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One stop shop: backbones trees for important phytopathogenic genera: I (2014)


Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

Contents and contributors (main contributors underlined)

  1. 1.

    BipolarisDS Manamgoda, KD Hyde

  2. 2.

    BotryosphaeriaceaeAJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

  3. 3.

    BotryosphaeriaAJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

  4. 4.

    BotrytisRB Terhem, M Hahn, JAL van Kan

  5. 5.

    ChoanephoraJ Pawłowska, G Walther, M Wilk, M Gorczak, M Wrzosek

  6. 6.

    ColletotrichumRS Jayawardena, DS Manamgoda, L Cai, XH Li, JY Yan, KD Hyde

  7. 7.

    CurvulariaDS Manamgoda, KD Hyde

  8. 8.

    DiaportheAJ Dissanayake, D Udayanga, JY Yan, XH Li, KD Hyde

  9. 9.

    Diplodia AJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

  10. 10.

    Dothiorella JK Liu, KD Hyde

  11. 11.

    FusariumB Summerell, MH Laurence

  12. 12.

    GilbertellaJ Pawłowska, G Walther, M Wilk, M Gorczak, M Wrzosek

  13. 13.

    Lasiodiplodia JK Liu, KD Hyde

  14. 14.

    MucorJ Pawłowska, G Walther, M Wilk, M Gorczak, M Wrzosek

  15. 15.

    Neofusicoccum AJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

  16. 16.

    PestalotiopsisSSN Maharachchikumbura SA Alias KD Hyde

  17. 17.

    PhyllostictaN Zhou, L Cai

  18. 18.

    PhytophthoraF Martin, JE Blair

  19. 19.

    PucciniaAR McTaggart, RG Shivas

  20. 20.

    PyrenophoraHA Ariyawansa, KD Hyde

  21. 21.

    PythiumCFJ Spies, TL Rintoul, AWAM de Cock, SL Glockling, CA Lévesque

  22. 22.

    RhizopusM Gorczak, G Walther, J Pawłowska, M Wilk, M Wrzosek

  23. 23.

    StagonosporopsisN Vaghefi, PWJ Taylor

  24. 24.

    UstilagoAR McTaggart, RG Shivas

  25. 25.

    VerticilliumPWJ Taylor, PVR Nair


Fungi form a large and heterogeneous eukaryotic kingdom with an estimated 1.5 million extant species. While all fungi are heterotrophic, a wide range of nutritional strategies is known in the kingdom. Most of the ca. 100,000 described species of fungi are associated with plants through interactions including symbiosis, endophytism, saprotrophy and parasitism (Stajich et al. 2009; Delaye et al. 2013; Persoh 2013; Hyde et al. 2013b). As plant parasites, fungi can cause significant economic loss with far-reaching social implications and consequences in agriculture, forestry and natural ecosystems (Fisher et al. 2012). They are also part of the natural ecosystem and play an important role in regulation of species (Hantsch et al. 2014).

The study of plant pathogenic fungi–their systematics, biology, and biological control–has a long and rich history (Udayanga et al. 2011; Maharachchikumbura et al. 2011; Manamgoda et al. 2011). The inconspicuous nature of most fungi makes their study difficult. For example, there are typically few discriminatory morphological characters, which often makes precise field- or laboratory-based identification problematic. Morphological characters that vary with the choice of host or environmental conditions form an additional, serious concern. Many species are difficult or impossible to keep alive in culture, which excludes them from physiological and often molecular tests that are available. The formation of sexual fruiting bodies is rarely observed in many plant pathogenic fungi, which has hampered their integration in the fungal tree of life, resulting in the proliferation of polyphyletic asexual genera. The biology of most plant pathogenic fungi remains poorly understood.

The last 25 years have witnessed the emergence of molecular data (DNA sequences) as a source of high fidelity information that has revolutionised mycology (Nilsson et al. 2014). DNA sequences offer a means by which to examine and compare fungi, independent of morphological plasticity, cultivability, and host-pathogen interactions. Since integration of molecular data in the study of plant pathogenic fungi in the early 1990s, there has been a much deeper understanding of the ecology, distribution, and systematics of plant pathogenic fungi (Bridge et al. 2005; Wingfield et al. 2012; Udayanga et al. 2013; Manamgoda et al. 2013). The use of molecular data in diagnostics and systematic studies is not without pitfalls and shortcomings that researchers must consider (Kang et al. 2010; Ko et al. 2011; Hyde et al. 2013a). Synonyms, homonyms, asexual-sexual relationships, ambiguous and misidentified specimens are rife in the plant pathology literature and public databases of DNA sequences, which posses an enormous challenge for the unwary. Equally challenging is the large number of unidentified and seemingly unidentifiable fungi and fungal sequences isolated from plants (Kõljalg et al. 2013; Unterseher et al. 2013). Certain plant pathogenic fungi require specialized extraction and PCR primers/protocols in order to amplify their DNA. Furthermore, the same genetic markers that give unparalleled phylogenetic resolution in some fungi may give none whatsoever in others. Many plant pathology studies focus on single species of fungi, and recent revisions or synopses at the generic or higher levels are lacking for the majority of plant pathogenic fungi.

The present study seeks to facilitate present and future studies of plant pathogenic fungi by providing phylogenetic backbone trees for as many groups of fungi as our expertise allowed. Our ambition is to synthesize all recent molecular data, recommendations on correct names, type material, geo/ecological observations, literature, and lineage-specific laboratory advice into a comprehensive, uniform molecular treatise for some of the largest and most widely encountered lineages of plant pathogenic fungi.

Material and methods

The phylogenetic analyses were performed based on up to date ex-type, ex-epitype or otherwise authentic sequence data available in GenBank as a concerted effort of the multiple contributors listed in authors section. By authentic sequence data we refer to those sequences used for names that are considered by the current working groups with the support of reliable publications in each genus as representative for each species. Sequences for the genes and genetic markers recommended for each genus were selected based on the current publications and have commonly been used for each of the genera (Table 1). The single gene sequence alignments were initially aligned with Clustal X and improved in MAFFTv. 7.017 (Katoh et al. 2002). Individual alignments were then concatenated and used to construct the backbone trees of each pathogenic genus listed. The phylogenetic analyses were performed for maximum parsimony in PAUP v. 4.0b10 (Swofford 2002), maximum likelihood in RAxML 7.4.2 Black Box or RAxMl GUI (Stamatakis 2006; Stamatakis et al. 2008), PhyML 3.0 (Guindon et al. 2010) or Bayesian inference in MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) as specified in the legend of each phylogenetic tree. The trees used to represent each genus were analyzed by multiple contributors based on the selection of genes in given publications under each description.

Table 1 Gene regions and primers
Table 2 Bipolaris. Details of the isolates used in the phylogenetic tree
Table 3 Botryosphaeriaceae. Details of the isolates used in the phylogenetic tree
Table 4 Botryosphaeria. Details of the ex-type and voucher isolates used in the phylogenetic tree
Table 5 Botrytis. Details of the isolates used in the phylogenetic tree
Table 6 Choanephora. Details of the isolates used in the phylogenetic tree
Table 7 Colletotrichum. Details of the isolates used in the phylogenetic tree
Table 8 Details of the isolates used in the phylogenetic tree
Table 9 Diaporthe. Details of the isolates used in the phylogenetic tree
Table 10 Diplodia. Details of the isolates used in the phylogenetic tree
Table 11 Dothiorella. Details of the isolates used in the phylogenetic tree
Table 12 Fusarium. Details of the isolates used in the phylogenetic tree
Table 13 Gilbertella. Details of the isolates used in the phylogenetic tree
Table 14 Lasiodiplodia. Details of the isolates used in the phylogenetic tree
Table 15 Mucor. Details of the isolates used in the phylogenetic tree
Table 16 Neofusicoccum. Details of the isolates used in the phylogenetic tree
Table 17 Pestalotiopsis. Details of the isolates used in the phylogenetic tree
Table 18 Phyllosticta . Details of the voucher and extype isolates used in the phylogenetic tree
Table 19 Pythium. Strain numbers, host information and GenBank accession numbers for species included in Fig. 21
Table 20 Pyrenophora. Details of the isolates used in the phylogenetic tree
Table 21 Puccinia. Details of the isolates used in the phylogenetic tree
Table 22 Rhizopus. Details of the isolates used in the phylogenetic tree
Table 23 Stagonosporopsis. Details of the isolates used in the phylogenetic tree
Table 24 Ustilago. Details of the isolates used in the phylogenetic tree
Table 25 Verticillium. Details of the isolates used in the phylogenetic tree

Backbone tree for important phytopathogens

Condensed synopses are provided for 25 important plant pathogenic group or genera. Each synopsis includes notes on background, species identifications and numbers, molecular phylogeny, recommended genetic markers, tables of species and the latest phylogenetic trees. We have not been able to include all important phytopathogenic genera (e.g. Alternaria, powdery mildews), but intend to update or add these in future publications. Interested parties should contact the corresponding author.



The genus Bipolaris belongs to the family Pleosporaceae of the Pleosporales in Dothideomycetes (Ascomycota). Bipolaris was introduced by Shoemaker (1959) and typified with B. maydis. Bipolaris species are pathogens, saprobes or endophytes mostly associated with grasses including cultivated cereals. Some species are important plant pathogens. The Bengal famine in 1943 was caused by B. oryzae and caused 90 % of crop losses in India as well as the loss of 1.5 million human lives (Scheffer 1997). In the 1970s, around 19 million metric tons of wheat were destroyed in the USA due to southern corn leaf blight caused by B. maydis. Bipolaris sorokiniana causes southern leaf blotch, seedling blight and crown rot. Bipolaris sorokiniana was confirmed as the most economically important foliar pathogen in warm areas by the conference “Wheat for the national warm areas” held in Brazil in 1990. Bipolaris species have also been recorded from other plant families such as Alliaceae, Anacardiaceae, Araceae, Euphorbiaceae, Fabaceae, Malvaceae, Rutaceae and Zingiberaceae (Manamgoda et al. 2011).

Species identification and numbers

Bipolaris species were formerly described in Helminthosporium, however, species associated with grasses were morphologically distinct from H. velutinum, the type species (Luttrell 1963; Ellis 1971; Alcorn 1988). In several taxonomic refinements, these graminicolous Helminthosporium species were segregated into four genera; Bipolaris, Curvularia, Drechslera and Exserohilum (Sivanesan 1987). Later Subramanian and Jain (1966) placed all Bipolaris species in Drechslera, but this transfer was not accepted by later authors (Sivanesan 1987; Alcorn 1988). After molecular data became available, Drechslera was shown to be a phylogenetically different genus from Bipolaris (Berbee et al. 1999). The sexual state of Bipolaris is Cochliobolus (Drechsler 1934). Cochliobolus is the older name but conservation of the name Bipolaris over Cochliobolus has been proposed to avoid numerous name changes and Bipolaris is the most common name among plant pathologists (Manamgoda et al. 2012a; Rossman et al. 2013).

Morphology-based classification of Bipolaris species is challenging as the asexual state has overlapping conidia and conidiophore dimensions (Sivanesan 1987). A few Bipolaris species are known to be host-specific, while most of the other species are generalists (Manamgoda et al. 2011). However, some of the host-specific species are known only from limited collections. Therefore, the information on host-specificity may change with further collections. Interspecific compatibility can be observed between some taxa. For example, successful hybridization leading to ascospore production has been reported between B. zeicola and B. victoriae (Nelson 1960a, b) as well as between B. maydis and B. oryzae (Alcorn 1988). However, the latter species are definitively distinct phylogenetic species and also they are commonly recorded pathogens, causing different symptoms on their respective hosts. Identification of Bipolaris species using morphological and biological species concepts is not always correct and it is essential to use molecular tools in identifying species. Lack of DNA sequences from type material/ex-type cultures (or other authentic material) in public sequence databases is a problematic issue regarding the molecular identification of the Bipolaris species (Nilsson et al. 2014). Currently there are 118 Bipolaris names listed in Index Fungorum (2014), but nine of them do not belong to this genus based on phylogenetic evidence.

Molecular phylogeny

The first phylogenetic analysis for Bipolaris with its sister genus Curvularia was carried out by Berbee et al. (1999) and Goh et al. (1998) using a combined ITS and GPDH analysis. These studies showed that Bipolaris species cluster in two clades. Combined ITS, GPDH, EF and LSU phylogenetic analysis for Bipolaris and Curvularia by Manamgoda et al. (2012a) showed that Bipolaris and Curvularia cluster into two major clades. Nine Bipolaris species clustered with the generic type, Curvularia lunata Boedijn, while other species of Bipolaris clustered with the generic type, Bipolaris maydis. Accordingly, the nine Bipolaris species were moved to Curvularia, and Bipolaris was maintained as a distinct genus based on the generic type and those species that clustered with it. In this section we provide a backbone tree (Table 2, Fig. 1) for Bipolaris using combined ITS and GPDH sequence data.

Fig. 1

Phylogram generated from parsimony analysis based on combined ITS and GPDH sequenced data of Bipolaris. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Curvularia lunata

Recommended genetic markers

GPDH is the best single genetic marker for the genus Bipolaris (Manamgoda et al. 2012a). Combined ITS, EF and GPDH can resolve almost all species of Bipolaris currently known from sequence data (Manamgoda et al. 2012a).


The family Botryosphaeriaceae is classified in the order Botryosphaeriales of the Dothideomycetes (Ascomycota). Members of the fungal family Botryosphaeriaceae were described in the 1820’s as species of Sphaeria (Fr.) (Crous et al. 2006; Schoch et al. 2006). There have subsequently been various treatments of the family. von Arx and Müller (1954) included 15 genera, but later reduced it to 14 genera (von Arx and Müller 1975). Barr (1987) included only nine genera, which are mostly different from those of von Arx and Müller (1954). Hawksworth et al. (1995) listed five genera. Lumbsch and Huhndorf (2010) included 11 genera, while Hyde et al. (2011) and Wijayawardene et al. (2012) listed 16 genera. Liu et al. (2012) included 17 genera in the family based on molecular data and examination of generic types. Species of Botryosphaeriaceae range in habit from saprobic to parasitic or endophytic (Smith et al. 1996; Denman et al. 2000; Phillips et al. 2006; Slippers and Wingfield 2007; Huang et al. 2008; Pérez et al. 2010; Ghimire et al. 2011; González and Tello 2011). Members are cosmopolitan in distribution and occur on a wide range of monocotyledonous, dicotyledonous and gymnosperm hosts; on woody branches, herbaceous leaves, stems and culms of grasses; and on twigs and in the thalli of lichens (Barr 1987; Denman et al. 2000; Mohali et al. 2007; Lazzizera et al. 2008; Marincowitz et al. 2008).

Species identification and numbers

Currently, more than 2,000 species names are linked to Botryosphaeriaceae, including sexual and asexual states of Diplodia, Botryosphaeria, Fusicoccum, Dothiorella, Lasiodiplodia and Sphaeropsis. Identification to genus and species is presently undergoing major revision and it is likely that many older names will not be used in modern treatments. Identification of species in Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia and Neofusicoccum are dealt separately under this family entry.

Molecular phylogeny

Recent advances in DNA-based molecular techniques have begun to provide efficient tools to characterize the presence and identity of species of the Botryosphaeriaceae (Slippers and Wingfield 2007). Studies applying these tools are revealing significantly greater diversity on some hosts than was previously realized. Recent studies on the taxonomy of Botryosphaeria have employed molecular methods to reveal phylogenetic relationships among species (Jacobs and Rehner 1998) and to resolve species complexes (Denman et al. 2003; Alves et al. 2004; Phillips et al. 2005). Two major clades corresponding to species with Diplodia and Fusicoccum asexual morphs were revealed based on ITS phylogenies (Jacobs and Rehner 1998; Denman et al. 2003). Later studies, including additional species and a larger suite of genetic markers, supported this grouping (Zhou and Stanosz 2001; Alves et al. 2004; Slippers et al. 2004d). Lasiodiplodia has been treated as a distinct genus from Diplodia by many authors due to its distinct phylogeny (usually ITS or EF-1α) and morphology (striated or smooth conidia and presence or absence of pseudoparaphyses). Pavlic et al. (2004) employed morphological and phylogenetic data to separate Lasiodiplodia from Diplodia. The value of the intron-dominated sequences of the ITS, β-tubulin and TEF markers (on which most previous studies were based) to infer phylogenetic relationships across the diversity of the genus is, however, unclear. The more conserved mtSSU data have, for example, suggested that B. dothidea and B. corticis (Demaree and Wilcox) are unrelated to Fusicoccum (Zhou and Stanosz 2001) even though they are typically assigned to this genus.

Most taxonomic studies on Botryosphaeriaceae using molecular data have employed ITS rDNA phylogenies, but this single marker can underestimate the species diversity among closely related or cryptic species. Multiple gene sequence concordance phylogenies have therefore been applied to identify cryptic or previously overlooked species of Botryosphaeriaceae (Slippers et al. 2004a, b, c; Burgess et al. 2005; Phillips et al. 2005). As the elongation fctor 1- alpha (TEF) gene is consistently more variable than the ITS rDNA region in these fungi, most commonly data from TEF have been combined with ITS sequence data. Unfortunately no single genetic region is sufficient to distinguish all species, because not all single nucleotide polymorphisms (SNPs) represent restriction sites, especially between some closely related species.

The Botryosphaeriaceae has been separated into numerous distinct genera (Crous et al. 2006; Liu et al. 2012). A natural classification is needed for a more stable and accurate taxonomic framework and this will strongly influence the understanding of the ecology of the Botryosphaeriaceae. In this part we provide a tree to the genera of Botryosphaeriaceae (Table 3, Fig. 2) and deal with the important genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia and Neofusicoccum in the following parts.

