Figure legends
Figure 1: The effects of PA treatment on tomato growth and susceptibility to root-knot nematodes.(a) Number of galls in the roots of tomato plants 28 days after inoculation with 250 M. incognita J2s. (b) Number of galls in the roots of rice plants 14 days after inoculation with 250M. graminicola J2s. (c-e) Effect of weekly PA treatment on the dry shoot mass (c), leaf area (b) and root system length (c) of tomato seedlings. Treatments with different letters differ significantly (P < 0.05). N = 8. (f-g) Efficacy of PA (applied thrice as a 300 µM foliar spray) at controlling root-knot nematodes in tomato plants grown in soil naturally infested with a mixed population of M. incognita and M. javanica . Cedroz, a commercial nematicide, was used as reference treatment. PA was applied three times, Cedroz was applied six times according to the manufacturer’s instructions. Error bars indicate the standard error of the mean. (f) Disease severity (as indicated Zeck’s galling index) over the course of the trial. N = 4 plots per treatment (from each of which four plants were uprooted and evaluated at the first three time points and ten at the final time point). (g) Cumulative yield (in kg tomatoes per plot). N = 4 plots per treatment.
Figure 2: Efficacy of PA against foliar pests and diseases of tomato: Botrytis cinereaR16, Pseudomonas syringae DC3000, Tetranychus urticae, Aculops lycopersici and Frankliniella occidentalis . (a)Percentage of B. cinerea inoculation sites that led to successful colonization four days after inoculation (N = 32 inoculation sites on 8 leaves). (b) Fv/Fm(photosynthesis quantum yield) in B. cinerea infection sites.(c) chlorophyll images of B. cinerea inoculation sites in tomato leaves pre-treated with 300 µM piperonylic acid (PA) or a corresponding mock treatment (Control); black coloration indicatesB. cinerea -induced necrosis. (d) Number of P. syringae DC3000 specks per cm2 of leaf area five days after dip inoculation (N = 12). (e) Number of T. urticaeoffspring present on tomato plants ten days after inoculation with 10 adult females (N = 8). (f) Number of A. lycopersicioffspring present on tomato plants ten days after inoculation with 30 mixed-sex mites (N = 6). (g) Number of F. occidentalisoffspring present on tomato plants five days after ten adult females were given 24 hours to deposit eggs (N = 15).
Figure 3: (a) Principal component analysis scatterplot of samples from tomato roots and shoots harvested 24 hours after the second PA or mock treatment (CON), based on mRNA-sequencing based analysis of gene expression. Shaded ellipses represent 95% confidence ellipses.(b) Heatmap (showing normalized, centered and scaled expression values) and hierarchical clustering of samples and transcripts from tomato roots and shoots harvested after PA or mock treatment (CON), based on the same dataset as (a).
Figure 4: Gene expression in shoots (a) and roots(b) of tomato seedlings harvested 6, 24 and 72 hours after the second foliar treatment with 300 µM PA relative to mock-treated control plants. Relative expression values are log2-transformed; error bars indicate 95% confidence intervals. Asterisks indicate statistical significance (*: FDR-adjusted P < 0.05; **: P < 0.01; ***: P < 0.001). BEB: Basic Endochitinase B (Solyc10g055800.2), CHI9: Basic 30 kDa endochitinase CHI9 (Solyc10g055810.2), PR11: Acidic chitinase PR11 (Solyc05g050130.5), POX51: peroxidase , ortholog to A. thaliana peroxidase 51 (Solyc02g092580.3), NIMIN2:NIM-interacting 2-like (Solyc03g119590.1), SAD4: SAR deficient 4 (Solyc06g036330.1), WRKY40: WRKY40 (Solyc06g068460.3), ERF:Ethylene Response Factor D3 (Solyc01g108240.4), NBS: NBS-LRRclass disease resistance protein (Solyc07g056200.3), Twi1:Tomato Wound Induced 1 (Solyc01g107820.2), 23 kDa: 23 kDasubunit of oxygen evolving system of photosystem II (Solyc07g044860.3), CPK2: calcium-dependent protein kinase(Solyc03g033540.3)
Figure 5: Effect of PA treatment on reactive oxygen species metabolism. (a) Percentage of droplets in which DAB staining showed ROS accumulation at one, three, six and 24 hours post treatment. Treatments with different letters are significantly different (P < 0.05). Error bars indicate the standard error of the mean. N = 10 (ten leaflets per treatment, each with three droplets).(b) Peroxidase activity in shoot and root samples of tomato seedlings harvested 14 days after emergence (24 hours after the second PA treatment) and treated with PA or a corresponding control. Error bars indicate the standard error of the mean. (N = 6) (c)Representative images of PA-treated, DAB-stained leaflets from the experiment shown in Figure 4a. The white arrow shows the location where the PA droplet was applied.
Figure 6: (a) PCA plot of samples from tomato roots and shoots harvested after PA or mock treatment (Control), based on the features detected by negative mode UPLC-MS analysis. Shading represent 95% confidence ellipses. (b) Heatmap (showing normalized, centered and scaled feature abundances) and hierarchical clustering of samples and features from tomato roots and shoots harvested after PA or mock treatment (Control), based on the same dataset as used for Figure 6a(c) Number of galls in the roots of tomato plants 28 days post inoculation with 250 J2 juveniles of M. incognita wild type tomato plants (WT) or tomato plants expressing the NahG construct (NahG) and pre-treated with 300 µM piperonylic acid (PA) or a corresponding mock treatment (Control). Treatments with different letters differ significantly.
Figure 7: Effect of exposure to a dilution series of crude methanol extracts from PA or mock-treated tomato shoots and roots on the motility of M. incognita J2s. (a) after 4 hours of exposure (b) after 24 hours of exposure. Data are plotted with a logarithmic x-axis. Quasibinomial regression lines, and their associated 95% confidence intervals (shown as shaded bands, are shown.
Figure 8: Primed accumulation of phenylpropanoid pathway-related products in leaves, stems and roots of tomato plants pre-treated with PA or a corresponding mock treatment and subsequently inoculated with 400 M. incognita J2 juveniles (‘Mi’). Plants were harvested seven days after inoculation. (a) Quantity of free phenolic compounds and other reducing substances as determined via the Folin-Ciocalteu assay, expressed in mg gallic acid equivalent per g of fresh weight. (b) Quantity of cell-wall bound phenolic compounds and other reducing substances released after sodium hydroxide digestion as determined via the Folin-Ciocalteu reaction, expressed in mg gallic acid equivalent per g of fresh weight. (c) Amount of lignin as determined through the acetyl bromide method (expressed as % of total dry cell wall mass). ‘ns’ (not significant) indicates no differences between treatments in a tissue; where significant differences between treatments exist, letters are used to indicate which treatments differ significantly (P < 0.05). N = 15.