Zugl.: Bonn, Univ., Diss., 2007

Universiẗat, Diss., 2004--Bonn.

Non-mycorrhizal fungal endophytes are able to colonize internally roots without causing visible disease symptoms establishing neutral or mutualistic associations with plants. These fungi known as non-clavicipitaceous endophytes have a broad host range of monocot and eudicot plants and are highly diverse. Some of them promote plant growth and confer increased abiotic-stress tolerance and disease resistance. According to such possible effects on host plants, it was aimed to isolate and to characterize native fungal root endophytes from tomato (Lycopersicon esculentum Mill.) and to analyze their effects on plant development, plant resistance and fruit yield and quality together with the model endophyte Piriformospora indica. Fifty one new fungal strains were isolated from desinfected tomato roots of four different crop sites in Colombia. These isolates were roughly characterized and fourteen potential endophytes were further analyzed concerning their taxonomy, their root colonization capacity and their impact on plant growth. Sequencing of the ITS region from the ribosomal RNA gene cluster and in-depth morphological characterisation revealed that they correspond to different phylogenetic groups among the phylum Ascomycota. Nine different morphotypes were described including six dark septate endophytes (DSE) that did not correspond to the Phialocephala group. Detailed confocal microscopy analysis showed various colonization patterns of the endophytes inside the roots ranging from epidermal penetration to hyphal growth through the cortex. Tomato pot experiments under glass house conditions showed that they differentially affect plant growth depending on colonization time and inoculum concentration. Three new isolates (two unknown fungal endophyte DSE48, DSE49 and one identified as Leptodontidium orchidicola) with neutral or positiv effects were selected and tested in several experiments for their influence on vegetative growth, fruit yield and quality and their ability to diminish the impact of the pathogen Verticillium dahliae on tomato plants. Although plant growth promotion by all three fungi was observed in young plants, vegetative growth parameters were not affected after 22 weeks of cultivation except a reproducible increase of root diameter by the endophyte DSE49. Additionally, L. orchidicola increased biomass and glucose content of tomato fruits, but only at an early date of harvest and at a certain level of root colonization. Concerning bioprotective effects, the endophytes DSE49 and L. orchidicola decreased significantly disease symptoms caused by the pathogen V. dahliae, but only at a low dosis of the pathogen. In order to analyze, if the model root endophytic fungus Piriformospora indica could be suitable for application in production systems, its impact on tomato was evaluated. Similarly to the new fungal isolates, significant differences for vegetative growth parameters were only observable in young plants and, but protection against V. dahliae could be seen in one experiment also at high dosage of the pathogen. As the DSE L. orchidicola, P. indica increased the number and biomass of marketable tomatoes only at the beginning of fruit setting, but this did not lead to a significant higher total yield. If the effects on growth are due to a better nutrition of the plant with mineral element was analyzed in barley in comparison to the arbuscular mycorrhizal fungus Glomus mosseae. While the mycorrhizal fungus increased nitrogen and phosphate uptake of the plant, no such effect was observed for P. indica. In summary this work shows that many different fungal endophytes can be also isolated from roots of crops and, that these isolates can have positive effects on early plant development. This does, however, not lead to an increase in total yield or in improvement of fruit quality of tomatoes under greenhouse conditions. / Endophyten, die nicht zu den Mykorrhizapilzen gehören, können das Innere von Wurzeln ohne sichtbare Krankheitssymptome besiedeln und bilden so mit der Pflanze neutrale oder mutualistische Wechselwirkungen. Diese