Oregano essential oil vapour prevents Plasmopora viticola infection in grapevine (Vitis Vinifera) by triggering autoimmune metabolic pathways

The reduction of synthetic fungicides in agriculture a major challenge in maintaining sustainable production, protecting the environment and consumers’ health. Downy mildew caused by the oomycete Plasmopora viticola is the major pathogen in viticulture worldwide and responsible for up to 60% of pesticide treatments. Alternatives to reduce fungicides are thus utterly needed to ensure sustainable vineyard-ecosystems, consumer health and public acceptance. Essential oils (EOs) are amongst the most promising natural plant protection alternatives and have shown their antibacterial, antiviral and antifungal properties on several agricultural crops. However, the efficiency of EOs highly depends on timing, application method and the molecular interactions between the host, the pathogen and EO. Despite proven EO efficiency, the underlying processes are still not understood and remain a black box. The objectives of the present study were: a) to evaluate whether a continuous fumigation of a particular EO can control downy mildew in order to circumvent the drawbacks of direct application, b) to decipher molecular mechanisms that could be triggered in the host and the pathogen by EO application and c) to try to differentiate whether essential oils directly repress the oomycete or act as plant resistance primers. A custom-made climatic chamber was used for a continuous fumigation of potted vines with different EOs during long-term experiments. The grapevine (Vitis vinifera) cv Chasselas was chosen in reason of its high susceptibility to Plasmopara viticola. Grapevine cuttings were infected with P. viticola. and subsequently exposed to continuous fumigation of different EOs at different concentrations, during 2 application time spans (24 hours and 10 days). Experiments were stopped when infection symptoms were clearly observed on the leaves of the control plants. Plant physiology (photosynthesis and growth rate parameters) were recorded and leaves were sampled at different time points for subsequent RNA extraction and transcriptomics analysis. Strikingly, the Oregano vulgare essential oil vapour treatment during 24h post-infection proved to be sufficient to reduce downy mildew development by 95%. Total RNA was extracted from leaves of 24h and 10d treatments and used for whole transcriptome shotgun sequencing (RNA-seq). Sequenced reads were then mapped onto the V. vinifera and P. viticola genomes. Less than 1% of reads could be mapped onto the P. viticola genome from treated samples, whereas up to 30 % reads from the controls mapped onto the P. viticola genome, thereby confirming the visual observation of P. viticola absence in the treated plants. On average, 80 % of reads could be mapped onto the V. vinifera genome for differential expression analysis, which yielded 4800 modulated genes. Transcriptomic data clearly showed that the treatment triggered the plant’s innate immune system with genes involved in salicylic, jasmonic acid and ethylene synthesis and signaling, activating Pathogenenesis-Related-proteins as well as phytoalexin synthesis. These results elucidate EO-host-pathogen interactions for the first time and indicate that the antifungal efficiency of EO is mainly due to the triggering of resistance pathways inside the host plants. This is of major importance for the production and research on biopesticides, plant stimulation products and for resistance-breeding strategies Author Summary The reduction of synthetic plant protection products is a major concern of modern agriculture. The oomycete Plasmopora viticola which causes downy mildew in grapevine is amongst the most important grapevine pests and responsible for the dispersion of huge amounts of pesticides in vineyards. Among the evaluated alternatives to reduce or replace synthetic pesticides, plant volatile compounds could represent a sustainable solution. Some plant essential oils (EOs) have already shown antifungal capacities. However, their application is often difficult in terms of the right timing of treatment, degradation, bad rainfastness, mixability and phytotoxicity. The aim of the present work was to investigate whether the vapour phase, applied by a continuous fumigation of different EOs, might inhibit the development of downy mildew on grapevine, and in case of proven efficiency, to study the induced transcriptomic changes by RNA-sequencing in an attempt to elucidate the underlaying molecular interactions. Our results showed that the vapour phases of O. vulgare and T. vulgaris were highly efficient against the pathogen. The study of differentially expressed genes indicated that the EO vapour triggered the main mechanisms of the plant’s innate immune system such as PTI (Pattern-Triggered Immunity) and ETI (Effector Triggered immunity). For the first time these results highlight the effects of EOs vapour on plant genes expression, which is very valuable information for the development of new natural plant protection products, as well as for breeding disease resistant cultivars.

continuous fumigation of a particular EO can control downy mildew in order to circumvent the drawbacks of 24 direct application, b) to decipher molecular mechanisms that could be triggered in the host and the pathogen by 25 EO application and c) to try to differentiate whether essential oils directly repress the oomycete or act as plant 26 resistance primers.