Fig. 2

Phylogram generated from parsimony analysis based on combined SSU, LSU, TEF, β-tubulin and ITS sequence data of Botryosphaeriaceae. Parsimony bootstrap support values greater than 50 % and Bayesian posterior probabilities greater than 0.5 are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Melanops tulasnei CBS 116805

Fig. 3

Phylogram generated from parsimony analysis based on combined ITS, TEF, β- tubulin, LSU and SSU sequenced data of Botryosphaeria. Parsimony bootstrap support values greater than 50 % and Bayesian posterior probabilities greater than 0.5 are indicated near the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Macrophomina phaseolina CBS 227.33

Recommended genetic markers

  • LSU, SSU, β-tubulin and ITS–generic level

  • TEF–species level

LSU has been shown to be suitable for distinguishing many ascomycetes at the generic level due to its relatively conserved nature (Crous et al. 2006; Schoch et al. 2006; Hibbett et al. 2007). The study of Liu et al. (2012) suggested that the combined TEF and β- tubulin gene analysis is best for delimiting genera of Botryosphaeriaceae. It has also been recommended that the RPB2 gene should be considered in similar combined analyses of genus and species levels of Botryosphaeriaceae (Pavlic et al. 2009a, b).



The genus Botryosphaeria (Botryosphaeriaceae) was introduced by Cesati and de Notaris (1863), amended by Saccardo (1877), and is based on the type species Botryosphaeria dothidea (Barr 1972; Slippers et al. 2004c). Species in Botryosphaeria were described largely on the basis of the morphology of their ascomata and host associations, and this has led to a proliferation of names. von Arx and Müller (1954) examined 183 taxa of Botryosphaeriales and reduced them to 11 species, with extensive synonymies under B. dothidea and B. quercuum, together with nine new combinations. In later studies these synonymies were not always accepted (Shoemaker 1964; Sivanesan 1984; Slippers et al. 2004a). Slippers et al. (2004b) epitypified the type species Botryosphaeria dothidea based on morphology and phylogeny (combined ITS, TEF and β-tubulin analysis) and this enabled a better resolution of species. Species of Botryosphaeria occur on a wide range of monocotyledonous, dicotyledonous and gymnosperm hosts, on woody branches, herbaceous leaves and grasses (Barr 1987). The life styles may be saprobic, parasitic and endophytic (Smith et al. 1996; Denman et al. 2000), and species can cause die-back and canker diseases of numerous woody hosts (von Arx 1987). Species in the genus Botryosphaeria have hyaline to dark ascospores, multiloculate ascomata, and a wide range of asexual morphs that typically lack a mucoid sheath and apical appendage.

Species identification and numbers

More than 18 asexual genera have been associated with Botryosphaeria. A phylogenetic study based on part of the 28S ribosomal DNA gene together with morphological characters revealed that Botryosphaeria comprises several distinct lineages, each comprising individual genera (Crous et al. 2006). In that study, only B. dothidea and B. corticis were retained in Botryosphaeria, while most species were reduced to synonymy under Diplodia (conidia mostly ovoid, pigmented, thick-walled), or Fusicoccum (conidia mostly fusoid, hyaline, thin-walled). Studies have also linked Botryosphaeria to species with pigmented, septate ascospores and Dothiorella asexual morphs, or Fusicoccum asexual morphs with Dichomera synanamorphs. More recently B. agaves (which has been epitypified), B. fusispora (Liu et al. 2012), and B. schariffi (Abdollahzadeh et al. 2013) were described in the genus Botryosphaeria, while B. fabicerciana was illustrated from Eucalyptus sp. in southern China (Chen et al. 2011). Phylogenetically, B. fabicerciana is closely related to B. corticis, B. dothidea, B schariffi and B. ramosa. The present phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 4).

Molecular phylogeny

Recent studies on the taxonomy of Botryosphaeria have employed molecular methods to reveal phylogenetic relationships among species (Jacobs and Rehner 1998) and to resolve species complexes (Smith and Stanosz 2001; Phillips et al. 2002, 2005; Denman et al. 2003; Alves et al. 2004; Slippers et al. 2004c). Studies including additional species and a larger suite of DNA-based markers supported this grouping (Zhou and Stanosz 2001; Alves et al. 2004; Slippers et al. 2004c). Based on combined ITS and TEF sequence data seven species are currently recognised in Botryosphaeria (Phillips et al. 2013). The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 3.

Fig. 4

Phylogram generated from Maximum likelihood analysis based on combined sequences of G3PDH, HSP60 and RPB2 from 28 recognized Botrytis species. Bootstrap support values greater than 50 % are indicated above/below the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Monilinia fructigena and Sclerotinia sclerotiorum

Fig. 5

Phylogram generated from Maximum likelihood analysis based on ITS sequenced data of Choanephora. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold

Fig. 6

Phylogram generated from parsimony analysis based on combined ITS, GADPH, CHS-1, ACT, HIS and β- tubulin data of Colletotrichum. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Monilochaetes infuscans CBS 869.96

Fig. 7

Phylogram generated from parsimony analysis based on combined ITS, GADPH, CHS-1, ACT, HIS and β- tubulin sequenced data of Colletotrichum acutatum complex. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with C. orchidophilum

Fig. 8

Phylogram generated from parsimony analysis based on combined ITS, GADPH, CHS-1, ACT, HIS and β- tubulin sequenced data of Colletotrichum gloeosporioides complex. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with C. coccodes ITCC6079

Recommended genetic markers

  • LSU, SSU and ITS–generic level

  • β-tubulin and TEF–species level



Erected by Micheli in 1729, the genus Botrytis is one of the first described genera of fungi. Persoon (1801) designated five species under the binomial system of Linnaeus, validated the genus, and included one of Micheli’s species, B. cinerea, so named by Von Haller (1771). The genus name refers to the structure of the macroconidia, which rise and form clusters with the shape of grape bunches: ‘botryose’. Botrytis is the asexual stage of Botryotinia. The Botrytis community has in its recent meeting (Italy, 23–28 June 2013) unanimously recommended the exclusive use of the asexual name Botrytis over Botryotinia, the name of the sexual stage, since Botrytis is historically the oldest name and it is commonly used by plant pathologists, breeders and growers. In line with this recommendation, a list of generic names of fungi for protection under the International Code of Nomenclature has included this genus under the name Botrytis and not Botryotinia (Kirk et al. 2013). We therefore follow this recommendation in this paper and use Botrytis. Species of the genus Botrytis infect >250 host species, including major greenhouse and field crops such as tomato, grape, strawberry, onion and ornamentals such as rose, lily, and tulip (Staats et al. 2005). Most Botrytis species are necrotrophic pathogens that (are able to) kill the host tissue during infection. Interestingly, an endophytic species (B. deweyae) has recently been discovered, which under appropriate conditions can cause ‘spring sickness’ in ornamental Hemerocallis (daylily) hybrids (Grant-Downton et al. 2014). Botrytis cinerea is the best-studied species in the genus (Williamson et al. 2007) and was recently elected as the second most important plant pathogenic fungal species (Dean et al. 2012).

In the asexual state, Botrytis produces different tissues including mycelia, macroconidia, microconidia, and sclerotia. Macroconidia are ellipsoidal to obovoid shape and rise from conidiophore branches into botryose clusters. They are pale brown and range in size from 9–23 × 8–15 μm. Microconidia are more sphaerical and much smaller than macroconidia (about 1 μm), and function as male spermatia (Groves and Loveland 1953; Faretra et al. 1988; Beever and Parkes 1993; Fukumori et al. 2004). Sclerotia are irregularly hemispherical, convex and normally have a concave surface. They are usually black, with sizes ranging between 1 and 10 mm (Whetzel 1945), and function as survival structures during winter and serve as maternal parent in the production of apothecia.

The sexual state forms fruiting bodies called apothecia: a cup- or open saucer-shaped ascoma at the top of a stalk, that acts as a platform to discharge ascospores from the ascus. Botrytis apothecia vary in size depending on the species, between 1 and 25 mm high and 1–6 mm diam. (Hennebert and Groves 1963; Bergquist and Lorbeer 1972). Apothecia are brown and become darker when mature (Hennebert and Groves 1963; Bergquist and Lorbeer 1972; Faretra and Antonacci 1987). Generally multiple apothecia can develop on a single sclerotium. Mature apothecia normally can be observed 2 months after fertilization (Faretra et al. 1988; Hennebert and Groves 1963; Van Der Vlugt-Bergmans et al. 1993). In the genus Botrytis, both homothallic and heterothallic reproductive lifestyles have been reported. Homothallic (self-fertile) species can undergo sexual reproduction and form apothecia and generate progeny in the absence of a mating partner, e.g. B. porri and B. globosa (Buchwald 1953; Elliott 1964). By contrast, heterothallic (self-sterile, self-incompatible) species require isolates with compatible mating types in order to complete the sexual cycle. B. cinerea is considered a typical heterothallic fungus (Elliott 1964; Faretra et al. 1988). Mating is controlled by the mating type locus with two alleles, MAT1-1 and MAT1-2 (Faretra et al. 1988), each carrying two distinct, non-homologous genes (Amselem et al. 2011).

Species identification and numbers

Approximately half of the Botrytis species are named after the host that they are derived from (listed in Table 5). One hybrid species, B. allii which originated from hybridization between B. byssoidea and B. aclada (Nielsen and Yohalem 2001; Yohalem et al. 2003) could not be placed in the phylogeny (Staats et al. 2005) and was omitted from Table 3. The genus Botrytis predominantly comprises narrow host range pathogens that infect a single, or a few (often related) host species. There are two exceptions to this rule: B. cinerea can infect more than 200 host species (Jarvis 1977), and B. pseudocinerea has been isolated from several unrelated host species (Fournier et al. 2005; Leroch et al. 2013).

The taxonomic classification and nomenclature in Botrytis have rarely been comprehensively reviewed. Morphological descriptions of most species have been published in the 19th and first half of the 20th century in separate papers, many of which are not easily accessible. The most recent taxonomic compilation of the genus is in a monograph by Jarvis (1977), which also lists ~25 excluded or doubtful species, and briefly describes the historical debates between mycologists and the confusion in classification of Botrytis species. Morphological features were often inadequate to distinguish species and the variability among isolates of the same species further complicated the situation (Jarvis 1977). Recent studies have identified B. cinerea and B. pseudocinerea as species that are very similar in morphology, yet recognized as distinct taxa that diverged several million years ago (Walker et al. 2011). Even more puzzling, the morphology and narrow host range of B. fabae separate this species clearly from B. cinerea and B. pseudocinerea, but phylogenetic studies revealed it to be a sister species of B. cinerea (see below). These examples illustrate the limitations of morphological characters for Botrytis species identification.

Molecular phylogeny

Holst-Jensen et al. (1998) were the first to use nuclear ribosomal ITS sequences to infer a phylogeny of the family Sclerotiniaceae, including several members of the genus Botrytis. The relationships among many Botrytis species could not be resolved because of the limited number of informative characters, however the study permitted the conclusion that Botryotinia asexual morphs along with Botrytis sexual morphs constitute a monophyletic lineage (Holst-Jensen et al. 1998). The phylogeny of the Sclerotiniaceae was further refined by Andrew et al. (2012) using three protein-coding genes: calmodulin, glyceraldehyde 3-phosphate dehydrogenase G3PDH and heat shock protein HSP60.

Staats et al. (2005) performed a comprehensive phylogenetic analysis of the genus Botrytis, at that time comprising 22 recognized species and one hybrid. Using three protein-coding genes (G3PDH, HSP60 and the DNA-dependent RNA polymerase subunit II gene RPB2), they corroborated the morphological and host plant-based classification of Botrytis spp. and divided the genus into two (rather widely separated) clades. Clade I contained species that only infect eudicot plants, while Clade II contained species that can infect either eudicotyledonous or monocotyledonous plants. The use of the same three genes facilitated the discovery of Botrytis sinoallii, a new species infecting Allium spp., and its distinction from other Botrytis spp. infecting the same hosts (Zhang et al. 2010b); B. fabiopsis, a new species infecting broad bean, very distant from B. fabae (Zhang et al. 2010a); and B. caroliniana, a new species infecting blackberry (Li et al. 2012).

Two genes, encoding phytotoxic proteins NEP1 and NEP2, were shown to provide higher resolution in distinguishing species in the genus Botrytis because they seem to be the subject of higher evolutionary rates than the housekeeping genes G3PDH, HSP60 and RPB2 (Staats et al. 2007a). The NEP1 and NEP2 genes were shown to have evolved under positive selection which suggested a role of these proteins in the infection process (Staats et al. 2007a). One might therefore infer that such genes cannot serve as neutral phylogenetic markers. Functional analysis in B. cinerea and B. elliptica using targeted knockout mutants failed to reveal a role of NEP genes in virulence of these two species (Staats et al. 2007b; Cuesta Arenas et al. 2010), which would lend support to considering these genes as neutral markers and adequate tools in phylogeny.

The studies by Staats et al. (2005) revealed incongruence between the phylogenies of Botrytis spp. and their hosts. Species infecting the same host clustered in different (sub) clades, e.g. B. aclada, B. squamosa, B. porri, B. byssoidea and B. sinoallii all infecting Allium. Conversely, closely related species can infect very different hosts, e.g. B. elliptica infecting the monocotyledonous host Lilium and B. ficariarum infecting the dicotyledonous host Ficaria (Staats et al. 2005). More recently, similar incongruence has been reported for newly described species, e.g. B. fabiopsis infecting Vicia faba is very distant from B. fabae infecting the same host (Zhang et al. 2010a), and B. caroliniana infecting blackberries and strawberries is very distant from B. cinerea (Li et al. 2012).

Recently, Khan et al. (2013) combined data from ITS and IGS regions with the G3PHD gene, with the aim of improving molecular identification of Botrytis species that cause neck rot disease on onion. ITS and IGS regions were insufficiently informative to distinguish B. allii and B. byssoidea. The sequences of ITS and IGS for B. allii and B. byssoidea confirmed that they have a close relationship, but G3PDH sequences of several B. allii isolates were clearly distinct, some clustering with B. aclada and others clustering with B. byssoidea (Khan et al. 2013), as might be expected for a hybrid species.

Sequence analysis of the G3PDH and β-tubulin genes amplified from herbarium specimens of Botrytis collected from grey mould-infected apple (deposited in 1932) enabled O’Gorman et al. (2008) to corroborate the existence of B. mali, a species that had been published (Ruehle 1931), but by lack of description was considered doubtful.

Figure 4 shows a maximum likelihood tree of Botrytis spp., based on concatenated sequences of parts of the three genes G3PDH, HSP60 and RPB2 (amplified using primers defined by Staats et al. (2005). Five species described after publication of the phylogeny by Staats et al. (2005), i.e. B. caroliniana, B. deweyae, B. fabiopsis, B. pseudocinerea and B. sinoallii, clearly cluster within the genus and are genuine Botrytis species. Botrytis mali could not be included in the tree due to lack of sequences for the HSP60 and RPB2 genes. Based on G3PDH and ß-tubulin sequences it would cluster with B. paeoniae (O’Gorman et al. 2008).

The Botrytis cinerea species complex

The Botrytis ‘dicot’ clade I consists of B. cinerea, B. pelargonii, B. fabae, B. pseudocinerea and B. calthae. Molecular data do not fully support a separation between B. pelargonii and B. cinerea (Staats et al. 2005, 2007a; Plesken et al. 2014), and the existence of B. pelargonii as a separate species is therefore doubtful. As mentioned above, B. cinerea and B. pseudocinerea are morphologically very similar yet phylogenetically more distant from each other than B. cinerea and B. fabae. All genes tested so far place B. calthae as most remote to all other clade I species.

Botrytis cinerea not only has a broad host range, but also shows considerable phenotypic variability in vegetative growth, conidiation and sclerotium formation (Kerssies et al. 1997; Martinez et al. 2003; Schumacher et al. 2013). Numerous studies have documented a similar variability in genotypic characters, such as amplified restriction length polymorphism, detection of transposable elements and microsatellite heterogeneity. Recently, B. cinerea strains have been described that produce bikaverin, a reddish pigment. These strains contain an intact bikaverin biosynthesis gene cluster (presumably acquired by horizontal gene transfer from Fusarium), which is partially deleted and nonfunctional in most non-bikaverin producing B. cinerea strains (Campbell et al. 2012; Schumacher et al. 2013

A subdivision of B. cinerea into genetically distinct groups has proved to be difficult. Analysis of the presence or absence of two types of transposable elements, named Boty (Diolez et al. 1995) and Flipper (Levis et al. 1997), was adopted as a tool to divide isolates into four transposon types, Transposa (isolates having both elements), Vacuma (isolates having neither element), Boty and Flipper (Giraud et al. 1997, 1999). This classification led to the discovery of B. pseudocinerea, which is usually Vacuma, but the transposon-based classification turned out to be of limited use since B. cinerea populations appear to consist of mixtures of different transposon types. Intriguingly, predominance of a certain type appears to be influenced by the host. While on grapes, strawberries and tomatoes, Transposa types are predominant, whereas B. cinerea populations from kiwi and apples are dominated by Vacuma types (Esterio et al. 2011; Johnston et al. 2013; Muñoz et al. 2002; Samuel et al. 2012; M. Hahn, unpublished). Reasons for this observation are unknown.

Evidence for genetic differentiation of B. cinerea populations with different host preference was obtained with microsatellite markers. In France, isolates from grapes and blackberries were shown to be divergent, indicating limited gene flow between populations on these host plants (Fournier and Giraud 2008). A recent study on grey mould isolates from fungicide-treated strawberry fields revealed the existence of a predominant B. cinerea genotype, named group S, that is closely related to but distinct from the common genotype of B. cinerea (Leroch et al. 2013). Sequencing of the highly polymorphic MRR1 gene revealed that group S isolates show more than 4 % divergence from B. cinerea strains B05.10 and T4, which have MRR1 genes with 99.9 % identity. Further sequencing of HSP60 and NEP2, and of two FUNYBASE genes that are suitable for phylogenetic studies (Marthey et al. 2008), partially supported the genetic separation of group S isolates (Johnston et al. 2013; Leroch et al. 2013). Genome sequencing of several B. cinerea and group S strains, and the analysis of additional polymorphic genes in isolates collected from various host plants in different countries, revealed at least two subclades that could be separated from the common B. cinerea genotype (Plesken and Hahn, unpublished). In fungicide-treated strawberry fields group S isolates dominated, whereas grapes were infected almost exclusively by common B. cinerea genotypes. These data, together with those of putative new endophytic Botrytis taxa that grouped close to B. cinerea (Shipunov et al. 2008), support the idea that B. cinerea represents a species complex, comprising genetically and phenotypically distinct groups.

