Abstract
Ramularia is a species-rich genus in the order Capnodiales that harbours hyphomycetes with hyaline conidiophores and conidia with distinct, thickened, darkened, refractive conidiogenous loci and conidial hila. The sexual morph of Ramularia species belongs to Mycosphaerella but the number of experimentally proven links is small and some species may be true asexual holomorphs. Currently Ramularia species are accepted as being host-specific, although some exceptions are known. Most species are phytopathogenic and associated with leaf spots, necrosis or chlorosis, but some species can be saprobic or even hyperparasitic. The most important Ramularia plant pathogens are R. collo-cygni and R. beticola that cause severe economic losses to barley and sugar beet crops, respectively. Protecting crops from damage by weeds, animal pests and pathogens is of major importance in order to increase productivity to meet the global increase in demand for food, feed and bioenergy. The present study serves as a backbone for future studies on the taxonomy of Ramularia and allied genera since it includes the largest number of Ramularia isolates and species ever subjected to DNA sequence analyses. Combined with morphological descriptions and photo plates of several species, it provides a powerful tool to better understand and promote further research on Ramularia and allied genera. More than 1 000 names are known in Ramularia alone, and this study covered only 88 taxa, which means many species still need to be recollected and characterised based on their DNA sequence data. In addition, the present study aimed to clarify the phylogenetic position of the genera currently accepted to belong to Mycosphaerellaceae, thus providing a broad framework and phylogeny for the family and laying a foundation for additional genera and species to be recognised and described. Recent studies have already clearly defined several genera, but it was clear that genera such as Passalora, Zasmidium, Stenella and Ramichloridium remained para- and polyphyletic. Although the type species of several genera have been reliably identified and typified, many genera remain unresolved or are in need of a more in-depth study. What was known as Mycosphaerella sensu Aptroot (2006), now represents a great number of different genera accommodated in different families within Dothideomycetes. The fundamental work performed in this thesis will provide plant pathologists with the resources to facilitate a more reliable identification of the pathogens they work with, as well as provide a solid platform to base their research on, while at the same time also giving more stability to the names which are used to communicate about these fungi.
Chapter 1 gives a general introduction to the genera Mycosphaerella, Ramularia and allied genera. Their taxonomic history and their economic importance as plant pathogens are detailed. The importance of the morphological characteristics in identification versus the molecular approach is introduced. The introduction of the one species = one name and its impact on the taxonomy of Ramularia and allied genera is explained.
Chapter 2 provides an in-depth view on the Ramularia eucalypti species complex. Ramularia eucalypti was the only species of this genus known to infect Eucalyptus by causing severe leaf-spotting symptoms. Isolates of R. eucalypti obtained from other plant hosts, environmental samples and human clinical specimens were heterogeneous based on their ITS sequence data and morphology. Therefore, a polyphasic approach involving morphology, multi-gene phylogeny and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was applied in order to resolve the taxa representing this species complex. A six-gene alignment (ITS, actA, tef1-α, his3, gapdh and rpb2) including 44 isolates of R. eucalypti s. lat. and closely related species was used in both Bayesian and parsimony phylogenetic analysis. The resulting phylogenetic trees showed significant support for separation of seven species: two previously described species (R. eucalypti and R. miae), four newly introduced (R. haroldporteri, R. glennii, R. mali and R. plurivora) and one undescribed Ramularia species (sterile). There are now two Ramularia spp. known to infect Eucalyptus hosts, namely R. eucalypti and R. glennii. The pathogen responsible for causing lenticel rot in fruits of apple and pear in Italy is the newly described R. mali. The ITS barcode was not sufficient to achieve species level identification but any of the partial genes tef1-α, rpb2 or gapdh could be used as a secondary barcode to efficiently identify these species. The growth curve analysis of the studied isolates revealed that one strain of R. plurivora obtained from a clinical sample was able to grow at 40 o C by changing its morphology from a filamentous fungus to an arthroconidial yeast. The MALDI-TOF is a popular diagnostics tool in clinical samples that allows the identification of microorganisms by analysing their unique protein peak pattern and comparing it with a database of reference main mass spectra (MSPs). Main mass spectra (MSPs) of several R. eucalypti s. lat. strains were generated using MALDI-TOF MS and were compared through a Principal Component Analysis (PCA) dendogram. The PCA dendrogram supported three clades containing R. plurivora, R. glennii / R. mali and R. eucalypti / R. miae. Although the dendrogram separation of species differed from the phylogenetic analysis, the clinically relevant strains of R. plurivora and R. glennii were successfully identified by MALDI-TOF MS.
