Phylogeny and DNA-based identification in Phoma and related genera

This thesis treats the taxonomy of a generic complex presently known as PhomaSacc. emend Boerema & Bollen. This group of fungi comprises more than 200 taxa at species or variety level that are characterised by the production of hyaline, non-septate conidial spores in pycnidial conidiomata. The genus is omnipresent in the environment, and exponents can be found on a wide range of host substrates.

For many years the genus Phomawas the main research topic of a group of mycologists at the Dutch National Plant Protection Service. The studies conducted in the last decennia of the previous century culminated in a handbook that monographed the majority of the species in the above-mentioned generic complex. This handbook marked the end of the era in which the taxonomy of this genus mainly relied on morphological observations and cultural descriptions. However, it can also be regarded as the starting point of the present study. The aim of the present project was to integrate DNA-based identification methods into the taxonomic system established by the previously mentioned group of researchers. The major part of this study therefore deals with the validation of current generic and species concepts.

An extensive literature review of the biology, taxonomy and identification methods to the species in this genus is provided in Chapter 2, with specific reference to the progress that has been made in Phomataxonomy after the publication of the abovementioned handbook. The advantages and disadvantages of the current taxonomical system are discussed. Furthermore, this chapter describes the general biology of the species in this fungal group, including their life cycles, distribution and host substrates. The importance of the genus for plant health and quarantine issues is illustrated, and the development of a rapid and robust identification technique based on DNA barcodes is advocated.

Chapter 3treats species in Phomasection Peyronellaea. Species in this section are typified by the production of dictyochlamydospores, and thus have additional morphological characters to use in taxon delineation in comparison with species in the other Phomasections. All species in this group were subjected to a morphological re-examination and phylogenetic analyses employingITS, actin, and β-tubulin nucleotide sequences. Based on multi-gene analyses, Phomasection Peyronellaeacould not be maintained as a taxonomic entity, due to the polyphasic nature of taxa in this section. The morphological study revealed that for five species a taxonomic revision was required. A further five species appeared to be new to science, including Ph. microchlamydospora, Ph. omnivirens, and Ph. schachtii. Also the taxonomic noveltiesPh. coffeae-arabicaeand Ph. sancta are described here, and are allocated to the genus Peyronellaea, re-erected in Chapter 5.

In Chapter 4the diversity among species and varieties belonging to the Ph. exiguaspecies complex is investigated. The Ph. exiguaspecies complex includes nine taxa at varietal level and four species that have a high morphological similarity both in vivoand in vitro, whilst historical relations with plant hosts cannot be maintained. Among this group, both omnipresent saprobes as well as host-specific plant pathogens are present – including the potato pathogen Ph. foveata.

The diversity in this complex is studied by means of Internal Transcribed Spacer regions 1 & 2 and intervening 5.8S nrDNA (ITS) and actin nucleotidesequence analyses and a DNA fingerprinting technique rarely used to study fungal diversity. This technique, DNA Amplification Fingerprinting (DAF) employs short, arbitrary primers that form a loop, or a mini-hairpin, under specific temperature conditions and is frequently used in molecular plant breeding. The amplified DNA fragments were isolated and sequenced in order to develop taxon-specific markers and primer combinations based on the SCARs (Sequence Characterised Amplified Regions) and actin sequence data generated. These tools can aid rapid identification of this morphologically highly similar set of taxa.

Two separate taxa were recognised within the type variety Ph. exiguavar. exigua. In the following chapter these taxa are described and all species and varieties in this complex are recombined into the new genus Boeremia.

Chapter 5provides further details about the taxonomy and phylogeny of the species of interest, with a special focus on the taxa that are phylogenetically placed with Didymellaceae. In total 206 taxa were treated, of which 159 have affinities with Phoma.

The genus is circumscribed in the first section of this chapter. The phylogeny was reconstructed using 28S nrDNA (Large Subunit) and 18S nrDNA (Small Subunit) sequence data. It was shown that the currently used Boeremaean subdivision of the phomoid taxa and the phylogeny were inconsistent, as the genus was highly polyphyletic. Species belonging to the form-genus Phomawere retrieved in as much as six distinct clades within Pleosporales. These clades even represent different families. The majority of the phomoid taxa, including the type species Ph. herbarumand most exponents of the sections Macrospora, Peyronellaea, Heterospora, and Phyllostictioides, were found in a single clade that represented the Didymellaceae. Most species that are associated with the Phomasections Plenodomusand Pilosacluster with the Leptosphaeriaceaeand Pleosporaceaeclades. Furthermore, some species were also found to cluster in the Sporormiaceaeand Cucurbitariaceaeclades.

In the second part of this chapter, the phylogenetic variation of the species and varieties in Didymellaceaeis further assessed, using a phylogenetic reconstruction that is based on DNA sequences of the Large Subunit, ITS, and part of the β-tubulin (TUB) gene region. Besides the teleomorph genus Didymella, members of the teleomorph genera Leptosphaerulinaand Macroventuriawere also found to cluster in this clade. Based on the reconstructed phylogeny, Didymellaceaesegregate into at least 18 distinct clusters, of which many can be associated with specific morphological characters. Furthermore, a number of taxa did not match any of these clusters, suggesting that an evolutionary correct subdivision of the Phomaspecies in Didymellaceaeis even more complex. Taxa in four of these phylogenetic clusters were also defined well enough by means of morphology to elevate these groups to new or reinstalled genera, namely Stagonosporopsis, Epicoccum, Boeremiaand Peyronellaea. A total of 61 taxa were recombined and several new species of Phomawere introduced, namely Ph. brasiliensis, Ph. bulgarica, Ph. dactylidis, Ph. dimorpha, Ph. longicolla, Ph. minor, Ph. pedeiaeand Ph. saxea. Furthermore, two new varieties were described, Boeremia exigua var. gilvescensand B. exigua var. pseudolilacis.

Finally, the results presented in this dissertation are highlighted and discussed inChapter 6. In total 13 species of Phomaand two taxa at varietal level were newly described during the course of this study. Moreover the taxonomic status of species in the form-genus Phomawere further clarified, and insight provided into the phylogenetic status of Didymellaceae, a fungal family that was recently established, comprising most species of Phoma, Ascochytaand Didymella. All macro- and micromorphological data obtained in this study, as well as the DNA sequences, were placed online in a publicly available polyphasic identification database (www.q-bank.eu). This will enable scientists and institutes involved in plant health to correctly identify phomoid species. Rapid identification of these species based on the tools and data generated in this study, can facilitate swift clearing of plant material and arable products during export and import, and prevent the spread of quarantine organisms.