Recommended genetic markers

G3PDH, RPB2 and HSP60—placement within the Sclerotiniaceae and the ascomycetes

NEP1 and NEP2—for higher resolution within the genus Botrytis,

The NEP1 and NEP2 genes are under positive selection (Staats et al. 2007a) and potentially influence interactions with the host plants. The NEP genes should therefore be used with caution.

Research is ongoing to identify a set of highly polymorphic genes that better resolve the phylogeny of taxa in clade I (Hahn et al., unpublished). It remains to be established whether those gene are equally useful for resolving the clade II species, and whether universal primers can be designed before these genes can be employed to infer a comprehensive phylogeny of the entire genus.



The genus Choanephora belongs to family Choanephoraceae in the order Mucorales (former Zygomycota). The genus was introduced by Currey (1873) for C. cunninghamii, to replace the generic name of his newly described species Cunninghamia infundibulifera, as Cunninghamia already existed as a genus of conifers. Because the specific epithet could not be retained, Choanephora cunninghamia remained invalid, based on the same type as Cunninghamia infundibulifera. The proper name Choanephora infundibulifera was validly published by Saccardo (1891), so the correct authorship of the species is “(Currey) Sacc.” It is also the type species of the genus. Choanephora was monographed by Hesseltine (1953), Milko and Beljakova (1970) and Kirk (1984). Currently the genus is classified within the family Choanephoraceae which can be distinguished by the presence of a persistent sporangium wall that ruptures at preformed sutures. It is furthermore placed in the subfamily Choanephoroideae, which is characterized by the presence of apposed suspensors and smooth zygospores (Hoffmann et al. 2013).

Both species of the genus can grow as saprobes, but they frequently become plant pathogens causing various leaf and fruit rots and blights and are commonly reported from a wide range of plant hosts, including angiosperms (monocotyledons and dicotyledons) and gymnosperms (Farr and Rossman 2014). Their distribution is worldwide, however, disease development is more common in tropical and subtropical regions characterized by high temperatures and humidity. Choanephora cucurbitarum is the causal agent of fruit and blossom rot of various cucurbits, e.g. yellow crookneck squash (Kucharek and Simone 1983). This species is also known from crop plants such as green beans (McMillan 1972), garden peas (Oikawa et al. 1986), and okra (El-Sayed and El-Sajed 2013) and is reported as an agent of wet rot of Mesembryanthemum crystallinum in hydroponic greenhouse culture in Japan (Kagiwada et al. 2010). It is very common during rainy summers in the southeastern United States and globally in other regions with similar climates. Recently it was isolated also from cultivated Hyoscyamus muticus in Japan (Abdel-Motaal et al. 2010) and Withania in India (Saroj et al. 2012). Choanephora often attacks tissues that have been damaged mechanically by insects or otherwise; plants that are poorly adapted to a hot humid climate are particularly prone to infection by the genus. The general appearance of Choanephora rot is similar to that of blights caused by other Mucorales representatives. Signs of infections on fruits or leaves include water-soaked, necrotic lesions, which progress rapidly under wet conditions. As the fungus begins to produce spores, affected tissues become dark grey-brown and hairy. This specific appearance results from the tall sporangiophores that produce a cluster of brown, one-spored sporangiola at their tips (Turkensteen 1979).

Species identification and numbers

Although more than ten species (and many varieties) have been described within this genus, only two species (viz. Choanephora infundibulifera and Choanephora cucurbitarum) were finally recognized in a monograph of the genus (Kirk 1984). These two species can be distinguished by shape and ornamentation of indehiscent sporangiola. C. cucurbitarum produces ellipsoid sporangiola, which are usually distinctly longitudinally striate, whereas C. infundibulifera forms subglobose to obovoid sporangiola with usually smooth or faint striate ornamentation. The remaining species were synonymized under these taxa (e.g. C. mandshurica is currently a synonym of C. cucurbitarum) or were moved to other genera (e.g. C. persicaria is a synonym of Gilbertella persicaria). Choanephora circinans with its two varieties (C. circinans var. indica and C. circinans var. prolifera) were moved by Kirk (1984) to Poitrasia. Poitrasia was established for those species belonging to the family Choanephoraceae that do not form dehiscent or indehiscent sporangiola (Kirk 1984). Although Poitrasia is primarily a soil-borne genus, it has been isolated from Equisteum arvense (Rai 1990). Recent molecular studies confirmed the taxonomic position of Poitrasia proposed by Kirk (1984).

Molecular phylogeny

All Choanephora strains available in CBS culture collection (three strains of C. infundibulifera and five strains of C. cucurbitarum) have been sequenced for their ITS sequences and included in molecular analysis by Walther et al. (2013). These studies showed that the universal fungal DNA barcoding marker–the ITS region (Schoch et al. 2012)–is sufficient for Choanephora species identification (Table 6, Fig. 5). Multigene phylogenetic analysis including representatives of this genus was performed by Hoffmann et al. (2013).

Fig. 9

Phylogram generated from parsimony analysis based on combined ITS and GPDH sequenced data of Curvularia. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Alternaria alternata

Recommended genetic markers

  • The internal transcribed spacer (ITS)–generic and species level

  • The large and small subunits (LSU and SSU) of nrDNA–placement within the Mucorales order, higher-level phylogeny

  • The partial actin gene (ACT) and the partial translation elongation factor 1-alpha gene (TEF)–higher-level phylogeny



The genus Colletotrichum was introduced by Corda (1831) and belongs to the family Glomerellaceae (Glomerellales, Ascomycota). Colletotrichum is a coelomycetous phytopathogenic genus with a Glomerella sexual state that includes a number of important pathogens causing diseases of crops and fruits worldwide (Cai et al. 2009; Cannon et al. 2012; Doyle et al. 2013). Colletotrichum species have furthermore been recorded as endophytes in angiosperms, conifers, ferns, lichens and grasses (Hofstetter et al. 2012; Damm et al. 2012b; Cannon et al. 2012; McKenzie et al. 2009; Petrini et al. 1990; Manamgoda et al. 2013; Tao et al. 2013). This genus was voted the eighth most important group of plant pathogenic fungi in the world, based on perceived scientific and economic importance (Dean et al. 2012). Colletotrichum species commonly cause anthracnose resulting in sunken necrotic lesions on leaves, stems, flowers and fruits of numerous hosts, including important crops (Lenne 2002; Waller et al. 2002; Agrios 2005; Cai et al. 2009; Than et al. 2008; Peng et al. 2012; Doyle et al. 2013). It is therefore important to plant health disease practitioners, quarantine personnel and plant breeders to know what species infect which crops (Huang et al. 2013b; Lima et al. 2013; Giaretta et al. 2010; Sangeetha and Rawal 2010; Liu et al. 2009a; Akinbode and Ikotun 2008; Adegbite and Amusa 2008; Peres et al. 2002). Therefore, having a rigid and stable taxonomy for the identification of Colletotrichum species is a significant practical concern (Shenoy et al. 2007). Identification of Colletotrichum species has been difficult due to the lack of reliable morphological features and confused, ambiguous species boundaries (Hyde et al. 2009a, b; Cai et al. 2009). Difficulties in recognising Colletotrichum species has resulted from having a few and variable morphological characters, widespread host ranges and pathogenicity, lost specimens or type specimens in poor condition and incorrectly named sequences in NCBI (Freeman et al. 2000; Du et al. 2005; Thaung 2008; Crouch et al. 2009a, b; Damm et al. 2009; Cai et al. 2009).

Colletotrichum species are extensively studied as model organisms for research in genetics (Cannon et al. 2012). The pathogenicity genes of C. higginsianum were discovered by random mutagenesis (Huser et al. 2009). Genomes and transcriptomes of C. higginsianum and C. graminicola were studied through the use of two different infection strategies by O’Connell et al. (2012). Work on the genetics of pathogenicity in the C. orbiculare species aggregate led to transformation of pathogenic strains to endophytic forms (Cannon et al. 2012). Gene manipulation techniques such as Agrobacterium tumefacien-mediated transformation or protoplast transformation were established (Tsuji et al. 2003). Peroxisome biogenesis genes, PEX6 and PEX13 were identified and their pathogenesis was functionally analyzed (Fujihara et al. 2010). The importance of the pexophagy factor ATG26 for apressorium formation was discovered by Asakura et al. (2009). Whole genomes of C. higginsianum and C. graminicola have been sequenced (O’Connell et al. 2012). Correct species identification is essential in plant pathogenic genera. In order to have effective measures to prevent the unwanted entry of diseases in to a country, the plant pathologists should be able to name the Colletotrichum species confidently. Therefore, pathologists need to be able to clarify and identify the species of Colletotrichum using the wide genetic variation among the taxa (Cannon et al. 2000).

Species identification and numbers

Colletotrichum species have been traditionally named after their hosts. The history of naming Colletotrichum species has been reviewed in several key papers (Cannon et al. 2008, 2012; Hyde et al. 2009a). Cai et al. (2009) outlined the recent polyphasic protocols for species identification: A total of 25 Colletotrichum species have been epitypified, one has been neotypified and three have been lectotypified (Cannon et al. 2008; Damm et al. 2009, 2012a, b, 2013; Doyle et al. 2013; Liu et al. 2011a, b, 2013; Su et al. 2011; Weir and Johnston 2010; Weir et al. 2012). Significant changes to the understanding of Colletotrichum species took place with incorporation of these polyphasic approaches, especially the use of multi-marker phylogenetic analysis, classification and knowledge of species complexes, as well as epitypifications for many species (Cai et al. 2009; Cannon et al. 2012; Damm et al. 2012a, b, 2013; Doyle et al. 2013; Su et al. 2011; Weir et al. 2012). Cannon et al. (2012) studied nearly all presently sequenced species in the genus using a six-gene analysis, and revealed at least nine clades; 119 species previously thought to be well circumscribed proved to be polyphyletic. Colletotrichum gloeosporioides (Cannon et al. 2008; Phoulivong et al. 2010a, b; Weir et al. 2012), C. acutatum (Marcelino et al. 2008; Shivas and Tan 2009; Damm et al. 2012a), C. boninense (Moriwaki et al. 2003; Yang et al. 2009; Damm et al. 2012b), C. orbiculare (Damm et al. 2013) form important species complexes within Colletotrichum and well-resolved among all the nine clades. Further studies in the C. gloeosporioides species complex led to identification of C. murrayae (Peng et al. 2012), C. viniferum (Peng et al. 2013), C. citricola (Huang et al. 2013b), C. fructivorum (Doyle et al. 2013), C. melanocaulon (Doyle et al. 2013), C. temperatum (Doyle et al. 2013), C. endophyticta (Manamgoda et al. 2013) and C. syzygicola (Udayanga et al. 2013). Tao et al. (2013) introduced seven new species; four species belonging to the graminicola clade, two species belonging to the spaethianum clade and one singleton species. Damm et al. (2013) resolved C. orbiculare and introduced four new species. Crouch (2014) introduced a new species complex, C. caudatum, with five new species found on warm-season grasses, characterized by the conidial apex reducing into a filiform appendage. The current numbers of species recognised in the genus are listed in Table 7.

Molecular phylogeny

Some species such as Colletotrichum gloeosporioides were defined using ITS sequence data, but the outcome was not good partially due to prolific misidentification in GenBank and because ITS does not resolve Colletotrichum species well. In Colletotrichum, species definitions based on ITS sequence data, the “universal” DNA barcoding marker for fungal species has proved unsatisfactory (Du et al. 2005; Crouch et al. 2009b; García et al. 2009; Cannon et al. 2012; Doyle et al. 2013; Gunjan et al. 2013). Comparison of a phylogenetic tree of Colletotrichum species derived from ITS sequence alone and one generated from multi-marker data confirms that ITS resolves major clades well, although it does not reflect their higher-order relationships accurately in all cases (Cannon et al. 2012). Cannon et al. (2012) suggested that a robust sequence-based identification system for Colletotrichum species must therefore use an alternative molecular marker or a combination of markers. Damm et al. (2012a) indicated that the most diagnostic markers are β-tubulin and GPDH. β-tubulin performed marginally better than GPDH due to a larger overall number of base pair differences, but even so, some clades differed only by one base pair in the β-tubulin alignment. As single genes that were used are not efficient to differentiate the species, Cai et al. (2009) suggested using multiple markers. Cannon et al. (2012), Weir et al. (2012), and Damm et al. (2012a, b) used several genetic markers: actin (act), chitin synthase (chs1 β-tubulin and ITS which revealed that Colletotrichum comprises nine major clades as well as a number of small clusters and singleton species. Many recent studies used multimarker phylogeny including actin (act), chitin synthase (chs1), β-tubulin, calmodulin (cal), glyceraldehydes-3-phosphate dehydogenase (gadph), histamine (HIS3), glutamine synthetase (GS), DNA lyase (apn2), intergenic region of apn2 and MAT1-2-1 genes (ApMat) (Weir et al. 2012; Damm et al. 2012a, b; Cannon et al. 2012; Peng et al. 2012; Doyle et al. 2013; Gunjan et al. 2013) to understand the phylogenetic divergence of Colletotrichum species. There is, however, no agreement among mycologists as to which genetic markers should be used (Doyle et al. 2013; Gunjan et al. 2013). Silva et al. (2012) stressed the need to use ‘powerful genes’ such as ApMat and Apn25L. The Apmat marker provides better resolution as compared to the genetic markers used by Weir et al. (2012), Silva et al. (2012), Doyle et al. (2013) and Gunjan et al. (2013). Up to now it has been a better gene-marker for resolving species within C. gloeosporioides species complex (Doyle et al. 2013; Gunjan et al. 2013). Only ITS sequences are available for several species of Colletotrichum showing the need of sequencing the other important gene regions and those species were not included in this analysis. Here we present an analysis using six genetic markers for all the Colletotrichum species that are accepted (Fig. 6) and for the C. acutatum species complex (Fig. 7). Figure 8 presents the analysis of C. gloeosporioides species complex using the apmat gene. The whole genomes of several species of Colletotrichum have been sequenced, such that it is now possible to carry out whole-genome analysis, and compare this with single gene analysis to establish a gene (or gene combinations) that can really resolve species in the genus.

Fig. 10

Phylogram generated from parsimony analysis based on combined ITS, EF1-α, β- tubulin, and CAL sequenced data of Diaporthe. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Diaporthella corylina CBS 121124

Fig. 11

Phylogram generated from parsimony analysis based on combined ITS, TEF and β- tubulin sequenced data of Diplodia. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Lasiodiplodia theobromae CBS 164.96

Fig. 12

Phylogram generated from parsimony analysis based on combined ITS and TEF sequenced data of Dothiorella. Parsimony bootstrap support values greater than 50 % are indicated above the nodes, and branches with Bayesian posterior probabilities greater than 0.95 are given in bold. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Spencermartinsia viticola CBS 117009

Recommended genetic markers

  • ITS alone will not resolve species in the genus, but it can separate taxa to species complexes. Multigene analysis using the following genes has been recommended for a backbone tree for species of Colletotrichum:

  • GPDH–Glyceraldehyde-3-phosphate dehydrogenase- resolves to species level, more accurate.

  • β-tubulin–Beta-tubulin resolves to species level

  • ApMat–Intergenic region of apn2 and MAT1-2-1 genes can resolve within the C. gloeosporioides complex

  • GS–glutamine synthetase–CHS-1. HIS3–Histone3 and ACT–Actin–Placement within the genus and also some species-level delineation.

These marker combinations can resolve the phylogenetic positions of most species in the genus. GPDH alone can delineate the majority of species. However, research is ongoing to identify better genetic markers to resolve the phylogenetic position of many species of Colletotrichum.



Curvularia is a dematiaceous hyphomycete genus in the family Pleosporaceae, Pleosporales, Dothideomycetes (Ascomycota) (Boedijn 1933). It is typified by C. lunata. Curvularia species have been recorded as saprobes and also plant, human and animal pathogens. Bipolaris and Curvularia species are associated with Cochliobolus sexual states (Sivanesan 1987). Curvularia species are found as plant pathogens especially associated with the family Poaceae. Species such as C. lunata, C. tuberculata and C. trifolii cause leaf spots and leaf blights of some cereal crops such as maize, rice and horticultural crops such as Bermuda grasses and turf grasses (de Luna et al. 2002). The most frequent human and animal pathogens within the genus are C. aeria, C. geniculata, C. lunata, C. inaequalis, C. verrucosa and C. borreriae. These species cause keratitis, sinusitis, cutaneous and subcutaneous infections, peritonitis, onychomycosis, endocarditis, endophthalmitis, cerebral phaeohyphomycosis, and allergic bronchopulmonary as well as disseminated disease (da Cunha et al. 2013).

Species identification and numbers

Curvularia is morphologically characterized by its dark mycelium, geniculate conidiophores with sympodial, tretic conidiogenous cells, conidia with smooth to slightly verrucose wall and several false septa (distosepta). Morphological species identification of Curvularia species is challenging as many species have morphological similarities and have overlapping conidial dimensions. Most of the clinical isolates and common plant pathogens of Curvularia are recorded as C. lunata, which was recently epitypified (Manamgoda et al. 2012a). Following phylogenetic assessments, it was revealed that most of the sequences named as C. lunata in GenBank are incorrectly identified (Cai et al. 2011; da Cunha et al. 2013). Ellis (1971) and Sivanesan (1987) described 37 species in the genus Curvularia and currently there are 122 species epithets in Index Fungorum.

