The effect of an extract from mycelium of "Penicillium chrysogenum" on plant-pathogen interactions and characterisation of elicitors in this extract

In agriculture, infection of plants with microorganisms including fungi, bacteria and viruses can cause high losses of yield. Apart from a broad spectrum of indirect and direct techniques to protect plants from damage, the concept of induced resistance provides a promising strategy for the control of diseases. Preliminary studies suggested that an aqueous extract from the dry mycelium of the non-pathogenic ascomycete Penicillium chrysogenum, further called ‘Pen’, can enhance resistance of many plants against several pathogens. The objective of this thesis was to unravel whether Pen can be used as a plant activator in commercial agriculture, to study its mode of action and to narrow down the active principles in Pen.
Pen protected grapevine from downy and powdery mildew (P. viticola and U. necator), tomato from early blight (P. infestans), onion from downy mildew (P. destructor) and apple tree from apple scab (V. inaequalis) under greenhouse and field conditions without having a direct fungicidal effect. The efficacy of Pen was generally comparable to traditional fungicides such as copper and sulphur and equal to or even better than well-known inducers of resistance such as BABA or BTH. The raw material for extraction of Pen was of constant quality, a prerequisite for a future application in practice. However, Pen often caused phytotoxic side effects such as small necrotic spots or, more rarely, larger necrotic areas. The development of the phytotoxic symptoms was dependent on several parameters, including concentration of Pen, the number of applications, the persistance on the plant tissue, the plant species and variety and environmental conditions. A partially purified fraction of Pen was less toxic than the crude extract.
To study signal transduction pathways involved in Pen-mediated resistance, the model plant Arabidopsis thaliana was used, allowing a comparison with the mode of action of other well-known inducers. Pen protected A. thaliana from a broad range of pathogens, including an oomycete (Peronospora parasitica), two ascomycetes (Botrytis cinerea, Alternaria brassicicola) and a bacterium (Pseudomonas syringae pv. tomato DC3000). Pen was still fully protective against B. cinerea in Arabidopsis transgenes or mutants impaired in the salicylic acid (NahG, npr1), jasmonic acid (coi1), and ethylene (ein2) signalling pathway. Pen-mediated resistance against P. parasitica was reduced in the transgene NahG, but was not affected in the mutants npr1, coi1 or ein2, indicating that Pen induced resistance against P. parasitica on a salicylic acid-dependent, but NPR1-independent pathway.
Pen triggered early defense-related responses such as an extracellular alkalinisation, an oxidative burst and ethylene production in suspension-cultured cells as well as in intact leaf tissue of numerous mono- and dicotyledon plant species. Cells pretreated with chitin or ergosterol were refractory to a second treatment with the same stimulus but fully responsive to Pen, indicating that Pen contains at least one unidentified elicitor (the ‘Pen-elicitor’).
To develop new strategies for production of an extract without the undesired phytotoxic side effects, we aimed at purifying and characterizing the Pen-elicitor. Measuring early defense-related responses in suspension-cultured cells is a simple, fast and sensitive bioassay and was thus used as a tool for purification and characterization of the Pen-elicitor. The Pen-elicitor could only be isolated from a high but not from a low penicillin-producing strain of P. chrysogenum. The Pen-elicitor was sensitive to protease digestion, to basic hydrolysis, to oxidation by periodate and, to a less extent, to acidic hydrolysis. The Pen-elicitor was not affected by numerous other enzymes and by several chemical treatments. Reversed phase, ion exchange, size exclusion and affinity chromatography revealed that heterogeneity is a characteristic of the Pen-elicitor. Heterogeneity could not be reduced by treating Pen with several specific enzymes or chemicals which do not destroy elicitor-activity, preventing a further analysis.
In conclusion, in this thesis it was shown that Pen has interesting, unique characteristics for an application as a plant protection agent in organic agriculture, provided its phytotoxic side effects can be removed. Our work on Arabidopsis thaliana has revealed that Pen has the potential to protect a plant species against a broad range of pathogens, including biotrophic as well as necrotrophic microorganisms belonging to different classes. Furthermore, Pen seems to activate defense mechanisms by way of signal transduction pathways different from known plant activators. We hypothesize that the Pen-elicitor consists of a small, distinct
elicitor-active region, most likely a protein or peptide, which is part of a larger molecule varying in size and/or chemical composition. Although identification of the resistance-inducing substance would considerably facilitate to develop strategies for the preparation and processing of Pen, it is not necessarily a prerequisite for a future usage in practice. As an alternative, improved formulation as well as refined purification steps could make an application of the Pen-extract feasible.