Chapter 3 focuses on the resolution of the species complex of Ramularia endophylla and on the known links between asexual and sexual morphs among Ramularia species. Ramularia endophylla (syn. M. punctiformis) is an endophyte often associated with broad-leaved trees worldwide. The ITS sequences of several isolates from different hosts appeared to be heterogenous. In order to evaluate the presence of cryptic species a polyphasic approach involving morphology and multi-gene phylogeny was employed. A total of 81 isolates of R. endophylla s. lat. and 32 isolates representing 11 Ramularia species were targeted for the amplification of eleven genes (LSU, ITS, actA, tef1-α, his3, gapdh and rpb2, cmdA, tub2, MAT1-1-1 and MAT1-2-1). The amplification of cal and tub2 was often unsuccessful and the sequences obtained were not used in the multigene analysis. The amplification of the mating-type loci was not successful for all the strains and was particularly challenging for the MAT1-2-1 with the use of the available primers. A Bayesian phylogenetic analysis, as well as a parsimony analysis, was performed on a combined five-locus dataset and the resulting trees showed significant support for three species within the complex, including the previously described R. endophylla and R. vizellae, and the newly introduced Ramularia unterseheri. The ITS barcode alone proved to be insufficient for species level identification and the partial gene sequences of actA, rpb2 and gapdh individually proved to be good complementary phylogenetic markers since they successfully separated the three species. The parsimony analyses performed separately with the mating-type gene sequences (MAT1-1-1 and MAT1-2-1) generated trees that were in accordance with those of the multigene analysis. There are presently five Ramularia species with an experimentally confirmed link between the asexual ramularia-like and the sexual mycosphaerella-like morph (R. endophylla, R. grevilleana, R. inaequalis, R. phacae-frigidae and R. variabilis). A total of 15 other links found in literature are either doubtful or have not been experimentally proven and await further collections and study. The taxa identified as Mycosphaerella in much of the plant pathology literature needs to be revisited.
Chapter 4 treats the species within the genus Ramularia and its closest allied genera. Ramularia is a species-rich genus that harbours hyphomycetes with hyaline conidiophores and conidia with distinct, thickened, darkened, refractive conidiogenous loci and conidial hila. Because of its simple morphology closely related species can be difficult to distinguish and several allied genera are frequently confused with Ramularia. In the present study a polyphasic approach based on phylogenetic, morphological and cultural data were used in order to improve species and genus circumscription. A total of 420 isolates were targeted for the amplification and sequencing of six partial genes. Five congruent genes were used in the phylogenetic analysis based on three methods that included Bayesian, Maximum-Likelihood and Parsimony methods. Although Ramularia and Ramulariopsis proved to be monophyletic, Cercosporella and Pseudocercosporella were polyphyletic. The genus Phacellium is tentatively reduced to synonymy with Ramularia since all the studied isolates clustered in the Ramularia clade and the current phylogenetic position of the type species is unknown. Cercosporella and Pseudocercosporella isolates that were not congeneric with the ex-type strains of the type species of those genera were assigned to existing genera or to the newly introduced genera Teratoramularia and Xenoramularia, respectively. The genera Apseudocercosporella, Filiella and Neopseudocercosporella are newly introduced to include pseudocercosporella-like species non-congeneric with their purported type. The genus Fusoidiella was introduced to accommodate a passalora-like species closely related to Filiella. The genera Epicoleosporium and Mycosphaerelloides were newly introduced to accommodate ramularia-like species non-congeneric with Ramularia. Several isolates included in the genus Ramularia were morphologically and molecularly characterised, nine new species were described, 12 species were epitypified, two new combinations and two new names were proposed, and a new sexual-asexual link was observed in R. hydrangeae-macrophyllae. Based on the individual genes, ITS was able to distinguish 58 % of the species while tef1 -α recognised 62 %, actA 72%, gapdh 76 % and rpb2 84 % of the species. The K2P test showed that the ITS barcode has a lower ability to distinguish species than protein-coding genes and that the rpb2 gene would be a good candidate for a secondary barcode gene. Ramularia and allied genera are much undersampled and are frequently described without culture or DNA sequence data.
