Necrotrophic Growth Research Articles

SsPtc3 Modulating SsSmk1‐MAPK and Autophagy to Facilitate Growth and Pathogenicity in Sclerotinia sclerotiorum

ABSTRACTThe compound appressoria of Sclerotinia sclerotiorum can produce cell wall‐degrading enzymes, effectors and toxins, which promote penetration and the death of host cells. Subsequently, invasive hyphae (IH) branch rapidly as necrotrophic growth and disease symptoms are observed. S. sclerotiorum can respond to complex stresses and regulate its metabolism to adapt to the external environment. Here we demonstrated that type 2C Ser/Thr phosphatase (PP2C) SsPtc3 responds to nutritional, osmotic, cell wall and oxidative stresses. Loss of function ΔSsptc3 mutants displayed defects in mycelial growth, sclerotia formation and reduced virulence. Phosphoproteomic analyses revealed that SsPtc3 is involved in autophagy and MAPK signalling pathways. We obtained evidence that SsPtc3 negatively modulates the phosphorylation of SsSmk1. SsSmk1 is essential for mycelial growth, compound appressorium formation and pathogenicity, SsPtc3 modulated phosphorylation homeostasis of SsSmk1 to maintain hyphal growth. SsPtc3 interacted with SsAtg1 to influence autophagic flux under starvation. Taken together, these results reveal that SsPtc3 responds to various stresses that modulate autophagy and phosphorylation of SsSmk1‐MAPK, which facilitates the growth and virulence of S. sclerotiorum.

Functional characterization of extracellular and intracellular catalase-peroxidases involved in virulence of the fungal wheat pathogen Zymoseptoria tritici.

Understanding how pathogens defend themselves against host defence mechanisms, such as hydrogen peroxide (H2O2) production, is crucial for comprehending fungal infections. H2O2 poses a significant threat to invading fungi due to its potent oxidizing properties. Our research focuses on the hemibiotrophic fungal wheat pathogen Zymoseptoria tritici, enabling us to investigate host-pathogen interactions. We examined two catalase-peroxidase (CP) genes, ZtCpx1 and ZtCpx2, to elucidate how Z. tritici deals with host-generated H2O2 during infection. Our analysis revealed that ZtCpx1 was up-regulated during biotrophic growth and asexual spore formation invitro, while ZtCpx2 showed increased expression during the transition from biotrophic to necrotrophic growth and in-vitro vegetative growth. Deleting ZtCpx1 increased the mutant's sensitivity to exogenously added H2O2 and significantly reduced virulence, as evidenced by decreased Septoria tritici blotch symptom severity and fungal biomass production. Reintroducing the wild-type ZtCpx1 allele with its native promoter into the mutant strain restored the observed phenotypes. While ZtCpx2 was not essential for full virulence, the ZtCpx2 mutants exhibited reduced fungal biomass development during the transition from biotrophic to necrotrophic growth. Moreover, both CP genes act synergistically, as the double knock-out mutant displayed a more pronounced reduced virulence compared to ΔZtCpx1. Microscopic analysis using fluorescent proteins revealed that ZtCpx1 was localized in the peroxisome, indicating its potential role in managing host-generated reactive oxygen species during infection. In conclusion, our research sheds light on the crucial roles of CP genes ZtCpx1 and ZtCpx2 in the defence mechanism of Z. tritici against host-generated hydrogen peroxide.

The expression landscape and pangenome of long non-coding RNA in the fungal wheat pathogen Zymoseptoria tritici.

Long non-coding RNAs (lncRNAs) are regulatory molecules interacting in a wide array of biological processes. lncRNAs in fungal pathogens can be responsive to stress and play roles in regulating growth and nutrient acquisition. Recent evidence suggests that lncRNAs may also play roles in virulence, such as regulating pathogenicity-associated enzymes and on-host reproductive cycles. Despite the importance of lncRNAs, only a few model fungi have well-documented inventories of lncRNA. In this study, we apply a recent computational pipeline to predict high-confidence lncRNA candidates in Zymoseptoria tritici, an important global pathogen of wheat impacting global food production. We analyse genomic features of lncRNAs and the most likely associated processes through analyses of expression over a host infection cycle. We find that lncRNAs are frequently expressed during early infection, before the switch to necrotrophic growth. They are mostly located in facultative heterochromatic regions, which are known to contain many genes associated with pathogenicity. Furthermore, we find that lncRNAs are frequently co-expressed with genes that may be involved in responding to host defence signals, such as oxidative stress. Finally, we assess pangenome features of lncRNAs using four additional reference-quality genomes. We find evidence that the repertoire of expressed lncRNAs varies substantially between individuals, even though lncRNA loci tend to be shared at the genomic level. Overall, this study provides a repertoire and putative functions of lncRNAs in Z. tritici enabling future molecular genetics and functional analyses in an important pathogen.

