Chemical warfare between the host and the pathogen plays a crucial role in plant-necrotrophic pathogen interactions, but examples of its involvement in quantitative disease resistance in plants are poorly documented. In the Daucus carota-Alternaria dauci pathosystem, the novel toxin aldaulactone has been identified as a key factor in both fungal pathogenicity and the carrot's partial resistance to the pathogen. Bioinformatic analyses have pinpointed a secondary metabolism gene cluster that harbors two polyketide synthase genes, AdPKS7 and AdPKS8, that are likely responsible for the biosynthesis of aldaulactone. Here, we present the functional validation of AdPKS7 and AdPKS8 as genes responsible for aldaulactone production in A. dauci. We generated A. dauci knock-out mutants for AdPKS7 and AdPKS8 by replacing essential domains with a hygromycin resistance gene, marking the first reported case of genetic manipulation in A. dauci. Following transformation, the mutants were analyzed for toxin production via HPLC-UV and assessed for pathogenicity in planta. Aldaulactone production was abolished in all PKS mutants, which also exhibited significantly reduced pathogenicity on H1-susceptible carrot leaves. These findings confirm the roles of AdPKS7 and AdPKS8 in aldaulactone biosynthesis and their contribution to fungal pathogenicity.

Plant microbiota appear more and more as potential sources of antagonistic microorganisms. However, the seed microbiota associated with wild plant species has rarely been explored. To identify fungal antagonists to the seed-borne pathogen Alternaria brassicicola, seeds were collected in natural populations of three Brassicaceae species, Arabidopsis thaliana, Capsella bursa-pastoris and Draba verna. A large number of fungal strains reduced the growth of A. brassicicola. The most antagonistic strains belonged to Alternaria, Apiospora, Trichoderma and Aspergillus. Seed-associated fungi tolerated host plant defenses and exhibited lower sensitivity compared to A. brassicicola to indolic compounds such as the phytoalexin camalexin and the glucosinolates (GLS)-breakdown compound indole-3-carbinol. By contrast, antagonistic strains were as inhibited as A. brassicicola in presence of allyl-isothiocyanates (ITC) derived from aliphatic GLS, and more inhibited by benzyl-ITC derived from aromatic GLS. However, all defense compounds could enhance the antagonistic effects of some of the isolated strains on A. brassicicola. The observed potential synergistic effects between defense compounds and seed-associated antagonistic strains emphasize the need for further studies to elucidate the molecular bases of the interactions. A better understanding of the interactions between host plants, pathogens and fungal endophytes is also needed to develop sustainable biocontrol strategies.

Since 2015, necrosis caused by Fusarium spp. has been regularly observed in carrot seed crops in France. A collection of 53 Fusarium isolates was gathered from lesions on carrot between 2016 and 2019. Morphological characterization at both the macroscopic and microscopic levels and molecular typing based on the ACL1, RPB2 and EF1-α partial gene sequences resulted in the identification of two main groups, corresponding to 22 F. avenaceum and 22 F. tricinctum isolates. In addition, three isolates of F. graminearum, two each of F. oxysporum and F. acuminatum, and one isolate of F. solani were also identified. Dual cultures of Trichoderma spp. (T. atroviride and T. harzianum) and Fusarium spp. (F. tricinctum isolate FT001 and F. avenaceum isolate FA002) did not indicate any antibiosis capabilities of Trichoderma. The in vitro sensitivity of isolates FT001 and FA002 to two fungicides and one natural product was similar, with an efficacy depending on the active ingredient. Prothioconazole + tebuconazole was more effective (mycelial growth reduced by 97% for FT001 and 99.8% for FA002) than fluopyram + trifloxystrobin (mycelial growth reduced by 58% and 56%, respectively). Allium extracts seemed to be a promising alternative to fungicides, since they were almost as efficient as prothioconazole + tebuconazole. Artificial inoculation of durum wheat using three isolates from carrot lesions (FT001, FA002 and F. graminearum FG001) induced plant necrosis. This result suggests that Fusarium pathogens of carrot seed crops may be transmitted to wheat, which is commonly used in rotation with carrot.

