Refine
Document Type
- Doctoral Thesis (8)
Language
- English (8)
Has Fulltext
- yes (8)
Is part of the Bibliography
- no (8)
Keywords
- Aphanomyces astaci (1)
- Ustilaginomycotina (1)
- environmental DNA (1)
- freshwater crayfish (1)
- sage downy mildew (1)
- smut fungi (1)
Institute
Biodiversity is threatened worldwide because of ongoing habitat loss and fragmentation, overexploitation, pollution, biological invasions and a changing global climate. Due to the major importance of biological diversity for modern human living, efficient conservation and management strategies are required to protect endangered habitats and species. For this purpose, ambitious multilateral agreements on regional and global scale were declared to prevent biodiversity loss.
Efficient biomonitoring methods are required to adequately implement these biodiversity conventions. Species monitoring as a core activity in biodiversity research is an effective tool to assess the status of species and trends within habitats. Data collection can be obtained with visual, electronic or genetic surveys. Still, these monitoring programs can be expensive, laborious and inefficient for accurate species assessments. New techniques based on environmental DNA (eDNA) allows for the detection of DNA traces in environmental samples (soil, sediment, water and air samples) and open up new possibilities for species monitoring. The eDNA methodology enables detection of single species in a qualitative (presence/absence) or (semi-) quantitative way. eDNA metabarcoding approaches can be an effective community structure assessment method.
This thesis, located at the interface between experimental and applied research, illustrates the suitability of the eDNA methodology in applied biomonitoring using the example of the water-borne crayfish plague pathogen Aphanomyces astaci (Schikora 1906). The obtained results provide new insights into A. astaci sporulation dynamics in natural water courses. A. astaci sporulation is influenced by seasonal variation of water temperatures and life history traits (molting, activity, mating) of infected crayfish. The results also imply a high transmission risk of A. astaci spores during the complete year. This thesis compares two eDNA methods, which are successfully and consistently detecting A. astaci spores. Each approach is suitable for different biomonitoring tasks due to the method-specific requirements. The obtained results also reveal spatial variation in A. astaci occurance in the tested water bodies. A. astaci spore estimates are positively correlated with population density and pathogen loads of captured A. astaci- positive crayfish. eDNA results show a downstream zoospore transport of up to three kilometres distance from a distribution hot spot area of A. astaci-infected crayfish. The eDNA methodology is helpful in gaining reliable information on A. astaci occurrence in large water bodies. This information is urgently needed to initiate efficient management decisions for the conservation of European crayfish species.
eDNA-based methods such as for A. astaci detection are a useful complement for conventional monitoring and should have a strong impact on conservation policy. eDNA methodology will be helpful for the practical implementation of the main aims of key conservation agreements and thus will make important contributions to biodiversity protection.
This study comprises a survey on ecology, morphology and taxonomy of parasitic fungi infecting Pteridophytes and Orchidaceae found by the author on several field trips to Western Panama as part of the project plant parasitic micro-fungi of Western Panama (ppMP). In Panama, approximately 9500 species of vascular plants are found. Of these, Orchidaceae are with ca. 1150 (ca. 12%) species by far the most speciose family. The Pteridophytes in Panama comprise ca. 940 species in 31 families. Most fungal pathogens on Orchidaceae in tropical regions were described from plants in culture or from material intercepted at borders by plant quarantine services and not from their natural habitats. Therefore, little is known about distribution and ecology of these pathogens in their natural range. The author determined and classified several hundred Orchidaceae-species and Pteridophytes at the sites selected in the context of the project. This work facilitated the identification of many host plants (at least to genus-level) even in sterile condition in the field. About 65 species of Pucciniales are known to infest Orchidaceae and ca. 38% of them are described from tropical America. All available types of Pucciniales on Orchidaceae in tropical America were studied and compared with 91 specimens of rust fungi on orchids collected by the author in Panama. Several hundred additional specimens housed in the BPI, almost all intercepted from plant quarantine services, were used for comparison. As result of this work, it is suggested to combine Uromyces stenorrhynchi Henn. to Sphenospora and, as this is the oldest epithet, to synonymize S. kevorkianii Linder, S. mera Cumm. and S. saphena