Modelling drinking water biofilms: Bacterial adhesion and Legionella pneumophila necrotrophic growth

We propose a mathematical model to simulate the establishment and growth of a drinking water distribution system biofilm, focusing on the influence of ionic strength on bacterial adhesion and persistence of Legionella pneumophila. Specifically, we consider how ionic strength affects interaction energies during the initial phase of biofilm formation and we include the dependence of the attachment flux, modelled as a linear rate with respect to free floating cells, on it. The model also incorporates a novel necrotrophic kinetics to simulate the Legionella pneumophila metabolism. The biofilm is modelled as a 1D free boundary domain, and its evolution is governed by hyperbolic–parabolic PDEs. The initial attachment phase is modelled by considering a vanishing initial value for the free boundary. The model is investigated numerically highlighting the impact of the necrotrophic kinetic parameters, the influence of ionic strength on the initial stage of biofilm formation through bacterial attachment, and how variations in nutrient levels affect system dynamics. The numerical results demonstrate that: the ionic strength mainly governs bacterial adhesion for young biofilms and affects the biofilm ecology also in presence of a biocide; the necrotrophic metabolism of Legionella pneumophila favours its persistence in oligotrophic biofilms.

MIF-like domain containing protein orchestrates cellular differentiation and virulence in the fungal pathogen Magnaporthe oryzae

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of an MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results suggest that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

Distinct roles for different autophagy-associated genes in the virulence of the fungal wheat pathogen Zymoseptoria tritici

The fungal wheat pathogen Zymoseptoria tritici causes major crop losses as the causal agent of the disease Septoria tritici blotch. The infection cycle of Z. tritici displays two distinct phases, beginning with an extended symptomless phase of 1–2 weeks, before the fungus induces host cell death and tissue collapse in the leaf. Recent evidence suggests that the fungus uses little host-derived nutrition during asymptomatic colonisation, raising questions as to the sources of energy required for this initial growth phase. Autophagy is crucial for the pathogenicity of other fungal plant pathogens through its roles in supporting cellular differentiation and growth under starvation. Here we characterised the contributions of the autophagy genes ZtATG1 and ZtATG8 to the development and virulence of Z. tritici. Deletion of ZtATG1 led to inhibition of autophagy but had no impact on starvation-induced hyphal differentiation or virulence, suggesting that autophagy is not required for Z. tritici pathogenicity. Contrastingly, ZtATG8 deletion delayed the transition to necrotrophic growth, despite having no influence on filamentous growth under starvation, pointing to an autophagy-independent role of ZtATG8 during Z. tritici infection. To our knowledge, this study represents the first to find autophagy not to contribute to the virulence of a fungal plant pathogen, and reveals novel roles for different autophagy-associated proteins in Z. tritici.

The Role of the Wheat Reduced height (Rht)-DELLA Mutants and Associated Hormones in Infection by Claviceps purpurea, the Causal Agent of Ergot.

Partial resistance to the biotrophic fungal pathogen Claviceps purpurea, causal agent of ergot, has been found that colocates with mutant alleles of the wheat Reduced height (Rht) loci on chromosomes 4B and 4D. These Rht loci represent the wheat orthologs of the Arabidopsis Della genes. To investigate the role of the Rht mutant DELLA proteins in ergot resistance, we assessed C. purpurea infection in wheat near-isogenic lines (NILs) carrying the gibberellic acid (GA)-insensitive semidwarf alleles Rht-B1b and Rht-D1b and the severe dwarf alleles Rht-B1c and Rht-D1c. NILs of the GA-sensitive alleles Rht8 (chromosome 2D) and Rht12 (chromosome 5A) were also included. A general trend toward increased resistance to C. purpurea, with smaller and lighter sclerotia, was observed on the NILs Rht-B1b, Rht-D1b, Rht-B1c, and Rht-D1c, and also on Rht8. Levels of the bioactive GA4 and the auxin indole-3-acetic acid increased after inoculation with C. purpurea, following similar patterns and implicating a potential auxin-mediated induction of GA biosynthesis. In contrast, jasmonic acid (JA) levels fell in the parental lines 'Mercia' and 'Maris Huntsman' after inoculation with C. purpurea, but increased in all the Rht-mutant NILs. Inoculation with C. purpurea did not show any informative changes in the levels of salicylic acid. Our results suggest that GA-mediated degradation of the DELLA proteins and down-regulation of JA-signaling pathways supports infection of wheat by C. purpurea. As these responses are generally associated with necrotrophic fungal pathogens, we propose that the biotroph C. purpurea may have a necrotrophic growth stage.