Alternaria dauci is a Dothideomycete fungus, causal agent of carrot leaf blight. As a member of the Alternaria genus, known to produce a lot of secondary metabolite toxins, A. dauci is also supposed to synthetize host specific and non-host specific toxins playing a crucial role in pathogenicity. This study provides the first reviewing of secondary metabolism genetic basis in the Alternaria genus by prediction of 55 different putative core genes. Interestingly, aldaulactone, a phytotoxic benzenediol lactone from A. dauci, was demonstrated as important in pathogenicity and in carrot partial resistance to this fungus. As nothing is known about aldaulactone biosynthesis, bioinformatic analyses on a publicly available A. dauci genome data set that were reassembled, thanks to a transcriptome data set described here, allowed to identify 19 putative secondary metabolism clusters. We exploited phylogeny to pinpoint cluster 8 as a candidate in aldaulactone biosynthesis. This cluster contains AdPKS7 and AdPKS8, homologs with genes encoding a reducing and a non-reducing polyketide synthase. Clusters containing such a pair of PKS genes have been identified in the biosynthesis of resorcylic acid lactones or dihydroxyphenylacetic acid lactones. AdPKS7 and AdPKS8 gene expression patterns correlated with aldaulactone production in different experimental conditions. The present results highly suggest that both genes are responsible for aldaulactone biosynthesis.

The transmission of seed-borne pathogens by the germinating seed is responsible for major crop diseases. The immune responses of the seed facing biotic invaders are poorly documented so far. The Arabidopsis thaliana/Alternaria brassicicola patho-system was used to describe at the transcription level the responses of germinating seeds and young seedling stages to infection by the necrotrophic fungus. RNA-seq analyses of healthy versus inoculated seeds at 3 days after sowing (DAS), stage of radicle emergence, and at 6 and 10 DAS, two stages of seedling establishment, identified thousands of differentially expressed genes by Alternaria infection. Response to hypoxia, ethylene and indole pathways were found to be induced by Alternaria in the germinating seeds. However, surprisingly, the defense responses, namely the salicylic acid (SA) pathway, the response to reactive oxygen species (ROS), the endoplasmic reticulum-associated protein degradation (ERAD) and programmed cell death, were found to be strongly induced only during the latter post-germination stages. We propose that this non-canonical immune response in early germinating seeds compared to early seedling establishment was potentially due to the seed-to-seedling transition phase. Phenotypic analyses of about 14 mutants altered in the main defense pathways illustrated these specific defense responses. The unexpected germination deficiency and insensitivity to Alternaria in the glucosinolate deficient mutants allow hypothesis of a trade-off between seed germination, necrosis induction and Alternaria transmission to the seedling. The imbalance of the SA and jasmonic acid (JA) pathways to the detriment of the JA also illustrated a non-canonical immune response at the first stages of the seedling.

A procedure was developed for in vitro propagation of Rosa genotypes along with an efficient de novo shoot organogenesis (DNSO) method. We tested, on one genotype (hybrid of Rosa wichurana), the effects of MS basal medium complemented with two growth regulators to achieve either shoot elongation or shoot multiplication of plants. These media were complemented with carbohydrate concentrations from different sources. Then, the impacts of various carbohydrates (fructose, glucose, maltose, sorbitol, sucrose) on the growth and development of several rose genotypes during donor plant subculturing were studied on SMM. The results showed high variability in growth and development between genotypes. Contrary to other members of the Rosaceae family, no correlation was found between the shoot size and number when the amount of sorbitol was increased. Murashige and Skoog medium supplemented with 3.0 mg L-1 BAP and containing fructose or glucose at 30 g L-1 was chosen to induce leaf explants for the DNSO experiments. MS basal medium complemented with TDZ/IBA at three ratios and the same range of carbohydrate sources were tested for DNSO. Significant genotypic variations with regard to the percentage of regeneration was demonstrated with six genotypes. For two genotypes, a hybrid of Rosa wichurana and Rosa 'White Pet', we defined the conditions required to obtain 100% DNSO. For Rosa chinensis 'Old blush' and the rootstock genotype Rosa 'Natal Briar', we obtained 74 and 87.5% DNSO and only 56.67% and 37.5% for Rosa GUY SAVOY (R) ('Delstrimen') and Rosa Felicite et Perpetue' respectively. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies in Rosa.

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.

