Susceptible Host Genotypes Research Articles

Ectopical expression of bacterial collagen-like protein supports its role as adhesin in host-parasite coevolution.

For a profound understanding of antagonistic coevolution, it is necessary to identify the coevolving genes. The bacterium Pasteuria and its host, the microcrustacean Daphnia, are a well-characterized paradigm for co-evolution, but the underlying genes remain largely unknown. A genome-wide association study suggested a Pasteuria collagen-like protein 7 (Pcl7) as a candidate mediating parasite attachment and driving its coevolution with the host. Since Pasteuria ramosa cannot currently be genetically manipulated, we used Bacillus thuringiensis to express a fusion protein of a Pcl7 carboxy-terminus from P. ramosa and the amino-terminal domain of a B. thuringiensis collagen-like protein (CLP). Mutant B. thuringiensis (Pcl7-Bt) spores but not wild-type B. thuringiensis (WT-Bt) spores attached to the same site of susceptible hosts as P. ramosa. Furthermore, Pcl7-Bt spores attached readily to susceptible host genotypes, but only slightly to resistant host genotypes. These findings indicated that the fusion protein was properly expressed and folded and demonstrated that indeed the C-terminus of Pcl7 mediates attachment in a host genotype-specific manner. These results provide strong evidence for the involvement of a CLP in the coevolution of Daphnia and P. ramosa and open new avenues for genetic epidemiological studies of host-parasite interactions.

Impacts of Continued Exposure to a Susceptible Host Genotype on Aggressiveness of Phytophthora nicotianae Isolates Adapted to Multiple Sources of Partial Resistance.

Pathogen adaptation can threaten the durability of partial resistance. Mixed plantings of susceptible and partially resistant varieties may prolong the effectiveness of partial resistance, but little is known about how continued exposure to a susceptible genotype can change the aggressiveness of pathogen isolates adapted to a source of partial resistance. The objective of this study was to examine the effects of continued exposure to a highly susceptible tobacco genotype on isolates of Phytophthora nicotianae that had been adapted to partial resistance. Isolates of P. nicotianae previously adapted to two sources of partial resistance were continually exposed to either the original host of adaptation or a susceptible host. After six generations of host exposure, isolates obtained from the partially resistant and the susceptible hosts were compared for their aggressiveness on the resistant host and for differences in expression of genes associated with pathogenicity and aggressiveness. Results suggested that exposure to the susceptible tobacco genotype reduced aggressiveness of isolates adapted to partial resistance in K 326 Wz/- but not of isolates adapted to partial resistance in Fla 301. Quantification of pathogenicity-associated gene expression using qRT-PCR suggested the rapid change in aggressiveness of isolates adapted to Wz-sourced partial resistance may have resulted from modification in gene expression in multiple genes.

First report of oospore formation in Plasmopara viticola, the causal agent of grapevine downy mildew, in highland regions of southern Brazil

AbstractThis work evaluates the formation of oospores of Plasmopara viticola, the causal agent of grape downy mildew (DM), in highland regions in southern Brazil. Leaves of susceptible and resistant grape genotypes naturally infected with the pathogen were collected in the autumn of 2017, 2018, and 2019 from vineyards located in the highlands of Santa Catarina state. Leaf tissues were evaluated by light microscopy and scanning electron microscopy. Oospores of P. viticola were identified in both susceptible and resistant host genotypes. They were concentrated in the central regions of the DM lesions, close to the leaf veins, and exhibited a rounded shape, yellowish colour, thick wall, and a diameter ranging from 16.28 to 49.15 µm. The formation of oospores is strong evidence that sexual reproduction is occurring in P. viticola in the climatic conditions of the highlands of southern Brazil. Sexual reproduction contributes to the maximization of genetic diversity via meiosis. Populations with high genetic variability are more likely to break resistance mechanisms conferred by resistance genes and to develop resistance to fungicides applied for disease control. To our knowledge, this is the first scientific study to prove the formation of P. viticola oospores in Brazil. The results presented provide a solid basis for further studies on sexual recombination in P. viticola. Genetic improvement programmes for grapevines, disease management methods, and disease prediction models need to consider the sexual reproduction of this pathogen, otherwise their effectiveness may be compromised.