Thermal and chemical disinfection of water and biofilms: only a temporary effect in regard to the autochthonous bacteria.

Thermal and chemical disinfection of technical water systems not only aim at minimizing the level of undesired microorganisms, but also at preventing excessive biofouling, clogging and interference with diverse technical processes. Typically, treatment has to be repeated in certain time intervals, as the duration of the effect is limited. The transient effect of disinfection was demonstrated in this study applying different treatments to water and biofilms including heat, chlorination, a combination of hydrogen peroxide and peracetic acid and monochloramine. Despite the diverse treatments, the reduction in live bacteria was followed by regrowth in all cases, underlining the universal validity of this phenomenon. The study shows that autochthonous bacteria can reach the concentrations given prior to treatment. The reason is seen in the nutrient concentration that has not changed and that forms the basis for regrowth. Nutrients are released by disinfection from lysed cells or are still fixed in dead biomass that is subsequently scavenged by necrotrophic growth. Treatment cycles therefore only provide a transient reduction of water microbiology if nutrients are not removed. When aiming at greater sustainability of the effect, biocidal treatment has to be equally concerned about nutrient removal by subsequent cleaning procedures as about killing efficiency.

Sequence and Gene Expression Analysis of Recently Identified NLP from Plasmopara viticola.

Grapevine downy mildew, evoked by the obligate biotrophic oomycete Plasmopara viticola, is one of the most challenging diseases in viticulture. P. viticola establishes an infection by circumvention of plant immunity, which is achieved by the secretion of effector molecules. One family of potential effectors are the necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). NLP are most abundant in plant pathogenic microorganisms and exist in cytotoxic and non-cyctotoxic forms. Cytotoxic NLP often act as virulence factors and are synthesized in necrotrophic or hemibiotrophic pathogens during the transition from biotrophic to necrotrophic growth. In addition to these cytotoxic NLP, many non-cytotoxic NLP have been identified; their function in biotrophic pathogens is still unknown. In 2020, eight different NLP coding genes were identified in P. viticola and named PvNLP1 to PvNLP8 (Plasmopara viticola NLP 1–8). In the present study, PvNLP4 to PvNLP8 were characterized by using qPCR analysis and transient expression in the model plant Nicotiana benthamiana. Gene expression analysis showed high PvNLP expression during the early stages of infection. Necrosis-inducing activity of PvNLP was not observed in the nonhost N. benthamiana.

Fusarium head blight resistance in European winter wheat: insights from genome-wide transcriptome analysis

BackgroundFusarium head blight (FHB) is a devastating disease of wheat worldwide. Resistance to FHB is quantitatively controlled by the combined effects of many small to medium effect QTL. Flowering traits, especially the extent of extruded anthers, are strongly associated with FHB resistance.ResultsTo characterize the genetic basis of FHB resistance, we generated and analyzed phenotypic and gene expression data on the response to Fusarium graminearum (Fg) infection in 96 European winter wheat genotypes, including several lines containing introgressions from the highly resistant Asian cultivar Sumai3. The 96 lines represented a broad range in FHB resistance and were assigned to sub-groups based on their phenotypic FHB severity score. Comparative analyses were conducted to connect sub-group-specific expression profiles in response to Fg infection with FHB resistance level. Collectively, over 12,300 wheat genes were Fusarium responsive. The core set of genes induced in response to Fg was common across different resistance groups, indicating that the activation of basal defense response mechanisms was largely independent of the resistance level of the wheat line. Fg-induced genes tended to have higher expression levels in more susceptible genotypes. Compared to the more susceptible non-Sumai3 lines, the Sumai3-derivatives demonstrated higher constitutive expression of genes associated with cell wall and plant-type secondary cell wall biogenesis and higher constitutive and Fg-induced expression of genes involved in terpene metabolism. Gene expression analysis of the FHB QTL Qfhs.ifa-5A identified a constitutively expressed gene encoding a stress response NST1-like protein (TraesCS5A01G211300LC) as a candidate gene for FHB resistance. NST1 genes are key regulators of secondary cell wall biosynthesis in anther endothecium cells. Whether the stress response NST1-like gene affects anther extrusion, thereby affecting FHB resistance, needs further investigation.ConclusionInduced and preexisting cell wall components and terpene metabolites contribute to resistance and limit fungal colonization early on. In contrast, excessive gene expression directs plant defense response towards programmed cell death which favors necrotrophic growth of the Fg pathogen and could thus lead to increased fungal colonization.