Cumm. with it. Further, it could be demonstrated that Uredo aurantiaca Montemartini, U. cyrtopodii Syd. & P. Syd., U. epidendri Henn., U. guacae Mayor, U. gynandrearum Corda, U. lynchii (Berk.) Plowr., U. neopustulata Cumm. (≡U. pustulata Henn.), U. nigropuncta Henn., U. oncidii Henn., U. ornithidii F. Kern., Cif. & Thurst., and presumably U. scabies Cke., are anamorphs of this variable species. U. gynandrearum is the oldest anamorph-name for all these taxa. Therefore, it can be established that this rust infects more than 80 species of Orchidaceae in three subfamilies. In total, the anamorph of this species was collected by the author on 17 different species of Orchidaceae in Panama which, apart from one species, are all new hosts to science. The molecular data obtained by the author confirm this view, although more data, especially from material from the whole range of distribution of U. gynandrearum, are necessary. Puccinia spiranthicola Cumm. was found to be a synonym of P. cinnamomea Diet. & Holw. and was found by the author on three different Orchidaceae in two subfamilies. Uredo pleurothallidis Keissl. is now considered a synonym of U. wittmackiana Henn. and the latter as the anamorph of Puccinia oncidii Cumm. In the anamorph genus Uredo, a new species was found infecting at least five different species of Sobralia and Elleanthus (Sobraliinae) at different localities. Molecular data indicate it to be related to the currently polyphyletic Phakopsoraceae. For the rusts with suprastomatal sori on Orchidaceae, now separated from Hemileia and placed in the genus Desmosorus (nom. inval.), the current concept with only one taxon is rejected and the establishment of three subspecies is suggested. The complicated taxonomy is discussed and makes it necessary to validate the genus-name and make a new combination. Another Hemileia-anamorph species was found by the author and is considered to be new to science. This is the first species of this alliance in America on Orchidaceae. Molecular data obtained by the author confirm the separation of Desmosorus from Hemileia and the position of the new species. For rusts on Pteridophytes, a new species of Milesia, (teleomorph: Milesina) and a new anamorphic species of Uredinopsis was found, both on hosts hitherto not known. In Calidion, the presumable anamorph-genus of Uncol, the species C. cf. cenicafeae Salazar & Buriticá was found on several new hosts. Further, the teleomorph was found. Morphologically, this teleomorph did not agree with the description of Uncol by the author of the genus, although the anamorph characteristics left no doubt that it is Calidion. Apparently, the description of Uncol is inadequate, but cannot be improved, as the type is unavailable. Molecular data obtained by the author show this species to be closest to Desmosorus. For Uredo superficialis Speg., the anamorph of Desmella, nine new hosts in eight different fern families were found by the author and the collaborators of the ppMP-project. Ecological data indicate that this species includes different host specific races, which, however could not be distinguished morphologically. For all these rusts, a thorough discussion of the ecology in their habitats is given. In total, 21 LSU rDNA sequences from 6 different rust species on Orchidaceae and Pteridophytes were obtained and analyzed with the Maximum Parsimony and Minimum Evolution method. Here, the position of several groups could be confirmed, and some anamorphs could be assigned to different teleomorphic relationships. Within the Ascomycota and their anamorphs, several hitherto unknown species and species not known from these hosts or not known from Panama were found and analyzed. On Orchidaceae, the following fungi belonging to the Ascomycota are described, illustrated and discussed: In the Phyllachorales, a hitherto not known Phyllachora sp. was found on Oncidium warszewiczii Rchb. f. and was compared with the other species of this order currently known from Orchidaceae. In the Asterinaceae s. l. Lembosia cf. epidendri Meir. Silva & O. R. Pereia was found on Maxillaria crassifolia (Lindl.) Rchb. f., which is a new host and new host alliance for this fungus hitherto only known from Brazil. The fungus is described and compared with all species of Asterinaceae currently known on Orchidaceae. In the Meliolaceae, Meliola orchidacearum Cif. was found on Camaridium biolleyi (Schltr.) Schltr. and an Epidendrum sp. which are new hosts and new host alliances of this fungus which was hitherto only known from the Caribbean Islands. It is described, illustrated and compared with the type. In the Glomerellaceae, Glomerella cingulata and its anamorph Colletotrichum gloeosporioides were found on several hosts. The species is illustrated, described and compared with data from literature. In the anamorphic Mycosphaerellaceae, Pseudocercospora odontoglossii (Prill. & Delacr.) U. Braun, a species currently only known from culture, was found on the new host Pleurothallis imraei Lindl. It is illustrated, described and compared with data from literature. On