- Open Access
Diaporthe is paraphyletic
IMA Fungus volume 8, pages153–187(2017)
Previous studies have shown that our understanding of species diversity within Diaporthe (syn. Phomopsis) is limited. In this study, 49 strains obtained from different countries were subjected to DNA sequence analysis. Based on these results, eight new species names are introduced for lineages represented by multiple strains and distinct morphology. Twelve Phomopsis species previously described from China were subjected to DNA sequence analysis, and confirmed to belong to Diaporthe. The genus Diaporthe is shown to be paraphyletic based on multi-locus (LSU, ITS and TEF1) phylogenetic analysis. Several morphologically distinct genera, namely Mazzantia, Ophiodiaporthe, Pustulomyces, Phaeocytostroma, and Stenocarpella, are embedded within Diaporthe s. lat., indicating divergent morphological evolution. However, splitting Diaporthe into many smaller genera to achieve monophyly is still premature, and further collections and phylogenetic datasets need to be obtained to address this situation.
Species of Diaporthe are known as important plant pathogens, endophytes or saprobes (Udayanga et al. 2011, Gomes et al. 2013). They have broad host ranges, and occur on many plant hosts, including cultivated crops, trees, and ornamentals (Diogo et al. 2010, Thompson et al. 2011, Gomes et al. 2013, Huang et al. 2015). Some Diaporthe species are responsible for severe diebacks, cankers, leaf-spots, blights, decay or wilts on different plant hosts, several of which are economically important (Mostert et al. 2001, Van Rensburg et al. 2006, Thompson et al. 2011, Gomes et al. 2013), leading to serious diseases and significant yield losses (Santos et al. 2011). For example, Diaporthe helianthi is the cause of one of the most important diseases of sunflower (Helianthus annuus) worldwide, and has reduced production by up to 40% in Europe (Masirevic & Gulya 1992, Thompson et al. 2011). Diaporthe neoviticola and D. vitimegaspora, the causal agents of leaf-spot and swelling arm, are known as severe pathogens of grapevines (Vitis vinifera) (Van Niekerk et al. 2005). Ürbez-Torres et al. (2013) indicated that D. neoviticola was one of the most prevalent fungi isolated from grapevine perennial cankers in declining vines. Diaporthe scabra has been reported causing cankers and dieback on London plane (Platanus acerifolia) in Italy (Grasso et al. 2012). Symptoms of umbel browning and necrosis caused by D. angeliace have been regularly observed on carrots in France, resulting in seed production losses since 2007 (Ménard et al. 2014). Avocado (Persea americana), cultivated worldwide in tropical and subtropical regions, is threatened by branch cankers and fruit stem-end rot diseases caused by D. foeniculina and D. sterilis (Guarnaccia et al. 2016). Furthermore, species of Diaporthe are commonly introduced into new areas as endophytes or latent pathogens along with plant produce. For instance, Torres et al. (2016) reported D. rudis causing stemend rot in avocados in Chile, which was imported via avocado fruit from California (USA). Some endophytes have been shown to act as opportunistic plant pathogens. Diaporthe foeniculina (syn. P. theicola), which is a common endophyte, has been shown to cause stem and shoot cankers on sweet chestnut (Castanea sativa) in Italy (Annesi et al. 2015, Huang et al. 2015). Because of this unique ecology and potential role as plant pathogens, it is of paramount importance to accurately identify species of Diaporthe to facilitate disease surveillance, control, and trade.
The initial species concept of Diaporthe based on the assumption of host-specificity, resulted in the introduction of more than 1000 names (http://www.indexfungorum.org/Names/Names.asp); (Gomes et al. 2013, Gao et al. 2016). In recent years, however, a polyphasic approach employing multi-locus DNA data together with morphology and ecology has been employed for species delimitation in the genus (Udayanga et al. 2011, Gomes et al. 2013). The nuclear ribosomal internal transcribed spacer (ITS), the translation elongation factor 1-α (TEF1), β-tubulin (TUB), histone H3 (HIS), and calmodulin (CAL) genes are the most commonly used molecular loci for the identification of Diaporthe spp. (Dissanayake et al. 2015, Udayanga et al. 2015, Huang et al. 2015, Santos et al. 2017). Furthermore, molecular marker aids are being used to rapidly identify Diaporthe species which tend to be morphologically conserved (Udayanga et al. 2012, Tan et al. 2013, Lombard et al. 2014, Thompson et al. 2015, Huang et al. 2015). However, defining species boundaries remains a major challenge in Diaporthe (Huang et al. 2015), which may be a consequence of limited sampling or the use of DNA loci with insufficient phylogenetic resolution (Liu et al. 2016). It has therefore been proposed that new species in the genus should be introduced with caution, and that multiple strains from different origins should be subjected to a multi-gene phylogenetic analysis to determine intraspecificvariation (Liu et al. 2016).
The generic relationships of Diaporthe with other genera in Diaporthaceae remain unclear. The family name Diaporthaceae was established by Wehmeyer (1926) to accommodate Diaporthe, Mazzantia, Melanconis, and some other genera, mainly based on morphological characters such as the position, structure, and arrangement of ascomata, stroma, and spore shapes. Castlebury et al. (2002) reported that Diaporthaceae comprised Diaporthe and Mazzantia based on LSU DNA sequence data, removing other genera to different families in Diaporthales. Additional genera subsequently placed in the Diaporthaceae include Leucodiaporthe (Vasilyeva et al. 2007), Stenocarpella (Crous et al. 2006), Phaeocytostroma (Lamprecht et al. 2011), Ophiodiaporthe (Fu et al. 2013), and Pustulomyces (Dai et al. 2014). All the above genera were represented by a few species or are monotypic. Although they appeared to be morphologically divergent from Diaporthe, their phylogenetic relationships remain unclear.