Transcriptome analysis of two lines of Brassica oleracea in response to early infection with Xanthomonas campestris pv. campestris

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is a devastating disease of cruciferous crops worldwide and a threat to the economic production of Brassica oleracea L. in China. Strategies to control this disease are limited. In addition, knowledge of the molecular mechanisms of pathogenicity and host resistance are still only poorly understood. In this study, we performed transcriptomic analyses of the leaves of two contrasting B. oleracea lines (Xcc-resistant line QP07 and Xcc-susceptible line DBP71) to study the early defence response induced by infection with Xcc. We identified 3357 upregulated and 4091 downregulated genes between QP07 and DBP71 at 0, 12, 24, 48 and 96 h post-inoculation (hpi). A functional annotation pathways analysis of the differentially expressed genes indicated that the glucosinolate biosynthetic and catabolic pathways were enhanced, and ROS scavenging, hormonal, receptor-kinase-related genes and nucleotide binding site (NBS)-encoding resistance genes were expressed during the early infection period. In addition, we found that the host plants may respond to Xcc infection by actively regulating photosynthetic energy metabolism. The results provide new insights to help understand the different responses to Xcc in resistant and susceptible host genotypes, and to aid in the development of effective strategies for the prevention of black rot.

Salivary proteins of Phloeomyzus passerinii, a plant-manipulating aphid, and their impact on early gene responses of susceptible and resistant poplar genotypes

Successful plant colonization by parasites requires the circumvention of host defenses, and sometimes a reprogramming of host metabolism, mediated by effector molecules delivered into the host. Using transcriptomic and enzymatic approaches, we characterized salivary glands and saliva of Phloeomyzus passerinii, an aphid exhibiting an atypical feeding strategy. Plant responses to salivary extracts of P. passerinii and Myzus persicae were assessed with poplar protoplasts of a susceptible and a resistant genotype, and in a heterologous Arabidopsis system. We predict that P. passerinii secretes a highly peculiar saliva containing effectors potentially interfering with host defenses, biotic stress signaling and plant metabolism, notably phosphatidylinositol phosphate kinases which seemed specific to P. passerinii. Gene expression profiles indicated that salivary extracts of M. persicae markedly affected host defenses and biotic stress signaling, while salivary extracts of P. passerinii induced only weak responses. The effector-triggered susceptibility was characterized by downregulations of genes involved in cytokinin signaling and auxin homeostasis. This suggests that P. passerinii induces an intracellular accumulation of auxin in susceptible host genotypes, which is supported by histochemical assays in Arabidopsis. This might in turn affect biotic stress signaling and contribute to host tissue manipulation by the aphid.

Time-Sampled Population Sequencing Reveals the Interplay of Selection and Genetic Drift in Experimental Evolution of Potato Virus Y.

