU392260
Diplodia seriata	CBS 112555	Vitis vinifera	Montemor-o-Novo, Portugal	A.J.L. Phillips	AY259093	AY573219
	CBS 119049	Vitis vinifera	Italy	L. Mugnai	DQ458889	DQ458874
	CAP 154	Vitis vinifera	France	P. Larignon	EU392303	EU392280
	CAP 171	Olea europaea	Ruffano, Lecce, Puglia, Italy	S. Frisullo	EU392305	EU392282
	CAP 276	Malus sylvestris	Monte da Caparica, Portugal	A.J.L. Phillips	GQ923876	GQ923844
	CBS 124137	Prunus domestica	Plovdiv, Bulgaria	S. Bobev	GQ923877	GQ923845
	CBS 124138	Malus sylvestris	Plovdiv, Bulgaria	S. Bobev	GQ923878	GQ923846
	CAP 337	Cotoneaster bullatus	Plovdiv, Bulgaria	S. Bobev	GQ923879	GQ923847
	CBS 124139	Vitis vinifera	Plovdiv, Bulgaria	S. Bobev	GQ923880	GQ923848
	CBS 121885	Olea europaea	Casarano, Lecce, Puglia, Italy	S. Frisullo	EU392289	EU392266
	CAP 217	Olea europaea	Ruffano, Lecce, Puglia, Italy	S. Frisullo	EU392293	EU392270
	CAP 220	Olea europaea	Felline, Lecce, Puglia, Italy	S. Frisullo	EU392294	EU392271
Diplodia pseudoseriata	UY107	Myrcianthes cisplatensis	Uruguay	C. Perez	EU080914	EU863178
	CBS124907	Hexachlamis edulis	Uruguay	C. Perez	EU080922	EU863179
	CBS 124906	Blepharocalyx salicifolius	Uruguay	C. Perez	EU080927	EU863181
	UY1263	Myrciaria tenella	Uruguay	C. Perez	EU080933	EU863182
Diplodia alatafructa	CBS 124931	Pterocarpus angolensis	Sudwala Caves area, South Africa	J. Mehl & J. Roux	FJ888460	FJ888444
	CBS 124932	Pterocarpus angolensis	Sudwala Caves area, South Africa	J. Mehl & J. Roux	FJ888461	FJ888445
	CBS 124933	Pterocarpus angolensis	Buffelskloof Nature Reserve, South Africa	J. Mehl & J. Roux	FJ888478	FJ888446
Lasiodiplodia theobromae	CBS164.96	Fruit along coral reef coast	New Guinea	A. Aptroot	AY640255	AY640258
	CBS124.13	Unknown	USA	J.J. Taubenhaus	DQ458890	DQ458875

¹ Acronyms of culture collections: CAA: A. Alves, Universidade de Aveiro, Portugal; CAP: Alan J.L. Phillips, Universidade Nova de Lisboa, Portugal; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CMW: M.J. Wingfield, FABI, University of Pretoria, South Africa.

² Sequence numbers in italics were retrieved from GenBank. All other sequences were obtained in this study.

³ Ex-type strains in bold face.

Fig. 1.

One of the 500 most parsimonious trees resulting from the combined analysis of ITS and EF1-α nucleotide sequence data. ML/MP/BI bootstrap support and posterior probabilities values are given at the nodes. The values are shown only for those nodes that received support in at least two of the phylogenetic inference methods. Ex-type isolates are in bold.

Four main clades were identified and in Fig. 1 they are labelled 1–4. Clade 1 is composed of species with brown, aseptate conidia that occasionally develop one or two septa. Six species can be resolved in this clade, although bootstrap support (MP and ML) for some of them was generally low. One of these species is described here as new. Clade 2 consists of five species with hyaline, aseptate conidia that later become brown and one septate. Clade 3 includes three species, one of which is here described as new, that have conidia that are hyaline or pale brown and become one-septate. Clade 4 is composed only of D. corticola whose conidia are similar to those of species in clade 2.

Taxonomy

A group of isolates from Malus in clade 2 lay within a distinct sub-clade separate from all other species and was supported by high bootstrap value and posterior probability. Morphologically these isolates correspond in all ways with the isotype of D. malorum. Therefore this name is re-instated for the species that is found on Malus, and an epitype is designated here. A group of isolates from Malus and one from Cydonia, morphologically similar to D. pinea, lay within a distinct sub-clade in clade 1 and were considered to represent a distinct species, which is described here as Diplodia intermedia sp. nov. A further species in clade 3 sister to D. cupressi and ‘B.’tsugae does not correspond to any known species and is described here as D. bulgarica sp. nov.

