Scherf, Andrea (2012)
Licorice, cucumber, downy mildew: tracing the secret Interactions between the plant extract, the host and the pathogen.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung
Kurzbeschreibung (Abstract)
In ancient times, right after they started to domesticate plants, humans had to become plant protectors. The first protection strategies were mainly against herbivorous animals of any kind, arthropods as well as vertebrates. Thousands of years later humans discovered microorganisms like bacteria and fungi and soon correlated them with the disease they, their livestock and their plants had to suffer. The control agents they used against plant pests were mainly inorganic substances such as sulfur, arsenic or mercury. Their own diseases, in contrast, were fought with medicinal plants for thousands of years, more or less successfully. After problems caused by chemical pesticides, like DDT, became obvious (enrichment in the food chain and with that in the human tissues as well), the search for alternatives started. In more recent times more and more scientific studies investigate the potential of medicinal plants as plant protection agents. The aim of this thesis was to investigate the potential of ethanolic extract of leaves of the medicinal plant Glycyrrhiza glabra (licorice) as a control agent against one of the main pests in cucumbers, the Oomycete Pseudoperonospora cubensis (downy mildew). The first question addressed was: Has the licorice extract a potential as alternative control agent, not only in the laboratory, but also under commercial conditions in greenhouses? The second question addressed was: If it shows a potential, what is the active ingredient? And the final question addressed was: What is the mode of action? Under semi-commercial conditions the licorice extract has a high potential as control agent against cucumber downy mildew (efficacy up to 83.0%, application interval 7 -11 days, 3% extract concentration). Besides this, in semi-commercial trials a dark green color of licorice extract treated plants was observed. The crude licorice extract was fractionated by shake-out procedure in 6 fractions. The active ingredient was found to be part of extract fraction F6, which contains acidic substances. This fraction reached efficacies up to 97.6% in bioassays on treated cumber plants. Three flavanoids were detected in sub-fractions of fraction F6 and identified as glabranin, licoflavanon and pinocembrin. All three are known for their antimicrobial effect and were effective against another Oomycete, Phytophthora infestans in in vitro assays. Also, it is known that G. glabra extract kills zoospores of P. cubenis in vitro (Schuster et al. 2010) which may be due to the detected flavanoids. However, the effect of fraction F6 is not explainable by the action of the antimicrobials glabranin, licoflavanon and pinocembrin alone. Sub-fractions not containing those three substances showed efficacies up to 89.2%. This together with the observed dark green color of treated plants in semi-commercial trials led to the assumption that licorice leaf extract has not only a direct effect on the pathogen but influences the plant itself. This assumption was supported by the finding of elevated chlorophyll and anthocyanin content in treated plants and a positive effect on the stress indicator chlorophyll fluorescence. Even in highly infected fraction F6 treated plants (disease severity 92.5%) the Fv/Fm value (0.80) was stable during the whole measuring period, whereas the Fv/Fm value of also highly infected water treated plants decreased (disease severity 100.0%) from 0.80 to 0.69 in the same time. Furthermore, an elevated H2O2 level in the leaf tissues of licorice extract treated uninfected cucumber plants and an up-regulation of PR-1 mRNA could be found. The level of PR-1 mRNA was approximately 4 times higher than in water treated plants. Both, an elevated H2O2 level and an up-regulation of Pr-1mRNA expression are strong indicators for induced resistance. Since PR-1 has antifungal activity, this high amount of PR-1 is a possible explanation for inhibition of germ tube growth of P. cubensis zoospores on licorice treated leaf discs found in this thesis. The germ tube length on water treated leaf discs was 17.3-26.6 µm, whereas the germ tube length on licorice treated leaf disc was only 1.6-6.4 µm. Based on these results it was concluded that the ethanolic leaf extract of G. glabra is a highly potent control agent against cucumber downy mildew (P. cubensis) and its mode of action is a combination of direct effects on the pathogen development and induced resistance.