Investigating the cell and developmental biology of plant infection by the rice blast fungus Magnaporthe oryzae

Magnaporthe oryzae is the causal agent of rice blast disease, the most widespread and serious disease of cultivated rice. Live cell imaging and quantitative 4D image analysis have provided new insight into the mechanisms by which the fungus infects host cells and spreads rapidly in plant tissue. In this video review article, we apply live cell imaging approaches to understanding the cell and developmental biology of rice blast disease. To gain entry to host plants, M. oryzae develops a specialised infection structure called an appressorium, a unicellular dome-shaped cell which generates enormous turgor, translated into mechanical force to rupture the leaf cuticle. Appressorium development is induced by perception of the hydrophobic leaf surface and nutrient deprivation. Cargo-independent autophagy in the three-celled conidium, controlled by cell cycle regulation, is essential for appressorium morphogenesis. Appressorium maturation involves turgor generation and melanin pigment deposition in the appressorial cell wall. Once a threshold of turgor has been reached, this triggers re-polarisation which requires regulated generation of reactive oxygen species, to facilitate septin GTPase-dependent cytoskeletal re-organisation and re-polarisation of the appressorium to form a narrow, rigid penetration peg. Infection of host tissue requires a further morphogenetic transition to a pseudohyphal-type of growth within colonised rice cells. At the same time the fungus secretes an arsenal of effector proteins to suppress plant immunity. Many effectors are secreted into host cells directly, which involves a specific secretory pathway and a specialised structure called the biotrophic interfacial complex. Cell-to-cell spread of the fungus then requires development of a specialised structure, the transpressorium, that is used to traverse pit field sites, allowing the fungus to maintain host cell membrane integrity as new living plant cells are invaded. Thereafter, the fungus rapidly moves through plant tissue and host cells begin to die, as the fungus switches to necrotrophic growth and disease symptoms develop. These morphogenetic transitions are reviewed in the context of live cell imaging studies.

The RsRlpA Effector Is a Protease Inhibitor Promoting Rhizoctonia solani Virulence through Suppression of the Hypersensitive Response.

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.

Interactive Gene Expression Patterns of Susceptible and Resistant Lens ervoides Recombinant Inbred Lines and the Necrotroph Ascochyta lentis.

Ascochyta lentis is a foliar pathogen of Lens species and is of worldwide importance in cultivated lentil production. High levels of resistance were identified in the wild species Lens ervoides. This resistance was explored through histopathology, qPCR estimation of fungal biomass and transcriptome sequencing in a susceptible and a resistant recombinant inbred line (RIL) of L. ervoides infected with an aggressive isolate of A. lentis. Necrotrophic growth was delayed in the resistant RIL compared to accelerated necrotrophy of A. lentis in the susceptible RIL. Analysis of the fungal secretome indicated that the early activation of cell wall-degrading enzymes contributed to increased virulence of A. lentis. On the host side, gene co-expression analysis revealed that the invasion by A. lentis caused mRNA, DNA and protein decay in infected plants regardless of the level of resistance in the host. The resistant RIL exhibited a stronger gene co-expression in lipid localization and sulfur processes, and cellular responses to nutrients and stimuli than the susceptible RIL. In addition, differential gene analysis revealed that the repression of both, gibberellin signaling and cell death associated with the hypersensitive response (HR), were associated with enhanced A. lentis resistance.

Characterization of the Barley Net Blotch Pathosystem at the Center of Origin of Host and Pathogen.

Net blotch (NB) is a major disease of barley caused by the fungus Pyrenophora teres f. teres (Ptt), and P. teres f. maculata (Ptm). Ptt and Ptm infect the cultivated crop (Hordeum vulgare) and its wild relatives (H. vulgare ssp. spontaneum and H. murinum ssp. glaucum). The main goal of this research was to study the NB-causing pathogen in the crop center of origin. To address this, we have constructed a Ptt (n = 15) and Ptm (n = 12) collection isolated from three barley species across Israel. Isolates were characterized genetically and phenotypically. Aggressiveness of the isolates was determined based on necrotrophic growth rate on detached leaves of barley. In addition, isolates were genetically characterized by the mating type, followed by phylogenetic analysis, clustering them into seven groups. The analysis showed no significant differentiation of isolates based on either geographic origin, host of origin or form (Ptt vs. Ptm). Nevertheless, there was a significant difference in aggressiveness among the isolates regardless of host species, geographic location or sampling site. Moreover, it was apparent that the isolates derived from wild hosts were more variable in their necrotrophic growth rate, compared to isolates sampled from cultivated hosts, thereby suggesting that NB plays a major role in epidemiology at the center of barley origin where most of the diversity lies. Ptm has significantly higher necrotrophic and saprotrophic growth rates than Ptt, and for both a significant negative correlation was found between light intensity exposure and growth rates.