About 991 names of Diaporthe and 979 of Phomopsis have been established to date (http://www.indexfungorum.org/Names/Names.asp). Among them, many old epithets lack molecular data, and few morphological characters can be used in species delimitation, making it difficult to merge these names to advance to the one name scenario (Rossman et al. 2014, 2015). In China, more than 50 plant pathogenic Phomopsis species have been published to date (Chi et al. 2007). In order to stabilize these species names in the genus Diaporthe, here we introduce 12 new combinations for Phomopsis species that have been subjected to DNA sequencing, and whose phylogenetic position has been resolved in Diaporthe in the present study.
The objectives of this study were: (1) to examine the phylogenetic relationships of Diaporthe with other closely related genera in Diaporthaceae; (2) to introduce new species in Diaporthe; and (3) to transfer Phomopsis species described from China to Diaporthe based on morphological and newly generated molecular data.
Material and Methods
Strains were isolated from leaves of both symptomatic and healthy plant tissues from Yunnan, Zhejiang, and Jiangxi Provinces in China. A few other strains were obtained via the Ningbo Entry-Exit Inspection and Quarantine Bureau, which were isolated from imported plants from other countries. Single spore isolations were conducted from diseased leaves with visible fungal sporulation following the protocol of Zhang et al. (2013), and isolation from surface sterilized leaf tissues was conducted following the protocol of Gao et al. (2014). Fungal endophytes were isolated according to the method described by Liu et al. (2015). The Diaporthe strains were primarily identified from the other fungal species based on cultural characteristics on PDA, spore morphology, and ITS sequence data. Type specimens of new species were deposited in the Mycological Herbarium, Microbiology Institute, Chinese Academy of Sciences, Beijing, China (HMAS), with ex-type living cultures deposited in the China General Microbiological Culture Collection Center (CGMCC).
Cultures were incubated on PDA at 25 °C under ambient daylight and growth rates were measured daily for 7 d. To induce sporulation, isolates were inoculated on PNA (pine needle agar; Smith et al. 1996) containing double-autoclaved (30 min, 121°C, 1 bar) healthy pine needles and incubated at a room temperature of ca. 25 °C (Su et al. 2012). Cultures were examined periodically for the development of conidiomata and perithecia. Conidia were taken from pycnidia and mounted in sterilized water. The shape and size of microscopic structures were observed and noted using a light microscope (Nikon Eclipse 80i) with differential interference contrast (DIC). At least 10 conidiomata, 30 conidiophores, alpha and beta conidia were measured to calculate the mean size and standard deviation (SD).
DNA extraction, PCR amplification and sequencing
Isolates were grown on PDA and incubated at 25 °C for 7 d. Genomic DNA was extracted following the protocol of Cubero et al. (1999). The quality and quantity of DNA was estimated visually by staining with GelRed after 1% agarose gel electrophoresis. The primers ITS5 and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA gene operon, including the 3’ end of the 18S nrRNA, the first internal transcribed spacer region, the 5.8S nrRNA gene; the second internal transcribed spacer region and the 5’ end of the 28S nrRNAgene. The primers EF1-728F and EF1-986R (Carbone & Kohn 1999) were used to amplify part of the translation elongation factor 1-α gene (TEF1), and the primers CYLH3F (Crous et al. 2004) and H3-1b (Glass & Donaldson 1995) were used to amplify part of the histone H3 (HIS) gene. The primers T1 (O’Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson 1995) were used to amplify the beta-tubulin gene (TUB); the additional combination of Bt2a/Bt2b (Glass & Donaldson 1995) was used in case of amplification failure of the T1/Bt2b primer pair. The primer pair CAL228F/CAL737R (Carbone & Kohn 1999) and LR0R/LR5 primer pair (Rytas & Mark 1990) were used to amplify the calmodulin gene (CAL) and the LSU rDNA, respectively. Amplification reactions of 25 µL were composed of 10 × EasyTaq buffer (MgCl2+ included; Transgen, Beijing), 50 µM dNTPs, 0.2 µM of each forward and reverse primers (Transgen), 0.5 U EasyTaq DNA polymerase (Transgen) and 1–10 ng of genomic DNA. PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s (52 °C for ITS and LSU, 56 °C for CAL, HIS, TEF1 and TUB), extension at 72 °C for 30 s and a final elongation step at 72 °C for 10 min. DNA sequencing was performed by Omegagenetics Company, Beijing.
The DNA sequences generated with forward and reverse primers were used to obtain consensus sequences using MEGA v. 5.1 (Tamura et al. 2011), and subsequently aligned using MAFFT v. 6 (Katoh & Toh 2010); alignments were manually edited using MEGA v. 5.1 when necessary. Two datasets were employed in the phylogenetic analyses. LSU, ITS and TEF1 loci were selected to infer the generic relationships within Diaporthaceae (Table 1), with Valsa ambiens as outgroup. All available sequences of Diaporthe species were included in the dataset of combined ITS, HIS, TEF1, TUB, and CAL regions to infer the interspecific relationships within Diaporthe (Table 2) with Diaporthella corylina as outgroup. Maximum likelihood (ML) gene trees were estimated using the software RAxML v. 7.4.2 Black Box (Stamatakis 2006, Stamatakis et al. 2008). The RAxML software selected the GTR model of nucleotide substitution with the additional options of modelling rate heterogeneity (Γ) and proportion invariable sites (I). Bayesian analyses (critical value for the topological convergence diagnostic set to 0.01) were performed on the concatenated loci using MrBayes v. 3.2.2 (Ronquist et al. 2012) as described by (Crous et al. 2006) using nucleotide substitution models for each data partition selected by jModeltest (Darriba et al. 2012) and MrModeltest v. 2.3 (Nylander 2004). Bayesian analyses were launched with random starting trees for 10 000 000 generations, and Markov chains were sampled every 1000 generations. The first 25% resulting trees were discarded as burn-in. The remaining trees were summarized to calculate the posterior probabilities (PP) of each clade being monophyletic. Trees were visualized in FigTree v. 1.1.2 (http://tree.bio.ed.ac.uk/software/). New sequences generated in this study were deposited in NCBI’s GenBank nucleotide database (www.ncbi.nlm.nih.gov; Table 1).