Molecular phylogeny

Phylogenetic recognition is crucial for species identification in Curvularia. Former morphological identifications do not correlate with the phylogeny (Manamgoda et al. 2012a, b). Combined ITS and GPDH analysis for Curvularia and its sister genus Bipolaris by Berbee et al. (1999) revealed that some Bipolaris species cluster within the genus Curvularia. Curvularia was therefore redefined by Manamgoda et al. (2012a) based on a combined phylogenetic analysis of ITS, GPDH, TEF and LSU. Nine Bipolaris species clustering within Curvularia were transferred and their nomenclature redefined (Manamgoda et al. 2012a). Lack of ex-type cultures and epitypifications form limitations for phylogenetic species recognition. In this paper we present a phylogenetic tree with combined ITS and GPDH sequences obtained from available type material and voucher cultures (Table 8, Fig. 9). This can be used as a backbone in the identification of Curvularia species.

Fig. 13

Fusarium The single most parsimonious tree inferred from a combined RPB1 and RPB2 dataset indicating the phylogenetic relationships among species complexes in the genus Fusarium. Branches with bootstrap intervals greater than 70 % and Bayesian posterior probabilities greater than 0.95 are indicated in bold. The NRRL (Agricultural Research Service Culture Collection, Peoria, Illinois USA) and RBG (Royal Botanic Gardens Trust Culture Collection, Sydney, New South Wales, Australia) numbers are indicated for all reference taxa

Recommended genetic markers

  • GPDH is the best single genetic marker for the genus Bipolaris (Manamgoda et al. 2012a). It is recommended to use a combination of ITS and GPDH. Another useful gene is TEF.



Diaporthe (=Phomopsis) is a cosmopolitan genus of fungi comprised of endophytes, plant pathogens, and saprobes occurring on a wide range of annual and perennial hosts, including economically important crops (Uecker 1988; Farr and Rossman 2014; Udayanga et al. 2011). The genus belongs to class Sordariomycetes, order Diaporthales and the family Diaporthaceae, typified by the species Diaporthe eres Nitschke (Wehmeyer 1933). With the change to one scientific name for fungi (McNeill et al. 2012), Diaporthe has priority, being the older generic name compared to Phomopsis. Many species are able to colonise diverse hosts as opportunists; some species are host specific and multiple species can even co-occur on the same host (Mostert et al. 2001; Farr et al. 2002a; Crous and Groenewald 2005). Species of Diaporthe cause cankers, diebacks, root rots, fruit rots, leaf spots, blights and wilts on a wide range of plant host including some economically important hosts and have been the subject of considerable phytopathological research. Examples of diseases on major crops include Diaporthe/Phomopsis complex causing soybean seed decay, pod and stem blight and cankers, sunflower stem canker (D. helianthi), dead arm of grapevines (D. ampelina) and melanose in Citrus (D. citri) (Van Niekerk et al. 2005; Santos et al. 2011; Thompson et al. 2011; Udayanga et al. 2014a, b). In addition, several species of Diaporthe are known from clinical reports of immuno-compromised patients, although these pathogens are only provisionally identified to species level (Garcia-Reyne et al. 2011; Mattei et al. 2013). Diaporthe comprises a major component of endophytes in tropical and temperate trees, and several species have been used in secondary metabolite research (Isaka et al. 2001; Li et al. 2010a, b; Kaul et al. 2012).

Species identification and numbers

The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) has been applied in the genus Diaporthe to define the species boundaries in recent studies (Udayanga et al. 2012b; Gomes et al. 2013; Tan et al. 2012). Therefore species delimitation is currently based on DNA sequence data and comparison of morphological characters (Santos and Phillips 2009; Santos et al. 2010; Diogo et al. 2010; Udayanga et al. 2014a, b). Although the genus Diaporthe has received much attention, few phylogenetic studies have thus far been conducted; hence the taxonomy of some of the species in this genus is still uncertain including many of the common plant pathogens. Index Fungorum lists 892 Diaporthe names and 983 Phomopsis names whereas MycoBank (2014) lists 919 Diaporthe names and 1,040 Phomopsis names. However, the names available in the literature are mostly applied based on host association and morphology except fewer species described in last two decades based on DNA sequence data. Ex-type cultures are available for less than 100 species known despite the large number of species listed in databases and literature. The delimitation of species within the genus Diaporthe improved once DNA sequence data were incorporated (Castlebury and Mengistu 2006; Van Rensburg et al. 2006; Santos et al. 2010; Udayanga et al. 2012b, 2014a, b), since this facilitates obtaining detailed insight into complex evolutionary relationships.

Molecular phylogeny

Since the first molecular phylogenetic study in Diaporthe (Rehner and Uecker 1994), rDNA ITS, partial sequences of translation elongation factor 1-α (TEF) and mating type genes (MAT 1-1-1/1-2-1) have commonly been used in molecular phylogenetic studies in this genus (Van Niekerk et al. 2005; Van Rensburg et al. 2006; Santos et al. 2010; Udayanga et al. 2011; Sun et al. 2012). Udayanga et al. (2012a) used ITS, TEF, β- tubulin and CAL genes with a selected set of ex-type cultures and additional isolates to infer the phylogeny of the genus. In a parallel study, a multi-marker phylogeny was effectively used to describe novel species in Diaporthe based on fresh collections from Thailand (Udayanga et al. 2012b). Gomes et al. (2013) used a Brazilian collection of isolates and existing ex-type cultures for a combined phylogenetic analysis of five genetic markers which included ITS, TEF, β- tubulin, CAL and HIS. They introduced several novel taxa from Brazilian collections from medicinal plants with one epitype for Diaporthe anarcardi from Kenya. Udayanga et al. (2014a, b) revisited the Diaporthe species associated with Citrus worldwide with comprehensive assessment of the genes including ITS, TEF, β- tubulin, CAL and ACT. The study revisited several important phytopathogens including D. citri, D. cytosporella, D. forniculina and D. rudis, with the epitypes designated with modern descriptions. The clarification of D. foeniculaina and D. rudis revealed the potential extensive host association of some species.

Udayanga et al. (2014a) further emphasized that ITS alone can cause much confusion in defining closely related taxa, which has also been noted by several previous researchers regarding closely related species in Diaporthe (Farr et al. 2002a, b; Murali et al. 2006; Santos et al. 2010). The variation of ITS sequences can result in superfluous, multiple terminal branches in combined analyses, even when other gene regions do not support these distinctions (Udayanga et al. 2014a, b). The TEF gene is informative when it comes to clarifying species limits in Diaporthe (Table 9, Fig. 10).

Fig. 14

Phylogram generated from Maximum likelihood analysis based on ITS sequenced data of Gilbertella. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold


ITS and TEF are recommended for preliminary identification of the species (Castlebury et al. 2001; Castlebury 2005; Santos and Phillips 2009; Santos et al. 2010). ITS, TEF, β- tubulin, CAL, HIS and ACT should be used in combined analysis (selection of 4–5 genes), with recommended primers in relevant publications (Udayanga et al. 2012b, 2014a, b; Gomes et al. 2013).



Species of Diplodia (Botryosphaeriaceae) are endophytes, pathogens, or saprobes associated with cankers, dieback and fruit rot (Crous et al. 2006; Slippers and Wingfield 2007) in a wide range of hosts of agricultural and forestry importance (Farr and Rossman 2014). Cryptic speciation is common in the genus Diplodia, which makes species identification difficult if only based on morphological characters (Phillips et al. 2012, 2013). Denman et al. (2000) suggested that Lasiodiplodia could be a synonym of Diplodia, however recent studies accepted them as distinct genera (Pavlic et al. 2004; Burgess et al. 2006; Damm et al. 2007; Alves et al. 2008).

The genus Diplodia was introduced by Montagne (1834) with concepts altering over the years and has been regarded as including species with dark brown, 1-septate conidia (Phillips et al. 2005). Diplodia is defined by having uni or multilocular conidiomata lined with conidiogenous cells that form hyaline, aseptate, thick-walled conidia at their tips (Phillips et al. 2005). Diplodia mutila is the type species of Diplodia (Montagne 1834; Fries 1849), however, there are no living cultures linked to the holotype. As this has severely hampered studies on taxonomy and phylogeny of Diplodia, Alves et al. (2004) provided a detailed description of this species based on one isolate from grapevines in Portugal (CBS 112553).

Species identification and numbers

Diplodia is a large genus and a search in MycoBank (2014) revealed 1,317 names. Species in Diplodia were described, often based on host association, which later resulted in a proliferation of species names. According to Slippers et al. (2004d), host is not of primary importance in species differentiation, thus, many of the names in Diplodia are likely to be synonyms.

Based on DNA sequence data (single or multimarker) and minor differences in conidial morphology, there are currently about 20 Diplodia species (de Wet et al. 2003; Alves et al. 2004, 2006; Gure et al. 2005; Damm et al. 2007; Lazzizera et al. 2008; Pérez et al. 2010; Jami et al. 2012; Phillips et al. 2012, 2013; Linaldeddu et al. 2013; Lynch et al. 2013). The phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 10).

Molecular phylogeny

Studies on the taxonomy and phylogeny of Diplodia were hampered by a lack of an ex-type culture linked to the generic type, D. mutila. A collection of D. mutila from Populus with an ex-type culture was designated as epitype by Alves et al. (2014). They obtained a large collection of Diplodia strains from ash and other woody hosts showing V-shaped cankers and branch dieback. These strains were identified based on morphological characters and DNA sequence data. Since 2003 several new species have been described in Diplodia and these species were recognized mainly from DNA sequence data. Diplodia scrobiculata was differentiated from D. sapinea on the basis of multiple gene genealogies inferred from six protein coding genes and six microsatellite loci (de Wet et al. 2003). Diplodia africana (Damm et al. 2007), D. olivarum (Lazzizera et al. 2008) and D. cupressi (Alves et al. 2006) have been differentiated from D. mutila on the basis of formation of distinct clades in phylogenies based on ITS and TEF sequence data and due to their unique conidial morphology (Phillips et al. 2012).

Combined morphological and phylogenetic analyses of DNA sequence data from ITS and TEF (Alves et al. 2014) showed that the Fraxinus isolates from Italy, Netherlands, Portugal and Spain belong to three distinct species namely Diplodia fraxini, D. mutila and D. subglobosa. The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 11.

Recommended genetic markers

  • LSU and SSU–generic level

  • ITS, TEF and β-tubulin–species level

ITS, TEF and β-tubulin are the common genetic markers used in identification of Diplodia species. Combined ITS and TEF genes provide satisfactory resolution for resolving species.



Dothiorella (Botryosphaeriaceae) was proposed by Saccardo in 1880 (Crous and Palm 1999) with D. pyrenophora as the generic type. The delimitation of the genus has been in a state of flux since it was introduced, and detailed explanations of its taxonomy have been given by Sutton (1977), Crous and Palm (1999) and Phillips et al. (2008, 2013). Crous and Palm (1999) examined the holotype of D. pyrenophora and synonymised Dothiorella under Diplodia based on a broad morphological concept of Diplodia. That treatment was followed by Denman et al. (2000), Zhou and Stanosz (2001) and Slippers et al. (2004a). Phillips et al. (2005) re-examined the type of D. pyrenophora and found that the conidia become brown and 1-septate when they are still attached to the conidiogenous cells, while in Diplodia the conidia are hyaline and become dark and septate only after discharge from the conidiomata. Crous et al. (2006) confirmed these morphological differences by re-examining types of both Diplodia and Dothiorella. The sexual state of the species is rarely found in nature and no sexual morph was formed in culture for any of the species, except for D. sarmentorum and D. iberica. Therefore, differentiation of species is mostly derived based on the asexual morphs and cultural characteristics.

Species identification and numbers

As members of Botryosphaeriaceae, species of Dothiorella are known as endophytes, pathogens and saprobes in association with various woody plants, and species in Dothiorella were mostly described based on host association, much as for other members of Botryosphaeriaceae. This led to the introduction of many species names, and there are 368 epithets for Dothiorella in Index Fungorum (2014) and 393 species names in MycoBank (2014). Slippers et al. (2013) suggested that host association should not be considered an important factor in species definition of the Botryosphaeriaceae, therefore most of these names are likely synonyms. There are 19 described species with available cultures, and with the exception of D. sarmentorum all have been described after 2005. The phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 11).

Molecular phylogeny

Phillips et al. (2005) broadened the concept of Botryosphaeria and included Dothiorella in Botryosphaeria based on ITS analysis. Crous et al. (2006) recognised ten lineages within Botryosphaeriaceae corresponding to different genera based on phylogenetic analysis of 28S rDNA, and the three species D. iberica, D. sarmentorum and D. viticola formed a clade within Botryosphaeriaceae. These were assigned to Dothidotthia. Subsequently, Phillips et al. (2008) showed that Do. symphoricarpa (the type species of Dothidotthia) belongs in a distinct family within the Pleosporales, while D. sarmentorum, D. iberica and D. viticola fall within two separate genera in the Botryosphaeriaceae and a new genus, Spencermartinsia was introduced to accommodate D. viticola. Phillips et al. (2013) listed all cultures of available Dothiorella species, and provided a key to species, as well as a phylogenetic tree. Abdollahzadeh et al. (2014) introduced five new Dothiorella species which were associated with woody plants in Iran, New Zealand, Portugal and Spain. The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 12.

Recommended genetic markers

  • ITS–placement within the Botryosphaeriaceae (the generic level), and also some specific delineation.

  • TEF–the generic level and inter-specific delineation.

  • β-tubulin–inter-specific delineation.

Slippers et al. (2013) suggested that all of the known species of Dothiorella in culture can be separated based solely on ITS, but bootstrap support values for some of the internal nodes are quite low. Due to the studies on the other members of Botryosphaeriaceae, therefore, we strongly recommend that it is necessary to combine ITS and TEF (or intended β-tubulin gene) when molecular studies are carried out on Dothiorella.



The genus Fusarium was described by Link (1809) and later became a sanctioned name (Fries 1821). It is based on the type species Fusarium sambucinum (Nirenberg 1995). Species in Fusarium were described largely on the basis of the morphology of the canoe shaped septate conidia produced by most species as well as the shape and formation of other asexual spores Leslie and Summerell 2006). The sexual morphs (ascospores produced in perithecia) have played little role in the differentiation of most species as they are rare, if produced at all (Seifert 2001). Fusarium includes a number of species that are very important plant pathogens, some that are potent producers of an array of mycotoxins and several species or species complexes that are involved in diseases of humans (Leslie and Summerell 2006). There are also many species that are apparently endophytic in plants as well as species that are saprobes in soil and in organic matter.

Two species, F. graminearum and F. oxysporum, were included in an assessment of the top 10 fungal plant pathogens by Dean et al. (2012). Fusarium graminearum is the cause of head blight of wheat (Windels 2000), and F. oxysporum causes wilt diseases in a range of crops including bananas, tomatoes and other vegetables as well as cotton (Beckman 1987). Other species of Fusarium cause stalk and cob rots in maize and sorghum, canker diseases in woody plants and root and crown diseases across a vast spectrum of plant species (Summerell et al. 2011). Species of Fusarium produce a very large number of secondary metabolites, but two toxin groups, trichothecenes and fumonisins, are particularly detrimental to livestock and humans (through consumption) and as such are heavily regulated in many parts of the world (Desjardins 2005). As a result of the importance of these diseases, the genus is one of the most heavily researched of all genera of fungi and an enormous body of work on all facets of its biology exists (Leslie and Summerell 2006).

Several sexual morph genera are associated with Fusarium, the most important of which is Gibberella (Desjardins 2003). Most Fusarium species, particularly the plant pathogenic species, have a Gibberella sexual morph. Other sexual morph genera include Albonectria, Haematonectria and Neocosmospora as well as a number of other generic names (Gräfenhan et al. 2011). With the changes to the International Code of Nomenclature for Algae, Fungi and Plants providing the opportunity to have a single name for fungi of this nature there has been a strong consensus amongst the community of researchers working on Fusarium that this name be used for all the fungi in the so-called terminal Fusarium clade (Geiser et al. 2013). The end result of this is that species of Fusarium such as F. solani, F. decemcellulare and F. dimerum are included with species with Gibberella sexual morphs in the current generic definition of Fusarium (Geiser et al. 2013).

Species identification and numbers

It is difficult to accurately quantify the number of extant, currently recognized species of Fusarium. Over 1,500 names are listed in MycoBank; Leslie and Summerell (2006) documented 72 species, although this was not intended as a monograph, and many of species have been described in the intervening period (e.g. Jacobs et al. 2010; Laurence et al. 2011; Schroers et al. 2009; Walsh et al. 2010). Recent investigations into a number of important species (e.g. F. graminearum, F. incarnatum, F. oxysporum, F. solani) have provided evidence that they are complexes of phylogenetically distinct lineages that have been, or will eventually be described as species (Aoki et al. 2005; O’Donnell et al. 2004, 2008, 2009).

