Formulated hydroxy fatty acids from fruit pomaces reduce apple scab development caused by Venturia inaequalis through a dual mode of action

Abstract

The outermost hydrophobic layer of plants, i.e. the cuticle, is mainly composed of cutin, a polyester of hydroxy fatty acids with reported eliciting and/or antimicrobial activities for some of them. By-products of the fruit processing industry (fruit pomaces), often strongly enriched in cuticular material, are therefore a potential source of bioactive compounds for crop protection against pathogen attack. We investigated the utilization of tomato and apple pomaces in the development of a cutin-based biocontrol solution against apple scab, a major apple disease caused by Venturia inaequalis. Several cutin monomer extracts obtained through different strategies of depolymerization and purification were first compared for their ability to induce a targeted set of defense genes in apple seedlings after foliar application. After a step of formulation, some extracts were chosen for further investigation in planta and in vitro. Our results show that formulated cutin monomers could trigger a significant transcriptome reprogramming in apple plants and exhibit an antifungal effect on V. inaequalis. Cutin monomers-treated apple seedlings were significantly protected against infection by the apple scab agent. Altogether, our findings suggest that water-dispersed cutin monomers extracted from pomaces are potential new bio-based solutions for the control of apple scab.

Keywords: apple; apple scab; cutin; defense; hydroxy fatty acids; pomaces; protection; tomato.

Figure 1

Normalized attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) spectra of the cutin monomer fractions.

Figure 2

Defense-eliciting activity of crude tomato cutin monomer extracts (E1 to E6, 0.5 g/l) on apple seedlings. (A) Cumulative histograms of log₂ ratios (extracts vs. water) obtained for 29 defense genes (grouped by defense categories) in leaves, 3 days after the second treatment. Mean values obtained from 4 biological replicates in 2 independent experiments (n = 4). (B) Statistical analysis of gene expression data. Letters indicate statistical classes (Kruskal-Wallis and LSD, P < 0.05). (C) Radar graph detailing the log₂ ratio (extract vs. water) obtained for each gene with extracts E1 and E3. ACCO, 1-aminocyclopropane-1-carboxylic acid oxidase; AGG, agglutinin; APOX, ascorbate peroxidase; BIS2, biphenyl synthase 2; CalS, callose synthase; CHS, chalcone synthase; CAD, cinnamyl alcohol dehydrogenase; CSL, cysteine sulfoxide lyase; DFR, dihydroflavonol reductase; EIN3, ethylene insensitive 3; EDS1, enhanced disease susceptibility 1; FPPS, farnesyl pyrophosphate synthase; GST, glutathione-S-transferase; HMGR, hydroxymethyl glutarate-CoA reductase; JAR, jasmonate resistant 1; LOX, 13-lipoxygenase; PAL, phenylalanine ammonia-lyase; PR1-2-4-5-8-10-14, pathogenesis-related proteins; PECT, pectin methyl esterase; POX, peroxidase; PPO, polyphenol oxidase; TPS, terpene synthase; WRKY, WRKY transcription factor 53; W, water control.

Figure 3

Dose-response to the formulated tomato cutin monomer extracts E1F and E3F. Cumulative histograms showing log₂ ratios (extracts or FB vs. water) obtained for 10 defense genes in apple leaves, 3 days after the second treatment with the different doses of extracts (ranging from 0.25 to 2 g/l). Mean values obtained from 4 biological replicates in 2 independent experiments (n = 4). AGG, agglutinin; BIS2, biphenyl synthase 2; CSL, cysteine sulfoxide lyase; PR1-2-4-8-10-14, pathogenesis-related proteins; WRKY, WRKY transcription factor 53; FB, formulation blank.

Figure 4

Defense-eliciting activity of the formulated tomato (E3F) and apple (E7F) cutin monomer extracts (1.5 g/l). Cumulative histograms showing log₂ ratios (extracts vs. water) obtained for 10 defense genes in apple leaves, 3 days after the second treatment. Mean values obtained from 6 biological replicates in 3 independent experiments (n = 6) with P-values of LSD test (* P < 0.05 and ** P < 0.01) between water and other groups. AGG, agglutinin; BIS2, biphenyl synthase 2; CSL, cysteine sulfoxide lyase; PR1-2-4-8-10-14, pathogenesis-related proteins; WRKY, WRKY transcription factor 53; FB, formulation blank.

Figure 5

Apple leaf transcriptome response to the formulated tomato cutin monomer extract E3F (1.5 g/l). (A) Volcano plot representing gene expression modulation 3 days after the second treatment. Each scattered point represents a single gene: the x coordinates correspond to the log fold change (logFC) of the expression ratio between E3F- and FB-treated plants; the y coordinates correspond to –log₁₀ of the p-value (n = 3 samples per treatment from 3 independent experiments). (B) Distribution of the number of genes by logFC ranges with main enriched Gene Ontology (GO) categories of biological process. D.E, differentially expressed.

Figure 6

Activities of the formulated tomato cutin monomer extracts E1F and E3F (1.5 g/l) on V. inaequalis. (A) Protection tests with the extracts applied as two preventive treatments. Sporulating lesions recorded on treated or untreated apple leaves 2 (bright colors) and 3 (bright colors) weeks post-inoculation. Mean values ± 95% confidence intervals obtained from 90 plants in 3 independent experiments (n = 90). Letters indicate statistical classes (Kruskal-Wallis and LSD, P < 0.05). The pictures show different levels of disease severity on apple seedling leaves (B) In vitro biocidal effect (mycelium growth of V. inaequalis). Mycelium mats (diameter) measured 12 days after transferring inoculum plugs in Petri dishes containing solid medium supplemented either with E1F, E3F, FB, water or captan. Three Petri dishes with 3 plugs per condition and per experiment in at least 2 independent experiments (18 ≤ n ≤ 27). The initial diameter of the plugs is represented by the dashed line (9 mm). FB, formulation blank.

Figure 7

Hypothetical immune response to cutin monomers treatment. Cutin monomers are probably recognized by RLK/RLP receptors whose expression is notably up-regulated after recognition (these receptors could recognize cutin monomers released by the cutinase activity of Venturia inaequalis and also chitin monomers from the fungus). Recognition of these motifs triggers a PTI-like response, including the initiation of an oxidative burst. JA synthesis is induced, triggering the expression of defense-related genes. The biosynthesis of antimicrobial PR proteins and phytoalexins is then initiated, along with that of the cell wall, cuticle and wax components, leading to the establishment of physical and chemical barriers against the fungus. ABCG, ABC transporter G; BIS, biphenyl synthase; CER, eceriferum; CYP450s, cytochrome P450 proteins; DAMPs/PAMPs, damage/pathogen-associated molecular patterns; GDSL, GDSL esterase/lipase; GST, glutathione-S-transferase; KCS, ketoacyl-CoA synthase; LAC15, laccase 15; LOX, 13-lipoxygenase; LTP, lipid transfer protein; MYB15, MYB transcription factor 15; MYC4, MYC transcription factor 4; NMD, monoterpene synthase; PAE, pectin acetyl esterase; PECT, pectin esterase inhibitor; POX, peroxidase; PPO, polyphenol oxidase; PR1-2-4-8-10-14, pathogenesis-related proteins; PTI, PAMP-triggered immunity; RhaT, rhamnosyltransferase; RLK/RLP, receptor-like protein kinase/receptor-like protein; ROS, reactive oxygen species; TIFY/JAZs, jasmonate ZIM-domain protein; TPS, terpene synthase; WRKYs, WRKY transcription factors.