ferns, the following other fungi are described, illustrated and discussed: A conspicuous undescribed form of Polycyclus was found by the author on Elaphoglossum ciliatum (C. Presl.) T. Moore (Dryopteridaceae) and Serpocaulon loriceum (L.) A. R. Sm. (Polypodiaceae). A conspectus of Parmulariaceae infecting ferns is given and demonstrated that Polycyclina should be synonymized under Polycyclus. Summing up, it can be assessed, especially for the Pucciniales, that the most speciose plant family in Panama carries remarkable few species of specific parasites, and that many of them seem to be distributed over a wide range of species which often are not closely related. One reason amongst others seems to be that parasites need a minimum density of host plants in a habitat to survive. As orchid species often occur with only few (and often small) individual plants at a given locality, the probability for a specific pathogen to infect a plant gets too low, hence high diversity by low abundance of hosts might be an impediment for specific pathogens. In this case, unspecific parasites, or such which are infecting larger alliances, are in advantage. Other reasons could be specific traits of orchids, like succulence and mycotrophy which might hamper fungal infections.
Smut fungi (Ustilaginomycotina) were previously defined as plant parasites that produced blackish or brownish masses of teliospores in or on various organs of plants. Each teliospore germinates to form a single basidium with usually four basidiospores that subsequently grow as a saprobic, yeast-like, haploid stage. The Ustilaginomycotina are a highly diverse group with about 1,700 species in 115 different genera. All of the species were united in a single order, the Ustilaginales, in late 19th century. These teliospore producing fungi are now considered the classic smut fungi. Towards the end of the 20th century, new ideas were brought into this classification system. Most notable was the comparative work regarding the ultrastructure of septal pores and the anatomy of the interaction zones between host and parasite. This work changed the whole concept of smut fungi and their evolutionary relationships. These results were subsequently supported by molecular phylogenetic studies. Both lines of investigation led to the classification of the smut fungi into four different classes, Ustilaginomycetes, Exobasidiomycetes, Malasseziomycetes and Moniliellomycetes (see chapter 1.3).
A reliable taxonomy that reflects phylogenies needed in order to estimate the diversity and the relationships between the diverse groups of smut fungi. In the last 20 years, molecular investigations based mostly on rDNA loci, e.g. ITS (internal transcribed spacer) or LSU (large subunit), have revealed the evolutionary relationships between many taxa of smut fungi. However, there are few phylogenetic studies available for smut fungi (see chapter 1.5.1), and much work is needed to develop backbone phylogenetic trees and to resolve species complexes of many smut fungi.
This thesis reports the results of six different studies that aimed to develop new and improved tools for the phylogenetic analyses of smut fungi, and then apply these methods to selected groups of smut fungi. The first study (Kruse et al. 2017a, Chapter 3) developed a method to improve the amplification of ITS sequences of some smut fungi. Due to its high discrimination value, the ITS gene region is widely used as a barcoding locus for species delimitation of fungi. For this purpose, the general ITS primers ITS1 and ITS4 or more specific modifications, e.g. ITS1F for Ascomycota, ITS4B for Basidiomycota or M-ITS1 for smut fungi, were used. As these primer combinations often yielded unsatisfactory results, due to coamplification of other (contaminant) fungi or the host plant DNA, improvement of the amplification of the ITS region was needed. In order to design new smut specific primers for the ITS region, a representative set of several sequences of the flanking regions of the ITS region (LSU and SSU) of smut fungi, plants and other fungi were downloaded from GenBank. A set of primers was designed on this dataset. These primers were tested on a representative set of about 70 different smut genera under different PCR conditions. Finally, three different primers, one forward primer, smITS-F, and two reverse primers, smITS-R1 and -R2, were selected as the best ones. The following tests with different combinations of these primers, and also under inclusion of the M-ITS1 primer, showed only slight differences in the number of different genera that successfully amplified. But there were some differences regarding the genera that amplified. A broader test on 205 samples in 39 genera showed that the PCR efficiency of the newly designed primers was much better than the primer set ITS4/M-ITS1. With the primers designed in this study almost no non-target ITS was amplified, giving new opportunities especially for amplifying ancient DNA or DNA from older herbarium samples. However, many species groups remain unresolved by only one gene region.