Collection of Diaporthe strains
Twenty-one Diaporthe strains including presumed plant pathogens and endophytes were isolated from 11 different host plant species (Table 2) collected from three provinces (Jiangxi, Yunnan, Zhejiang) in the northern part of China. In addition, 28 strains were isolated from the plant samples inspected by Jiangsu Entry-Exit Inspection and Quarantine Bureau.
The paraphyly of Diaporthe
Phylogenetic analysis was conducted with 224 sequences derived from 76 ingroup taxa from Diaporthaceae with Valsa ambiens as the outgroup (Table 1). The combined alignment comprised 1 817 characters including gaps (795 for LSU, 558 for ITS, 464 for TEF1). Based on the results of the Mrmodeltest, the following priors were set in MrBayes for the different data partitions: GTR+G models with gamma-distributed rates were implemented for LSU and ITS, HKY+I+G model with invgamma-distributed rates were implemented for TEF1. The Bayesian analysis lasted 7 × 108 generations and the consensus tress and posterior probabilities were calculated from the trees left after discarding the first 25% generations for burn-in (Fig. 1).
The generic relationships of Mazzantia, Ophiodiaporthe, Phaeocytostroma, Pustulomyces, and Stenocarpella with Diaporthe from this analysis are shown in Fig. 1. The topology and branching order of the phylogenetic trees inferred from ML and Bayesian methods were essentially similar. Five genera from Diaporthaceae did not form discrete clades from Diaporthe species but are scattered in the latter, although the family remains monophyletic. The paraphyletic nature of Diaporthe, however, is demonstrated (Fig. 1). Ophiodia-porthe formed a well resolved and distinct clade represented by strain YMJ 1364, and clustered together with the ex-type culture of D. sclerotioides (CBS 296.67) (BPP 0.99, MLBS: 90). Stenocarpella, represented by S. maydis and S. mac-rospora, was well supported (BPP 1, MLBS = 96) and closely related to several species of Phaeocytostroma. Mazzantia, however, was poorly supported for its phylogenetic position in Diaporthaceae (Fig. 1).
Phylogenetic analyses of the combined datasets of Diaporthe species
In total, 1089 sequences derived from 273 ingroup taxa were combined and Diaporthella corylina was used as outgroup. A total of 2783 characters including gaps (568 for CAL, 554 for HIS, 523 for ITS, 636 for TEF1 and 456 for TUB) were included in the multi-locus dataset, comprising sequences generated from this study and others downloaded from GenBank (Table 2). For the Bayesian inference, GTR+I+G model was selected for CAL, HIS and ITS, HKY+I+G for TEF1 and TUB through the analysis of Mrmodeltest. The maximum likelihood tree conducted by the GTR model confirmed the tree topology and posterior probabilities of the Bayesian consensus tree.
The topology and branching order for the phylogenetic trees inferred from ML and Bayesian methods were essentially similar (Fig. 2). Based on the multi-locus phylogeny and morphology, 49 strains were assigned to 13 species, including eight taxa which we describe here as new (Fig. 2).
Diaporthe acutispora Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the acute spores.
Diagnosis: Diaporthe acutispora is phylogenetically distinct and morphologically differs from species reported from the host genera Coffea and Camellia in the larger conidiophores and alpha conidia (Table 3).
Type: China: Yunnan Province: Aini Farm, on healthy leaves of Coffea sp., 20 Sep. 2014, W.J. Duan (HMAS 247086 — holotype, dried culture; CGMCC 3.18285 = LC 6161 -ex-type culture).
Description: On PNA: Conidiomata pycnidial, globose, brownish, embedded in tissue, erumpent at maturity, 99–173 µm diam, often with a yellowish conidial cirrus exuding from the ostioles. Conidiophores 10–34.5 × 2–3 µm, cylindrical, hyaline, septate, branched, straight or slightly curved, tapering towards the apex. Alpha conidia abundant in culture, 7–10.5 × 2–3 µm (x̅ = 8.4 ± 0.7 × 2.6 ± 0.2, n = 30), aseptate, hyaline, ellipsoidal to fusoid, multi-guttulate. Beta conidia not observed.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 7.5 mm diam/d. Colony entirely white at surface, reverse with pale brown pigmentation, white, fluffy aerial mycelium.
Additional material examined: China: Yunnan Province: Xishuangbanna, on healthy leaves of Camellia sasanqua, 20 Sep. 2014, W.J. Duan, culture LC 6142; ibid. culture LC 6160.
Diaporthe elaeagni-glabrae Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the host species Elaeagnus glabra.
Diagnosis: Diaporthe elaeagni-glabrae can be distinguished from the closely related species D. elaeagni (96% in ITS, 93% in TEF1, 94% in TUB, 96% in HIS, and 94% in CAL) and D. stictica (96% in ITS, 95% in TEF, 97% in TUB, 96% in HIS, and 96% in CAL) (Fig. 2). Diaporthe elaeagni-glabrae differs from other species recorded from Elaeagnus in the significantly longer alpha conidia (Table 3).