ABSTRACTRNA viruses are one of the fastest-evolving biological entities. Within their hosts, they exist as genetically diverse populations (i.e., viral mutant swarms), which are sculpted by different evolutionary mechanisms, such as mutation, natural selection, and genetic drift, and also the interactions between genetic variants within the mutant swarms. To elucidate the mechanisms that modulate the population diversity of an important plant-pathogenic virus, we performed evolution experiments with Potato virus Y (PVY) in potato genotypes that differ in their defense response against the virus. Using deep sequencing of small RNAs, we followed the temporal dynamics of standing and newly generated variations in the evolving viral lineages. A time-sampled approach allowed us to (i) reconstruct theoretical haplotypes in the starting population by using clustering of single nucleotide polymorphisms' trajectories and (ii) use quantitative population genetics approaches to estimate the contribution of selection and genetic drift, and their interplay, to the evolution of the virus. We detected imprints of strong selective sweeps and narrow genetic bottlenecks, followed by the shift in frequency of selected haplotypes. Comparison of patterns of viral evolution in differently susceptible host genotypes indicated possible diversifying evolution of PVY in the less-susceptible host (efficient in the accumulation of salicylic acid).IMPORTANCE High diversity of within-host populations of RNA viruses is an important aspect of their biology, since they represent a reservoir of genetic variants, which can enable quick adaptation of viruses to a changing environment. This study focuses on an important plant virus, Potato virus Y, and describes, at high resolution, temporal changes in the structure of viral populations within different potato genotypes. A novel and easy-to-implement computational approach was established to cluster single nucleotide polymorphisms into viral haplotypes from very short sequencing reads. During the experiment, a shift in the frequency of selected viral haplotypes was observed after a narrow genetic bottleneck, indicating an important role of the genetic drift in the evolution of the virus. On the other hand, a possible case of diversifying selection of the virus was observed in less susceptible host genotypes.

Mutations That Determine Resistance Breaking in a Plant RNA Virus Have Pleiotropic Effects on Its Fitness That Depend on the Host Environment and on the Type, Single or Mixed, of Infection.

Overcoming host resistance in gene-for-gene host-virus interactions is an important instance of host range expansion, which can be hindered by across-host fitness trade-offs. Trade-offs are generated by negative effects of host range mutations on the virus fitness in the original host, i.e., by antagonistic pleiotropy. It has been reported that different mutations in Pepper mild mottle virus (PMMoV) coat protein result in overcoming L-gene resistance in pepper. To analyze if resistance-breaking mutations in PMMoV result in antagonistic pleiotropy, all reported mutations determining the overcoming of L(3) and L(4) alleles were introduced in biologically active cDNA clones. Then, the parental and mutant virus genotypes were assayed in susceptible pepper genotypes with an L(+), L(1), or L(2) allele, in single and in mixed infections. Resistance-breaking mutations had pleiotropic effects on the virus fitness that, according to the specific mutation, the host genotype, and the type of infection, single or mixed with other virus genotypes, were antagonistic or positive. Thus, resistance-breaking mutations can generate fitness trade-offs both across hosts and across types of infection, and the frequency of host range mutants will depend on the genetic structure of the host population and on the frequency of mixed infections by different virus genotypes. Also, resistance-breaking mutations variously affected virulence, which may further influence the evolution of host range expansion. A major cause of virus emergence is host range expansion, which may be hindered by across-host fitness trade-offs caused by negative pleiotropy of host range mutations. An important instance of host range expansion is overcoming host resistance in gene-for-gene plant-virus interactions. We analyze here if mutations in the coat protein of Pepper mild mottle virus determining L-gene resistance-breaking in pepper have associated fitness penalties in susceptible host genotypes. Results show that pleiotropic effects of resistance-breaking mutations on virus fitness depend on the specific mutation, the susceptible host genotype, and the type of infection, single or mixed, with other virus genotypes. Accordingly, resistance-breaking mutations can have negative, positive, or no pleiotropic effects on virus fitness. These results underscore the complexity of host range expansion evolution and, specifically, the difficulty of predicting the overcoming of resistance factors in crops.

Defence gene expression profiling to Ascochyta rabiei aggressiveness in chickpea.

Significant differences in defence pathway-related gene expression were observed among chickpea cultivars following A. rabiei infection. Differential gene expression is indicative of diverse resistances, a theoretical tool for selective breeding. A high number of Ascochyta rabiei pathotypes infecting chickpea in Australia has severely hampered efforts towards breeding for sustained quantitative resistance in chickpea. Breeding for sustained resistance will be aided by detailed knowledge of defence responses to isolates with different aggressiveness. As an initial step, the conserved and differential expressions of a suit of previously characterised genes known to be involved in fungal defence mechanisms were assessed among resistant and susceptible host genotypes following inoculation with high or low aggressive A. rabiei isolates. Using quantitative Real-Time PCR (qRT-PCR), 15 defence-related genes, normalised with two reference genes, were temporally differentially expressed (P<0.005) as early as 2h post inoculation of Genesis090 (resistant) or Kaniva (susceptible). The highly aggressive isolate, 09KAL09, induced vastly different expression profiles of eight key defence-related genes among resistant and susceptible genotypes. Six of these same genes were differentially expressed among ten host genotypes, inclusive of the best resistance sources within the Australian chickpea breeding program, indicating potential use for discrimination and selection of resistance "type" in future breeding pursuits.