Genome-wide DNA methylation and transcriptomic profiles in the lifestyle strategies and asexual development of the forest fungal pathogen Heterobasidion parviporum

ABSTRACTHeterobasidion parviporum is the most devastating fungal pathogen of conifer forests in Northern Europe. The fungus has dual life strategies, necrotrophy on living trees and saprotrophy on dead woods. DNA cytosine methylation is an important epigenetic modification in eukaryotic organisms. Our presumption is that the lifestyle transition and asexual development in H. parviporum could be driven by epigenetic effects. Involvements of DNA methylation in the regulation of aforementioned processes have never been studied thus far. RNA-seq identified lists of highly induced genes enriched in carbohydrate-active enzymes during necrotrophic interaction with host trees and saprotrophic sawdust growth. It also highlighted signaling- and transcription factor-related genes potentially associated with the transition of saprotrophic to necrotrophic lifestyle and groups of primary cellular activities throughout asexual development. Whole-genome bisulfite sequencing revealed that DNA methylation displayed pronounced preference in CpG dinucleotide context across the genome and mostly targeted transposable element (TE)-rich regions. TE methylation level demonstrated a strong negative correlation with TE expression, reinforcing the protective function of DNA methylation in fungal genome stability. Small groups of genes putatively subject to methylation transcriptional regulation in response to saprotrophic and necrotrophic growth in comparison with free-living mycelia were also explored. Our study reported on the first methylome map of a forest pathogen. Analysis of transcriptome and methylome variations associated with asexual development and different lifestyle strategies provided further understanding of basic biological processes in H. parviporum. More importantly, our work raised additional potential roles of DNA methylation in fungi apart from controlling the proliferation of TEs.

Highly flexible infection programs in a specialized wheat pathogen.

Many filamentous plant pathogens exhibit high levels of genomic variability, yet the impact of this variation on host–pathogen interactions is largely unknown. We have addressed host specialization in the wheat pathogen Zymoseptoria tritici. Our study builds on comparative analyses of infection and gene expression phenotypes of three isolates and reveals the extent to which genomic variation translates into phenotypic variation. The isolates exhibit genetic and genomic variation but are similarly virulent. By combining confocal microscopy, disease monitoring, staining of ROS, and comparative transcriptome analyses, we conducted a detailed comparison of the infection processes of these isolates in a susceptible wheat cultivar. We characterized four core infection stages: establishment, biotrophic growth, lifestyle transition, and necrotrophic growth and asexual reproduction that are shared by the three isolates. However, we demonstrate differentiated temporal and spatial infection development and significant differences in the expression profiles of the three isolates during the infection stages. More than 20% of the genes were differentially expressed and these genes were located significantly closer to transposable elements, suggesting an impact of epigenetic regulation. Further, differentially expressed genes were enriched in effector candidates suggesting that isolate‐specific strategies for manipulating host defenses are present in Z. tritici. We demonstrate that individuals of a host‐specialized pathogen have highly differentiated infection programs characterized by flexible infection development and functional redundancy. This illustrates how high genetic diversity in pathogen populations results in highly differentiated infection phenotypes, which fact needs to be acknowledged to understand host–pathogen interactions and pathogen evolution.

Drinking water bacterial communities exhibit specific and selective necrotrophic growth

Physicochemical water disinfection methods result in the reduction of bacterial concentrations by orders of magnitude, but not in the total elimination of the bacterial community. As such, the dead bacterial biomass may act as a carbon and nutrient source for the survivor populations. The ability of bacterial strains to grow on dead bacterial cells has been described before as necrotrophy. We investigated the impact of killed bacterial biomass of two different bacterial strains on the growth potential of natural drinking water microbial communities. Many indigenous bacterial taxa could grow on dead biomass, with the total bacterial concentration increasing from 104 to 108 cells/ml. Necrotrophic growth was specific (43 enriched taxa) and selective (i.e. enriched taxa were dependent on the type of dead biomass). The potential of natural water communities to grow necrotrophically has remained underexplored. Nevertheless the phenomenon can have a big impact in water quality and deserves more attention.