The second study (Kruse et al. 2017c, Chapter 4) found new loci and suitable primers that better resolved multi-locus trees. To date, the most frequently used loci for making multi-locus trees are SSU (small subunit), LSU (large subunit) and ITS (internal transcribed spacer). While the LSU is not always sufficient to distinguish between closely related species, it is highly discriminative above the species level. In an effort to increase the phylogenetic resolution of smut phylogenies, some protein-coding genes were used, including rpb1, rpb2, and atp6 with varying success (see Chapter 2.1.2). As most of these loci are seldom used or sometimes only work on pure cultures because of their low specifity, new protein-coding loci were identified that produced reliable phylogenetic trees. Based on five available genomes, potential gene loci were filtered for possible primers. Initially, 40 different primer combinations for 14 gene loci were tested on a set of twelve different genera of smut fungi. The best candidates were selected and optimized during further tests. Finally, 22 different forward primers and 17 different reverse primers for nine different gene regions were developed, with each differentiating at least one genus of smut fungi (preferably for Ustilaginomycetes). The different primers showed varying discriminative power for different smut genera. They worked best for the Ustilaginaceae, based on the primer designed from Ustilaginomycetes genomes. These new primer sets and loci have the potential to resolve different species groups within the smut fungi and furthermore to produce reliable phylogenetic trees with high resolution. To prove their applicability, three species complexes were investigated in-depth, two from the Ustilaginomycetes and one from the Exobasidiomycetes.
...
Downy mildew of common sage (Salvia officinalis), caused by Peronospora salviae-officinalis, has become a serious problem in sage production worldwide. The causal agent of the disease belongs to the Pe. belbahrii species complex and was described as a species of its own in 2009. Nevertheless, very little is known about its infection biology and epidemiology. The aims of the current study were therefore to unravel the life cycle of this downy mildew and gain deeper insights into the epidemiology of the disease, as well as to clarify the species boundaries in the Pe. belbahrii species complex.
Infection studies showed that temperatures between 15 and 20 °C were most favourable for infection and disease progress. At 5 °C Pe. salviae-officinalis is still able to infect sage plants, but sporulation was only observed at higher temperatures. Furthermore, Pe. salviae-officinalis needs two events of leaf wetness or high humidity, a first one of at least three hours for conidial germination and penetration of the host, and a second one for sporulation. Additionally, contamination of sage seeds by Pe. salviae-officinalis was proven by seed washing and by PCR and DNA sequence comparisons, suggesting that infested seeds might play a major role in the fast spread of sage downy mildew, which is an important finding for phytosanitary or quarantine measures.
A protocol for fluorescence staining and confocal laser scanning microscopy was established and the whole life cycle of Pe. salviae-officinalis was tracked including oospore formation. The method was also used to examine samples of Pe. lamii on Lamium purpureum and Pe. belbahrii on Ocimum basilicum demonstrating the usefulness of this method for studying the infection process of downy mildews in general.