A collection of 102 Diaporthe isolates was compiled from lesions on carrot, parsley and wild Apiaceae species in France from 2010 to 2014. Molecular typing based on ITS rDNA sequences resulted in the identification of 85 D. angelicae and 17 D. eres isolates. Based on sequences of the 30 part of the IGS rDNA, intraspecific variability was analysed for 17 D. angelicae and 13 D. eres isolates from diverse plant species, locations in France, and plant tissues. The genetic diversity was greater for D. angelicae isolates than D. eres isolates. In vitro sensitivity of five D. angelicae and four D. eres isolates to each of nine fungicides was similar for isolates of both species, with a marked variation in fungicide sensitivity depending on the active ingredient. To assess the pathogenicity of D. angelicae and D. eres isolates on carrot, one isolate of each species was inoculated onto umbels in a controlled environment. Typical lesions were observed for both isolates. Carrot crop debris collected from a seed production field in France and placed in controlled conditions produced perithecia and ascospores typical of Diaporthe, that were further characterized molecularly as belonging to D. angelicae. Detection of Diaporthe species on seed lots from three carrot production fields in France was investigated. Both species were detected on seeds by conventional PCR assay, with a greater frequency for D. angelicae than D. eres (67% vs 33%, respectively). Overall, the results highlighted that umbel browning in carrot seed crops in France was mainly caused by D. angelicae.

Alternaria leaf blight, caused by the necrotrophic fungus Alternaria dauci, is the most damaging foliar disease in carrot production. Fungicide use has variable efficiency, and presents economic and ecological costs. Popular partially resistant cultivars exist, but their resistance level is still unsatisfactory. A better knowledge on carrot A. dauci resistance mechanisms may help to produce more resistant cultivars faster. More generally, partial resistance mechanisms in plants are still poorly understood. In order to better understand partial resistance to leaf blight in carrot, we implemented an a priori approach, investigating the role of different resistance mechanisms in the carrot-A. dauci interaction. It has been suggested that Alternaria dauci produces toxins, such as zinniol. The work presented here is centered on the role played by these toxins in the plant-fungus interaction, and more especially plant resistance. Plant cell suspensions from several genotypes were treated with raw fungal extracts. A good correlation was found between whole plant resistance to the fungus and metabolic activity of the cell suspensions after 48 h of exposure. Similar results were obtained using different techniques and different exposure times. Additionally, it was found that the toxicity of fungal extracts was due to secreted apolar compounds, which did not include zinniol. Zinniol is only produced when the fungus is grown in anoxic conditions. Nevertheless, fungal growth condition and zinniol content do not impact extract toxicity. Moreover, zinniol was not toxic to plant cells at physiological concentration.

The host range of Alternaria dauci was investigated in a greenhouse. Two strains were inoculated onto nine cultivated Apiaceae species, two wild Daucus species and six cultivated non-Apiaceae species representing six botanic families. Lesions of various intensities were observed on all dicot species, but no symptoms developed on the two monocots studied. Typical A. dauci conidia were observed after in vitro incubation of necrotic leaves. Fungal strains were isolated from lesions and A. dauci was diagnosed on the basis of conidium morphology and specific conventional PCR findings. Genotyping of individual isolates performed with microsatellite markers confirmed the presence of the inoculated strain. Our results strongly suggest that species other than carrot can be alternative hosts. Those results are important to consider for a better management of Alternaria leaf blight in carrot fields.

Early blight is a common disease of Solanaceae crops worldwide. The occurrence of Alternaria spp. was studied during three epidemics on tomato in northwestern Algeria. Alternaria was detected in more than 80 % of the diseased plant samples and accounted for more than 50 % of the total fungal isolates recovered from these samples. Morphological and molecular investigations revealed that small-spored isolates producing beaked conidia, i.e. belonging to the section alternaria, were prominent in most of the surveyed locations representing more than 80 % of the total Alternaria isolates in three locations (Mascara, Ain Témouchent and Sidi Belabbèsse). Based on their sporulation patterns they were recognized as A. alternata and A. tenuissima. Small-spored isolates producing conidia without beak and assigned to A. consortialis were also found at a low frequency (< 1 %). Large-spored isolates producing conidia ended by typical long beaks and identified as A. linariae (syn. A. tomatophila), A. solani and A. grandis were also recovered from all the sampled areas and represented 33.8 %, 6.3 % and 1.3 % of the total Alternaria isolates, respectively. Pathogenicity tests on tomato with a selection of 85 strains representative of the isolates collection revealed that all the tested isolates were able to produce extending lesions on inoculated leaves albeit with variable intensity. Large-spored species included the most aggressive isolates. Small-spored Alternaria, although less aggressive than large-spored Alternaria, had the ability to provoke brown necrotic spots and circumstantially developed synergistic interactions in mixed infections with moderately aggressive isolates of A. linariae.