Pilze, auch als nicht-clavicipetale Endophyten bekannt, haben ein breites Wirtsspektrum von mono- und dikotyledonen Pflanzen und weisen eine hohe Diversität auf. Einige von ihnen fördern Pflanzenwachstum und erhöhen Resistenz und Toleranz gegenüber biotischem und abiotischem Stress. Ausgehenden von diesen möglichen Effekten auf ihre Wirtspflanzen war das Ziel der vorliegenden Arbeit die Isolierung und Charakterisierung neuer pilzlicher Wurzelendophyten der Tomate (Lycopersicon esculentum Mill.) und die Analyse ihres Einflusses auf Pflanzenentwicklung und Pflanzenresistenz, sowie auf Ertrag und Fruchtqualität unter Einbeziehung des Modellendophyten Piriformospora indica. Aus vier verschiedenen Anbaugebieten in Kolumbien konnten 51 neue Pilzstämme von oberflächensterilisierten Tomatenwurzeln isoliert werden. Diese Isolate wurden vorcharakterisiert und 14 potentielle Endophyten bezüglich ihrer Taxonomie, ihrer Besiedlungsmuster und ihres Einfluss auf das Pflanzenwachstum näher untersucht. Sequenzierung der ITS Region des ribosomalen RNA Genclusters und genaue morphologische Charakterisierung zeigten, dass sie zu verschiedenen phylogenetischen Gruppen innerhalb der Ascomycota gehören. Neun Morphotypen ließen sich beschreiben, wobei sechs zu den ‚Dark Septate Endophytes’ (DSEs) gehören, aber nicht mit der bekannten Phialocephala Gruppe verwandt waren. Ausführliche konfokale mikroskopische Untersuchungen ergaben sehr verschiedene Besiedelungsmuster der Wurzelendophyten vom Endringen in die Epidermis bis zum Hyphenwachstum durch den Kortex. Topfexperimente unter Gewächshausbedingungen zeigten dass die Isolate in Abhängigkeit von der Inokulumkonzentration und der Zeit der Besiedlung das Wachstum der Tomaten sehr unterschiedlich beeinflussten. Drei neue Isolate (die beiden unbekannte pilzlichen Endophyten DSE48 und DSE49 und eines identifiziert als Leptodontidium orchidicola) mit neutralen oder positiven Effekten wurden für weitere Versuche ausgewählt. In mehreren Experimenten sollte ihr Einfluss auf das vegetative Wachstum, auf Ertrag und auf Fruchtqualität untersucht werden, sowie ihre Fähigkeit die Auswirkungen des Pathogens Verticillium dahliae auf Tomatenpflanzen zu vermindern. Obwohl wachstumsfördernde Effekte durch alle drei Pilze in jungen Pflanzen beobachtet wurden, waren vegetative Wachstumsparameter nach 22 Wochen der Besiedlung nicht mehr beeinflusst bis auf ein signifikante Erhöhung des Wurzeldurchmessers durch den Endophyten DSE49. L. orchidicola dagegen erhöhte die Biomasse und den Glukosegehalt der Früchte, aber nur zu frühen Ernteterminen und bei einer bestimmten Intensität der Wurzelbesiedelung. Hinsichtlich eines schützenden Effekts, konnten die Endophyten DSE49 und L. orchidicola die Krankheitssymptome, die durch V. dahliae verursacht wurden, vermindern, aber nur bei einem geringen Pathogendruck. Um zu überprüfen, ob der Modellendophyt P. indica in Produktionssytemen eingesetzt werden kann, wurde seine Auswirkungen auf Tomaten untersucht. Ähnlich wie die neuen pilzlichen Isolate, zeigte aber auch er seinen fördernden Einfluss nur auf das frühe vegetative Wachstum. Schützende Effekte gegen V. dahliae konnten ebenfalls nur bei niedrigem Pathogendruck konstant beobachtet werden. Wie L. orchidicola erhöhte P. indica die Biomasse an marktfähigen Tomaten am Anfang des Fruchtansatzes, was nicht zu einem insgesamt höheren Ertrag führte. Ob die beobachteten Effekte auf ein verbesserte Nährstoffversorgung der Pflanze zurückzuführen seien, wurde in Gerste im Vergleich mit dem arbuskulären Mykorrhizapilz Glomus mosseae untersucht. Während der Mykorrhizapilz sowohl Phosphat wie Stickstoffaufnehme der Pflanze erhöhte, konnte dies für P. indica nicht festgestellt werden. Zusammenfassend zeigt diese Arbeit, dass auch aus Wurzeln von Kulturpflanzen viele verschiedene pilzliche Endophyten isoliert werden können, und dass einige von diesen durchaus einen positiven Effekt auf die frühe Pflanzenentwicklung aufweisen. Zumindest für Tomate unter Gewächshausbedingungen führen diese Effekte aber nicht zu einer Erhöhung des Gesamtertrags oder einer nachhaltigen Verbesserung der Fruchtqualität.