Peronospora species parasitizing S. sclarea, S. pratensis, O. basilicum, and Plectranthus scutellarioides were studied using light microscopy and molecular phylogenetic analyses based on six loci (ITS rDNA, cox1, cox2, ef1a, hsp90 and β-tubulin). The downy mildew on S. pratensis was shown to be distinct from Pe. salviae-officinalis and closely related to Pe. glechomae, and is herein described as a new taxon, Peronospora salviae-pratensis. The downy mildew on S. sclarea was found to be caused by Peronospora salviae-officinalis. The multi-gene phylogeny revealed that the causal agent of downy mildew on coleus is distinct from Pe. belbahrii on basil, and is herein described as a new taxon, Pe. choii.
Deciphering the ecological functions of fungal root endophytes based on their natural occurrence
(2017)
Plants are colonized by a large diversity of fungi, some residing on the surface and others penetrating the plant tissues, the latter referred to as fungal endophytes (endon Gr., within; phyton, plant; de Bary 1879). Despite the saprotrophic potential of fungal endophytes, they are not found to cause visible disease symptoms to the host. Plants are colonized simultaneously by various fungal species, which form rich and diverse endophytic assemblages. Although it is hypothesized that fungal endophytes contribute to the fitness of their hosts and to the functioning of ecosystems, the ecological function of fungal endophytic assemblages remains cryptic. The aims of this doctoral thesis are to gain insight to the ecological functions of root fungal endophytes, by deciphering their roles in ecosystems based on their natural occurrence and the structure of their assemblages. The thesis focuses on studying the diversity and structure of the endophytic mycobiome within roots of two annual and widespread plant hosts Microthlapsi perfoliatum and M. erraticum (Brassicaceae) in several locations across northern Mediterranean and central Europe. The thesis is composed by six Chapters, with a primary focus on Chapter 1, 2 and 3.
Chapter 1 (Glynou et al., 2016) aimed at characterizing the diversity of fungal endophytes in roots at a continental scale and at assessing the factors affecting the structure of endophytic assemblages with the use of cultivation-based methods. For that, root samples were collected from 52 plant populations, along with a collection of soil, bioclimatic, geographic and host data. Cultivation of surface-sterilized root samples on culture media and isolation of fungal colonies in pure culture generated 1,998 fungal colonies. Grouping of sequences into Operational Taxonomic Units (OTUs), based on the 97% similarity of the isolates’ rDNA Internal Transcribed Spacer (ITS) sequence, generated in total 296 OTUs, representing taxa mostly within the phylum Ascomycota with a minor representation of Basidiomycota. Endophytic assemblages were mostly correlated with variation in bioclimatic conditions. Interestingly, despite the large diversity revealed, the assemblages were dominated by only six OTUs related to the orders Hypocreales, Pleosporales and Helotiales, which had a widespread distribution across populations but with some following patterns of ecological preferences.
Chapter 2 aimed at characterizing the uncultivable fraction of the root fungal endophytic diversity, which was not possible to capture in Chapter 1. High-throughput sequencing via the
Illumina Miseq platform was implemented in 43 of the 52 original populations and mostly in the same root samples. In comparison with the cultivation-based approach, the HTS managed to cover the overall diversity within samples. It revealed a large non-cultivated endophytic diversity but the same cultivable fungi dominated assemblages. Moreover, the endophytic diversity was grouped mostly within fungal orders with demonstrated ability to grow in culture and taxonomically related groups were found to have divergent ecological preferences.
The genetic identity of the most abundant OTUs was further investigated in Chapter 3 (Glynou et al., 2017), aiming to unravel genotypic variability, which was possibly overlooked due to the use of lTS, as a universal genetic marker, and could explain their high abundance and widespread distribution. Multi-locus gene sequencing and AFLP profiling for the five most abundant OTUs suggested a low within-OTU genetic variability and show that these fungi have ubiquitous distribution and are not limited by environmental conditions within the ecological ranges of the study. A selection of endophytes frequently isolated in Chapter 1 was functionally characterized in Chapter 4 (Kia et al., 2017) based on the isolates’ traits and interactions with plants. In Chapter 5 (Cheikh-Ali et al., 2015) fungal cultures of Exophiala sp. with differential colony structure where investigated for their production of secondary metabolites. Moreover, Chapter 6 (Maciá-Vicente et al., 2016) comprises the description of the new species Exophiala radicis based on morphological and molecular characteristics.