Type: China: Jiangxi Province: on diseased leaves of Elaeagnus glabra, 5 Sep. 2013, Y.H. Gao (HMAS 247089 — holotype, dried culture; CGMCC 3.18287 = LC 4802 — ex-type culture).
Description: On PDA: Conidiomata globose, to 330–;1170 µm, erumpent, with slightly elongated black necks, yellowish or dirty white, spiral conidial cirri extruding from ostioles. Conidiophores 16–;28 × 1.5–;2.5 µm, cylindrical, phialidic, septate, branched, sometimes inflated. Alpha conidia 6–;13 × 1.5–;3 µm (x̅ = 8.3 ± 1.4 × 2.2 ± 0.3, n = 30), hyaline, fusiform or oval, usually biguttulate. Beta conidia 7.5–;22.5 × 1–;2 µm (x̅ = 15.1 ± 3.5 × 1.2 ± 0.2, n = 40), hyaline, filiform, smooth, curved, base truncate.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 7 mm diam/d. Colony pale yellowish, greenish to brownish at the centre, reverse pale yellowish and brownish at the centre with age. Aerial mycelium white, sparse, fluffy, with irregular margin and visible conidiomata at maturity.
Additional material examined: China: Jiangxi Province: on diseased leaves of Elaeagnus glabra, 5 Sep. 2013, Y.H. Gao, culture LC 4806.
Diaporthe helianthi Munt.-Cvetk. et al., Nova Hedwigia 34: 433 (1981).
Description: Sexual morph not produced. Conidiomata pycnidial globose to subglobose, dark brownish to black, erumpent or immersed in medium, translucent conidia exuded from the ostioles, 110–;380 µm diam. Conidiophores cylindrical, straight or sinuous, apical or base sometimes swelling, 11.5–;23.5 × 1.8–;3.5 µm (x̅ = 16 ± 3 × 2.5 ± 0.5, n = 30). Beta conidia filiform, hamate or slightly curved, base truncate, tapering towards one apex, 11.5–;32 × 0.5–;2 µm (x̅= 20±7.5×1± 0.4, n = 20). Alpha conidia not observed.
Culture characters: Cultures on PDA at 25 °C in dark, with 12/12 h alternation between daylight and darkness pure white (surface) and pale yellow to cream (reverse). Colony pellicular, forming less pigmented sectors, with concentric rings of gummy mycelium. Growth rate was 10.5 mm diam/d.
Material examined: Ukraine: from seeds of Helianthus annuus, 30 Oct. 2015, W.J. Duan culture LC 6173. — Japan: Lagerstroemia indica, 30 Oct. 2015, W.J. Duan, culture LC 6185.
Notes: Diaporthe helianthi, responsible for stem canker and grey spot disease of sunflower (Helianthus annuus) (Muntanola-Cvetkovic et al. 1981), has been listed in the Chinese quarantine directory. There is increasing evidence that this serious sunflower pathogen is being quickly and globally disseminated with international trade. The cases reported here were intercepted from imported sunflower seeds from Ukraine and Lagerstroemia indica from Japan.
Diaporthe incompleta Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the absence of alpha conidia.
Diagnosis: Diaporthe incompleta is phylogenetically distinct and differs morphologically from other species recorded from Elaeagnus and Camellia in the much longer beta conidia (Table 3).
Type: China: Yunnan Province: Xishuangbanna, on diseased of Elaeagnus glabra, 19 Apr. 2015, F Liu (HMAS 247088 — holotype, dried culture; CGMCC 3.18288 = LC 6754 — ex-type culture).
Description: Conidiomata pycnidial, subglobose to globose, brownish to black, 207–;650 µm diam, cream to pale luteous conidial droplets exuding from the central ostioles. Conidiophores 8–;22 × 1–;2.5 µm, cylindrical, hyaline, septate, unbranched, smooth, slightly curved, tapering towards apex. Alpha conidia not observed. Beta conidia 19–;44 × 0.5–;1.5 µm (x̅ = 30.5 ° 8.7 × 1.1 ± 0.4, n = 30), smooth, hyaline, filiform, base subtruncate, straight or curved.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 16.5 mm diam/d. Colony entirely white, flat, reverse pale yellowish, becoming brownish zoned at the centre with age. Aerial mycelium white, cottony, margin lobate, conidiomata visible at maturity.
Additional material examined: China: Yunnan Province: Xishuangbanna, on diseased leaves of Camellia sinensis, 19 Apr. 2015, F. Liu, culture LC 6706.
Diaporthe podocarpi-macrophylli Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the host plant Podocarpus macrophyllus.
Diagnosis: Diaporthe podocarpi-macrophylli can be distinguished from the phylogenetically closely related species D. pseudophoenicicola (97% identity in ITS, 90% in TEF1, 98% in TUB, 97% in HIS, and 97% in CAL). Morphologically, D. podocarpi-macrophylli differs from other species occurring on the host genera Podocarpus and Olea, i.e. D. cinerascens and Phomopsis podocarpi in its wider and shorter alpha conidia and the presence of beta conidia (Chang et al. 2005, Gomes et al. 2013; https://nt.ars-grin.gov/fungaldatabases/).
Type: Japan: on healthy leaves of Podocarpus macrophyllus, 20 Sep. 2014, W.J. Duan (HMAS 247084 — holotype, dried culture; CGMCC 3.18281 = LC 6155-ex-type culture).