Chapter 5 introduces a revision of the current taxonomic knowledge of the genera within the Mycosphaerellaceae. The Mycosphaerellaceae contains numerous genera that include thousands of fungal species. Most of these species are plant pathogens and some can cause significant harm to crops we depend on for food, feed and fuel. Nevertheless, the taxonomy of many genera belonging to this family remains unclear to this day mostly due to the scarcity of cultures and the difficulty of identification based on morphological characters. Therefore, a multigene phylogenetic analysis was performed in order to resolve the phylogenetic relationships among the genera currently recognised within the family and to clarify the position of the cercosporoid fungi among them. The alignment was based on three genes (LSU, ITS and rpb2), contained 415 isolates representing 297 taxa and included ex-type strains when available. Based on the analysis, the genera Passalora, Zasmidium, Stenella and Ramichloridium are shown to be para- and polyphyletic. As a consequence, several old generic names including Cercosporidium, Fulvia, Mycovellosiella, Phaeoramularia and Raghnildiana are resurrected to accommodate the species non-congeneric with the Passalora type and 19 genera are newly introduced for the remaining passalora-like species (e.g. Graminopassalora, Pleuropassalora). Previous generic definitions can no longer be applied to these genera in their current circumscription, and the description of new species is strongly reliant on the availability of DNA sequence data. New passalora-like species to be described cannot be assigned without molecular data and, if molecular data are not available, should tentatively be assigned to Passalora s. lat. Species of Ramichloridium and Stenella in Mycosphaerellaceae which were not congeneric with the respective type species currently in Teratosphaeriaceae, were combined into existing genera (e.g. Zasmidium), or newly described genera (e.g. Pachyramichloridium). The genera Periconiella and Verrucisporota were combined under a broader concept of Zasmidium, due to strong phylogenetic support of the basal branches and morphological similarity of the species involved. The genus Phaeophleospora was polyphyletic and species non-congeneric with the type were reassigned to the new genus Pseudophaeophleospora. The rpb2 gene proved to be effective in both species and genera separation within the family and is recommended for future phylogenetic work as a secondary barcode. Based on MycoBank, the Mycosphaerellaceae contains 213 genera but, based on the phylogenetic data from the present study only 120 genera are known to belong in the family. The phylogenetic position of the remaining genera remains unresolved until fresh collections and DNA data are obtained.
Chapter 6 discusses the results of the performed studies for the present thesis. The main focus is the impact of the use of molecular tools in modern classification as well as how it impacts other scientific fields besides taxonomy and systematics.
Chapter 1 gives a general introduction to the genera Mycosphaerella, Ramularia and allied genera. Their taxonomic history and their economic importance as plant pathogens are detailed. The importance of the morphological characteristics in identification versus the molecular approach is introduced. The introduction of the one species = one name and its impact on the taxonomy of Ramularia and allied genera is explained.
