Table_1_Aldaulactone – An Original Phytotoxic Secondary Metabolite Involved in the Aggressiveness of Alternaria dauci on Carrot.DOCX

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.

自基因对基因假说（gene-for-gene hypothesis）提出以来，植物定性抗病机制与病原菌致病性已得到广泛研究。调控病原菌侵袭力（aggressiveness）与植物部分抗性（partial resistance）这类数量性状的分子机制仍未得到充分解析。尽管如此，这类互作模式在多数植物-死体营养病原菌（necrotrophic pathogen）互作体系中普遍存在，包括野胡萝卜（Daucus carota）与胡萝卜链格孢（Alternaria dauci）的互作体系。在这类互作体系中，病原菌产生的植物毒性代谢物在其侵袭力中发挥关键作用。本研究旨在探究胡萝卜链格孢侵袭力与其毒素产生能力之间的关联。我们采用不同侵袭力水平的胡萝卜链格孢菌株分泌液，分别处理来自感病基因型H1以及两个部分抗病基因型I2与K3的胡萝卜胚性细胞培养物。有趣的是，仅本研究中侵袭力最强的菌株ITA002的分泌液，会对携带胡萝卜链格孢抗性的胡萝卜基因型产生毒害效应。本研究结果揭示了胡萝卜链格孢侵袭力与其真菌分泌液细胞毒性之间的正相关关系。我们推测该真菌分泌液的毒性与其所产生的毒性化合物总量密切相关。值得注意的是，该真菌产生的有机分泌物含量与其侵袭力呈正相关。因此，我们进一步采用高效液相色谱（HPLC）对真菌有机提取物进行分析，并检测了色谱峰强度与真菌侵袭力之间的相关性。我们发现某一色谱峰的强度与真菌侵袭力呈现高度相关。我们成功纯化得到该色谱峰对应的化合物，经核磁共振（NMR）分析鉴定，其为一种新型10元苯二醇内酯类聚酮化合物，我们将其命名为aldaulactone。我们采用一种新型自动化图像分析方法，证实aldaulactone在对应浓度下对离体培养的植物细胞具有毒性。相较于感病基因型H1的胡萝卜细胞，aldaulactone与真菌有机提取物对I2抗病基因型胡萝卜细胞的毒害效应均更弱。综上，本研究结果表明：（1）aldaulactone是一种新型植物毒素；（2）胡萝卜链格孢所产生的aldaulactone含量与其侵袭力密切相关；（3）胡萝卜对胡萝卜链格孢的抗性机制包含对aldaulactone的抗性。