Induction of plant defence and secretion of volatile compounds by Colletotrichum acutatum in leaf infection assay of woodland strawberry

Abstract: Consumers demand healthy food with less residues of pesticides, in addition to the global awareness of the negative impact such chemicals have caused to the environment during the last decades. Farmers and other agricultural participants are far away from completely end up the usage of pesticides but currently used practice could stepwise be improved in pest management. One major step in this progress is to improve tolerance in our existing cultivars and/or breed new cultivars based on improvements in research in such way that includes plant-pest interactions on the genomic, transcriptomic, and metabolomic level. Plant tolerance (in stronger sense resistance) against pests is a great area of research aiming for reduction in usage of pesticide. One possibility to improve our knowledge of tolerance/resistance, which partially is based on plant produced antimicrobial compounds, is to bioassay such compounds (or secondary metabolites) with plants collected from nature. Woodland strawberry (Fragaria vesca) is a close relative to garden strawberry (Fragaria x ananassa). Fragaria vesca co-existis with important pathogens to F. x ananassa and this wild relative is used as the model plant for research in pathogen/pest-interactions of F. x ananassa. In this work, different fungal pathogens were bio assayed to determine tolerance traits of one F. vesca genotype whose detached leaves has (in previous infection assay) showed to be resistant against the fungal pathogen Colletotrichum acutatum, causing anthracnose disease of F. x ananassa. An inhibition assay was performed with F. vesca leaf extract, resulting in inhibition of the fungal pathogen Botrytis cinerea (causing grey mould on F. x ananassa fruit). This experiment was practicing the well-established theory of pathogen associated molecular pattern (PAMP)-Triggered Immunity (PTI), with responses like de novo production of antimicrobial compounds. Moreover, C. acutatum has been shown to secret βcaryophyllene (a volatile sesquiterpenoid) in resent infection assay with strawberry fruits. Interestingly, β-caryophyllene is also produced by a large number of different plant species. Some plant-secreted volatiles repeals and some attract different lifeforms (e.g. insects) to plants and this phenomenon indicates that volatiles could be essential for the pathogen lifestyle. However, C. acutatum was used for infection assay of F. vesca leaves to determine if β-caryophyllene is secreted during this particular (leaf infection) interaction. Gas Chromatography Mass Spectrometry (GC-MS) analysis was performed for detection of volatiles, resulting in higher abundancy of βcaryophyllene and other volatile sesquiterpenoids in infected leaf samples, in comparison with uninfected leaves (control). RNA extraction of the same leaf samples which were used for the GC-MS analysis was also performed to reveal that the increased abundancy of β-caryophyllene in infected samples, was due to the pathogen secretion and not by the leaf itself. The enzyme which catalyzes the biosynthesis of this product (β-caryophyllene) is coded by the C. acutatum Sesquiterpenoid synthase gene (CaTPS), the gene of interest in this study. CaTPS RNA was not detected during PCR step, most likely due to fail in cDNA synthesis which relies on the purity level of the RNA sample taken from the infected F. vesca leaf tissue.