Chapter 2 provides an in-depth view on the Ramularia eucalypti species complex. Ramularia eucalypti was the only species of this genus known to infect Eucalyptus by causing severe leaf-spotting symptoms. Isolates of R. eucalypti obtained from other plant hosts, environmental samples and human clinical specimens were heterogeneous based on their ITS sequence data and morphology. Therefore, a polyphasic approach involving morphology, multi-gene phylogeny and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was applied in order to resolve the taxa representing this species complex. A six-gene alignment (ITS, actA, tef1-α, his3, gapdh and rpb2) including 44 isolates of R. eucalypti s. lat. and closely related species was used in both Bayesian and parsimony phylogenetic analysis. The resulting phylogenetic trees showed significant support for separation of seven species: two previously described species (R. eucalypti and R. miae), four newly introduced (R. haroldporteri, R. glennii, R. mali and R. plurivora) and one undescribed Ramularia species (sterile). There are now two Ramularia spp. known to infect Eucalyptus hosts, namely R. eucalypti and R. glennii. The pathogen responsible for causing lenticel rot in fruits of apple and pear in Italy is the newly described R. mali. The ITS barcode was not sufficient to achieve species level identification but any of the partial genes tef1-α, rpb2 or gapdh could be used as a secondary barcode to efficiently identify these species. The growth curve analysis of the studied isolates revealed that one strain of R. plurivora obtained from a clinical sample was able to grow at 40 o C by changing its morphology from a filamentous fungus to an arthroconidial yeast. The MALDI-TOF is a popular diagnostics tool in clinical samples that allows the identification of microorganisms by analysing their unique protein peak pattern and comparing it with a database of reference main mass spectra (MSPs). Main mass spectra (MSPs) of several R. eucalypti s. lat. strains were generated using MALDI-TOF MS and were compared through a Principal Component Analysis (PCA) dendogram. The PCA dendrogram supported three clades containing R. plurivora, R. glennii / R. mali and R. eucalypti / R. miae. Although the dendrogram separation of species differed from the phylogenetic analysis, the clinically relevant strains of R. plurivora and R. glennii were successfully identified by MALDI-TOF MS.
Chapter 3 focuses on the resolution of the species complex of Ramularia endophylla and on the known links between asexual and sexual morphs among Ramularia species. Ramularia endophylla (syn. M. punctiformis) is an endophyte often associated with broad-leaved trees worldwide. The ITS sequences of several isolates from different hosts appeared to be heterogenous. In order to evaluate the presence of cryptic species a polyphasic approach involving morphology and multi-gene phylogeny was employed. A total of 81 isolates of R. endophylla s. lat. and 32 isolates representing 11 Ramularia species were targeted for the amplification of eleven genes (LSU, ITS, actA, tef1-α, his3, gapdh and rpb2, cmdA, tub2, MAT1-1-1 and MAT1-2-1). The amplification of cal and tub2 was often unsuccessful and the sequences obtained were not used in the multigene analysis. The amplification of the mating-type loci was not successful for all the strains and was particularly challenging for the MAT1-2-1 with the use of the available primers. A Bayesian phylogenetic analysis, as well as a parsimony analysis, was performed on a combined five-locus dataset and the resulting trees showed significant support for three species within the complex, including the previously described R. endophylla and R. vizellae, and the newly introduced Ramularia unterseheri. The ITS barcode alone proved to be insufficient for species level identification and the partial gene sequences of actA, rpb2 and gapdh individually proved to be good complementary phylogenetic markers since they successfully separated the three species. The parsimony analyses performed separately with the mating-type gene sequences (MAT1-1-1 and MAT1-2-1) generated trees that were in accordance with those of the multigene analysis. There are presently five Ramularia species with an experimentally confirmed link between the asexual ramularia-like and the sexual mycosphaerella-like morph (R. endophylla, R. grevilleana, R. inaequalis, R. phacae-frigidae and R. variabilis). A total of 15 other links found in literature are either doubtful or have not been experimentally proven and await further collections and study. The taxa identified as Mycosphaerella in much of the plant pathology literature needs to be revisited.
