Taking Advantage of Pathogen Diversity and Immune Priming to Minimize Disease Prevalence in Host Mixtures: A Model.

Host mixtures are a promising method for agroecological plant disease control. Plant immunity is key to the success of host mixtures against polymorphic pathogen populations. This immunity results from priming-induced cross-protection, whereby plants able to resist infection by specific pathogen genotypes become more resistant to other pathogen genotypes. Strikingly, this phenomenon was absent from mathematical models aiming at designing host mixtures. We developed a model to specifically explore how priming affects the coexistence of two pathogen genotypes in host mixtures composed of two host genotypes and how it affects disease prevalence. The main effect of priming is to reduce the coexistence region in the parameter space (due to the cross-protection) and to generate a singular mixture of resistant and susceptible hosts corresponding to the maximal reduction disease prevalence (in absence of priming, a resistant pure stand is optimal). The epidemiological advantage of host mixtures over a resistant pure stand thus appears as a direct consequence of immune priming. We also showed that there is indirect cross-protection between host genotypes in a mixture. Moreover, the optimal mix prevents the emergence of a resistance-breaking pathogen genotype. Our results highlight the importance of considering immune priming to design optimal and sustainable host mixtures.

A comparison of PTI defense profiles induced in Solanum tuberosum by PAMP and non-PAMP elicitors shows distinct, elicitor-specific responses.

The induction of general plant defense responses following the perception of external elicitors is now regarded as the first level of the plant immune response. Depending on the involvement or not of these molecules in pathogenicity, this induction of defense is called either Pathogen-Associated Molecular Pattern (PAMP) Triggered Immunity or Pattern Triggered Immunity-both abbreviated to PTI. Because PTI is assumed to be a widespread and stable form of resistance to infection, understanding the mechanisms driving it becomes a major goal for the sustainable management of plant-pathogen interactions. However, the induction of PTI is complex. Our hypotheses are that (i) the recognition by the plant of PAMPs vs non-PAMP elicitors leads to specific defense profiles and (ii) the responses specifically induced by PAMPs target critical life history traits of the pathogen that produced them. We thus analyzed, using a metabolomic approach coupled with transcriptomic and hormonal analyses, the defense profiles induced in potato foliage treated with either a Concentrated Culture Filtrate (CCF) from Phytophthora infestans or two non-PAMP preparations, ß-aminobutyric acid (BABA) and an Ulva spp. Extract, used separately. Each elicitor induced specific defense profiles. CCF up-regulated sesquiterpenes but down-regulated sterols and phenols, notably α-chaconine, caffeoyl quinic acid and rutin, which decreased spore production of P. infestans in vitro. CCF thus induces both defense and counter-defense responses. By contrast, the Ulva extract triggered the synthesis of a large-spectrum of antimicrobial compounds through the phenylpropanoid/flavonoid pathways, while BABA targeted the primary metabolism. Hence, PTI can be regarded as a heterogeneous set of general and pathogen-specific responses triggered by the molecular signatures of each elicitor, rather than as a uniform, non-specific and broad-spectrum set of general defense reactions.

The Effectiveness of Induced Defense Responses in a Susceptible Potato Genotype Depends on the Growth Rate of Phytophthora infestans.

Phytophthora infestans causes the devastating potato late blight disease, which is widely controlled with fungicides. However, the debate about chemical control is fueling a promotion toward alternative methods. In this context, the enhancement of natural plant immunity could be a strategy for more sustainable protection. We previously demonstrated that a concentrated culture filtrate (CCF) of P. infestans primes defense reactions in potato. They are genotype-dependent and metabolites produced decrease pathogen growth in vitro but not in vivo on tubers. Induced potato defenses are assumed to affect P. infestans life history traits depending on strains. This assumption was studied in vivo through induced leaflets on a susceptible genotype inoculated with four P. infestans strains differing for lesion growth rate. This study combines both defenses mechanistic analysis and ecological observations. Defense-gene expressions were thus assessed by quantitative reverse transcription-polymerase chain reaction; pathogen development was simultaneously evaluated by measuring necrosis, quantifying mycelial DNA, and counting sporangia. The results showed that CCF pretreatment reduced the pathogenicity differences between slow- and fast-growing strains. Moreover, after elicitation, PR-1, PR-4, PAL, POX, and THT induction was strain-dependent. These results suggest that P. infestans could develop different strategies to overcome plant defenses and should be considered in biocontrol and epidemic management of late blight.