Molecular phylogeny

There has been substantial work on understanding the phylogenetic relationships within Fusarium, and in defining generic boundaries (e.g. Geiser et al. 2013; O’Donnell et al. 2013). This has provided refined concepts for several important plant pathogenic species (e.g. F. graminearum, F. pseudograminearum, F. subglutinans, F. verticillioides) and it has also shown that several important plant pathogens (especially F. oxysporum and F. solani) are in fact species complexes (Laurence et al. 2014; O’Donnell et al. 2008). A genus-wide phylogeny was inferred using the RNA polymerase largest subunit (RPB1) and RNA polymerase second largest subunit (RPB2) (O’Donnell et al. 2013), as these genes are very informative from a phylogenetic perspective across the whole genus (Table 12, Fig. 13)

Fig. 15

Phylogram generated from parsimony analysis based on combined ITS and TEF sequenced data of Lasiodiplodia. Parsimony bootstrap support values greater than 50 % are indicated above the nodes, and branches with Bayesian posterior probabilities greater than 0.95 are given in bold. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Diplodia mutila CBS 112553

Recommended genetic markers

The recommended and most frequently used gene for identification of species of Fusarium is the translation elongation factor 1α gene (TEF) and this is generally used for routine identifications, effectively performing a DNA barcoding function, and forms a significant component of the FUSARIUM-ID database (; Geiser et al. 2004). This database provides a similar facility to GenBank but is based on sequences from accurately identified and validated cultures held in reference collections (Geiser et al. 2004). Using a standard approach (Summerell et al. 2003), sequencing the TEF gene and comparing the sequence with the FUSARIUM-ID database makes it possible to rapidly and accurately identify most pathogenic Fusarium species. The ITS region is less informative in Fusarium from both a barcoding and phylogenetic perspective and as a result it has not been used extensively. This is primarily because there are nonorthologous copies of ITS2 that are incongruent with species phylogenies derived from other unlinked loci in species of economic importance {O’Donnell and Cigelnik (1997) #1278}. As a consequence it is not recommended that ITS be used for differentiation or identification of Fusarium species (Summerell et al. 2003).



The monotypic genus Gilbertella belongs to the family Choanephoraceae and subfamily Gilbertelloideae (Mucorales, former Zygomycota). It was established by Hesseltine (1960) for species described earlier as Choanephora persicaria by Eddy (1925), and consequently the type species of the genus is Gilbertella persicaria. Benny (1991) proposed a new family, Gilbertellaceae to accommodate this genus. Currently, the genus belongs to the family Choanephoraceae and subfamily Gilbertelloideae that can be distinguished from Choanephoroideae (Voigt and Kirk 2012) by ornamented zygospores and opposed suspensors (Voigt 2012). Although G. persicaria was originally described as Choanephora persicaria (Eddy 1925), its separate position within the family has been confirmed in several studies (Papp et al. 2003; Hoffmann et al. 2013).

In tropical and subtropical regions Gilbertella is a common postharvest pathogen, causing rots of pears (Mehrotra 1963a), peaches (Hesseltine 1960; Mehrotra 1963b; Ginting et al. 1996) and tomatoes (Mehrotra 1966). It was reported by Butler et al. (1960) and Hesseltine (1960) from mulberry (Morus sp.) in USA. It was also recently isolated from pitaya fruits (Hylocereus undatus, Cactaceae) in Japan (Taba et al. 2011) and China (Guo et al. 2012).

Species identification and numbers

Currently, Gilbertella persicaria is the only species within the genus. Although another species–Gilbertella hainanensis–has been described (Cheng and Hu 1965), after recent molecular studies of its ITS sequence, it is not currently recognized as a separate species (Walther et al. 2013). Two varieties of G. persicaria have been described: G. persicaria var. persicaria and G. persicaria var. indica, however only the former was accepted in the monograph published by Benny (1991).

Gilbertella persicaria produces sporangia with a persistent wall that ruptures at preformed sutures in two halves. Ellipsoid, smooth-walled, hyaline sporangiospores with polar appendages are released in droplet of fluid. Light brown ornamented zygospores are formed on opposed suspensors (Hesseltine 1960). Examination of morphology is usually enough for correct species identification. Moreover, the morphological identification may be easily confirmed by ITS sequencing (Table 13, Fig. 14).

Molecular phylogeny

The phylogenetic relationships based on the complete ITS region of Gilbertella representatives and related Mucorales taxa was completed by Papp et al. (2003). All Gilbertella cultures available in the CBS culture collection have been sequenced for their ITS region and were included in a molecular analysis by Walther et al. (2013). These studies showed that the universal fungal DNA barcoding marker–the ITS region (Schoch et al. 2012)–is sufficient for Gilbertella species identification (Fig. 16). The multi-marker phylogenetic analysis including representatives of this genus performed by Hoffmann et al. (2013), confirmed a distinct, well-supported position of Gilbertella within Choanephoraceae family.

Fig. 16

Maximum likelihood tree based on partial LSU sequences for Mucor species and main groups within the genus. Detailed phylogenetic trees for each group may be found in Walther et al. (2013)

Recommended genetic markers

  • The internal transcribed spacer (ITS) region–generic and species level

  • The large and small subunits (LSU and SSU) of nrDNA–placement within the Mucorales order, higher-level phylogeny



Lasiodiplodia (Botryosphaeriaceae) was introduced by Ellis in 1894 with L. tubericola as the type species. Clendenin (1896) provided a description of the genus and the species, but did not refer to any type or other specimens of the genus or species. Pavlic et al. (2004) could not locate the types, nor find any specimens from the original hosts or origins, but gave a clear concept of the genus and the type. A new status for the type species of Lasiodiplodia has been proposed by Phillips et al. (2013) and they designated CBS 164.96 as ex-neotype culture, and deposited a dried specimen as neotype with convincing reasons, although this specimen was collected from an unidentified fruit in Papua New Guinea, whereas the type was collected in Ecuador on cocoa plant. Twenty new species have been described since 2004; however the generic application of the name, L. theobromae, has not been resolved.

Species identification and numbers

Lasiodiplodia differs from Diplodia species in having striations on the conidia, and differs from Neodeightonia as Lasiodiplodia has conidiomatal paraphyses. Barriopsis differs as it has unique striate conidia, with the striations present on immature, hyaline conidia. A sexual morph has been reported for L. theobromae, which has been linked to Botryodiplodia rhodina (Cooke) Arx, but this link has not been unequivocally proven (Alves et al. 2008; Phillips et al. 2008). Phillips et al. (2013) transferred Auerswaldia lignicola (Liu et al. 2012) to Lasiodiplodia, and this is the only species where the asexual morph and sexual have been definitively linked. There are 30 epithets of Lasiodiplodia recorded in Index Fungorum (2014) and 32 species names in MycoBank (March 2014), and 24 species are currently kept in culture. Species can be differentiated based on conidial morphology (especially dimensions) and morphology of the paraphyses. The phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 14).

Molecular phylogeny

Denman et al. (2000) suggested that Lasiodiplodia could be a possible synonym of Diplodia based on the ITS data analysis. However, phylogenetic studies by Zhou and Stanosz (2001), Slippers et al. (2004a) and Phillips et al. (2008) show that it clusters separately from Diplodia. As more genes and molecular data have become available, more complex sections within Botryosphaeriaceae have been resolved. By combining TEF and β-tubulin genes with ITS, Phillips et al. (2005, 2008) reinstated the genus Neodeightonia in the Diplodia/Lasiodiplodia complex and also showed that the latter asexual genera are morphologically and phylogenetically distinct. Most of the known species with available cultures have been described based on at least two genetic markers (ITS, TEF/ β-tubulin). The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 15.

Recommended genetic markers

  • ITS–placement within the Botryosphaeriaceae (the generic level), and also some species-level delineation.

  • TEF–generic level and inter-specific delineation.

  • β-tubulin–generic level and inter-specific delineation, mostly for inter-specific delineation.

In most cases, a combination of ITS and TEF will separate all species and a minimal requirement for Lasiodiplodia species separation. However, for some groups, such as L. theobromae, β-tubulin is needed.



The genus Mucor belongs to the Mucoraceae, which is the largest and the most diverse family within Mucorales (former Zygomycota; Hoffmann et al. 2013). It was described by Fresenius (1850). The type species of the genus is Mucor mucedo, although the name Mucor had been used long before also by other authors to describe species currently classified as Rhizopus stolonifer (syn. Mucor mucedo L. 1753 or Mucor mucedo (Tode) Pers. 1801).

There has been no comprehensive molecular phylogenetic study in the genus Mucor and consequently its taxonomy is still widely based on morphological characters. Mucor representatives produce nonapophysate sporangia arising directly from the substrate and they do not form stolons. Rhizoids were also considered to be absent in Mucor, but it is now known that they can be produced under certain conditions (Walther et al. 2013).

Mucor representatives are saprotrophs that can be found mainly in soil or on plant debris. They are also known as postharvest plant pathogens, e.g. M. mucedo (Moline and Millner 1981) and M. piriformis (Michailides and Spotts 1990a). In case of peach and nectarine rots, Michailides and Spotts (1990b), Spotts (1990) and Michailides et al. (1992) regarded flies (especially Drosophila melanogaster) and nitidulid beetles (Carpophilus hemipterus and C. freemani) as effective vectors. Mucor rot symptoms include softening of juicy decayed tissue, often with a sweet odour, lesions with a sharp margin and eventually developing of grey mycelium with sporangia. Mucor isolated from several different plant hosts, angiosperms and gymnosperms, monocots as well as dicotyledons. USDA Fungus-Host Database reports 375 cases of Mucor infections from plants from approximately 40 countries in Europe, Central and South-East Asia, Australia, Africa, and North and South America (Farr and Rossman 2014). Mucor circinelloides causes rots in tomatoes (Smith et al. 1976), mangoes (Johnson 2008), yam (Amusa et al. 2003) and peaches (Restuccia et al. 2006). Mucor hiemalis can be pathogenic on guavas (Kunimoto et al. 1977), carrots and cassava (Snowdon 1991). Mucor piriformis is a destructive pathogen of fresh strawberries (Snowdon 1990; Pitt and Hocking 2009) and a major cause of rotting of cold-stored pears, apples, peaches, nectarines and tomatoes (Smith et al. 1979; Bertrand and Saulie-Carter 1980; Michailides and Spotts 1986; Michailides 1991; Mari et al. 2000; Pitt and Hocking 2009; Ukeh and Chiejina 2012), plums (Børve and Vangdal 2007), sweet persimmons (Kwon et al. 2004) and yams (Amusa and Baiyewu 1999; Iwama 2006). Mucor piriformis may infect the stem, calyx or wounds on the skin of fruits (Michailides and Spotts 1990a, b). Mucor mucedo was reported as important postharvest pathogen of strawberries (Dennis and Davis 1977), and from tomatoes (Moline and Kuti 1984). Mucor racemosus was noted causing soft rot of cherry tomato fruits in Korea (Kwon and Hong 2005). Some Mucor species (e.g. M. circinelloides) are also human opportunistic pathogens, especially dangerous to immunodeficient patients (Walther et al. 2013).

Species identification and numbers

The last extensive studies of the genus Mucor (Schipper 1973, 1975) are from the pre-molecular era. Based on morphological features and mating experiments Schipper (1976, 1978) recognized 39 species, 4 varieties and 11 formae. In the following years further species were described (e.g. Watanabe 1994; Zalar et al. 1997). Molecular phylogenetic analyses of the entire Mucorales revealed the polyphyly of the genus (Voigt and Wöstemeyer 2001; O’Donnell et al. 2001). The study of Walther et al. (2013) on the genetic diversity within the Mucorales based on sequences of the nuclear ribosomal internal transcribed spacer region (ITS) and the large ribosomal subunit (LSU) strongly supported the polyphyly of Mucor. The genus was split into several morphological groups differing in the size of the sporangia and the branching mode of the sporangiophores that are widely in agreement with the intrageneric classification of Schipper (1973). However, ?in molecular analyses these groups are intermingled by other sporangia-forming genera such as Pilaira und Pirella and sporangiola-forming genera such as Ellisomyces, Chaetocladium, Helicostylum and Thamnidium (Walther et al. 2013). The position of the Mycotyphaceae and the Choanephoraceae in relation to the Mucoraceae is still not resolved (Hoffmann et al. 2013).

Recently, the introduction of new species or changes of the taxonomic status were supported by sequence analyses of the ITS and/or rDNA genes (Jacobs and Botha 2008; Budziszewska et al. 2010; Álvarez et al. 2011; Madden et al. 2011). Several studies on certain species or species complexes (Li et al. 2011; Lu et al. 2013) or a particular ecological group (Hermet et al. 2012) used multi-marker approaches for phylogenetic species recognition in the genus Mucor. However, a comprehensive study on the entire genus is still lacking. As a consequence, species and even generic boundaries are still unclear for Mucor. Currently 58 species are recognised within the genus (Walther et al. 2013) (Table 15, Fig. 16).

Molecular phylogeny

The ITS region allows identification to species level for most mucoralean representatives (Walther et al. 2013). Detailed molecular species identification is currently not possible for species complexes such as M. circinelloides or M. flavus because of unclear species boundaries (Walther et al. 2013).

Along with the ITS region for species identification, the LSU (e.g. Fig. 16, Álvarez et al. 2011) or the SSU (e.g. Budziszewska et al. 2010) genes have frequently been used in molecular phylogenetic analyses of Mucor because the ITS is too variable to be confidently aligned across the entire genus (Walther et al. 2013). In addition, the RNA polymerase subunit gene (rpb1) was successfully used for multi-marker studies at the species level (Li et al. 2011; Hermet et al. 2012; Lu et al. 2013). Hermet et al. (2012) also used the fragment of a mini-chromosome maintenance protein (MCM7) and of the 20 S rRNA accumulation protein (tsr1). The multi-marker analysis of the entire Mucorales including representatives of genus Mucor by Hoffmann et al. (2013) were based on partial genes of actin and the translation elongation factor 1-alpha in addition to the rRNA genes.

Fig. 17

Phylogram generated from parsimony analysis based on combined ITS, TEF, β- tubulin, LSU and SSU sequenced data of Neofusicoccum. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Spencermartinsia viticola CBS 117009

Recommended genetic markers

  • The internal transcribed spacer (ITS)–genus and species level

  • The RNA polymerase II largest subunit gene (RPB1)–species level

  • The large and small subunits (LSU and SSU) of nrDNA–placement within the Mucorales order, higher-level phylogeny

  • The mini-chromosome maintenance proteins gene (MCM7–higher-level phylogeny)



Pennycook and Samuels (1985) listed Fusicoccum parvum as the asexual morph when they described Botryosphaeria parvum. Neofusicoccum was introduced by Crous et al. (2006) for species that have an asexual morph that occurs with a “Dichomera” like synanamorph (morphologically similar, but phylogenetically distinct from Botryosphaeria). They suggested the name as it provides more information of the morphological state.

Species identification and numbers

On the basis of conidial dimensions and pigmentation, pigment production in media and ITS sequence data, 22 species are currently recognized in Neofusicoccum, although some of these characters have recently been questioned (Abdollahzadeh et al. 2013). Four new species, N. batangarum, N. cordaticola, N. kwambonambiense and N. umdonicola were identified in this complex based on congruence between genealogies of multiple genes (Pavlic et al. 2009a, b; Begoude et al. 2010). Though many species of Neofusicoccum are morphologically similar and can be very difficult to distinguish from one another, an attempt has been made to differentiate all species in a key by Phillips et al. (2013) (Table 16).

Molecular phylogeny

Crous et al. (2006) proposed new combinations for 13 species based on the sequence data from cultures. Based on DNA sequence data for five nuclear markers, Pavlic et al. (2009a, b) identified three new species of Neofusicoccum within the N. parvum/N. ribis species complex in South Africa. N. batangarum was described from Terminalia catappa by Begoude et al. (2010). Analysis of TEF, β-tubulin and LSU gene sequences (Alves et al. 2008; Abdollahzadeh et al. 2010) and Genealogical Sorting Index (GSI) has been used to resolve the asexual morph of Neofusicoccum (Sakalidis et al. 2011) (Fig. 17).

Recommended genetic markers

  • LSU, SSU and ITS–genus level

  • β-tubulin and TEF–species level

Common genetic markers that are used for the identification of Botryosphaeriaceae species are ITS, TEF, β- tubulin, LSU and SSU. Recent studies have shown that the combination of TEF, ITS and β- tubulin is sufficient to characterize species in this lineage. However, even when using only the TEF gene, it is possible to identify distinct species. The unavailability of the TEF sequence of several type species makes species identification using molecular phylogeny problematic. Therefore, in future research, it is recommended to use the combination of TEF, ITS and β- tubulin for better species level delimitation.



Pestalotiopsis is an appendage-bearing conidial asexual coelomycetous genus in the family Amphisphaeriaceae, Xylariales, Sordariomycetes (Ascomycota) (Barr 1975; Kang et al. 1998) that is common in tropical and temperate ecosystems (Maharachchikumbura et al. 2011, 2012). The sexual state is Pestalosphaeria and only 13 species are known as compared to the asexual state (253 species names). Species of Pestalotiopsis cause a variety of disease in plants, including canker lesions, shoot dieback, leaf spots, needle blight, tip blight, grey blight, scabby canker, severe chlorosis, fruit rots and leaf spots (Espinoza et al. 2008; Maharachchikumbura et al. 2013a, b; Tagne and Mathur 2001). Species belonging to the genus Pestalotiopsis are thought to be a rich source for bioprospecting, and chemical exploration of endophytic Pestalotiopsis species is on the increase (Aly et al. 2010; Xu et al. 2010, 2014). Pestalotiopsis species have been recorded as saprobes where they are recyclers of dead plant material (Maharachchikumbura et al. 2012) and are also known to cause human and animal infections (Pestalotiopsis clavispora) (Monden et al. 2013).

Most Pestalotiopsis names in the literature are described based on host association. However, molecular data have shown that the genus needs revision (Maharachchikumbura et al. 2011, 2012; Zhang et al. 2013c), and many of the traditional species may be spurious. There are also numerous cryptic species, very few distinct species, species with wide host ranges, those with cosmopolitan distribution and some species being opportunistic pathogens. This calls for critical re-examination of the genus, using both morphological studies and a multi-marker phylogeny based on ex-type and ex-epitype cultures (Maharachchikumbura et al. 2012, 2013c).