Multiparametric analysis of diversity in Botrytis cinerea isolates from Israel

The necrotrophic fungus Botrytis cinerea, known as the causal agent of gray mold, is ranked second for its phytopathological global-impact. The disease is controlled by cultural means and fungicides, however, these techniques have variable effect on different fungal isolates due to the plasticity of the pathogen populations. Here we studied and characterized the genetic and virulence associated variability of B. cinerea isolates from different sources. Starting from initial survey of 31 B. cinerea isolates, collected from seven hosts in different locations of Israel, we have focused on 10 isolates that exhibited potential phytopathogenic variability. Two genetic markers (microsatellite) Bc1 and Bc7, were able to differentiate between these isolates. Our analysis demonstrates significant variability in saprophytic growth rate, necrotrophic growth rate on tomato leaves and stems, and in the incidence of infection on leaves of whole plants. Following the observation of significant correlation between saprophytic growth rate, and necrotrophic growth on leaves, we have studied normalized (by saprophytic growth) virulence. Utilization of normalized necrotrophic growth rate enabled to indicate on the presence of virulence mechanisms other than growth rate, for several isolates. Exploration of this direction illustrated variability in resistance to paraquat (associated with resistance to oxidation), which was associated with high and low superoxide dismutase (SOD) gene expression for selected isolates showing high or low paraquat resistance, respectively. Finally, we have used unsupervised learning (clustering analysis) to explore patterns in the multivariable space, which demonstrated two modes of pathogenicity in the tested B. cinerea isolates.

Oligosaccharides successfully thwart hijacking of the salicylic acid pathway by Phytophthora infestans in potato leaves

Potato growing is severely threatened by the late blight agent Phytophthora infestans, which is usually controlled by massive amounts of fungicides. While variety resistance is often bypassed by the pathogen, the plant innate immunity opens the way to new biological plant protection tools, e.g. the COS‐OGA elicitor. This oligosaccharide composition mimics the interaction between plants and fungal pathogens as it combines chitosan oligomers (COS) with pectin‐derived oligogalacturonides (OGA). Two different COS‐OGA elicitors were evaluated against potato late blight: FytoSave, mainly efficient against powdery mildews, and FytoSol, a new composition still under development. In addition, a comparative study of plant defence induction was performed, focusing on the effect of repeated sprayings as well as on the stimulation of salicylic acid (SA), jasmonic acid and ethylene‐related pathways during the biotrophic and necrotrophic growth stages of the pathogen. The FytoSave elicitor strongly increased the SA content but failed to induce sufficient protection against late blight, while FytoSol maintained or even decreased the free SA content in the presence of P. infestans and was completely efficient. Surprisingly, the necrotrophic development of P. infestans occurred along with a strong leaf accumulation of free SA and SA‐related transcripts. This may represent an attempt by P. infestans to divert plant defences for its own benefit. Preventive sprayings with FytoSol but not FytoSave completely impeded this hijacking. FytoSol seemed to keep the SA pathway under control, thereby preventing its diversion by P. infestans.

A Dispensable Chromosome Is Required for Virulence in the Hemibiotrophic Plant Pathogen Colletotrichum higginsianum.

The hemibiotrophic plant pathogen Colletotrichum higginsianum infects Brassicaceae and in combination with Arabidopsis thaliana, represents an important model system to investigate various ecologically important fungal pathogens and their infection strategies. After penetration of plant cells by appressoria, C. higginsianum establishes large biotrophic primary hyphae in the first infected cell. Shortly thereafter, a switch to necrotrophic growth occurs leading to the invasion of neighboring cells by secondary hyphae. In a forward genetic screen for virulence mutants by insertional mutagenesis, we identified mutants that penetrate the plant but show a defect in the passage from biotrophy to necrotrophy. Genome sequencing and pulsed-field gel electrophoresis revealed that two mutants were lacking chromosome 11, encoding potential pathogenicity genes. We established a chromosome loss assay to verify that strains lacking this small chromosome abort infection during biotrophy, while their ability to grow on artificial media was not affected. C. higginsianum harbors a second small chromosome, which can be lost without effects on virulence or growth on agar plates. Furthermore, we found that chromosome 11 is required to suppress Arabidopsis thaliana plant defense mechanisms dependent on tryptophan derived secondary metabolites.