Identification of three elicitins and a galactan-based complex polysaccharide from a concentrated culture filtrate of Phytophthora infestans efficient against Pectobacterium atrosepticum.

The induction of plant immunity by Pathogen Associated Molecular Patterns (PAMPs) constitutes a powerful strategy for crop protection. PAMPs indeed induce general defense responses in plants and thus increase plant resistance to pathogens. Phytophthora infestans culture filtrates (CCFs) are known to induce defense responses and decrease the severity of soft rot due to Pectobacterium atrosepticum in potato tubers. The aim of this study was to identify and characterize the active compounds from P. infestans filtrate. The filtrate was fractionated by gel filtration, and the protection effects against P. atrosepticum and the ability to induce PAL activity were tested for each fraction. The fraction active in protection (F1) also induced PAL activity, as did the whole filtrate. Three elicitins (INF1, INF4 and INF5) were identified in F1b, subfraction of F1, by MALDI-TOF-MS and MS/MS analyses. However, deproteinized F1b still showed biological activity against the bacterium, revealing the presence of an additional active compound. GC-MS analyses of the deproteinized fraction highlighted the presence of a galactan-based complex polysaccharide. These experiments demonstrate that the biological activity of the CCF against P. atrosepticum results from a combined action of three elicitins and a complex polysaccharide, probably through the activation of general defense responses.

Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors.

KEY MESSAGE: Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

Nicotiflorin, rutin and chlorogenic acid: phenylpropanoids involved differently in quantitative resistance of potato tubers to biotrophic and necrotrophic pathogens.

Physiological and molecular mechanisms underlying quantitative resistance of plants to pathogens are still poorly understood, but could depend upon differences in the intensity or timing of general defense responses. This may be the case for the biosynthesis of phenolics which are known to increase after elicitation by pathogens. We thus tested the hypothesis that differences in quantitative resistance were related to differential induction of phenolics by pathogen-derived elicitors. Five potato cultivars (Solanum tuberosum, L.) spanning a range of quantitative resistance were treated with a concentrated culture filtrate (CCF) of Phytophthora infestans or purified lipopolysaccharides (LPS) from Pectobacterium atrosepticum. The kinetic of phenolics accumulation was followed and a set of typical phenolics was identified: chlorogenic acid, phenolamides and flavonols including rutin (quercetin-3-O-rutinoside) and nicotiflorin (kaempferol-3-O-rutinoside). Our results showed that CCF but not LPS induced differential accumulation of major phenolics among cultivars. Total phenolics were related with resistance to P. atrosepticum but not to P. infestans. However, nicotiflorin was inversely related with resistance to both pathogens. Rutin, but not nicotiflorin, inhibited pathogen growth in vitro at physiological concentrations. These data therefore suggest that (i) several phenolics are candidate markers for quantitative resistance in potato, (ii) some of these are pathogen specific although they are produced by a general defense pathway, (iii) resistance marker molecules do not necessarily have antimicrobial activity, and (iv) the final content of these target molecules-either constitutive or induced-is a better predictor of resistance than their inducibility by pathogen elicitors.

Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level.

A direct, label-free immunosensor was designed for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ngml(-1) with a detection limit of 20 ngml(-1). The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ngml(-1) for a total assay time of 25 min.