Species identification and numbers

According to Index Fungorum (2014) there are 253 Pestalotiopsis names, while in MycoBank (2014) there are 264 names. The reason for the large number of names is historical and may not reflect the actual number of species (Jeewon et al. 2004). Kohlmeyer and Kohlmeyer (2001) described P. juncestris, which was isolated from the host Juncus roemerianus; this species is morphologically similar to P. versicolor and several other species of Pestalotiopsis, but the taxon was described as a new species based on the host occurrence. However, recent molecular data have shown that host association and geographical location is less informative for distinguishing taxa (Jeewon et al. 2004; Hu et al. 2007). Isolation of endophytic Pestalotiopsis strains for bioprospecting for new biochemical compounds has shown that the same species can be found in a range of hosts. It has been shown that most of the key conidial characters used in species level separation are not stable and vary with host range, generation, culture and other environmental conditions (Hu et al. 2007). Furthermore, the arrangement of species by Steyaert (1949) and Guba (1961) in various coloured groupings is problematic because this character has been shown to be variable within a species (Liu et al. 2010). Thus, most species in the above arrangements may be confused and many species are probably synonyms. Therefore, most of the species recorded in checklists and the literature may not reflect what actually occurs. Thus, many names assigned to Pestalotiopsis probably lack any accurate taxonomic basis, leaving the taxonomy of the genus markedly confused. Until 1990, phylogenetic understanding of the taxonomy associated with Pestalotiopsis and allied genera was based mainly on conidial characters (Steyaert 1949; Guba 1961; Nag Rag 1993), conidiogenesis (Sutton 1980) and sexual state association (Barr 1975). More recently, some new species have been introduced based on host occurrence, plus morphological and molecular data (Maharachchikumbura et al. 2012, 2013a, b; Strobel et al. 2000). Furthermore, currently only 36 Pestalotiopsis species have either ex-type or ex-epitype sequences.

Molecular phylogeny

Recently, many Pestalotiopsis species have been defined using ITS sequence data, however, there are only a few type cultures available for Pestalotiopsis. For example, Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae have numerous ITS sequences in GenBank. However, in phylogenetic studies all these species scattered throughout the phylogram and there appears to be no living ex-type strain for any of these species (Maharachchikumbura et al. 2011). Therefore it is unwise to use GenBank sequences to represent any of these names. Rapid development in molecular phylogeny has had a great impact on Pestalotiopsis taxonomy. For example, random amplification of polymorphic DNA (RAPD) can be used to detect genetic diversity in the genus (Tejesvi et al. 2007). Watanabe et al. (2012) evaluated the use of the ITS2 region and showed that it is conserved at the level of secondary structure rather than the level of primary sequence, which can be used for classification of the Pestalotiopsis. Hu et al. (2007) showed that the ITS region is less informative than the β-tubulin gene in differentiating endophytic species of Pestalotiopsis in Pinus armandii and Ribes spp. A combination of β-tubulin and ITS gave improved phylogenetic resolution, and they suggested that at least two genetic markers should be used to resolve the phylogeny of species of Pestalotiopsis. However, Liu et al. (2010) disagreed with above statement concerning the ITS region as being less informative when compared to the β-tubulin region. They indicated that alignment of the ITS region can be a useful character in grouping Pestalotiopsis to different types of pigmentation, which can be used as a key character for the phylogeny of the species. In order to select suitable markers for better species resolution, Maharachchikumbura et al. (2012) analyzed a combined ACT, β-tubulin, CAL, GPDH, GS, ITS, LSU, RPB 1, SSU and TEF dataset. They compared the morphological data versus the sequence data from each gene to establish which characters satisfactorily resolve the species. They narrowed down the 10 gene regions to three most applicable regions (ITS, β-tubulin and TEF), which were tested individually and in combination, to evaluate the differences between species. The species sequenced with ITS had a high PCR and sequence success rate and β-tubulin and TEF gene regions proved to be favourable taxonomic markers for Pestalotiopsis since they resolved the taxonomic relationships of most species studied. Further, TEF had better PCR amplification success rates and was found to be superior to β-tubulin. TEF is therefore a powerful tool to resolve lineages within Pestalotiopsis. Because of the better PCR and sequencing success rate and fewer difficulties with alignment, editing and better resolution, the TEF gene appears to be a very good molecular marker for phylogenetic investigation of Pestalotiopsis. Furthermore, a combination of ITS, β-tubulin and TEF gene data gave the best resolution as compared to any single marker (Table 17, Fig. 18). In addition to the above three markers, the authors also tested LSU, SSU, ACT and GPDH (low resolution), GS and RPB1 (cannot be synthesized using available primers or multiple copies) and CAL (species resolution is high, PCR success rate low).

Fig. 18

Strict consensus combined (ITS + β-tubulin + TEF) tree from Bayesian analysis of the analyzed Pestalotiopsis. Thickened lines indicate Bayesian posterior probabilities (PP) of 100 %. Strain accession numbers (ex-type are in bold) are followed by the species name. The scale bar represents the expected changes per site. The tree is rooted to Seiridium spp. (D96)

Recommended genetic markers

  • The large subunits of nrDNA (LSU)–placement within the Amphisphaeriaceae (generic level)

  • The internal transcribed spacer (ITS), β-tubulin and TEF–species level (as outlined in Maharachchikumbura et al. 2012)



Phyllosticta is an important plant pathogenic genus with coelomycetes asexual states. It was previously placed in Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes, Ascomycota. However following phylogenetic analysis, Wikee et al. (2013c) placed this genus in Phyllostictaceae which is sister to the Botryosphaeriaceae. Phyllosticta species are known to cause leaf spots and various fruit diseases worldwide on a diverse range of hosts including some economically important crops and ornamentals such as citrus, banana, apple, grapes, cranberry, orchids, mai dong and maple (Uchida and Aragaki 1980; Paul and Blackburn 1986; Baayen et al. 2002; McManus 1998; Olatinwo et al. 2003; Paul et al. 2005; Liu et al. 2009b; Wikee et al. 2011, 2012; Shivas et al. 2013b). Some species such as P. capitalensis are endophytes and weak pathogens (Baayen et al. 2002; Glienke et al. 2011; Wikee et al. 2013a), while others such as P. cocoicola are saprobes (Punithalingam 1974; Taylor and Hyde 2003). Phyllosticta species have been also used as bio-control agents and produce novel bioactive metabolites such as phyllostine and phyllostoxin (Yan et al. 2011; Evidente et al. 2008a, b; Wikee et al. 2011, 2013b).

The sexual state of Phyllosticta was named Guignardia which comprises 353 records in MycoBank (Hyde 1995; Crous et al. 1996; Hyde et al. 2010). Phyllosticta species have sometimes been named in Leptodothiorella after their spermatial state (Van der Aa 1973). Most species of Phyllosticta and Guignardia have been described independently, and only a few Phyllosticta species have been linked to their Guignardia sexual morphs (Wulandari et al. 2010). On the other hand, the host ranges of many diseases are poorly understood (Van der Aa and Vanev 2002; Wikee et al. 2011). It has been recommended that Phyllosticta which is the older, more commonly used and more species-rich, should have priority over Guignardia (Zhang et al. 2013a, b, c; Wikee et al. 2013c).

Phyllosticta species have been historically indentified based on morphology, culture characters as well as host association, which has resulted in several taxonomic revisions (Van der Aa 1973; Van der Aa and Vanev 2002). Fresh collections and future molecular analyses should help resolve species relationships (Hyde et al. 2010). Phylogenetic analysis has been routinely used in species identification, in combination with morphological characters (Crous and Groenewald 2005; Hyde et al. 2010; Wikee et al. 2013c). To create a stable and workable taxonomy, neo- or epitypification are required for many species of Phyllosticta (Hyde et al. 2010; Wikee et al. 2013c).

Species identification and numbers

The genus Phyllosticta was first introduced as the generic name for Sphaeria lichenoides by Persoon (1818). Desmazieres (1847) re-defined Phyllosticta, in which he did not restrict the genus to species with one-celled conidia. Consequently, many fungi with one-celled or septate conidia were named as Phyllosticta (Desmazieres 1847; Van der Aa 1973). Saccardo (1878) however, restricted Phyllosticta to species with one-celled conidia, and after that Phyllosticta was further restricted to leaf inhabiting species (Saccardo 1878, 1884; Van der Aa 1973; Petrak and Sydow (1927) published a compilation of Phyllosticta names, and gave extensive descriptions of 28 species. Van der Aa (1973) proposed a morphological identification criterion for the genus and detailed 46 Phyllosticta species based mostly on material collected in Europe and North America. The genus was revised by Van der Aa and Vanev (2002) and they accepted 141 species. The currently used generic circumscription of Phyllosticta is: “pycnidia globose, subglobose or tympaniform, conidiogenous cells holoblastic, with percurrent proliferation, conidia hyaline, 1-celled, ovoid, overate, ellipsoid, short cylindrical, or globose to subglobose, usually bearing a slime layer and an apical appendage” (Van der Aa 1973; Van der Aa and Vanev 2002). During 2002–2014, about 30 new species were described (Motohashi et al. 2008; Wulandari et al. 2009, 2010; Glienke et al. 2011; Wang et al. 2012; Su and Cai 2012; Wong et al. 2012; Wikee et al. 2012, 2013c; Zhang et al. 2013b; Shivas et al. 2013b), with the currently accepted species possibly being more than 171. Unfortunately, molecular data are currently available for about only 69 species (Table 18).

Molecular phylogeny

Phylogenetic analysis has become a standard approach in fungal identification and has been well applied in several other coelomycetous genera such as Colletotrichum (Cai et al. 2009; Crouch et al. 2009b, c; Hyde et al. 2009a, b) and Phoma (Aveskamp et al. 2008, 2010; de Gruyter et al. 2010). Recent reports on Phyllosticta have shown that molecular phylogenetic tools have significantly improved species identification and delimitation; similarly it has improved the resolution in species complexes (Wulandari et al. 2009; Glienke et al. 2011; Wicht et al. 2012).

Baayen et al. (2002) evaluated the P. citricarpa sensu lato from Citrus and associated hosts based on ITS sequence analysis and found that two phylogenetically distinct groups existed: a slowly growing pathogenic group and morphologically similar but fast-growing, non-pathogenic group which latter proved to be P. capitalensis. Wicht et al. (2012) used a polyphasic approach including morphological, molecular and proteomic techniques to analyze samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins, and showed that P. ampelicida isolated from grapevine cultivars should be split into two species.

Recent studies have provided clear phylogenetic relationships in the group. These efforts primarily used intron-dominated genes (ITS, ACT, TEF), and highly conserved gene coding regions (LSU, GPDH) that can recognize cryptic species in traditionally morphologically circumscribed species complexes, e.g. P. citricarpa on citrus, P. musarum on banana, P. vaccinii on Vaccinium, G. philoprina on Rhododendron, Hedera, Ilex, Magnolia and Taxus (Glienke et al. 2011; Wang et al. 2012; Wulandari et al. 2009; Wikee et al. 2013c, a, b, c; Wong et al. 2012; Zhang et al. 2013a) (Fig. 19).

Fig. 19

Phylogram generated from parsimony analysis based on combined ITS, TEF, GPDH and ACT sequenced data of Phyllosticta. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold


  • The large and small subunits of nrDNA (LSU and SSU)–placement within the ascomycetes (generic and family level)

  • The internal transcribed spacer (ITS)–generic level

  • Combined ITS, TEF, GPDH and ACT–inter-specific delineation



While resembling Eumycotan fungi with the production of hyphae, the genus is placed in the kingdom Straminipila, class Oomycetes, order Peronosporales, and family Peronosporacae. The type species is P. infestans described by de Bary in 1876. Since this time over 128 species have been described, many of which are important plant pathogens capable of significantly impacting agricultural production and natural ecosystems. Some species have a rather narrow host range (P. infestans, P. lateralis, P. sojae) while others are capable of infecting a wide range of plant host species (P. cinnamomi, P. nicotianae, P. ramorum). From a historical perspective, most investigations on the genus have focused on the impact of the genus on agricultural production systems, however, more recently there has been an increased interest in investigating the role this genus plays in natural ecosystems as exemplified by the number of publications concerning species such as P. ramorum and P. alni, as well as the description of many new species recovered from environmental sampling (Martin et al. 2012).

Although Phytophthora species resemble Eumycotan fungi with the production of hyphae, evolutionarily they are more closely related to chromophyte algae and plats than to Eumycotan fungi (Wainright et al. 1993). They have cell walls that are primarily cellulose rather than chitin as observed in fungi and they are incapable of synthesizing β-hydroxysterols (which are required for synthesis of hormones regulating sexual reproduction). In addition, Oomycetes are diploid throughout their life cycle in contrast to most true fungi.

An excellent overview of the ecology, biology and taxonomy of the genus (although missing more recently described species) can be found in Erwin and Ribeiro (1996), a review of the recent taxonomic status in Kroon et al. (2012) and an overview of the genus, including molecular identification and diagnostics, in Martin et al. (2012). There are several publically available databases that provide a wealth of up to date information on the genus, along with sequences useful for species identification via BLAST analysis, including the Phytophthora Database (, Phytophthora ID ( and Q-Bank ( Cline et al. (2008) have published an online list of Phytophthora spp. with a hyperlink for each species to the USDA SMML database that includes host range, distribution and supporting literature.

Species identification and numbers

A complicating factor when trying to identify Phytophthora species or investigate phylogenetic relationships is hybridization among distinct evolutionary lineages. While this does not appear to be a common occurrence, several stable hybrid species have been identified, e.g. P. andina (Goss et al. 2011; Blair et al. 2012); P. alni (Brasier et al. 1999); P. x pelgrandis (Nirenberg et al. 2009); P. x serindipita (Man in ’t Veld et al. 2012) as well as hybrid clade 1 species recovered from the field (Man in ’t Veld et al. 1998, 2007; Hurtado-Gonzales et al. 2009; Bonants et al. 2000). While conducting a detailed evaluation of clade 6 Phytophthora spp. from natural ecosystems in Australia, Burgess et al. (2010) observed ‘hybrid swarms’ that contained mixtures of parent, offspring, and intermediate isolates with high tendencies for back-crossing and out crossing. The authors’ concluded that the presence of such hybrid swarms was indicative of sexual and somatic hybridization events; the high proportion of these variant isolates within the population also suggested that these hybridization events were not uncommon. Recently four interspecific hybrid clade 6 species have been recovered from riparian ecosystems in Australia and South Africa that reflect outcrossing between P. amnicola, P. thermophila and P. taxon PgChlamydo (Nagel et al. 2013). Additional putative interspecific hybrids from riparian ecosystems in Australia were reported by Hüberli et al. (2013). Hybridization is a topic that requires a more detailed investigation as it could have a profound influence on gene flow among species and the evolution of new species with an expanded host range that could impact agricultural and natural ecosystems (as observed with P. alni).

Traditional classification to species level has been based on morphological characterization of reproductive structures (reviewed in Martin et al. 2012). This includes the sporangium (asexual) and oospore (sexual) as well as the production of chlamydospores (asexual structure not produced by all species). Important features of the sporangium include their dimensions (length and breadth), shape, thickening at the terminus (papilla), length of stalk (pedicle), whether or not the sporangium can be easily dislodged from the sporangiophore (caducity), and proliferation of sporangia (internal, external or nested).

The sexual reproductive structures consist of the antheridium and oogonium (paternal and maternal gametangia, respectively) and are produced when cultures are grown on the appropriate sterol-containing medium. Their fusion leads to the formation of an oogonium that matures into a thick-walled resting structure referred to as an oospore. While most species are homothallic and form oospores in single culture, there are heterothallic species where pairing with opposite mating types is essential to stimulate production of sexual reproductive structures. Since Phytophthora is sexually dimorphic (an isolate of a heterothallic species can function either as the maternal or paternal parent depending on the isolate it is paired with) it is advisable to pair self-sterile isolates with two tester isolates of opposite mating type. While the use of tester isolates of the same species is advisable, isolates of other heterothallic species (such as P. cryptogea or P. cambivora) may also be used. Characteristics such as the diameter of the oogonium and oospore, thickness of the oospore wall, whether or not the oospore fills the oogonium (plerotic), ornamentation on the oogonial wall, and mode of attachment of the antheridium are useful for species classification.

In an effort to simplify isolate identification and establish groupings of isolates for comparison of morphological features (but not phylogenetic relationships), Waterhouse (1963) introduced the concept of morphological groups I through VI based on a number of characteristics, and is still useful today. Unfortunately a dichotomous key that includes recently described species is not available for identification of isolates but there are several recent efforts to simplify morphological identification of species, including a manual for identification of 60 species of Phytophthora by integration of a dichotomous key with a DNA fingerprinting technique based on PCR-single strand conformational polymorphism (SSCP) (Gallegly and Hong 2008). A LUCID key for identification of 55 common Phytophthora spp. is available (Ristaino 2011) and an expanded LUCID key including most described species should be available on a dedicated website in the near future (G. Abad and Y. Balci, personal communication). A tabular presentation of morphological features enabling comparison among 117 species may be found in Martin et al. (2012; a downloadable file of the table alone is available on the journal website).

In 1999 the number of described species in the genus Phytophthora was approximately 55 (Brasier 2007) but since then there has been a significant increase., Brasier (2007) reported a doubling in number to 105 described species, with this number recently increasing to 117 (Martin et al. 2012). Additional species have recently been described; P. lacustris (Nechwatal et al. 2012) P. pluvialis (Reeser et al. 2013), P. mississippiae (Yang et al. 2013), P. cichorii, P. dauci and P. lactucae (Bertier et al. 2013), P. pisi (Heyman et al. 2013), P. stricta and P. macilentosa (Yang et al. 2014) and the hybrid species P. x serendipita and P. x pelgrandis (Man in ’t Veld et al. 2012), bringing the total to at least 128 described species. With the number of provisional species names used in the literature, and research efforts to evaluate the distribution of this genus in natural ecosystem, this number is likely to continue to increase in the future.

Molecular phylogeny

Historically the genus Phytophthora has been placed in the Pythialeswith Pythium and related genera but more recent phylogenetic analysis with the large (LSU) or small (SSU) rDNA sequences or cox2 gene has indicated a closer affiliation with downy mildews and white rusts (Albugo.) in the Peronosporales (Beakes and Sekimoto 2009; Thines et al. 2009). However, additional multigene analyses with a larger number of downy mildew species is needed to better characterize this relationship and the placement of Phytophthora spp. in clade 9 and 10 (Blair et al. 2008). The relationship between the Peronosporales and Pythium (Pythiales) needs clarification as well. A new genus, Phytopythium, was erected to accommodate an inconsistency between taxonomic and phylogenetic grouping for certain “intermediate” Pythium species (Bala et al. 2010), and it is likely that additional taxonomic revisions of the Peronosporomycetidae will be needed to fully resolve taxonomic conflicts.

