The aggressiveness of Alternaria dauci isolates was investigated in greenhouse conditions. Twenty-seven isolates were preselected from a large collection to represent high diversity according to geographic or host origins and intergenic spacer (IGS) polymorphism. IGS sequence analysis revealed that isolates were grouped within three different clusters. Eleven isolates were selected and inoculated on a susceptible carrot cultivar. Three criteria (mean lesion number, mean necrotic leaf area and mean disease index) were used to assess the aggressiveness of isolates. Continuous variation in aggressiveness was shown and no clear division into isolate classes was evident. For the host range study, two isolates were inoculated under greenhouse conditions onto nine cultivated Apiaceae species, two wild Daucus species and six cultivated non-Apiaceae species representing six botanical families. Lesions varying in severity were observed on all dicot species (Apiaceae and non-Apiaceae), but no symptoms developed on the two monocots studied (leek and sweetcorn). Plant species were also differentiated on the basis of expanding lesions (cultivated and wild carrot, dill and fennel) or non-expanding lesions (other dicot species). Typical A. dauci conidia were observed after in vitro incubation of leaves with symptoms. Fungal structures were isolated from lesions and A. dauci was confirmed on the basis of conidial morphology and specific conventional PCR results. Genotyping of individual isolates performed with microsatellite markers confirmed the presence of the inoculated isolate. The results clearly showed that, in controlled conditions, the host range of A. dauci is not restricted to carrot.

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.

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.