Table_2_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，植物定性抗性机制与病原菌毒力已得到广泛深入研究。但与病原菌致病力及植物部分抗性的数量性状相关的机制，至今仍未得到充分解析。尽管如此，这类机制在多数死体营养型病原菌与植物的互作体系中广泛存在，其中包括胡萝卜（Daucus carota）-胡萝卜链格孢（Alternaria dauci）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致病力中发挥关键作用。本研究旨在探究胡萝卜链格孢致病力与毒素产生之间的关联。我们采用不同致病力水平的胡萝卜链格孢菌株分泌物，处理来自感病基因型H1以及两个部分抗病基因型I2和K3的胡萝卜胚性细胞培养物。有趣的是，仅本研究中致病力最强的菌株ITA002的分泌物，对胡萝卜链格孢抗性基因型产生了影响。本研究结果表明，胡萝卜链格孢的致病力与其分泌物的细胞毒性呈正相关。我们推测，该真菌分泌物的毒性与其产生的有毒化合物的含量密切相关。值得注意的是，该真菌产生的有机分泌物的量与其致病力呈正相关。因此，我们采用高效液相色谱（High Performance Liquid Chromatography，HPLC）对该真菌的有机提取物进行了分析，并检测了所得峰强度与真菌致病力之间的相关性。其中一个检测到的峰的强度与真菌致病力呈显著相关。我们成功纯化了该峰对应的组分，经核磁共振（Nuclear Magnetic Resonance，NMR）分析显示，该纯化化合物为一种新型10元环苯二醇内酯，属于聚酮类化合物，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在对应浓度下对体外培养的植物细胞具有毒性。与H1型胡萝卜细胞相比，aldaulactone与真菌有机提取物对I2型抗病胡萝卜细胞的毒性作用均较弱。综上，本研究结果表明：（i）aldaulactone是一种新型植物毒素；（ii）胡萝卜链格孢产生的aldaulactone含量与其致病力存在关联；（iii）胡萝卜对胡萝卜链格孢的抗性机制包含对aldaulactone的抗性。