Early efforts to understand phylogenetic relationships in Phytophthora focused on the use of the nuclear encoded rDNA, primarily the ITS region (Förster et al. 2000; Cooke and Duncan 1997; Crawford et al. 1996). Cooke et al. (2000) published the first comprehensive phylogenetic analysis of the genus using the ITS region to examine the phylogeny of 50 species. Most isolates grouped within eight primary clades (numbered 1 to 8) with several other species placed in two additional clades (clades 9 and 10). Kroon et al. (2004) expanded this analysis using two nuclear (translation elongation factor 1α, β-tubulin) and two mitochondrial (cox1 and nad1) genes. While in general the results were congruent with those reported by Cooke et al. (2000), there were some notable differences in the grouping of some species. Subsequent analysis by Blair et al. (2008) using seven nuclear genes (60S ribosomal protein L10, ß-tubulin, enolase, heat shock protein 90, large subunit rDNA, TigA gene fusion and translation elongation factor 1α) representing 8.1 kb of sequence data for 82 Phytophthora spp. clarified these differences. This larger, multi-marker analysis supported the observations of Cooke et al. (2000) with eight main clades plus two additional closely affiliated clades (clades 9 and 10) as the sister clades to the rest of the genus. More recently, Martin et al. (2014) expanded on this analysis by adding four mitochondrial genes (cox2, nad9, rps10 and secY) and additional species. The resulting phylogeny from this 11-marker analysis (10,828 bp per isolate) was similar to the prior observations of Blair et al. (2008) and subsequent analysis indicated that similar results could be obtained when using only five markers (LSU, β-tubulin, cox2, nad9 and rps10).

While the ITS region may be useful for species identification (see below), length variation among species makes it impossible to construct an unambiguous alignment across the entire genus, thus hampering the utility of this marker for phylogenetic analysis. Likewise, the translation elongation factor 1α has been used for phylogenetic analysis, but recent analysis of Phytophthora genomic data indicates that the gene is duplicated; divergence among duplicates may complicate phylogenetic interpretations of species evolution (J. E. Blair, unpublished).

While the above noted phylogenetic analyses have provided insight into the broader evolutionary relationships within the genus, there is still ambiguity when examining some closely related species and species complexes. Significant progress has been made with the clarification of the P. megasperma complex and other clade six species (Brasier et al. 2003; Durán et al. 2008; Hansen et al. 2009; Jung et al. 2011a, b) but there are still several provisional species awaiting more comprehensive analysis (for example, P. taxon PgChlamydo, P. taxon raspberry, P. taxon canalensis, P. taxon erwinii, P. taxon hungarica, P. taxon oregonsis and P. taxon paludosa). While there have been advances in understanding the relationships among some clade 2 species, there is need for additional analysis to clarify species complexes such as P. citricola and P. citrophthora. One clade 8 species complex where phylogenetic resolution has been elusive is P. cryptogea and the closely related species P. drechsleri. The multigene analysis of Mostowfizadeh-Ghalamfarsa et al. (2010) confirmed that while P. drechsleri was monophyletic, the P. cryptogea complex formed three well-defined phylogenetic groups with group I closely affiliated with P. erythroseptica and group II and III as a separate clade (group III isolates have been reported as the provisional species, P. sp. kelmania; Martin et al. 2014). Some isolates were placed intermediate between groups II and III and exhibited a greater amount of heterozygosity than the other isolates, suggesting possible outcrossing between these groups. Using a parsimony-based ancestral recombination graph and genealogies inferred from the β-tubulin and translation elongation factor 1-α genes from greenhouse recovered isolates, Olson et al. (2011) suggested that divergence between P. cryptogea and P. drechsleri was recent and that speciation is still in progress.

In addition to the choice of markers to use for phylogenetic analysis, another important consideration is the type of analysis used for estimating phylogenetic relationships or for the description of new species. While traditional methods of phylogenetic analysis (maximum likelihood, neighbour-joining, Bayesian) have adequately described relationships among most species, they have been unable to fully resolve the deeper relationships among the ten Phytophthora clades or among related genera. A recent study by Martin et al. (2014) used a novel variation of a multispecies coalescent approach to evaluate the ten clades; in general support was higher than that observed in the phylogenetic analysis for the recovered relationships, but the position of certain clades (Clade 3 and the unique grouping of P. sp. ohioensis and P. quercina) remained ambiguous. Here we present an analysis using a more powerful and complex Bayesian method (Drummond et al. 2012) with five genetic markers (Fig. 20), and recover strong support for basal relationships among the clades that are quite similar to the 11-marker study of Martin et al. (2014). Newer phylogenetic methods may allow for more complex modelling of the evolutionary process, however they are still sensitive to the accuracy of a priori information provided by the user. Additional studies will be needed to provide more basic information on the tempo of molecular evolution within this group.

Fig. 20

Bayesian analysis of phylogenetic relationships within Phytophthora. Asterisks on nodes indicate posterior probabilities greater than 0.95 (95 %) generated from an analysis of five genes (nuclear LSU and β-tubulin; mitochondrial cox2, nad9, rps10). Evolutionary rates were estimated under a GTR + I + G model for nuclear markers and an HKY + I + G model for mitochondrial gene; each marker was treated as a separate partition. The analysis was run twice with 50 million generations under a strict clock model in BEAST v1.7.5. A 20 % burn in was removed before the maximum clade credibility tree was constructed. Ex-type isolates are shown in bold. Separate isolate numbers are shown for those few species that did not have sequence data available for both nuclear and mitochondrial genes from a single isolate

The description of new species is also an area were traditional phylogenetic methods may not accurately describe species relatedness. Aside from morphological characterization, recent species descriptions typically contain molecular evidence from one or a few genetic markers (primarily ITS and perhaps cox1 or 2). However, as described above, alignment ambiguity and the presence of intraspecific polymorphisms can seriously impact the recovered phylogeny; recent hybridization events and incomplete lineage sorting of ancestral polymorphisms also violate the assumptions made by traditional phylogenetic methods. The use of coalescent-based approaches to estimate species trees from a collection of gene trees has been gaining popularity among many other taxonomic groups, but has seen little attention in Phytophthora or oomycete research in general. The recent description of P. pisi (Heyman et al. 2013) employed a multispecies coalescent approach, which confirmed the individual analyses of ITS and cox2 data. In addition, a recent study of the hybrid species P. andina (Blair et al. 2012) used several coalescent methods to determine the likely parental lineages of this species, one of which was clearly P. infestans. In the future, the use of more complex phylogenetic methods as well as coalescent-based approaches will be needed to clarify relationships at both ends of the spectrum, from deep basal nodes to recently evolved and potentially interbreeding species complexes.

A common observation among all phylogenetic studies is there is no consistent correlation between phylogenetic grouping and morphological features (Cooke et al. 2000; Kroon et al. 2004, 2012; Blair et al. 2008; Martin et al. 2014). While there is some correlation with sporangial type (clade 4, 5, and 10 have primarily papillate sporangia while clade 3 has primarily semipapillate sporangia and clades 6, 7, and 9 primarily nonpapillate sporangia), other clades show combinations of these features (clade 1, 2 and 8). Characteristics such as oogonial ornamentation, heterothallism, and mode of antheridial attachment are all polyphyletic.

Because of the large number of species, intraspecific variation of some morphological features, and overlapping morphology among closely related species, traditional methods of species identification can be challenging and require some level of expertise to be effective. The use of molecular criteria has simplified this task and provides a tool for delineating distinct taxa within morphologically similar species complexes. The most accurate molecular method for species identification is sequence analysis of specific markers. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA) has been widely used and a large number of sequences are currently available in public databases. However, this marker may not be ideal for the identification of all species, especially those that are closely related. For example, many clade 1C species (P. infestans, P. mirabilis) cannot be distinguished using this marker alone, nor can P. fragariae and P. rubi. More recently a portion of the cox1 gene, along with the ITS region, have been proposed as the markers to use in the Barcode of Life Database ( and representative sequences for all described and some provisional species have been deposited (Robideau et al. 2011).

Several nuclear (60S ribosomal protein L10, β-tubulin, enolase, heat shock protein 90, large subunit rRNA, TigA gene fusion, translation elongation factor 1α; (Blair et al. 2008; Kroon et al. 2004; Villa et al. 2006)) and mitochondrial (cox1, nad1, cox2, nad9, rps10 and secY; (Kroon et al. 2004; Martin 2008; Martin and Tooley 2003a, b; Martin et al. 2014) markers have been sequenced for phylogenetic analysis of Phytophthora and can also be used for species identification. Background information for amplification and sequencing of many of these markers, as well as the capability for BLAST searches against a curated database for isolate identification, may be found at the Phytophthora Database ( A dataset for ITS and cox1 and 2 spacer sequences is also available at Phytophthora ID ((Grünwald et al. 2011), and sequence data for several markers (ITS, β-tubulin, elongation factor 1 alpha, and cox1), along with pictures of morphological features, may be found at Q-Bank (

There are several caveats to consider when using BLAST analysis to identify isolates to species level to prevent misidentification (Kang et al. 2010; Nilsson et al. 2012). BLAST scores are dependent on the length of the aligned sequences as well as the level of sequence identity; instances where high levels of sequence identity occur for only a portion of the target sequence may result in incorrect species identification. Also, it is common to encounter situations where scores are similar among multiple species, making it difficult to draw conclusions about an isolate’s identity (this can be especially problematic for isolates within or related to species complexes). In addition, the use of markers known to contain intraspecific polymorphisms may lead to inaccurate species identifications due to potentially lower similarities among closely related sequences. Heterozygosity in nuclear markers may also complicate identification efforts; while the presence of distinct alleles may indicate outcrossing (as Phytophthora is a known diploid), heterozygosity may also result from hybridization events between distinct lineages (as described above). Phylogenetic analysis of several markers is therefore suggested to confirm species identification, especially when working with species complexes. Additional gel based techniques, such as PCR-RFLP, SSCP, random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLP) and simple sequence repeat (SSR) analysis, for species identification and population analysis are reviewed in Martin et al. (2012).

Recommended genetic markers

The following genetic markers have been found to amplify well across all species and provided a similar level of phylogenetic resolution as a concatenated dataset of seven nuclear and four mitochondrial genes (Martin et al. 2014). Information on amplification and sequencing primers for these genes may be found at the Phytophthora Database (

Nuclear genes–LSU, β-tubulin

Mitochondrial–cox2, nad9, rps10

Phytophthora Data

Sequence alignments of the seven nuclear and four mitochondrial markers used in Martin et al. (2014) and Fig. 20 may be downloaded at TreeBASE ( A table with additional information on isolates used in the analysis may be found in Martin et al. (2014) with GenBank accession numbers listed in the supplementary material of this citation. These sequences can also be downloaded from the Phytophthora Database (



Pythium is classified as belonging to the family Pythiaceae sensu lato (s.l.), order Peronosporales s.l., class Peronosporomycetes, phylum Oomycota, and kingdom Straminipila (Beakes et al. 2014). Although many species are considered to be saprobes, the genus is known primarily for its parasitic interactions with plants. Several species also parasitize algae (green and red), fungi, other oomycetes, nematodes, insects, crustaceans, and fish. One species, P. insidiosum, is the causal agent of pythiosis in mammals, including humans (Van der Plaats-Niterink 1981; de Cock et al. 1987). Plant pathogenic Pythium species often target young below-ground plant parts such as fine roots, germinating seeds and emerging growth, resulting in damping-off, root rot and poor crop stands with stunted plants and reduced yield. Some species can also cause fruit rot, and at least one species, P. vexans, has been associated with trunk cankers of rubber trees (Van der Plaats-Niterink 1981; Zeng et al. 2005). Although some species have a limited host range, such as P. arrhenomanes that seems to be exclusively associated with gramineous crops, species like P. aphanidermatum, P. irregulare and P. ultimum are known for being highly virulent on an extensive range of plant hosts (Van der Plaats-Niterink 1981). However, not all Pythium species have a negative impact on the plants they are associated with. Besides saprobes, others can benefit plants by acting as biocontrol agents that parasitize pathogenic fungi and/or induce host resistance, e.g. Pythium oligandrum (Benhamou et al. 1997). Other species of Pythium have also been reported to stimulate plant growth (Mazzola et al. 2002). Recent genome sequencing of six Pythium species found high levels of variation in the number of CRN (“Crinkler”) effectors found in the different species, possibly suggesting species-specific infection strategies (Adhikari et al. 2013) that may contribute to the range of interactions of Pythium species with their hosts. Such species-specific host-interactions along with the ubiquitous nature of the genus in soils all over the world make accurate species identification necessary to facilitate disease diagnosis and management.

Debates regarding possible genera within Pythium were initially sparked by differences in sporangial morphology. Based on these characters some of the novel genera that have been proposed are Nematosporangium (for species with filamentous zoosporangia), Rheosporangium (species with lobulate zoosporangia), and Sphaerosporangium (species with ovoid, spherical or citriform sporangia) (Schröter 1897; Sparrow 1931). The legitimacies of these genera have been questioned for various reasons (Sideris 1931a; Sparrow 1932; Van der Plaats-Niterink 1981), and aside from some attempts at transferring Pythium species to Nematosporangium (Jaczewski and Jaczewski 1931; Sideris 1931b) the scientific community has stuck with the generic classification of Pythium versus these genera. As molecular taxonomy became a more popular approach to studying systematics, the paraphyletic nature of Pythium became apparent and the debate on splitting the genus was rekindled. Early sequence-based phylogenies provided strong arguments for P. vexans to be part of a separate genus (Briard et al. 1995; Cooke et al. 2000). The ITS and 28S phylogenies of Lévesque and de Cock (2004) divided Pythium into 11 clades (A-K) of which clade K (including P. vexans) is more closely related to Phytophthora than to the rest of the Pythium clades (Villa et al. 2006). A new genus, Phytopythium, was subsequently erected to include all clade K species, with Phytopythium sindhum as type species (Bala et al. 2010), although the official transfer of all clade K Pythium species to Phytopythium has not yet been published. The remaining ten clades (A-J) can be divided into two groups: species with filamentous zoosporangia (clades A-D) and species with globose zoosporangia (clades E-J) (Lévesque and de Cock 2004), calling to mind early suggestions of splitting the genus based on this character (Schröter 1893; Sparrow 1931). This division is echoed to varying degrees by phylogenies of the 28S rRNA, ITS, cytochrome c oxidase subunits 1 and 2 (cox1 and cox2), and β-tubulin, although the different gene trees are often incongruent and support for internal nodes low or absent (Martin 2000; Riethmüller et al. 2002; Villa et al. 2006; Hulvey et al. 2010; Robideau et al. 2011). Despite the shortcomings of these gene regions, Uzuhashi et al. (2010) used 28S and cox2 phylogenies to divide Pythium into five genera: Pythium (clades A-D), Globisporangium (clades E-G, I and J), Elongisporangium (clade H), Ovatisporangium (clade K, syn. Phytopythium), and Pilasporangium (distinct from any of the aforementioned 11 clades). Although this division is more or less in agreement with previous phylogenetic studies, it is problematic with regards to a lack of bootstrap support for the Pythium and Globisporangium clades, and the relationship and distinction between Elongisporangium and Globisporangium is not resolved with support (Fig. 21, Uzuhashi et al. 2010). Additionally, the genera Pythiogeton and Lagena seem to be phylogenetically situated within, or closely related to Pythium emend Uzuhashi, Tojo and Kakishima (Fig. 21, Huang et al. 2013a), so even this revised version of Pythium is paraphyletic. For these reasons investigators have generally been slow to adopt the proposed genera. Following this trend references to “Pythium” in this manuscript refer to Pythium s.l. (i.e. Pythium Pringsheim) unless stated otherwise.

Fig. 21

Maximum likelihood phylogeny of Pythium s.l. and related genera based on the concatenated 18S rRNA, ITS, 28S rRNA, cytochrome c oxidase subunit 2 (cox2), and β-tubulin regions. Bootstrap support values below 60 % are not indicated. Strains in bold typeface represent type-derived material, authentic strains or strains used by Van der Plaats-Niterink (1981) for descriptions. The 11 clades (A–K) of Lévesque and de Cock (2004) and the genera erected by Bala et al. (2010) and Uzuhashi et al. (2010) are indicated on the right along with related taxa such as Phytophthora, Lagenidium, Lagena, and Pythiogeton

Species identification and numbers

A combined list of Pythium species in MycoBank (2014) and Index Fungorum (2014) includes a total of 328 names of which several are either synonyms, orthographic variants or varieties that are rarely referred to and are possibly synonyms of other species (i.e. all varieties excluding varieties of P. ultimum). Excluding such cases along with putative synonyms based on cox1 and ITS sequence homology as identified by Robideau et al. (2011) leaves more or less 230 species of Pythium. Undoubtedly this number still includes species that should be synonymized and/or transferred to genera other than Pythium (Van der Plaats-Niterink 1981; Dick 1990, 2001), but for now this should serve as a rough estimate of the number of actual Pythium species discovered to date. Of these species 152 (66 %) are known to be represented by sequence(s) in GenBank, including at least 123 (53 %) species for which type-material, ex-type strains or strains described by Van der Plaats-Niterink (1981) were used to generate sequence data (Table 19, Fig. 21).