Description: Conidiomata pycnidial in culture on PDA, solitary or aggregated, deeply embedded in the PDA, erumpent, dark brown to black, 222–;699 µm diam, yellowish translucent conidial drops exuding from the ostioles. Alpha conidiophores 6–;18 × 1.5–;3 µm (x̅ = 12.3 ± 2.6 × 2.1 ± 0.3, n = 30), hyaline, septate, branched, cylindrical, straight to sinuous, sometimes inflated, occurring in dense clusters. Beta conidiophores 10.5–;27 × 1.5–;2.5 µm (x̅ = 15.3 ± 4.3 × 2.1 ± 0.3, n = 30), cylindrical to clavate, hyaline, septate, branched, smooth, straight. Alpha conidia 3.5–;8.5 × 1–;3 µm (x̅ = 6.3 ± 1.7 × 2.1 ± 0.7, n = 50), unicellular, aseptate, fusiform, hyaline, usually biguttulate and acute at both ends. Beta conidia 8.5–;31.5 × 0.5–;2 µm (x̅ = 19.5 ± 7.1 × 1.1 ± 0.4, n = 30), hyaline, aseptate, eguttulate, filiform, curved, tapering towards both ends, base truncate.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 12.5 mm diam/d. Colony at first white, becoming cream to yellowish, flat, with dense and felted mycelium, reverse pale brown with brownish dots with age, with visible solitary or aggregated conidiomata at maturity.
Additional material examined: Japan: on healthy leaves of Podocarpus macrophyllus, 20 Sep. 2014, W.J. Duan, culture LC 6141; ibid. culture LC 6144; ibid. culture LC 6156; ibid. culture LC 6157. — China: Zhejiang Province: on healthy leaves of P. macrophyllus, 10 Jul. 2015, W.J. Duan, culture LC 6194; ibid. culture LC 6195; ibid. culture LC 6196; ibid. culture LC 6197; ibid. culture LC 6198; ibid. culture LC 6199; ibid. culture LC 6200; ibid. culture LC 6201; ibid. culture LC 6202; ibid. culture LC 6235. — Italy: on healthy leaves of Olea europaea, 20 Sep. 2014, W.J. Duan, culture LC 6229.
Diaporthe undulata Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the colony’s undulate margin.
Diagnosis: Diaporthe undulata differs from the most closely related species, D. biconispora, in several loci (94% in ITS, 84% in TEF1, and 93% in TUB), and from other Diaporthe species in the obpyriform conidiophores and shorter and wider alpha conidia (Table 3).
Type: China-Laos border: on diseased leaves of unknown host, 19 Apr. 2014, F Liu (HMAS 247091 — holotype, dried culture; CGMCC 3.18293 = LC 6624 — ex-type culture).
Description: Conidiomata pycnidial, irregular, embedded in the needle, erumpent, necks, hairy, 282–;543 µm long, coated with short hyphae, one to several necks forming from a single pycnidium. Conidiophores obpyriform, hyaline, phiailidic, septate, branched, 5–;17.5 × 2–;3 µm (x̅ = 9.7 ± 4.0 × 2.4 ± 0.5, n = 20). Alpha conidia ellipsoid, hyaline, biguttulate, rounded at both ends, 5–;6.5 × 2–;3 (x̅ = 5.8 ± 0.4 × 2.3 ± 0.3, n = 50). Beta conidia not observed.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 10.5 mm diam/d. Colony entirely white, reverse pale yellowish and dark brownish at the centre with age. Aerial mycelium white, cottony, dense, with undulate margin and visible conidiomata at maturity.
Additional material examined: China-Laos border: unknown host, 19 Apr. 2014, F. Liu, culture LC 8110; ibid. culture LC 8111.
Diaporthe velutina Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the felted colony.
Diagnosis: Diaporthe velutina is distinguished from D. anacardii in the ITS, TEF1, TUB and HIS loci (99% in ITS, 95% in TEF1, 99% in TUB, and 98% in HIS), and from other Diaporthe species reported from Camellia sinensis in the more variable size of the alpha conidia (Table 3).
Type: China: Jiangxi Province: on diseased leaves of Neolitsea sp., 5 Sep. 2013, Y.H. Gao (HMAS 247087 — holotype, dried culture; CGMCC 3.18286 = LC4421 — ex-type culture).
Description: Conidiomata pycnidial, globose, black, embedded in PDA, aggregated in clusters, 69–;428 µm diam, cream translucent drop of conidia exuded from the central ostioles. Conidiophores 10–;23 × 1–;2.5 µm, cylindrical, hyaline, branched, densely aggregated, slightly tapering towards the apex, sometimes slightly curved. Alpha conidia 5.5–;10 × 2–;2.5 µm (x̅ = 6.9 ± 0.9 × 2.2 ± 0.2, n = 50), unicellular, aseptate, hyaline, fusoid to ellipsoid or clavate, bi-guttulate or multi-guttulate. Beta conidia 11–;27.5 × 0.5–;1.5 µm (x̅ = 16.1 ± 5.0 × 0.8 ± 0.4, n = 30), smooth, hyaline, apex acutely rounded, curved.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 18.75 mm diam/d. Colony entirely white, surface mycelium greyish to brownish at the centre, dense, felted, conidiomata erumpent at maturity, reverse centre yellowish to brownish.
Additional material examined: China: Jiangxi Province: Yangling, on diseased leaves of Neolitsea sp., 5 Sep. 2013, Y.H. Gao, culture LC 4419; ibid. culture LC 4422; Gannan Normal University, unknown host, 23 Apr. 2013, Q. Chen, culture LC 4788; Fengshan, on diseased leaves of Callerya cinerea, 5 Sep. 2013, Y.H. Gao, culture LC 4641. Yunnan Province: Xishuangbanna, on diseased leaves of Camellia sinensis, 19 Apr. 2015, F. Liu, culture LC 6708; loc. cit., on healthy leaves of C. sinensis, 21 Apr. 2015, F. Liu, culture LC 6519.