Host manipulation by a herbivore optimizes its feeding behaviour

Plant-manipulating herbivores can modify their host nutrients and defensive compounds to their own advantage. This generally results in enhanced performance of herbivores on modified tissues, but the anatomical and biochemical modifications undergone by host tissues could also modify the herbivores' behaviour. Phloeomyzus passerinii induces a reaction tissue in the bark of its susceptible poplar hosts, suggesting a host–plant manipulation by the aphid. We investigated whether the formation of this reaction tissue, confirmed by histological sections, affected the performance and feeding behaviour of the aphid. In addition we studied how host resistance affected these parameters by comparing two host genotypes of different susceptibility. Aphids developed faster on the preinfested susceptible host genotype, whereas no effect was detected on the resistant one, confirming an improvement in the host's nutritional value due to the preinfestation, in agreement with the nutrition hypothesis. The DC-EPG technique indicated that in both host genotypes, preinfestation shortened the delay until the first probe and increased both the rate and duration of sustained ingestion, and consequently host acceptance. This suggests that host manipulation can adjust not only the nutritional value of plant tissues at an optimal level for development but also host acceptance by the herbivores. In the resistant host genotype though, the number of probes and the duration of parenchyma exploration increased in the galled tissue, which was congruent with extensive cell wall modifications indicated by more extended induced lignification. The adaptive consequences of this modified feeding behaviour are discussed.

Trade‐offs in host range evolution of plant viruses

Understanding host range evolution in plant viruses is central for the control of viral diseases: changes in host range resulting in host switches or host range expansions are at the root of virus emergence either in new host species or in host genotypes previously resistant to the virus. Theory predicts that different selection pressures in different hosts will result in fitness trade‐offs across hosts, leading to specialization. This paper reviews the evidence in support of this prediction. A large fraction of plant viruses are multihost parasites: evidence for across‐species trade‐offs has been reported both from the analysis of field virus populations and, more often, from passage experiments resulting in host adaptation. More abundant evidence for across‐host trade‐offs derives from the analysis of the performance of resistance‐breaking virus genotypes in resistant and susceptible host genotypes. Data indicate that overcoming dominant resistance is coupled to severe fitness costs, while overcoming recessive resistance may be associated with fitness costs according to the specific virus genotype. There is ample evidence that antagonistic pleiotropy is a major cause of the reported across‐host trade‐offs.

Determination of genetic and pathological variance among Tilletia indica isolates and monosporidial lines using PCR based markers and host differentials

Karnal bunt of wheat caused by Tilletia indica is an important international quarantine disease in many countries. In this investigation, genetic and pathological variation among the 10 isolates and 15 monosporidial (Ms) lines belonged to different locations of North-West India was studied. Depending upon the pathogenic potential, most virulent and least aggressive isolate was found from Chaksu (Rajasthan) and Tarau (HP), which scored coefficients of infection 70.98 and 6.22, respectively, on susceptible host genotype HD 2009 under artificially inoculated conditions. Fifteen Ms lines were inoculated in 20 combinations. Most virulent compatible combination was found KB2MsD × KB6MsA, which scored co-efficient of infection 74.91%. Out of 32 Inter Simple Sequence Repeats based molecular markers, 28 were polymorphic generating 192 reproducible bands for all the T. indica isolates and Ms lines in this study. A grouping analysis using the unrooted neighbour – joining method was consistent with DARwin software and winboot analysis and combination approach suggested that self-paired Ms lines exhibit narrow genetic diversity. This result will be useful for developing integrated strategies for disease management and breeding programmes for improvement of the varieties.