Identification of Pythium isolates to the species level is generally straightforward when comparing both ITS and cox1 sequences to that of ex-type, authentic or other reliable representative strains. For this purpose the sequences generated by Lévesque and de Cock (2004) and Robideau et al. (2011) are excellent resources. Using only the ITS region would more often than not allow suitably accurate species identification, but some species are indistinguishable using ITS and require cox1 sequences for further identification (see Text S1A of Robideau et al. 2011). Several other species are indistinguishable even when both ITS and cox1 sequences are compared (see Text S1B of Robideau et al. 2011), and many of these should probably be formally synonymized pending more thorough investigations with multiple hypervariable genes. This approach should also resolve species complexes found in the group formed by P. irregulare, P. paroecandrum, P. cylindrosporum, P. cryptoirregulare and P. mamillatum (Barr et al. 1997; Matsumoto et al. 2000; Garzón et al. 2007; Spies et al. 2011a), the varieties of P. ultimum (Barr et al. 1996), and the P. vexans and P. cucurbitacearum group (Spies et al. 2011b). Some species epithets have been applied to multiple phylogenetic species due to imprecise species descriptions and/or misidentifications. Examples of these include P. iwayamai, P. okanoganense and P. violae (Lévesque and de Cock 2004; McLeod et al. 2009; Bahramisharif et al. 2013). Mislabelling or contamination of reference strains and/or data cause similar problems, as illustrated by the case of P. terrestre (published as “terrestris”) of which the holotype strain ITS sequence published with the description suggests phylogenetic placement in clade E (Paul 2002), while the ITS and cox1 sequences generated for the ex-type strain available from the Centraal Bureau voor Schimmelcultures (CBS) suggests phylogenetic placement in clade F (Robideau et al. 2011). Species identification within genetically diverse species complexes (see Text S1C of Robideau et al. 2011 for a partial list) can also be tricky, more due to uncertain species boundaries than due to the ineffectiveness of ITS and/or cox1 as barcoding regions. The onus is on the investigator to keep such issues in mind when identifying strains to the species level and to consider the identification in context of the taxonomic history of the species and its closest relatives.

Molecular phylogeny

The first molecular phylogenies of Pythium were inferred from sequences of the 28S, ITS, and cox2 regions respectively, and although each analysis included only a few species, the observed variation merited speculation regarding the polyphyletic nature of Pythium at least for the ITS and 28S phylogenies (Briard et al. 1995; Cooke et al. 2000; Martin 2000). The first study to provide an extensive DNA sequence based phylogeny of Pythium was that of Lévesque and de Cock (2004) who sequenced the 28S region of 51 species and complete ITS region (ITS1-5.8S rRNA-ITS2) region of 116 species. Although a two-marker phylogeny of the ITS-28S region was presented by Lévesque and de Cock (2004), these markers are adjacent multi-copy markers that might not accurately represent the evolutionary relationships in Pythium. Villa et al. (2006) used multiple markers (ITS, cox2, β-tubulin) in individual phylogenetic analyses with 39 species and confirmed previous suggestions of an intermediate evolutionary position of clade K species between Pythium and Phytophthora, but also suggested that clade H species (represented by Phytophthora undulataPythium undulatum) occupy a similar intermediate position, which contrasted the position of this clade nestled among clades E, F, G, I, and J as suggested by Lévesque and de Cock (2004). The multi-marker phylogeny (18S-ITS-28S, cox2 and β-tubulin) of 152 Pythium species and some related taxa presented here confirms the association of clade K with Phytophthora, but fails to provide support for the evolutionary association of clade H with any of the other recognized groups within Pythium (Fig. 21). Furthermore, organisms such as the obligate root pathogen Lagena radicicola and strains resembling Lagenidium form an unresolved cluster of taxa related to clade C (Fig. 21). In itself this phylogenetic placement of the genus Lagena necessitates a further taxonomic revision of the genus Pythium that can only be achieved once the internal nodes of the Pythium phylogeny have been resolved with support. Despite the fact that the phylogeny in Fig. 21 represents the most extensive sampling of taxa and genetic markers in a multi-marker phylogeny of Pythium to date, it still fails to achieve this goal. Phylogenetic markers additional or alternative to those currently used in Pythium systematics are needed to resolve these issues and elucidate taxon boundaries.

Recommended genetic markers

  • The 18S (small subunit, SSU) and 28S (large subunit, LSU) nuclear rRNA genes–generic level phylogenies within Pythium s.l.

  • The internal transcribed spacers (ITS including ITS1, 5.8S rRNA, and ITS2), cytochrome c oxidase subunit 2 (cox2)–sub-generic, inter- and intra-specific level phylogenies

  • ITS and cox1–non-phylogenetic species identification

Mitochondrial regions such as cox1 and cox2 should be used with consideration of the fact that they mainly reflect evolution of maternal lineages and can produce incongruent phylogenies. This is especially true for cox1, which is why this region was not included in Fig. 21. The β-tubulin region has also been used to a limited extent in Pythium phylogenies (Villa et al. 2006; Belbahri et al. 2008; Spies et al. 2011a, b). Although this region fails to resolve Pythium into the genera observed when using the dataset from Fig. 21 (data not shown) and has limited power in resolving species-level phylogenies (Spies et al. 2011a, b), it amplifies and sequences well for most Pythium species and is an easy resource for use in concatenated datasets (e.g. Bahramisharif et al. 2013; Fig. 21).



Pyrenophora represents a genus of plant pathogenic fungi associated with a wide variety of substrates. Fries (1849) list the genus as Pyrenophora typified with Pyrenophora phaeocomes. The genus Pyrenophora clusters in the suborder Pleosporineae of the family Pleosporaceae (Berbee 1996; Zhang and Berbee 2001; Hyde et al. 2013a, b; Zhang et al. 2012; Ariyawansa et al. 2014). Recent studies using multi-gene analysis and some coupled with morphology have provided the groundwork for classification of species in Pyrenophora (Berbee 1996; Zhang and Berbee 2001; Hyde et al. 2013a, b; Zhang et al. 2012).

Pyrenophora has been linked to asexual morphs in Drechslera. Pyrenophora species are important plant pathogens as well as saprobes. Many species cause disease on their graminicolous hosts and are usually present in their asexual state (Drechslera) (Zhang and Berbee 2001). Species of Pyrenophora are serious plant pathogens (Zhang and Berbee 2001). Pyrenophora teres (Drechslera teres) is a necrotrophic pathogen of economically important crops, such as barley (Gupta and Loughman 2001; Kingsland 1991). Pyrenophora graminea (Drechslera graminea) causes barley stripe resulting in significant yield losses (Tekauz 1983, 1990). Pyrenophora graminea lives within barley kernels as mycelium, and when seeds germinate, hyphae enter the seedling through the coleorrhiza, causing a systemic infection (Platenkamp 1976; Porta-Puglia et al. 1986). Pyrenophora tritici-repentis causes tan spot of wheat (Lamari and Bernier 1989) which occurs in all the major wheat-growing areas of the world and causes 3 to 50 % yield losses (Ballance et al. 1996). Its prevalence has increased recently.

Some Pyrenophora species have been used as biocontrol agents. Bromus tectorum is a dominant winter annual weed in cold deserts of the western United States (Meyer et al. 2007). Together with other annual brome grasses it has invaded many ecosystems of the western United States creating near-monocultures in which the native vegetation cannot compete (Meyer et al. 2007). Pyrenophora semeniperda has be used as a biocontrol agent to kill the dormant seeds of Bromus tectorum (Meyer et al. 2007). Several studies have assessed chemical production by Pyrenophora species. A new phytotoxic sesquiterpenoid penta-2,4-dienoic acid (pyrenophoric acid) was isolated from solid wheat seed culture of P. semeniperda.

Species identification and numbers

Pyrenophora is characterized by immersed, erumpent to nearly superficial ascomata, indefinite pseudoparaphyses, clavate to saccate asci usually with a large apical ring, and muriform terete ascospores. Morphologically, the terete ascospores of Pyrenophora can be easily distinguished from Clathrospora and Platyspora. The indefinite pseudoparaphyses and smaller ascospores of Pyrenophora can be clearly separated from those of Pleospora (Sivanesan 1984). Pyrenophora species can easy be distinguished from species in Cochliobolus and Setosphaeria on the basis of the shape, septation and colour of the ascospores (Zhang and Berbee 2001). Drechslera species were initially categorized in Helminthosporium on the basis of their dark colour, transversely septate conidia and a graminicolous habitat (Shoemaker 1959). Consequently, graminicolous Helminthosporium species were segregated into three genera, Bipolaris, Drechslera, and Exserohilum, defined based on their association with their sexual states Cochliobolus, Pyrenophora, or Setosphaeria, respectively (Zhang and Berbee 2001). Currently 198 species of Pyrenophora and 135 species of Drechslera are listed in Index Fungorum (2014).

Molecular phylogeny

Rapid identification of diseases caused by Pyrenophora has been determined via different DNA markers. Identification of molecular genetic markers in Pyrenophora teres f. teres associated with low virulence on ‘Harbin’ barley was assessed by random amplified polymorphic DNA (RAPD) (Weiland et al. 1999) and five RAPD markers were obtained that were associated in coupling with low virulence. The data suggested that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres (Weiland et al. 1999). Specific polymerase chain reaction (PCR) primers were developed from amplified fragment length polymorphism (AFLP) fragments of P. teres, in order to distinguish the two forms, P. teres f. teres (which cause net form blotch on barley leaves) and P. teres f. maculata (which causes spot form); the two forms are morphologically very similar in culture (Leisova et al. 2005). The PCR assay was certified with 60 samples of Pyrenophora species. The amplification with four designed PCR primer pairs provided P. teres form-specific products. No cross-reaction was observed with DNA of several other species, such as P. tritici-repentis and P. graminea (Leisova et al. 2005). Pyrenophora graminea is the causal agent of barley leaf stripe disease (Mokrani et al. 2012). Two leaf stripe isolates PgSy3 (exhibiting high virulence on the barley cultivar ‘Arabi Abiad’) and PgSy1 (exhibiting low virulence on Arabi Abiad), were mated and 63 progeny were isolated and phenotyped for the reaction on Arabi Abiad (Mokrani et al. 2012). From 96 AFLP markers, three AFLP markers, E37M50-400, E35M59-100 and E38M47-800 were linked to the virulence locus VHv1 in isolate PgSy3. Lubna et al. (2012) suggested that the three markers are closely linked to VHv1 and are unique to isolates carrying the virulence locus. Pecchia et al. (1998) developed an efficient PCR protocol for amplification of the IGS region in P. graminea and to characterize this region by restriction fragment analysis. During the study based on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb, two groups of isolates were identified from six cultures i.e. I3/88 (Italy; CBS 100862), I7/88 (Italy; CBS100861), 60/93 (Austria; CBS 100866), I10/95 (Tunisia; CBS 100863), I28/95 (Tunisia; CBS 100864), I33/95 (Tunisia; CBS 100865). The RFLP patterns of isolates obtained with the 6-base cutting enzymes ApaI, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and different between the two groups (Pecchia et al. 1998). Restriction patterns of IGS-PCR products digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their IGS-PCR product length (Pecchia et al. 1998).

Molecular studies of Pyrenophora/Drechslera species have detailed the taxonomic placement of the genus. Initially the 18S rRNA gene was used for the classification of Pyrenophora/Drechslera and related genera (Berbee 1996). Phylogenetic analysis based on 18S rRNA showed Pyrenophora to cluster within the Pleosporaceae (Zhang and Berbee 2001) rather than in Pyrenophoraceae (Zhang and Berbee 2001). Later, phylogenetic analysis of the ITS and gdp data showed that Pyrenophora is monophyletic (Zhang and Berbee 2001), and the asexual state Drechslera clustered with their predicted sexual relatives (Table 20, Fig. 22).

Fig. 22

Phylogram generated from parsimony analysis based on combined of ITS, gdp and LSU sequenced data of Pyrenophora. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Pleospora herbarum CBS 276.37

Recommended genetic markers

  • Large small subunits of nrDNA (LSU)–generic level

  • ITS and gdp–inter-specific delineation

Based on our phylogeny, we observed that gdp gives high resolution compared to ITS and LSU, such that it can be readily used to determine the placement of Pyrenophora species.



Puccinia is the type genus of the family Pucciniaceae in the order of rust fungi, Pucciniales (Basidiomycota). Puccinia has approximately 4,000 named species (Kirk et al. 2008), and is a widespread genus of plant pathogens that has shaped history. For example, Puccinia graminis, the type species of Puccinia, was investigated as a biological warfare agent in the cold war (Line and Griffith 2001). It was the impetus for breeding wheat cultivars resistant to disease that started the Green Revolution, lead by 1970 Nobel Laureate, Norman Borlaug (Zeyen et al. 2014). Epidemics of stem rust of wheat caused by P. graminis remain a threat with the emergence of races such as Ug99 (Singh et al. 2011). Other species of Puccinia are also serious pathogens of grasses (Poaceae), including P. coronata and P. striiformis (Kirk et al. 2008). Rusts of Asteraceae, e.g., P. helianthi, and rusts of Fabaceae in the closely related genus Uromyces, e.g., U. viciae-fabae, U. appendiculatus and U. ciceris-arietini, are important pathogens of cultivated fodder and food crops.

Among the ca. 120 to 160 genera of rust fungi (Cummins and Hiratsuka 2003; Kirk et al. 2008), Puccinia is readily recognized by the two-celled teliospores and the shape of the spermogonia (Cummins and Hiratsuka 2003). Uromyces with one-celled teliospores is typically differentiated from Puccinia, although some species of Puccinia have both one-celled (mesospores) and two-celled teliospores, e.g., P. lagenophorae. Teliospore morphology is homoplasious, and Puccinia and Uromyces were polyphyletic in systematic studies based on the LSU and SSU regions of nuclear ribosomal DNA (Maier et al. 2007; Aime 2006), and the two nuclear genes: elongation factor and β- tubulin (Van der Merwe et al. 2007). Some rust fungi have teliospores morphologically similar to Puccinia, but are not closely related or have an uncertain systematic position. For example, Allodus podophylli has two-celled teliospores convergent with Puccinia. A systematic analysis based on the nLSU and nSSU regions of rDNA determined Allodus and Puccinia were unrelated (Minnis et al. 2012). Puccinia psidii, which spread from South America to much of the Pacific region and South Africa, now infects 30 genera of Myrtaceae out of its natural host range (Pegg et al. 2013). It has two-celled teliospores, but its placement within the Pucciniales is unknown. Phylogenetic analyses of the nLSU and nSSU (Pegg et al. 2013) and the protein coding gene beta-tubulin (Van Der Merwe et al. 2008) indicated that P. psidii was sister to the Pucciniaceae. Several families and genera of rust fungi are polyphyletic, namely the Raveneliaceae, Phakopsoraceae and Pucciniaceae. These polyphyletic families and genera await resolution by molecular phylogenetic analyses.

Species identification and numbers

Rust fungi are usually considered host specific (Cummins and Hiratsuka 2003), although some, e.g., Puccinia psidii and P. lagenophorae, infect multiple host genera (McTaggart et al. 2014; Pegg et al. 2013). Some species of rust fungi are heteroecious, requiring two hosts in different families to complete their life cycle, e.g., P. graminis on Triticum (Poaceae) and Berberis (Berberidaceae).

Rust fungi have a complicated life cycle with up to five spore states (Cummins and Hiratsuka 2003). Consequently, up to three names have been proposed for the same taxon based on different life cycle stages. To add to the confusion, there are two systems of terminology that describe these spore states, one based on morphology (Laundon 1967), and the other on ontogeny (Arthur and Kern 1926; Cummins and Hiratsuka 2003; Hiratsuka 1973). These systems of terminology were summarised by Hennen and Hennen (2000).

Species of rust fungi are often identified on the basis of their host specificity, and monographs were organised by plant family (Sydow and Sydow 1904; McAlpine 1906; Cummins 1971, 1978). Morphological characters of the teliospores and urediniospores, such as size, apex shape and wall thickness, ornamentation, and germ pore position and number, are useful for species identification.

Molecular diagnostic tools have been developed for some species of Puccinia based on the ITS region of rDNA, e.g., P. coronata (Beirn et al. 2011; Pfunder et al. 2001), P. kuehnii (Glynn et al. 2010) and P. psidii (Langrell et al. 2008). The ITS region has successfully distinguished phylogenetic species in Uromyces (Barilli et al. 2011) and it was used in combination with TEF to resolve the taxonomy of P. melampodii (Seier et al. 2009). However, the ITS region was polymorphic in Puccinia lagenophorae (Littlefield et al. 2005; Scholler et al. 2011), and Morin et al. (2009) discovered a paralagous copy of the ITS region, which may have resulted from a hybridization event. A paralagous copy of the ITS region was also reported in P. kuehnii in the study by Virtudazo et al. (2001). Polymorphisms and paralogous copies are caveats for studies based on the ITS region in rust fungi.

Molecular phylogeny

Large-scale systematic studies of rust fungi have focused mainly on the SSU and LSU regions of rDNA (Aime 2006; Beenken et al. 2012; Dixon et al. 2010; Maier et al. 2003, 2007; Minnis et al. 2012; Wingfield et al. 2004; Yun et al. 2011) (Table 21). Protein coding genes such as beta-tubulin (Morin et al. 2009; Van der Merwe et al. 2007, 2008) and elongation factor (TEF) (Seier et al. 2009; Van der Merwe et al. 2007) were successfully used at the family, genus and species level in rust fungi, although beta-tubulin required cloning rather than direct sequencing of PCR product. Liu et al. (2013) included ITS, beta-tubulin, ribosomal polymerase subunit 2 (RPB2) and cytochrome c oxidase subunit 1 (COI) in a systematic study to resolve the P. coronata species complex. They discussed the difficulty of PCR amplification of older herbarium specimens, and that DNA repair was successful in some cases. Vialle et al. (2009) compared mitochondrial genes to rDNA markers in two genera of rusts, Chrysomyxa and Melampsora. They found rDNA had better species resolution than mitochondrial genes. Mitochondrial genes were since used in studies of the genera Chrysomyxa (Feau et al. 2011) and Dasyspora (Beenken et al. 2012), but have not yet been used for Puccinia