Diaporthe xishuangbanica Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the locality, Xishuangbanna.
Diagnosis: Diaporthe xishuangbanica can be distinguished from the phylogenetically closely related D. tectonigena in several loci (98% in ITS, 90% in TEF1, and 96% in TUB) (Fig. 2), and from other Diaporthe species reported from Camellia in the longer conidiophores and alpha conidia (Table 3).
Type: China: Yunnan Province: Xishuangbanna, on diseased leaves of Camellia sinensis, 19 Apr. 2015, F. Liu (HMAS 247083 — holotype, dried culture; CGMCC 3.18283 = LC 6744 — ex-type culture).
Description: Conidiomata pycnidial, globose, 180–;310 µm diam, scattered on the pine needle. Conidiophores cylindrical, 13–;34.5 × 1.5–;3 µm (x̅ = 20.9 ± 5.2 × 2.1 ± 0.3, n = 40), branched, septate, straight, sometimes sinuous or lateral. Alpha conidia 7–;9.5 × 2.5–;3.5 µm (x = 8.3 ± 0.7 × 2.8 ± 0.3, n = 30), fusiform, hyaline, multi-guttulate. Beta conidia not observed.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 17.5 mm diam/d. Colony entirely white, reverse pale yellowish to greenish. Aerial mycelium white, velvety, margin well defined, with visible conidiomata at maturity.
Additional material examined: China: Yunnan Province: Xishuangbanna, on diseased leaves of Camellia sinensis, 19 Apr. 2015, F. Liu, culture LC 6707 (CGMCC 3.18282).
Diaporthe yunnanensis Y.H. Gao & L. Cai, sp. nov.
Etymology: Named after the location where the fungus was collected, Yunnan Province.
Diagnosis: Diaporthe yunnanensis can be distinguished from the phylogenetically closely related D. siamensis (96% in ITS, 91% in TEF1, and 94% in TUB) (Fig. 2), and from other Diaporthe species reported on the genus Camellia in the smaller alpha conidia (Table 3).
Type: China: Yunnan Province: Xishuangbanna, on healthy leaves of Coffea sp., 20 Sep. 2014, W.J. Duan (HMAS 247096 — holotype, dried culture; CGMCC 3.18289 = LC 6168 — ex-type culture).
Description: Conidiomata pycnidial, 195–;880 µm diam, globose or irregular, erumpent, solitary or aggregated together, dark brown to black. Conidia exuding from the pycnidia in white to cream drops. Conidiophores cylindrical, straight or slightly curved. Alpha conidia 3–;6.5 × 1–;2.5 µm (x̅ = 5.5 ± 1 × 2 ± 0.5, n = 30), fusiform, hyaline, biguttulate, with one end obtuse and the other acute. Beta conidia 13.5–;33.5 × 1–;1.5 µm (x̅ = 27.5 ± 5.5 × 1.5 ± 0.3, n = 30), hyaline, aseptate, hamate or curved, base truncate.
Culture characters: Colonies on PDA flat, with a moderate growth rate of 5.5 mm diam/d, with abundant dirty white and yellowish pigmented mycelium, dry, felted, extensive thin, and in reverse the centre cream, with zone rings of pale to dark brownish pigmentation.
Additional material examined: China: Yunnan Province: Xishuangbanna, on healthy leaves of Coffea sp., 20 Sep. 2014, W.J. Duan, culture LC 8106; ibid. culture LC 8107.
Diaporthe sp. 1
Description: Conidiomata pycnidial, subglobose to globose, dark brown to black, deeply embedded in the substrate, scattered on the substrate surface, embedded in PDA, clusters in group of 2–;7 pycnidia, 268–;509 µm, yellowish drop of conidia diffusing from the central ostioles. Conidiophores 6.5–;19.5 × 1–;3 µm, cylindrical, hyaline, septate, branched, straight to sinuous, base inflated, slightly tapering towards the apex. Alpha conidia 7.5–;13.5 × 2–;3.5 µm (x̅ = 9.9 ± 1.4 × 2.8 ± 0.4, n = 30), unicellular, hyaline, fusoid to ellipsoid or clavate, two or several large guttulae observed, base subtruncate. Beta conidia 15–;10.5 × 1–;2.5 µm (x̅ = 26.0 ± 5.8 × 1.8 ± 0.5, n = 30), smooth, hyaline, curved, base subtruncate, tapering towards one apex.
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate 7.83 mm diam/day. Colony entire, white to dirty pink, cottony, sparse, brownish to black conidiomata erumpent at maturity, coated with white hypha, granular at margin, reverse pale brown, with brownish dots when maturity.
Material examined: China: Zhejiang Province: Gutianshan Nature Reserve (29°20′ N 18°14′ E), on leaves of Alnus mill, Jan. 2010, Y.Y. Su (culture CGMCC 3.18292 = LC 0771).
Notes: The present culture belongs to the Diaporthe eres complex, which is reported from a very wide range of host plants and includes mostly opportunistic pathogens or secondary invaders on saprobic host substrata (Udayanga et al. 2014a, Gao et al. 2016). Species delimitation in this complex is currently unclear. Udayanga et al. (2015) accepted nine phylogenetic species in the D. eres complex, including D. alleghaniensis, D. alnea, D. bicincta, D. celastrina, D. eres, D. helicis, D. neilliae, D. pulla, and D. vaccinia. Gao et al. (2016) examined 17 isolates belonging to the D. eres complex, and reported that many presented intermediate morphology among “species” and the phylogenetic analyses often resulted in ambiguous clades with short branch and moderate statistical support. The identification of taxa in this group remains unresolved.