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Aster tataricus (Asteraceae) is a plant native to Northern Asia and known for its use in the Traditional Chinese and Japanese Medicine. Beside many other secondary metabolites, it contains pentapeptides called astins from which some show an antitumor activity against different human cell lines. Astins are chlorinated, cyclic pentapeptides consisting of proteinogenic and non-proteinogenic amino acids. The astin structure indicates the involvement of non ribosomal peptide synthetases as well as flavin-dependent halogenases. Both enzymes are currently only known from bacteria and fungi. A new endophytic fungus Pelliciarosea asterica was isolated from A. tataricus which produces some of the astins found in the different plant organs. The nearest neighbors of P. asterica are ostropalean fungi from the Stictidaceae lineage (Stictidaceae, Ostropales, Lecanoromycetes, Pezizomycetes, Ascomycota). P. asterica is located in all plant organs of A. tataricus but the highest accumulation of the fungus is found in rhizomes and above-ground organs like leaves or inflorescences. In contrast, the highest astin concentration was found in the roots where nearly no fungus was detectable. P. asterica produces only one of the dichlorinated astins (astin C) in liquid culture, but in A. tataricus all three forms of the dichlorinated astins (A/B and C) were found. This indicates that either the plant is “using” the fungal astin C and metabolize it into one of the other astins or that the fungus, once living inside the plant, is itself producing the other astins. It was also searched for a candidate gene of a halogenase which is essential for the dichlorination of the astins with an antitumor activity. No halogenase could be found by PCR or Southern as well as colony blot, neither in A. tataricus nor in P. asterica. Even the genome sequencing of P. asterica revealed no candidate gene for a halogenase. Endophytes support the plant by suppressing pathogens (antibiosis) or by providing additional nutrients like phosphates or iron to the plant. P. asterica can solubilize different phosphate sources on agar plates. Different fungi are inhibited in growth by P. asterica on agar plates. The endophyte P. asterica from A. tataricus supports its host in different ways and produces secondary metabolites. These secondary metabolites seem to be fungal metabolites either used or degraded by the plant. P. asterica is therefore a good alternative for a possible large-scale production of such antitumor acting astins.

Aster tataricus (Asteraceae) is a plant native to Northern Asia and known for its use in the Traditional Chinese and Japanese Medicine. Beside many other secondary metabolites, it contains pentapeptides called astins from which some show an antitumor activity against different human cell lines. Astins are chlorinated, cyclic pentapeptides consisting of proteinogenic and non-proteinogenic amino acids. The astin structure indicates the involvement of non ribosomal peptide synthetases as well as flavin-dependent halogenases. Both enzymes are currently only known from bacteria and fungi. A new endophytic fungus Pelliciarosea asterica was isolated from A. tataricus which produces some of the astins found in the different plant organs. The nearest neighbors of P. asterica are ostropalean fungi from the Stictidaceae lineage (Stictidaceae, Ostropales, Lecanoromycetes, Pezizomycetes, Ascomycota). P. asterica is located in all plant organs of A. tataricus but the highest accumulation of the fungus is found in rhizomes and above-ground organs like leaves or inflorescences. In contrast, the highest astin concentration was found in the roots where nearly no fungus was detectable. P. asterica produces only one of the dichlorinated astins (astin C) in liquid culture, but in A. tataricus all three forms of the dichlorinated astins (A/B and C) were found. This indicates that either the plant is “using” the fungal astin C and metabolize it into one of the other astins or that the fungus, once living inside the plant, is itself producing the other astins. It was also searched for a candidate gene of a halogenase which is essential for the dichlorination of the astins with an antitumor activity. No halogenase could be found by PCR or Southern as well as colony blot, neither in A. tataricus nor in P. asterica. Even the genome sequencing of P. asterica revealed no candidate gene for a halogenase. Endophytes support the plant by suppressing pathogens (antibiosis) or by providing additional nutrients like phosphates or iron to the plant. P. asterica can solubilize different phosphate sources on agar plates. Different fungi are inhibited in growth by P. asterica on agar plates. The endophyte P. asterica from A. tataricus supports its host in different ways and produces secondary metabolites. These secondary metabolites seem to be fungal metabolites either used or degraded by the plant. P. asterica is therefore a good alternative for a possible large-scale production of such antitumor acting astins.