Diplodia bulgarica A.J.L. Phillips, J. Lopes & S.G. Bobev, sp. nov. — MycoBank MB519632; Fig. 2

Fig. 2.

Diplodia bulgarica. a. Culture growing on PDA; b. pycnidia developing on pine needles in culture; c. pycnidium on pine needle exuding conidia; d–g. conidiogenous cells with developing conidia; h. brown, aseptate conidia; i. brown aseptate conidia and a 2-celled conidium; j, k. conidium in two levels of focus showing finely verruculose inner surface of the conidium wall. — Scale bars: b = 500 μm; c = 200 μm; d–i = 10 μm; j, k = 5 μm.

Etymology. Named after Bulgaria where this species was first found.

Conidiomata pycnidial, produced on pine needles on WA after 7–21 d, solitary, immersed, partially erumpent when mature, dark brown to black, globose to ovoid, up to 600 μm diam and 700 μm high, mostly unilocular; wall composed of an outer layer of dark brown, thick-walled textura angularis, a middle layer of dark brown thin-walled cells, an inner layer of thin-walled hyaline cells. Ostiole central, circular, papillate. Conidiophores absent. Conidiogenous cells 9–18 × 2–5 μm, hyaline, smooth, thin-walled, cylindrical, slightly swollen at the base, holoblastic, forming a single conidium at the tip, discrete, indeterminate, proliferating internally giving rise to periclinal thickenings, or proliferating percurrently to form 1–5 annellations. Conidia aseptate, externally smooth, internally verruculose, thick-walled, oblong to ovoid, straight, both ends broadly rounded, (22.5–)24–27(–28) × (14.5–)15.5–18(–18.5) μm, 95 % confidence limits = 25–25.7 × 16.6–17 μm (mean ± S.D. of 50 conidia = 25.4 ± 1.2 × 16.8 ± 0.7 μm, L/W ratio = 1.5 ± 0.1), initially hyaline, soon becoming pale brown, later darkening and becoming 1-septate.

Specimens examined. BULGARIA, Plovdiv, Malus sylvestris, 2005, S.G.Bobev (CBS H-20189 holotype, culture ex-type CBS 124254). Additional isolates are given in Table 1.

Notes — This species is morphologically distinct from other Diplodia species reported from apples. Conidia are shorter and wider than both D. intermedia and D. malorum. Furthermore, the conidia are distinctive in that they become pale brown soon after they are formed. Phylogenetically this species is closely related to D. cupressi and ‘B.’ tsugae.

Diplodia intermedia A.J.L. Phillips, J. Lopes & A. Alves, sp. nov. — MycoBank MB519633; Fig. 3

Fig. 3.

Diplodia intermedia. a. Culture growing on PDA; b. pycnidia developing on pine needle; c. asci; d, e. ascus, ascospores and pseudoparaphyses; f–i. conidiogenous cells; j, k. conidia in two levels of focus to show finely verruculose inner surface of the conidium wall; l, m. conidia; n, o. microconidia. — Scale bars: b = 500 μm; c, d = 20 μm; e–m = 10 μm.

Etymology. Named after its morphology and phylogenetic position that are intermediate between D. pinea and D. seriata.

Ascomata unilocular, solitary or clustered, immersed, partially erumpent when mature, globose, up to 400 μm diam, dark brown to black, thick-walled, wall composed of outer layers of thick-walled, dark brown textura angularis, inner layers of thin-walled, hyaline textura angularis. Ostiole central, circular, non-papillate, periphysate. Pseudoparaphyses hyaline, branched, septate, constricted at the septum, 2–3 μm wide. Asci clavate, stipitate, bitunicate, 85–160 × 22–28 μm, containing eight ascospores biseriate in the ascus. Ascospores 32–37(–40) × 6–8 μm, fusiform, widest in the upper third, hyaline, thin-walled, smooth, aseptate. Conidiomata pycnidial, solitary or clustered, immersed in the host, partially erumpent at maturity, dark brown to black, ostiolate, nonpapillate, thick-walled, outer and inner layers composed of dark brown and thin-walled hyalinetextura angularis, respectively. Conidiogenous cells hyaline, thin-walled, smooth, cylindrical, swollen at the base, discrete, producing a single conidium at the tip, indeterminate, proliferating internally giving rise to periclinal thickenings or proliferating percurrently forming 2–3 annelations. Conidia aseptate, ovoid, widest in the middle, apex obtuse, base truncate or rounded, initially hyaline, becoming dark brown before release from the pycnidia, wall moderately thick, externally smooth, roughened on the inner surface, (24.6–)29–33.5(–36.9) × (10–)11–16(–17.5) μm, with 95 % confidence limits = 30.2–31.1 × 13–13.6 μm (mean ± S.D. of 150 conidia = 30.6 ± 1.9 × 13.3 ± 1.8 μm, L/W = 2.3 ± 0.3). Microconidiogenous cells not seen. Microconidia hyaline, aseptate, smooth, oblong, ends rounded, 5.5–9.5 × 4–6.5 μm.