Compilation of all results shows that the fungal endophytic diversity in roots of Microthlaspi spp. is high but few widespread OTUs dominate the assemblages, and have unlimited dispersal ability. These fungi seem also to have a wide niche breadth and are not affected by environmental filtering. The findings indicate that the local environment but also processes of competitive exclusion determine the structure of endophytic assemblages. In addition, the fungal endophytes associated with Microthlapsi spp. likely have saprotrophic activity however the interactions with plants are likely context-dependent. Further research is needed to assess the biotic interactions among endophytes and their effect on the structure of fungal endophytic assemblages. Ultimately, the findings of this thesis are useful to shed light on the processes underlying the structure of endophytic assemblages. They also upraise the need to describe diversity by combining genetic, metabolic and physiological data, in order to disentangle the elusive ecological roles of the endophytic mycobiome.
Fatty acids in oomycetes
(2021)
The early-diverging oomycetes contain a large number of holocarpic obligate parasites of diatoms, algae, aquatic phycomycetes, and invertebrate animals. These organisms are diverse and widespread. However, taxonomic placement most of the early-diverging oomycetes remains provisional and unresolved, since many have not been sequenced and studied for molecular phylogeny. Here, we report the taxonomy and phylogeny of several holocarpic oomycetes that we have rediscovered and newly classified, including several new species combinations. Phylogenetic reconstructions revealed that the type species of genus Ectrogella (E. bacillariacearum) is a member of the early-diverging Saprolegniales, while the type species of Olpidiopsis (O. saprolegniae) and Pontisma (P. lagenidioides) grouped within the early-diverging lineage of oomycetes forming distinct clades. Since the monophyletic red-algae parasitoids are unrelated to the Olpidiopsis, these were reclassified to the genus Pontisma, while genus Diatomophthora was introduced to accommodate all the diatom parasitoids that were previously assigned to Olpidiopsis. In addition, four new oomycete parasitoids, Miracula helgolandica, Miracula moenusica, Diatomophthora drebesii and Olpidiopsis parthenogenetica and a single rediscovered species, Diatomophthora gillii, are also classified here, including eight new species combinations of red-algae parasites (Pontisma bostrychiae, P. heterosiphoniae, P. muelleri, P. palmariae, P. porphyrae, P. pyropiae) and diatom parasitoids (Diatomophthora drebesii, D. gillii). The results obtained in this study have further improved the resolution and expanded the knowledge on the phylogeny of the earlydiverging oomycetes, leading to the establishment of three new orders (Miraculales, Diatomophthorales, Pontismatales) and one order (Anisolpidiales) being reintroduced.
Taxa under scrutiny in this thesis are Halophytophthora-like oomycetes. The genus Halophytophthora, proposed in 1990, is an assemblage of unrelated species grouped together on the basis habitat preference, i.e. the mangrove or saltmarsh biome, and morphological similarity to Phytophthora. The premise “Phytophthora-like species from the mangrove environment” became the genus concept for Halophytophthora and lasted for almost 2 decades which resulted to the addition of several species (i.e. H. elongata, H. exoprolifera, H. porrigovesica, H. kandeliae, H. masteri, and H. tartarea). At the onset of molecular phylogenetics, Halophytophthora was inferred as a highly polyphyletic taxon and the genus concept was found to be unsuitable. This thesis adds to this, since six Phytophthora spp. were isolated from the mangrove environment, two of which were found in the Philippines (Phytophthora elongata and Phytophthora insolita). After a thorough assessment of the morphologic and phylogenetic data of taxa included in this thesis, several taxonomic novelties were introduced – a new family (Salispinaceae), a new genus (Calycofera), new species (Calycofera cryptica, Phytopythium dogmae, Phytopythium leanoi, Salisapilia coffeyi, and Salispina hoi), and new combinations (Calycofera operculata, Salisapilia bahamensis, S. elongata, S. epistomia, S. masteri, S. mycoparasitica). In addition, Salisapiliaceae and Salisapilia were emended.