Identification of Lens culinaris defense genes responsive to the anthracnose pathogen Colletotrichum truncatum

BackgroundAnthracnose of lentil, caused by the hemibiotrophic fungal pathogen Colletotrichum truncatum is a serious threat to lentil production in western Canada. Colletotrichum truncatum employs a bi-phasic infection strategy characterized by initial symptomless biotrophic and subsequent destructive necrotrophic colonization of its host. The transition from biotrophy to necrotrophy (known as the biotrophy-necrotrophy switch [BNS]) is critical in anthracnose development. Understanding plant responses during the BNS is the key to designing a strategy for incorporating resistance against hemibiotrophic pathogens either via introgression of resistance genes or quantitative trait loci contributing to host defense into elite cultivars, or via incorporation of resistance by biotechnological means.ResultsThe in planta BNS of C. truncatum was determined by histochemical analysis of infected lentil leaf tissues in time-course experiments. A total of 2852 lentil expressed sequence tags (ESTs) derived from C. truncatum-infected leaf tissues were analyzed to catalogue defense related genes. These ESTs could be assembled into 1682 unigenes. Of these, 101 unigenes encoded membrane and transport associated proteins, 159 encoded proteins implicated in signal transduction and 387 were predicted to be stress and defense related proteins (GenBank accessions: JG293480 to JG293479). The most abundant class of defense related proteins contained pathogenesis related proteins (encoded by 125 ESTs) followed by heat shock proteins, glutathione S-transferase, protein kinases, protein phosphatase, zinc finger proteins, peroxidase, GTP binding proteins, resistance proteins and syringolide-induced proteins. Quantitative RT-PCR was conducted to compare the expression of two resistance genes of the NBS-LRR class in susceptible and partially resistant genotypes. One (contig186) was induced 6 days post-inoculation (dpi) in a susceptible host genotype (Eston) whereas the mRNA level of another ( LT21-1990) peaked 4 dpi in a partially resistant host genotype (Robin), suggesting roles in conditioning the susceptibility and conferring tolerance to the pathogen, respectively.ConclusionsData obtained in this study suggest that lentil cells recognize C. truncatum at the BNS and in response, mount an inducible defense as evident by a high number of transcripts (23% of the total pathogen-responsive lentil transcriptome) encoding defense related proteins. Temporal expression polymorphism of defense related genes could be used to distinguish the response of a lentil genotype as susceptible or resistant.

Differential selection pressures exerted by host resistance quantitative trait loci on a pathogen population: a case study in an apple × Venturia inaequalis pathosystem

Understanding how pathogens evolve according to pressures exerted by their plant hosts is essential for the derivation of strategies aimed at the durable management of resistant cultivars. The spectrum of action of the resistance factors in the partially resistant cultivars is thought to be an important determinant of resistance durability. However, it has not yet been demonstrated whether the pressures exerted by quantitative resistance are different according to their spectrum of action. To investigate selection pressures exerted by apple genotypes harbouring various resistance quantitative trait loci (QTLs) on a mixed inoculum of the scab disease agent, Venturia inaequalis, we monitored V.inaequalis isolate proportions on diseased apple leaves of an F1 progeny using quantitative pyrosequencing technology and QTL mapping. Broad-spectrum resistances did not exert any differential selection pressures on the mixed inoculum, whereas narrow-spectrum resistances decreased the frequencies of some isolates in the mixture relative to the susceptible host genotypes. Our results suggest that the management of resistant cultivars should be different according to the spectrum of action of their resistance factors. The pyramiding of broad-spectrum factors or the use of a mixture of apple genotypes that carry narrow-spectrum resistance factors are two possible strategies for the minimization of resistance erosion.