Quantitative resistance of potato to Pectobacterium atrosepticum and Phytophthora infestans: integrating PAMP-triggered response and pathogen growth.

While the mechanisms underlying quantitative resistance of plants to pathogens are still not fully elucidated, the Pathogen-Associated Molecular Patterns (PAMPs)-triggered response model suggests that such resistance depends on a dynamic interplay between the plant and the pathogen. In this model, the pathogens themselves or elicitors they produce would induce general defense pathways, which in turn limit pathogen growth and host colonisation. It therefore suggests that quantitative resistance is directly linked to a common set of general host defense mechanisms, but experimental evidence is still inconclusive. We tested the PAMP-triggered model using two pathogens (Pectobacterium atrosepticum and Phytophthora infestans) differing by their infectious processes and five potato cultivars spanning a range of resistance levels to each pathogen. Phenylalanine ammonia-lyase (PAL) activity, used as a defense marker, and accumulation of phenolics were measured in tuber slices challenged with lipopolysaccharides from P. atrosepticum or a concentrated culture filtrate from P. infestans. PAL activity increased following treatment with the filtrate but not with lipopolysaccharides, and varied among cultivars. It was positively related to tuber resistance to P. atrosepticum, but negatively related to tuber resistance to P. infestans. It was also positively related to the accumulation of total phenolics. Chlorogenic acid, the main phenolic accumulated, inhibited growth of both pathogens in vitro, showing that PAL induction caused active defense against each of them. Tuber slices in which PAL activity had been induced before inoculation showed increased resistance to P. atrosepticum, but not to P. infestans. Our results show that inducing a general defense mechanism does not necessarily result in quantitative resistance. As such, they invalidate the hypothesis that the PAMP-triggered model alone can explain quantitative resistance. We thus designed a more complex model integrating physiological host response and a key pathogen life history trait, pathogen growth, to explain the differences between the two pathosystems.

Arabidopsis thaliana cells: a model to evaluate the virulence of Pectobacterium carotovorum.

Pectobacterium carotovorum are economically important plant pathogens that cause plant soft rot. These enterobacteria display high diversity world-wide. Their pathogenesis depends on production and secretion of virulence factors such as plant cell wall-degrading enzymes, type III effectors, a necrosis-inducing protein, and a secreted virulence factor from Xanthomonas spp., which are tightly regulated by quorum sensing. Pectobacterium carotovorum also present pathogen-associated molecular patterns that could participate in their pathogenicity. In this study, by using suspension cells of Arabidopsis thaliana, we correlate plant cell death and pectate lyase activities during coinfection with different P. carotovorum strains. When comparing soft rot symptoms induced on potato slices with pectate lyase activities and plant cell death observed during coculture with Arabidopsis thaliana cells, the order of strain virulence was found to be the same. Therefore, Arabidopsis thaliana cells could be an alternative tool to evaluate rapidly and efficiently the virulence of different P. carotovorum strains.

Activation of defence reactions in Solanaceae: where is the specificity?

When a potential pathogen attempts to infect a plant, biochemical and molecular communication takes place and leads to the induction of plant defence mechanisms. In the case of efficient defence, visible symptoms are restricted and the pathogen does not multiply (incompatible interaction); when defence is inefficient, the plant becomes rapidly infected (compatible interaction). During the last 30 years, a growing body of knowledge on plant-pathogen interactions has been gathered, and a large number of studies investigate the induction of various plant defence reactions by pathogens or by pathogen-derived compounds. However, as most papers focus on incompatible interactions, there is still a lack of understanding about the similarities and differences between compatible and incompatible situations. This review targets the question of specificity in Solanaceae-pathogen interactions, by comparing defence patterns in plants challenged with virulent or avirulent pathogens (or with pathogen-associated molecular patterns from these). A special emphasis is made on analysing whether defence reactions in Solanaceae depend primarily on the type of elicitor, on the plant genotype/species, or on the type of interaction (compatible or incompatible).