Correlation coefficients for esterase activity ratios.

<p>Carrot cell suspensions with five different genotypes were tested for esterase relative specific activity in the presence of fungal extracts and toxins. The treatments were as follows: rA: <i>A. dauci</i> (strain FRA017) fungal culture raw extract; rM: uninoculated medium raw extract; aA: <i>A. dauci</i> fungal culture aqueous extract; aM, uninoculated medium aqueous extract; oA: <i>A.dauci</i> fungal culture organic extract; oM: uninoculated medium organic extract; DMSO: DMSO solution at a concentration corresponding to oM, z1, z2 and z3 treatments; z1: 0.025 µM zinniol; z2: 10 µM zinniol; z3: 500 µM zinniol. Correlation coefficients corresponding to significant (α = 0.05) linear regressions are in bold.</p

Influence of cultivar, fungal exudate fractions and zinniol on cell suspension integrity and somatic embryogenesis.

<p>Carrot cell suspensions with six different genotypes were tested for embryogenesis in the presence of fungal extracts and toxins. Embryogenesis was assessed 3 weeks after treatment.</p>1<p>Treatments were as follows: rA: <i>Alternaria dauci</i> (strain FRA017) fungal culture raw extract; rM: uninoculated medium raw extract; aA: <i>A. dauci</i> fungal culture aqueous extract; aM: uninoculated medium aqueous extract; oA: <i>A. dauci</i> fungal culture organic extract; oM: uninoculated medium organic extract; DMSO: DMSO solution, at a concentration corresponding to oM, z1, z2 and z3 treatments; z1: 0.025 µM zinniol; z2: 10 µM zinniol; z3: 500 µM zinniol. C: no treatment.</p>2<p>The signs are as follows: (−) no embryogenesis was visible and cells were damaged, (+) early-stage embryogenic masses were visible, (++) embryos were present, (+++) embryogenesis was profuse. +/− early-stage embryogenic masses were visible, or no embryogenesis was visible depending on the repetition.</p

Range of symptoms observed on leaves 13 days after inoculation.

<p>The symptom number was assessed at 7, 9 and 13(see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101008#pone-0101008-t001" target="_blank">Table 1</a>). The leaves shown here show a symptom severity representative of the plant partial resistance level. <b>A</b>: H1, <b>B</b>: Presto, <b>C</b>: K3, <b>D</b>: H4, <b>E</b>: Bolero, <b>F</b>: I2. H1, K3, H4 and I2 are breeding lines, while Presto and Bolero are widely cultivated Nantaise type carrot cultivars.</p

Range of embryogenic activity observed in cell suspensions 3 weeks after treatment.

<p>In order to assess carrot cell resistance to fungal toxins, carrot cell suspensions were tested for embryogenesis in the presence of fungal extracts and toxins. Embryogenesis was assessed 3 weeks after treatment, and compared to negative controls. Four levels of embryogenic activity were noted. <b>A</b>: (−) no embryogenesis was visible, cells were damaged, <b>B</b>: (+) early-stage embryogenic masses were visible, <b>C</b>: same as B, but after 6 weeks. <b>D</b>: (++) embryos were present, and <b>E</b>: (+++) embryogenesis was profuse.</p

Correlations between cell suspension reactions to <i>Alternaria dauci</i> raw extracts, organic extracts and low zinniol concentrations.

<p>Five carrot genotypes were tested for their metabolic activity when <i>A. dauci</i> raw (rA) or organic (oA) extract was added to the plant culture medium. The same experiments were conducted while adding uninoculated medium raw (rM) or organic (oM) extract and 0.025 µM zinniol to DMSO (z1) or DMSO. rA/rM denotes plant cell esterase activity variations due to the presence of fungal raw extracts, oA/oM denotes plant cell esterase activity variations due to the presence of fungal organic extracts, and z1/DMSO denotes plant cell esterase activity variations due to the presence of 0.025 µM zinniol in the medium. A: correlation plots of rA/rM, oA/oM and z1/DMSO by pairs. Bars represent standard errors. The three paired correlated activity indices presented here correspond to the most significant r² values (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101008#pone-0101008-t004" target="_blank">Table 4</a>). B: 3D correlation plot of rA/rM, oA/oM and z1/DMSO.</p

UHPLC detection of zinniol in fungal extracts.

<p>UHPLC chromatograms were obtained from different FRA017 <i>Alternaria dauci</i> fungal extracts and compared with an UHPLC chromatogram of pure synthetic zinniol. Retention times corresponding to main peaks are indicated <b>A</b>: UHPLC chromatogram of 10 µg synthetic zinniol. Observed zinniol retention time is 8.38 minutes <b>B</b>: UHPLC chromatogram of 13.4 µg organic extract of an <i>A. dauci</i> culture after 48 h under shaking conditions in carrot juice medium. Zinniol expected retention time of 8.38 minutes is indicated. <b>C</b>: UHPLC chromatogram of 13.4 µg organic extract of an <i>A. dauci</i> culture after 12 days without shaking (anoxic conditions) in V8 medium. A strong peak is visible, corresponding to zinniol retention time. <b>D</b>: UHPLC chromatogram of 13.4 µg organic extract of an <i>A. dauci</i> culture after 48 h under shaking conditions in V8 medium. Zinniol expected retention time of 8.38 minutes is indicated. Chromatograms C and D have the same scale. uAU: micro Absorption Units (optical density) at 233 nm.</p

Stability of zinniol over time.