Diaporthe sp. 2
Culture characters: Cultures incubated on PDA at 25 °C in darkness, growth rate, slow, 3.83 mm diam/d. Colony low, convex, entire white to yellowish, reverse brownish. Aerial mycelia white, dry, downy, with near-circular margin.
Material examined: Japan: on leaves of Acer sp., 20 Sep. 2014, W.J. Duan, culture CGMCC 3.18291 = LC 6140, culture LC 8112; ibid. culture LC 8113.
Notes: Although three isolates clustered in a clade distinctly different from known species in the genus included, they are not formally described because they were sterile. Diaporthe sp. 2 shares a low homology to the most closely related species, D. rhoina (95% in ITS, 87% in TEF1, 97% in TUB, 94% in HIS, and 95% in CAL). Five Diaporthe species are so far only known from the sterile state, including D. endophytica, D. inconspicua, D. infecunda, D. asheicola, and D. sterilis (Gomes et al. 2013, Lombard et al. 2014).
Diaporthe averrhoae (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis averrhoae C.Q. Chang et al., My-cosystema 24: 6 (2005).
Type: China: Fujian Province: on living branches of Averrhoa carambola, Y.H. Cheng (SCHM 3605 — holotype; AY618930, ITS sequence derived from the holotype SCHM 3605).
Diaporthe camptothecae (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis camptothecae C.Q. Chang et al., My-cosystema 24: 145 (2005).
Type: China: Hunan Province: on living branches of Camptotheca acuminate, L.J. Luo (SCHM 3611 — holotype; AY622996, ITS sequence derived from the holotype SCHM 3611).
Diaporthe chimonanthi (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis chimonanthi C.Q. Chang et al., My-cosystema 24: 146 (2005).
Type: China: Hunan Province: on living branches of Chimonanthus praecox, C.Q. Chang (SCHM 3614 — holotype; AY622993, ITS sequence derived from the holotype SCHM 3614).
Diaporthe eucommiae (F.X. Cao et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis eucommiae F.X. Cao et al., J. Middle-South China Forestry Coll. 10: 34 (1990); as ‘eucommi’
Type: China: Guangdong Province: from leaves of Eucommia ulmoides, F.X. Cao (SCHM 0020 — holotype; AY601921, ITS sequence derived from the holotype SCHM 0020).
Diaporthe eucommiicola (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis eucommiicola C.Q. Chang et al., My-cosystema 24: 147 (2005).
Type: China: Hunan Province: on living branches of Eucommia ulmoides and Styrax hypoglauca, L.J. Luo (SCHM 3607 — holotype; AY578071, ITS sequence derived from the holotype SCHM 3607).
Diaporthe glabrae (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis glabrae C.Q. Chang et al., My-cosystema 24: 8 (2005).
Type: China: Fujian Province: on living branches of Bougainvillea glabra, Y.H. Cheng (SCHM 3622 — holotype; AY601918, ITS sequence derived from the holotype SCHM 3622).
Diaporthe lagerstroemiae (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis lagerstroemiae C.Q. Chang et al., My-cosystema 24: 148 (2005).
Type: China: Hunan Province: on living branches of Lagerstroemia indica, C.Q. Chang (SCHM 3608 — holotype; AY622994, ITS sequence derived from the holotype SCHM 3608).
Diaporthe liquidambaris (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis liquidambaris C.Q. Chang et al., My-cosystema 24: 9 (2005).
Type: China: Fujian Province: on living branches of Liquidambar formosana, Y.H. Cheng (SCHM 3621 — holotype; AY601919, ITS sequence derived from the holotype SCHM 3621).
Diaporthe loropetali (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis loropetali C.Q. Chang et al., My-cosystema 24: 148 (2005).
Type: China: Hunan Province: on living branches of Loropetalum chinense, C.Q. Chang (SCHM 3615 — holotype; AY601917, ITS sequence derived from the holotype SCHM 3615).
Diaporthe magnoliicola Y.H. Gao & L. Cai, nom. nov.
Replaced name: Phomopsis magnoliae M.M. Xiang et al., My-cosystema 21 : 501 (2002).
Type: China: Guangdong Province: on leaves of Magnolia coco, Z.D. Jiang (SCHM 3001 — holotype; AY622995, ITS sequence derived from the holotype SCHM 3001).
Note: The epithet magnoliae is occupied, so Diaporthe magnoliicola is proposed as a replacement name.
Diaporthe michelina (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov.
Basionym: Phomopsis michelina C.Q. Chang et al., My-cosystema 24: 9 (2005); as ‘micheliae’.
Type: China: Fujian Province: on living branches of Michelia alba, Y.H. Cheng (SCHM 3603 — holotype; AY620820, ITS sequence derived from the holotype SCHM 3603).
Diaporthe phyllanthicola (C.Q. Chang et al.) Y.H. Gao & L. Cai, comb. nov. MycoBank MB821461
Basionym: Phomopsis phyllanthicola C.Q. Chang et al., My-cosystema 24: 10 (2005).
Type: China: Fujian Province: on living branches of Phyllanthus emblica, Y.H. Cheng (SCHM 3680 — holotype; AY620819, ITS sequence derived from the holotype SCHM 3680).