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A custom-made climatic chamber was used for a continuous fumigation of potted vines with different EOs during 28 long-term experiments. The grapevine (Vitis vinifera) cv Chasselas was chosen in reason of its high susceptibility 29 to Plasmopara viticola. Grapevine cuttings were infected with P. viticola. and subsequently exposed to onto the V. vinifera genome for differential expression analysis, which yielded 4800 modulated genes.

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Transcriptomic data clearly showed that the treatment triggered the plant's innate immune system with genes Introduction 67 68 Global food supply is highly dependent on industrial agriculture, which in turn would not be possible without the 69 intensive use of pesticides against fungal diseases and other pests. Responding to consumers' increasing 70 demands for a sustainable food production implies developing alternatives to conventional synthetic plant 71 protection products. Long-term fungicide applications have consequently led to increased resistances of 72 pathogens and detrimental impacts on ecosystems and humans (1), followed by a decreasing acceptance by 73 consumers (2). This is particularly true for grapevine (Vitis vinifera L.), which is highly sensitive to fungal diseases 7 149 a direct treatment with diffusers or in the form of co-plantations of EO-VOCs emitting plants could be considered 150 in integrated systems, able to control fungal diseases.

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The mechanisms underpinning the effect of antifungal EO-VOCs are not well understood so far. Some VOCs seem 152 to have a direct effect on pathogens, while others seem to elicit the plant innate immune system with its complex 153 mechanism. Several authors have found genes involved in the biosynthesis of phytoalexins, pathogenesis-related 154 (PR) proteins and cell wall proteins when VOCs were applied. (33). Understanding the mechanism involved in EO 155 efficacy against fungi could thus provide very valuable information when developing natural fungicides, plant 156 defense stimulation products, as well as providing genetic targets for the breeding of resistant varieties.

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The aims of the present study were thus to investigate if the vapour phase, applied by a continuous fumigation analysis, showed that the main constituents were carvacrol (21-60 %), p-cymene (6-20 %), γ-terpine (9-26%) 166 and thymol (9-26%) in EO of O. vulgare and thymol (24-45%), p-cymene (15-37%) and g-terpinene (6-24%) in T.  Only 40 genes were downregulated, while 37 genes were upregulated, being common to 24h and 10d   252  treatments. In the meantime, respectively 63 and 37 genes were inversely expressed between the 24h and 10d   253 treatments. This highlights a brief early response of the plant to EO vapour, which is only maintained during a 254 limited time span (0 to > 24h after treatment) and is then followed by an adaptation period, where many early 255 elicited genes return to the pre-treatment expression and a somehow long-term adaptation takes over. This early 256 transcriptomic reprogramming conditioned by EO vapour might thus base its main effect on an eventual plant 257 innate immunity priming.

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This is in some way confirmed by analysing the enriched functional categories (FC) within DEGs (Fig 6A and 6B),

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where for the 10d treatment, no significant enriched FC could be detected. This indicates that the long-term 260 (10d) treatment did trigger genes that are not, or less, collectively regulated within the main metabolic pathways.

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It would thus be likely that the observed gene deregulation by EO in the 10d treatment is somehow random.

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However, for the 24h treatment several categories were significantly and highly enriched in upregulated genes 263 ( Fig 6A) as well as in downregulated genes ( Fig 6B). This observation is discussed in subsequent sections below.

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Hierarchical clustering of DEGs, as illustrated by the heatmaps in Fig 7A and 7B, showed the higher number of 265 upregulated genes for each treatment, in both 24h and 10d experiment conditions. The stringent grouping of 266 genes detected in biological triplicates highlighted the rightness and strong significance of these results.  Visual observation as well as photosynthesis (PS) measurements (Fig 2) during EO vaporisation showed a 498 phytotoxic effect of EOs on vine physiology, which correlated with gene expression data.

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Several FC related to PS are enriched in repressed genes, as shown in Fig 6B (

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A direct phytotoxic effect of EO has previously been reported in several studies (41) and mainly attributed to 508 carvacrol and thymol (97-99), which are also the main terpenes of the applied EO in the present study.

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However, downregulation of PS and PS-related processes could also be part of the innate immune plant 510 response, which has been described in several studies. In particular, upregulation of MPK6, as discussed above, 511 has been associated with an altered expression of photosynthesis-related genes and inhibition of photosynthesis To what extent the inhibitory effect of EO vapour on P. viticola development was due to a direct toxicity of the 535 EO vapour on the pathogens or to the stimulation of the innate immune system of vines, could not be clearly 536 elucidated with the applied methodology.

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The study, however, provides important molecular data, such as target genes, gene networks and metabolic 538 pathways involved in the innate immune system of the plant and therefore important for future genetics studies 539 and resistance-breeding programmes (100-102).