Typ des Eintrags: |
Dissertation
|
Erschienen: |
2012 |
Autor(en): |
Scherf, Andrea |
Art des Eintrags: |
Erstveröffentlichung |
Titel: |
Licorice, cucumber, downy mildew: tracing the secret Interactions between the plant extract, the host and the pathogen |
Sprache: |
Englisch |
Referenten: |
Thiel, Prof. Dr. Gerhard ; Ullrich-Eberius, Prof. Dr. Cornelia |
Publikationsjahr: |
4 Juni 2012 |
Datum der mündlichen Prüfung: |
2 Februar 2012 |
URL / URN: |
urn:nbn:de:tuda-tuprints-29951 |
Kurzbeschreibung (Abstract): |
In ancient times, right after they started to domesticate plants, humans had to become plant protectors. The first protection strategies were mainly against herbivorous animals of any kind, arthropods as well as vertebrates. Thousands of years later humans discovered microorganisms like bacteria and fungi and soon correlated them with the disease they, their livestock and their plants had to suffer. The control agents they used against plant pests were mainly inorganic substances such as sulfur, arsenic or mercury. Their own diseases, in contrast, were fought with medicinal plants for thousands of years, more or less successfully. After problems caused by chemical pesticides, like DDT, became obvious (enrichment in the food chain and with that in the human tissues as well), the search for alternatives started. In more recent times more and more scientific studies investigate the potential of medicinal plants as plant protection agents. The aim of this thesis was to investigate the potential of ethanolic extract of leaves of the medicinal plant Glycyrrhiza glabra (licorice) as a control agent against one of the main pests in cucumbers, the Oomycete Pseudoperonospora cubensis (downy mildew). The first question addressed was: Has the licorice extract a potential as alternative control agent, not only in the laboratory, but also under commercial conditions in greenhouses? The second question addressed was: If it shows a potential, what is the active ingredient? And the final question addressed was: What is the mode of action? Under semi-commercial conditions the licorice extract has a high potential as control agent against cucumber downy mildew (efficacy up to 83.0%, application interval 7 -11 days, 3% extract concentration). Besides this, in semi-commercial trials a dark green color of licorice extract treated plants was observed. The crude licorice extract was fractionated by shake-out procedure in 6 fractions. The active ingredient was found to be part of extract fraction F6, which contains acidic substances. This fraction reached efficacies up to 97.6% in bioassays on treated cumber plants. Three flavanoids were detected in sub-fractions of fraction F6 and identified as glabranin, licoflavanon and pinocembrin. All three are known for their antimicrobial effect and were effective against another Oomycete, Phytophthora infestans in in vitro assays. Also, it is known that G. glabra extract kills zoospores of P. cubenis in vitro (Schuster et al. 2010) which may be due to the detected flavanoids. However, the effect of fraction F6 is not explainable by the action of the antimicrobials glabranin, licoflavanon and pinocembrin alone. Sub-fractions not containing those three substances showed efficacies up to 89.2%. This together with the observed dark green color of treated plants in semi-commercial trials led to the assumption that licorice leaf extract has not only a direct effect on the pathogen but influences the plant itself. This assumption was supported by the finding of elevated chlorophyll and anthocyanin content in treated plants and a positive effect on the stress indicator chlorophyll fluorescence. Even in highly infected fraction F6 treated plants (disease severity 92.5%) the Fv/Fm value (0.80) was stable during the whole measuring period, whereas the Fv/Fm value of also highly infected water treated plants decreased (disease severity 100.0%) from 0.80 to 0.69 in the same time. Furthermore, an elevated H2O2 level in the leaf tissues of licorice extract treated uninfected cucumber plants and an up-regulation of PR-1 mRNA could be found. The level of PR-1 mRNA was approximately 4 times higher than in water treated plants. Both, an elevated H2O2 level and an up-regulation of Pr-1mRNA expression are strong indicators for induced resistance. Since PR-1 has antifungal activity, this high amount of PR-1 is a possible explanation for inhibition of germ tube growth of P. cubensis zoospores on licorice treated leaf discs found in this thesis. The germ tube length on water treated leaf discs was 17.3-26.6 µm, whereas the germ tube length on licorice treated leaf disc was only 1.6-6.4 µm. Based on these results it was concluded that the ethanolic leaf extract of G. glabra is a highly potent control agent against cucumber downy mildew (P. cubensis) and its mode of action is a combination of direct effects on the pathogen development and induced resistance. |
Alternatives oder übersetztes Abstract: |
Alternatives Abstract | Sprache |