Genetic analysis and comparative virulence of isolates of Pyrenophora tritici-repentis from wheat in Argentina

Tan spot caused by the fungus Pyrenophora tritici-repentis, occurs in many wheat-growing regions worldwide and is considered among the major diseases of wheat in Argentina. In nature, this pathogen reproduces both sexually and asexually, but the extent of genetic variability in the P. tritici-repentis Argentinian population is unknown. The genetic structure of P. tritici-repentis populations was assessed with amplified polymorphic markers with sequence data from the internal transcribed spacer region of the ribosomal deoxyribonucleic acid (DNA) (ITS) analysis. Eighty-nine (89) P. tritici-repentis isolates were collected from 14 geographically distinct regions of the wheat-growing areas in Argentina. Clustered analysis of random amplified polymorphic DNAs (RAPDs) showed seven different groups of isolates at 43% similarity. Analyses of the ITS1 and ITS4 primers grouped the isolates in fifteen separated clusters and resulted in a sensible method by providing valuable information on the genotypic diversity detected in the populations. Additionally, diversity was assayed by analysis of severity and type of symptoms reaction indicating significant differences between cultivars and isolates. The utility of cleaved amplified polymorphic sequence analysis for molecular differentiation of pathotypes was demonstrated. It appears that the P. tritici-repentis population in Argentina is fairly diverse, and caution should be taken in any breeding strategy, since rare pathotypes may quickly become predominant if susceptible host genotypes are continuously grown. Key words: Tan spot, Pyrenophora tritici-repentis, wheat, genetic diversity, virulence.

The risks of hospitality

Cellular integrity is essential, because the chemical gradients necessary to sustain life would otherwise be subdued by entropy. No wonder that, already, the earliest life-forms have developed mechanisms to defend their integrity against invaders. However, there are two occasions where cells have to adopt a softer approach towards integrity: first, when a cell has to take up molecules or other cells to support its own metabolism (endocytosis in its variants); second, when two cells of the same species fuse to initiate sexual propagation. Whereas the uptake of food is perfectly compatible with defence, the defence machinery has to be quelled or at least modulated during sexual fusion in such a way that the DNA of the (male) “guest” is not damaged. This evolutionary reasoning already illustrates that any fusion or perforation at the outer membrane must be under the control of specific signals. Many pathogens have developed, during an evolutionary arms race with their hosts, means to manipulate this signalling to circumvent or suppress defence by so called effectors (Jones and Dangl 2006), which allows them to remodel the plasma membrane and to penetrate into the host cell without killing it. It is rather unlikely that these so called biotrophic pathogens have invented such a complex manipulation de novo—they probably just usurped a signal cascade that already functioned in the host cell in a different context: to downregulate defence during sexual fusion. Two contributions in the current issue provide surprising details of haustoria formation that link sexuality with defence. The work by Guan et al. (2011) in the current issue investigates resistance of the Chinese wild Vitis pseudoreticulata against powdery mildew (Uncinula necator). This biotrophic fungus penetrates into the epidermal cell and forms a haustorium which allows the invader to profit from the metabolism of the host. From the comparison of the resistant with a susceptible host genotype, they identified a glyoxal oxidase that is upregulated in the resistant host and localized in the apoplast. The function of this enzyme in plants is unclear; in wood-degrading fungi where it was originally discovered, glyoxal oxidase is secreted and seems to support the degradation process by generating hydroxy peroxides. The induction of glyoxal oxidase in the resistant V. pseudoreticulata might thus be either adaptive in nature or just a byproduct of defence. To test this, Guan et al. (2011) overexpressed the glyoxal oxidase in the susceptible host by agroinfiltration into young leaves that were subsequently infected with powdery mildew. They observed delayed and even partially arrested colonization in those leaf areas, where the transgene was expressed. Thus, the induction of glyoxal oxidase is sufficient to confer resistance to powdery mildew probably through the generation of extracellular reactive oxygen species that seem to suppress haustoria formation. The work by Hiratsuka and Terasaka (2011) in the current issue describes haustoria formation in a completely different context. The pollen tubes of many gymnosperms (that represent an evolutionary precursor of “modern” angiosperm pollen tubes) are branched. Some of these branches do not convey sperms, but penetrate into the extracellular matrix of the female nucellus acting as haustoria that support the male gametophyte during the often several months between pollination and sperm delivery. The transmitting tissue in the style secretes specific arabinogalactan proteins that guide pollen tube Handling Editor: Peter Nick