In this study, eight new species of Diaporthe are introduced, having been isolated from various plant hosts collected in different countries. Twelve Phomopsis species described from China were subjected to molecular analysis, and transferred to Diaporthe to conform to the “one fungus one name” rule (Udayanga et al. 2011, Rossman et al. 2016). To address the taxonomy of the other Phomopsis species described from China, neo- or epitypes will need to be designated to resolve their position and confirm their placement in Diaporthe.
Previous taxonomic studies in Diaporthe (syn. Phomopsis) have been primarily based on morphology, which has been shown to be unnatural in reflecting evolutionary history due to the simple and plastic morphological characters (Gao et al. 2015). The same applies to many other genera of ascomycetes. For example, species referred to Phoma have been shown to be highly polyphyletic and scattered throughout at least six families within Pleosporales (Aveskamp et al. 2010, Chen et al. 2015). Although Diaporthe was previously thought to be monophyletic based on its typical and unique Phomopsis asexual morph and diaporthalean sexual morph (Gomes et al. 2013), a paraphyletic nature is revealed in the present study (Fig. 1). Several genera, notably Ophiodiaporthe (Fu et al. 2013), Pustulomyces (Dai et al. 2014), Phaeocytostroma, and Stenocarpella (Lamprecht et al. 2011), are shown to be embedded in Diaporthe s. lat., none of which present an independent lineage from Diaporthe as currently circumscribed (Fig. 1). These genera were established based on their morphological characteristics (Vasilyeva et al. 2007, Lamprecht et al. 2011, Fu et al. 2013, Dai et al. 2014). For example, Ophiodiaporthe produces only one type of globose or subglobose conidia that differs from the dimorphic (fusiform and filiform) conidia of Diaporthe (Fu et al. 2013); Phaeocytostroma and Stenocarpella produce pigmented alpha conidia which differ from the hyaline conidia of Diaporthe (Lamprecht et al. 2011); Pustulomyces produces larger, straight or sigmoid conidia (Dai et al. 2014). Phaeocytostroma and Stenocarpella were originally suspected to be members of Botryosphaeriaceae (Botryosphaeriales) because of their pigmented alpha conidia and diplodia-like morphology (Crous et al. 2006). However, they were subsequently allocated to Diaporthales based on phylogenetic analysis (Lamprecht et al. 2011), which is confirmed in this study.
The large “Diaporthe” clade embedded with the heterogeneous genera Ophiodiaporthe, Pustulomyces, Phaeocytostroma, and Stenocarpella is probably a typical example of divergent evolution in morphological characters. Such an evolution could have been driven by host and/or environmental adaptations. For example, the monotypic Ophiodiaporthe is associated with Cyathea lepifera (a fern), while Pustulomyces is bambusicolous (Dai et al. 2014). On the contrary, none of the previously named over 1 900 Diaporthe / Phomopsis species was recorded from a fern or Bambusaceae (https://nt.ars-grin.gov/fungaldatabases/). It is therefore reasonable to speculate that the speciation of Ophiodiaporthe and Pustulomyces, as well as the distinctly different morphologies from their close Diaporthe allies, are the consequences of evolutionary adaption to new hosts. Similarly, Phaeocytostroma and Stenocarpella are mainly restricted to maize (Zea mays), causing root stalk and cob rot (Stovold et al. 1996, Lamprecht et al. 2011).
Splitting Diaporthe into many smaller genera would achieve monophyletic groupings, but would also create many additional problems. The “new genera” split from Diaporthe would have no recognisable morphological distinctions in either sexual or asexual morphs. In addition, splitting Diaporthe into many smaller genera will result in numerous name changes, which is certainly an unfavourable option for both mycologists and plant pathologists.
Diaporthe has long been well-known to include plant pathogens, some on economically important hosts, such as Helianthus annuus (sunflower; Thompson et al. 2011) and Glycine max (soybean; Santos et al. 2011). However, the number of known endophytic Diaporthe species has increased rapidly in recent years (Huang et al. 2015, Gao et al. 2016). Wang et al. (2013) concluded that our current knowledge of the ecology and biology of endophytic Diaporthe species is just the “tip of the iceberg”. In 2013, a new sterile endophytic species, Diaporthe endophytica, was formally named (Gomes et al. 2013). The research on Citrus conducted by Huang et al. (2015) recorded seven apparently undescribed endophytic Diaporthe species. Inspection of Diaporthe species on Camellia sinensis resulted in the description of four new and five known species, all occurring as endophytes (Gao et al. 2016). Because many of these plant pathogenic Diaporthe species are commonly encountered as sterile endophytes, a multigene DNA database will be essential to aid in theirfuture identification.
Accurate identification of fungal pathogens is the basis of quarantine and disease control (Udayanga et al. 2011). Thompson et al. (2011) reported significant damage to sunflower in Australia caused by Diaporthe helianthi which was originally only known from Europe (former Yugoslavia), and is apparently an invasive species in Australia. Diaporthe helianthi is listed in the Chinese quarantine directory, and has long been considered a predominant disease limiting production in Europe (Desanlis et al. 2013). Duan et al. (2016) reported this pathogen on sunflower seeds imported from Ukraine into China. Here, we report another interception of D. helianthi from Lagerstroemia indica imported from Japan to China. This serves as additional evidence of how quickly serious pathogens such as Diaporthe species can be distributed as endophytes or latent pathogens with global trade.
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We thank all the members in LC’s lab for help and assistance. This work was supported by grants from the National Natural Science Foundation of China (NSFC 31110103906), and the Ministry of Science and Technology, China (MOST 2014FY120100).
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Gao, Y., Liu, F., Duan, W. et al. Diaporthe is paraphyletic. IMA Fungus 8, 153–187 (2017) doi:10.5598/imafungus.2017.08.01.11