Specimens examined. PORTUGAL, Setúbal, Monte da Caparica, dead twigs of Malus sylvestris, Mar. 2006, A.J.L. Phillips (CBS H-20190 holotype, culture ex-type CBS 124462). Additional isolates listed in Table 1.

Notes — Phylogenetically this species is very closely related to D. pinea. However, on account of its smaller conidia, apparent preference for Rosaceae hosts, and the distinct clade it forms in the phylogenetic trees we consider it to represent a separate species.

Diplodia malorum Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 395. 1870. — MycoBank MB246351; Fig. 4

Fig. 4.

Diplodia malorum. a. Culture growing on PDA; b. pycnidia formed on pine needles; c–e. conidiogenous cells; f. hyaline conidia; g; hyaline and 1-septate brown conidia; h, i. brown conidia at different levels of focus to show the finely verruculose inner surface of the wall. — Scale bars: b = 500 μm; c–i = 10 μm.

Conidiomata pycnidial, immersed, erumpent, dark brown to black, aggregated, internally white, ostiolate, ostiole circular, central, short papilla. Conidiophores absent. Conidiogenous cells cylindrical, thin-walled, hyaline, holoblastic, indeterminate, proliferating at the same level to produce periclinal thickenings, or proliferating percurrently giving rise to 2–3 indistinct annelations. Conidia oblong with broadly rounded ends, smooth-walled, thick walled, hyaline, eguttulate, aseptate, becoming dark brown and 1-septate soon after release from the pycnidium, (24–)26–32(–36) × (12–)13–17.5(–18.5) μm, 95 % confidence intervals = 28–28.3 × 14.3–14.5 μm, (𝑥̄ ± S.D. of 700 conidia = 28.1 ± 2.4 ×14.43 ± 1.4 μm, L/W = 1.9 ± 0.24).

Specimens examined. GERMANY, Rhineland, on Malus, 1870, J. Fuckel, Fuckel, Fungi rhenani N° 1706 in K and M (isotypes). – PORTUGAL, Setúbal, Monte da Caparica, Malus sylvestris, Feb. 2006, A.J.L. Phillips (CBS H-201888 epitype designated herein, culture ex-epitype CBS 124130). Additional isolates given in Table 1.

Notes — Since the time that it was introduced by Fuckel (1870) the name D. malorum has been used infrequently, while the name D. mutila was applied to the apple pathogen. However, as shown here, D. malorum is morphologically and phylogenetically distinct from D. mutila. The conidia are larger than those of D. mutila and they frequently become brown and 1-septate soon after discharge from the pycnidia. The characteristics of the isolates studied here correspond with the isotypes (Fungi rhenani Nº 1706 in K and M).

Diplodia tsugae (A. Funk) A.J.L. Phillips & A. Alves, comb. nov. — MycoBank MB801409

Basionym. Botryosphaeria tsugae A. Funk, Canad. J. Bot. 42: 770. 1964.

Specimens examined. CANADA, British Columbia, near Bella Coola (Snootli Creek), on branches of Tsuga heterophylla, 11 Sept. 1963, A. Funk (DAVFP 15485 holotype, DAOM 96030 isotype, CBS H-6790 isotype, culture ex-isotype CBS 418.64).

Notes — When Funk (1964) introduced this species he did not apply a name to the anamorph, which he referred to as a Macrophoma species. When Crous et al. (2006) re-defined Botryosphaeria they removed this species to Diplodia but did not formally make a new combination. Since morphologically and phylogenetically this species is clearly a Diplodia species we recombine it in Diplodia.