La creation de varietes presentant un niveau de resistance fort et durable passe immanquablement par la mise en œuvre de methodes de phenotypage toujours plus discriminantes, reproductibles et pouvant etre mises en œuvre sur de grands effectifs. Les methodes actuelles de phenotypage font encore tres souvent appel a une notation visuelle des symptomes. Nous rapportons ici la mise en œuvre de differentes methodes de phenotypage alternatives permettant de caracteriser, soit la phase asymptomatique par l’utilisation de nouvelles technologies telles que la qPCR, la thermographie ou la mesure de fluorescence de chlorophylle, soit la phase symptomatique a l’aide de tests en conditions controlees. Les avantages et les limites de ces methodes sont abordes.

Liens [1] http://okina.univ-angers.fr/publications?f[author]=24358 [2] http://okina.univ-angers.fr/publications?f[author]=159 [3] http://okina.univ-angers.fr/publications?f[author]=15700 [4] http://okina.univ-angers.fr/publications?f[author]=157 [5] http://okina.univ-angers.fr/publications?f[author]=24359 [6] http://okina.univ-angers.fr/jeanjacques.helesbeux/publications [7] http://okina.univ-angers.fr/s.gagne/publications [8] http://okina.univ-angers.fr/p.richomme/publications [9] http://okina.univ-angers.fr/publications?f[author]=166 [10] http://okina.univ-angers.fr/philippe.simoneau/publications [11] http://okina.univ-angers.fr/pascal.poupard/publications [12] http://okina.univ-angers.fr/romain.berruyer/publications [13] http://okina.univ-angers.fr/publications/ua14422

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.

BACKGROUND:In order to select for quantitative plant resistance to pathogens, high throughput approaches that can precisely quantify disease severity are needed. Automation and use of calibrated image analysis should provide more accurate, objective and faster analyses than visual assessments. In contrast to conventional visible imaging, chlorophyll fluorescence imaging is not sensitive to environmental light variations and provides single-channel images prone to a segmentation analysis by simple thresholding approaches. Among the various parameters used in chlorophyll fluorescence imaging, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) is well adapted to phenotyping disease severity. Fv/Fm is an indicator of plant stress that displays a robust contrast between infected and healthy tissues. In the present paper, we aimed at the segmentation of Fv/Fm images to quantify disease severity.RESULTS:Based on the Fv/Fm values of each pixel of the image, a thresholding approach was developed to delimit diseased areas. A first step consisted in setting up thresholds to reproduce visual observations by trained raters of symptoms caused by Xanthomonas fuscans subsp. fuscans (Xff) CFBP4834-R on Phaseolus vulgaris cv. Flavert. In order to develop a thresholding approach valuable on any cultivars or species, a second step was based on modeling pixel-wise Fv/Fm-distributions as mixtures of Gaussian distributions. Such a modeling may discriminate various stages of the symptom development but over-weights artifacts that can occur on mock-inoculated samples. Therefore, we developed a thresholding approach based on the probability of misclassification of a healthy pixel. Then, a clustering step is performed on the diseased areas to discriminate between various stages of alteration of plant tissues. Notably, the use of chlorophyll fluorescence imaging could detect pre-symptomatic area. The interest of this image analysis procedure for assessing the levels of quantitative resistance is illustrated with the quantitation of disease severity on five commercial varieties of bean inoculated with Xff CFBP4834-R.CONCLUSIONS:In this paper, we describe an image analysis procedure for quantifying the leaf area impacted by the pathogen. In a perspective of high throughput phenotyping, the procedure was automated with the software R downloadable at http://www.r-project.org/. The R script is available at http://lisa.univ-angers.fr/PHENOTIC/telechargements.html.

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.

The in vitro effects of 6-MM and falcarindiol on the development of Alternaria dauci were analyzed. The results showed that A. dauci was significantly more susceptible to falcarindiol than to 6-MM. Experiments dealing with mixed solutions of 6-MM and falcarindiol indicated additive effects of the metabolites on growth inhibition. Permeabilization of the fungal plasma membrane was observed after falcarindiol treatment. In planta quantification of 6-MM/falcarindiol was performed on inoculated leaves of carrot genotypes. Significantly faster 6-MM production was observed in Bolero (partially resistant to A. dauci) than in Presto (susceptible). The falcarindiol levels were significantly higher in Bolero than in Presto. (C) 2012 Elsevier Ltd. All rights reserved.