<p>Synthetic zinniol was added to Gamborg medium in order to check its stability over time under our experimental conditions (dark, 22°C, shaking). HPLC was used to measure variations in the zinniol concentration over a time course. Three different HPLC analyses were performed for each time. Zinniol concentrations were divided by the initial zinniol concentration in the medium, giving a relative zinniol concentration (noted % t_o). Except for small (less than 2%) random variations, the zinniol concentration did not vary over time, indicating stability. Standard errors are not represented because they were smaller than the dots we used.</p

Table_2_Aldaulactone – An Original Phytotoxic Secondary Metabolite Involved in the Aggressiveness of Alternaria dauci on Carrot.DOCX

<p>Qualitative plant resistance mechanisms and pathogen virulence have been extensively studied since the formulation of the gene-for-gene hypothesis. The mechanisms involved in the quantitative traits of aggressiveness and plant partial resistance are less well-known. Nevertheless, they are prevalent in most plant-necrotrophic pathogen interactions, including the Daucus carota–Alternaria dauci interaction. Phytotoxic metabolite production by the pathogen plays a key role in aggressiveness in these interactions. The aim of the present study was to explore the link between A. dauci aggressiveness and toxin production. We challenged carrot embryogenic cell cultures from a susceptible genotype (H1) and two partially resistant genotypes (I2 and K3) with exudates from A. dauci strains with various aggressiveness levels. Interestingly, A. dauci-resistant carrot genotypes were only affected by exudates from the most aggressive strain in our study (ITA002). Our results highlight a positive link between A. dauci aggressiveness and the fungal exudate cell toxicity. We hypothesize that the fungal exudate toxicity was linked with the amount of toxic compounds produced by the fungus. Interestingly, organic exudate production by the fungus was correlated with aggressiveness. Hence, we further analyzed the fungal organic extract using HPLC, and correlations between the observed peak intensities and fungal aggressiveness were measured. One observed peak was closely correlated with fungal aggressiveness. We succeeded in purifying this peak and NMR analysis revealed that the purified compound was a novel 10-membered benzenediol lactone, a polyketid that we named ‘aldaulactone’. We used a new automated image analysis method and found that aldaulactone was toxic to in vitro cultured plant cells at those concentrations. The effects of both aldaulactone and fungal organic extracts were weaker on I2-resistant carrot cells compared to H1 carrot cells. Taken together, our results suggest that: (i) aldaulactone is a new phytotoxin, (ii) there is a relationship between the amount of aldaulactone produced and fungal aggressiveness, and (iii) carrot resistance to A. dauci involves mechanisms of resistance to aldaulactone.</p

Toxicity and resistance evaluations using fluorescence microscopy.

<p>Liquid cell cultures from two carrot genotypes were tested for mortality and metabolic activity when <i>Alternaria dauci</i> organic extract (oA) was added to the plant culture medium. The same experiments were conducted while adding uninoculated medium organic extract (oM). Seven and 14 days after adding extracts, membrane integrity and cell viability were evaluated by microscopy using a double staining method with fluoresceine diacetate (FDA) and propidium iodide (IP). The images shown are representative of results obtained from three independent experiments. oA treated K3 cell esterase activity, survival and embryogenesis could not be differentiated from oM treated cells. At 7 days, mortality was somewhat higher and esterase activity lower in oA- than oM-treated H1 cells. At 14 days, much greater observed differences followed a similar trend. High mortality was visible in oA treated H1 cells compared to oM-treated cells. Moreover, proembryogenic masses were visible in oM-treated H1 cultures, and not in oA treated cultures.</p

Comparison of two different carrot <i>A. dauci</i> colonization evaluation methods, symptom number assessment and qPCR-based fungal biomass evaluation.

<p>Carrot plants of six different genotypes were tested for <i>Alternaria dauci</i> resistance using two different methods simultaneously. Plants were grown in greenhouse conditions. The third leaf was inoculated after it was isolated in an incubation chamber without detaching it from the plant. The symptom number was assessed at 7, 9 and 13 dpi. At 13 dpi, leaves were detached and then subjected to DNA extraction and qPCR for fungal biomass evaluation. Log(AUDPC) was calculated from the visual assessments, log(I+1) from the qPCR experiments. Both were subjected to variance analysis followed by a Waller-Duncan multiple comparison. As could be expected, the two parameters were closely correlated (r² = 0.793). Interestingly, log(AUDPC) seemed to show a higher resolution, as the homogeneity groups appeared to be more numerous (4 vs 2).</p>1<p>Homogeneity goups were calculated using the Waller-Duncan multiple comparison following an ANOVA analysis.</p