DISCUSSION

In this work a collection of Diplodia isolates mainly from apples was studied in terms of morphology and phylogenetic position based on nucleotide sequence data from ITS and EF1-α loci. Two species are described as new, while one existing name that has rarely been used is recognised as valid and distinct. All species studied in this work lay within four distinct clades in a combined ITS plus EF1-α phylogeny. Each clade was characterised by distinct morphological features of the anamorphs. Isolates from apples and other hosts in Rosaceae were distributed between three of these clades.

The distinct morphologies associated with each clade deserves some comment. Clade 1 includes species with brown, aseptate conidia that occasionally develop one or two septa. Another distinctive feature of species in this clade is that their conidia become coloured at an early stage of development even before dehiscence from the conidiogenous cells. Within the Botryosphaeriaceae this feature is seen only in Dothiorella, Phaeobotryon and Spencermartinisia (Phillips et al. 2008). The main character used to separate species within this clade is conidial dimensions. In practice this can be difficult to apply on account of the variability within a species and the overlap of dimensions between the species. Nevertheless, they can all be distinguished phylogenetically, although for some species, such as D. pinea and D. intermedia, support for the branches is low.

Diplodia pseudoseriata was recently described from native Myrtaceae trees in Uruguay (Pérez et al. 2010) while D. alatafructa was described from Pterocarpus angolensis in South Africa (Mehl et al. 2011). In the phylogeny constructed in the present work isolates of both of these species clustered in a single clade suggesting that they represent a single phylogenetic species. Judging from the original descriptions these two species also appear to be morphologically indistinguishable. Therefore it seems that they should be regarded as synonyms and given that D. pseudoseriata was the first name to be published it takes priority over D. alatafructa. This possibility will be addressed in a future publication currently in preparation.

Both D. seriata (as Botryosphaeria obtusa) and D. mutila (as Botryosphaeria stevensii) have been implicated in fruit rot (black rot) and cankers of apples (Stevens 1933, Laundon 1973, Brown & Britton 1986, Brown-Rytlewski & McManus 2000). Stevens (1933) found that in the United States D. seriata was the most common cause of black rot while D. mutila was confined mainly to the west coast. In the present study the Diplodia species isolated from Malus and other Rosaceae were D. bulgarica, D. intermedia, D. malorum and D. seriata. This study has shown that D. seriata s.l. is a complex of species, two of which are associated with fruit rot and canker of apples and other Rosaceae, namely D. intermedia and D. seriata. This was based on five isolates of D. intermedia and only two of D. seriata, from three widely separate regions in Portugal and one sample from Bulgaria. Both species are easily confused on the basis of morphology only and therefore it is virtually impossible to know if previous reports regarding the black rot pathogen of apple were referring to D. intermedia, D. seriata or both. Given the data presented it may be premature to suggest that the cause of black rot of apples is D. intermedia and not D. seriata, but it does indicate that a more complete phylogenetic and pathogenicity study of the black rot fungus should be done on collections from a wider geographical range.

Although D. mutila has also been implicated as the cause of apple black rot and canker, the evidence presented here suggests that the name D. malorum is more correctly applied to isolates from Malus species. Slippers et al. (2007) initially regarded D. malorum to be a more appropriate name for the anamorph of ‘B.’obtusa but they rejected this possibility after studying the type specimen in G (Fungi rhenani 1706) and that was later confirmed by Phillips et al. (2007). The current study clearly supports this view and shows that D. malorum is morphologically and phylogenetically distinct from D. seriata, which is the anamorph of ‘B’. obtusa. It is likely that given the morphological similarity between D. malorum and D. mutila both species have been confused in the past. Again, the data presented here relates only to one geographical region in Portugal but the type specimen of D. malorum is on a rotten apple collected from Germany. On the other hand, the occurrence of D. mutila on apples and its association with cankers cannot be ruled out, since it has been reported in previous studies and the ITS sequences (GenBank accessions AF243406 and AF243407, Zhou & Stanosz 2001) from those isolates are 100 % identical to the ITS sequence of typical D. mutila (Alves et al. 2004). The lack of an ex-type culture, or other cultures linked to the holotype of D. mutila hampers this kind of study, but Alves et al. (2004) provided a detailed description of this species based on an isolate from grapevines in Portugal (CBS 112553), an isotype of D. mutila (K99664) and the lectotype of Physalospora mutila (BPI99153). They showed that CBS 112553 correlated closely with the morphology of D. mutila. This culture has subsequently been cited as typical of D. mutila and has been referred to as a standard isolate for this species (Alves et al. 2006, Damm et al. 2007, Lazzizera et al. 2008). In the future more studies should be done in order to confirm the occurrence and pathogenicity of D. mutila towards apples.