Chapter 4 treats the species within the genus Ramularia and its closest allied genera. Ramularia is a species-rich genus that harbours hyphomycetes with hyaline conidiophores and conidia with distinct, thickened, darkened, refractive conidiogenous loci and conidial hila. Because of its simple morphology closely related species can be difficult to distinguish and several allied genera are frequently confused with Ramularia. In the present study a polyphasic approach based on phylogenetic, morphological and cultural data were used in order to improve species and genus circumscription. A total of 420 isolates were targeted for the amplification and sequencing of six partial genes. Five congruent genes were used in the phylogenetic analysis based on three methods that included Bayesian, Maximum-Likelihood and Parsimony methods. Although Ramularia and Ramulariopsis proved to be monophyletic, Cercosporella and Pseudocercosporella were polyphyletic. The genus Phacellium is tentatively reduced to synonymy with Ramularia since all the studied isolates clustered in the Ramularia clade and the current phylogenetic position of the type species is unknown. Cercosporella and Pseudocercosporella isolates that were not congeneric with the ex-type strains of the type species of those genera were assigned to existing genera or to the newly introduced genera Teratoramularia and Xenoramularia, respectively. The genera Apseudocercosporella, Filiella and Neopseudocercosporella are newly introduced to include pseudocercosporella-like species non-congeneric with their purported type. The genus Fusoidiella was introduced to accommodate a passalora-like species closely related to Filiella. The genera Epicoleosporium and Mycosphaerelloides were newly introduced to accommodate ramularia-like species non-congeneric with Ramularia. Several isolates included in the genus Ramularia were morphologically and molecularly characterised, nine new species were described, 12 species were epitypified, two new combinations and two new names were proposed, and a new sexual-asexual link was observed in R. hydrangeae-macrophyllae. Based on the individual genes, ITS was able to distinguish 58 % of the species while tef1 -α recognised 62 %, actA 72%, gapdh 76 % and rpb2 84 % of the species. The K2P test showed that the ITS barcode has a lower ability to distinguish species than protein-coding genes and that the rpb2 gene would be a good candidate for a secondary barcode gene. Ramularia and allied genera are much undersampled and are frequently described without culture or DNA sequence data.
Chapter 5 introduces a revision of the current taxonomic knowledge of the genera within the Mycosphaerellaceae. The Mycosphaerellaceae contains numerous genera that include thousands of fungal species. Most of these species are plant pathogens and some can cause significant harm to crops we depend on for food, feed and fuel. Nevertheless, the taxonomy of many genera belonging to this family remains unclear to this day mostly due to the scarcity of cultures and the difficulty of identification based on morphological characters. Therefore, a multigene phylogenetic analysis was performed in order to resolve the phylogenetic relationships among the genera currently recognised within the family and to clarify the position of the cercosporoid fungi among them. The alignment was based on three genes (LSU, ITS and rpb2), contained 415 isolates representing 297 taxa and included ex-type strains when available. Based on the analysis, the genera Passalora, Zasmidium, Stenella and Ramichloridium are shown to be para- and polyphyletic. As a consequence, several old generic names including Cercosporidium, Fulvia, Mycovellosiella, Phaeoramularia and Raghnildiana are resurrected to accommodate the species non-congeneric with the Passalora type and 19 genera are newly introduced for the remaining passalora-like species (e.g. Graminopassalora, Pleuropassalora). Previous generic definitions can no longer be applied to these genera in their current circumscription, and the description of new species is strongly reliant on the availability of DNA sequence data. New passalora-like species to be described cannot be assigned without molecular data and, if molecular data are not available, should tentatively be assigned to Passalora s. lat. Species of Ramichloridium and Stenella in Mycosphaerellaceae which were not congeneric with the respective type species currently in Teratosphaeriaceae, were combined into existing genera (e.g. Zasmidium), or newly described genera (e.g. Pachyramichloridium). The genera Periconiella and Verrucisporota were combined under a broader concept of Zasmidium, due to strong phylogenetic support of the basal branches and morphological similarity of the species involved. The genus Phaeophleospora was polyphyletic and species non-congeneric with the type were reassigned to the new genus Pseudophaeophleospora. The rpb2 gene proved to be effective in both species and genera separation within the family and is recommended for future phylogenetic work as a secondary barcode. Based on MycoBank, the Mycosphaerellaceae contains 213 genera but, based on the phylogenetic data from the present study only 120 genera are known to belong in the family. The phylogenetic position of the remaining genera remains unresolved until fresh collections and DNA data are obtained.
Chapter 6 discusses the results of the performed studies for the present thesis. The main focus is the impact of the use of molecular tools in modern classification as well as how it impacts other scientific fields besides taxonomy and systematics.
| Original language | English |
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| Qualification | Doctor of Philosophy |
| Awarding Institution |
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| Supervisors/Advisors |
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| Award date | 16 Oct 2018 |
| Publisher | |
| Print ISBNs | 978-94-6343-494-2 |
| DOIs | |
| Publication status | Published - 16 Oct 2018 |