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Schon in historischer Zeit, als sie begannen Pflanzen zu domestizieren, wurden die Menschen zu Pflanzenschützern. Die ersten Strategien dienten vor allem dem Schutz gegen jegliche herbivore Tiere, sowohl Arthropoden als auch Vertebraten. Tausende Jahre später wurden Bakterien und Pilze erstmals mit den Krankheiten der Haustiere und Pflanzen in Zusammenhang gebracht. Die nun eingesetzten Schutzmittel waren vor allem anorganische Substanzen wie Schwefel, Arsen und Quecksilber. Die humanen Krankheiten jedoch wurden, mehr oder weniger erfolgreich, über tausende Jahre mit Medizinalpflanzen kuriert. Nachdem die Probleme, die durch chemische Pestizide, wie DDT, hervorgerufen sichtbar wurden (Anreicherung in der Nahrungskette und auch in menschlichem Gewebe) begann die Suche nach Alternativen. In jüngerer Zeit wurden nun mehr und mehr Studien durchgeführt, die das Potential von Medizinalpflanzen als Pflanzenschutzmittel bewerteten. Das Ziel dieser Arbeit war es dieses Potential eines ethanolischen Blattextraktes der Medizinalpflanze Glycyrrhiza glabra (Süßholz) gegen den Erreger des Falschen Mehltaus an der Gurke, den Oomyceten Pseudoperonospora cubensis, zu erforschen. Die erste Frage war: Zeigt der Süßholzextrakt ein Potential als alternatives Pflanzenschutzmittel nicht nur im Labor sondern auch unter kommerziellen Bedingungen im Gewächshaus? Die zweite Frage war: Wenn der Extrakt dieses Potential zeigt, welche Substanzen sind an der Wirkung beteiligt? Und die abschließende Frage war: Welcher Wirkmechanismus liegt vor? Unter semi-kommerziellen Bedingungen wurde das große Potential des Süßholzblattextraktes als alternatives Schutzmittel deutlich (Wirkungsgrade bis zu 83,0%, Applikationsintervall 7-11 Tage, 3%ige Extraktkonzentration). Zudem wurde in diesen semi-kommerziellen Versuchen eine dunklere Grünfärbung der Pflanzen nach der Behandlung mit Süßholzextrakt beobachtet. Die Fraktionierung des Extraktes erfolgte durch ein Ausschüttelverfahren im Scheidetrichter. Der Rohextrakt konnte in 6 Fraktionen unterteilt werden. Die Wirksubstanzen wurden in der sauren Fraktion F6 gefunden. Mit dieser Fraktion konnten in Biotests, an behandelten Gurkenpflanzen, Wirkungsgrade von bis zu 97,6% erzielt werden. Aus Unterfraktionen der Fraktion F6 konnten die drei Flavonoide, Pinocembrin, Licoflavanon und Glabranin, bestimmt werden. Alle drei Substanzen sind bekannt für ihre antimikrobielle Wirkung. Zudem zeigten sie in in vitro Tests eine Wirkung gegen den Oomyceten, Phytophthora infestans. Auch die von Schuster et al. (2010) in vitro beobachtete Mortalität von P. cubensis Zoosporen nach Kontakt mit G. glabra Extrakt scheint auf die drei Flavonoide zurückführbar zu sein. Dennoch kann die Wirkung von Fraktion F6 nicht alleine mit der Wirkung von Pinocembrin, Licoflavanon und Glabranin erklärt werden. Unterfraktionen, welche nicht die Flavonoide enthielten zeigten in Biotests Wirkungsgrade bis zu 89,2%. Diese Resultate, zusammen mit der beobachteten starken Grünfärbung der Pflanzen in den semi-kommerziellen Versuchen, legten die Vermutung nahe, dass der Extrakt neben dem direkten Effekt gegen das Pathogen auch die Pflanze selbst beeinflusst. Untermauert wurde diese Vermutung durch den Nachweis eines erhöhten Chlorophyll- und Anthocyan- Gehaltes in den Süßholz- behandelten Pflanzen. Zudem hatte die Behandlung der Gurken einen positiven Effekt auf den Stressindikator Chlorophyllfluoreszenz. In stark befallenen Fraktion F6 behandelten Pflanzen (Befall 92.5%) lag der Fv/Fm Wert bei 0,80 über die gesamte Versuchszeit. Der Fv/Fm Wert von ebenfalls stark befallenen Wasserkontrollpflanzen (Befall 100%) fiel in derselben Zeit von 0.80 auf 0,69. Auch konnte ein Anstieg von H2O2 und PR-1 mRNA im Gewebe von Süßholz-behandelten Pflanzen nachgewiesen werden. Der Gehalt von PR-1 mRNA war circa vierfach erhöht gegenüber den Wasserkontrollpflanzen. Sowohl ein Anstieg von H2O2 als auch die Überexpression von PR-1 mRNA kann als starker Indikator für eine Resistenzinduktion durch den Extrakt gewertet werden. Da PR-1 eine bekannte pilzhemmende Wirkung hat, ist dieser hohe Gehalt auch eine mögliche Erklärung für die zu beobachtende geringere Keimschlauchlänge von P. cubensis Zoosporen auf Süßholz-behandelten Blattscheiben. Die Keimschlauchlänge auf Wasser-behandelten Blattscheiben betrug 17.3-26.6 µm, während die Keimschläuche auf Süßholz-behandelten Blattscheiben lediglich eine Länge von 1.6-6.4 µm aufwiesen. Basierend auf all den genannten Ergebnissen ist festzuhalten, dass der Blattextrakt aus G. glabra ein hohes Potential als alternatives Schutzmittel gegen den Falschen Mehltau an der Gurke (P. cubensis) aufweist. Der Wirkmechanismus ist eine Kombination aus direkten Effekten des Extraktes auf die Pathogene als auch auf Induzierter Resistenz in den Gurkenblättern. | Deutsch |
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Freie Schlagworte: |
Biologischer Pflanzenschutz, induzierte Resistenz, Falscher Mehltau, Gurke |
Schlagworte: |
Einzelne Schlagworte | Sprache |
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Biological plant control, induced resistance, downy mildew, cucumber | Englisch |
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Sachgruppe der Dewey Dezimalklassifikatin (DDC): |
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie 500 Naturwissenschaften und Mathematik > 580 Pflanzen (Botanik) |
Fachbereich(e)/-gebiet(e): |
10 Fachbereich Biologie |
Hinterlegungsdatum: |
13 Jun 2012 08:49 |
Letzte Änderung: |
05 Mär 2013 10:01 |
PPN: |
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Referenten: |
Thiel, Prof. Dr. Gerhard ; Ullrich-Eberius, Prof. Dr. Cornelia |
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: |
2 Februar 2012 |
Schlagworte: |
Einzelne Schlagworte | Sprache |
---|
Biological plant control, induced resistance, downy mildew, cucumber | Englisch |
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