Molecular Characterization and Functional Analysis of MgNLP, the Sole NPP1 Domain–Containing Protein, from the Fungal Wheat Leaf Pathogen Mycosphaerella graminicola

Analysis of the fully sequenced genome of the wheat leaf-specific fungal pathogen Mycosphaerella graminicola identified only a single gene encoding a member of the necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein family (NLP). NLP proteins have frequently been shown to trigger cell death and the activation of defense signaling reactions in dicotyledonous plants. However, complete loss-of-function reverse genetics analyses for their importance in the virulence of eukaryotic plant pathogens are generally lacking. Real-time quantitative polymerase chain reaction on MgNLP demonstrated the gene to be specifically expressed in planta. Peak expression was observed during the immediate presymptomatic phase of colonization of a susceptible host genotype. This was followed by a dramatic decrease during disease lesion formation which, in this system, exhibits characteristics of host programmed cell death (PCD). No comparable peak in transcript levels was seen during an incompatible interaction with a host genotype exhibiting gene-for-gene-based disease resistance. Heterologously expressed MgNLP protein induced necrotic cell death and the activation of defense-related genes when infiltrated into Arabidopsis leaves but not in leaves of a susceptible wheat genotype. MgNLP infiltration also failed to stimulate wheat mitogen-activated protein kinase activities. Finally, targeted deletion of M. graminicola MgNLP caused no detectable reduction in plant pathogenicity or virulence, suggesting that this protein is not a major virulence determinant during fungal infection of its host plant. To our knowledge, this represents the first complete loss-of-function analysis of NLP in a eukaryotic plant pathogen and we discuss our findings in the context of possible functions for NLP in pathogens which only infect monocotyledonous plants.

Ecological and genetic determinants of multiple infection and aggregation in a microbial host-parasite system

The number of parasites colonizing a host (termed 'multiple infection') is an important determinant of host-parasite interactions. In theory, multiple infection is determined by random mass action in genetically and spatially homogeneous populations of host and parasite. In real populations, deviations from these assumptions may strongly influence levels of multiple infection. We carried out inoculation experiments in microcosms of the freshwater protozoan Paramecium caudatum and its bacterial parasite Holospora undulata. Increasing parasite dose produced higher levels of (multiple) infection; more susceptible host genotypes also were more multiply infected. An overall pattern of parasite aggregation (excess of uninfected individuals and of individuals carrying larger numbers of parasites) indicated deviations from random mass-action transmission. Homogenizing spatial distributions of parasite and host in our microcosms did not affect aggregation, whereas aggregation was more pronounced in old than in new host clones. Thus, variation in susceptibility may arise over time within clonal populations. When sequentially inoculated, already established infections increased the probability of additional infection in generally resistant host clones, but decreased it in more susceptible clones. Hence, the role of multiple infection as a driver of epidemiological or evolutionary processes may vary among populations, depending on their precise genetic composition or infection history.

Isolation and Variation in Virulence of Single-Spore Isolates of Plasmodiophora brassicae from Canada.

Clubroot of crucifers, caused by Plasmodiophora brassicae, is emerging as an important disease of canola (Brassica napus) in Alberta, Canada. Populations of the pathogen often consist of a mixture of different pathotypes. Therefore, a simple and efficient method to isolate single resting spores of P. brassicae was developed, based on serial dilution of spore suspensions. The virulence of 24 single-spore isolates, representing five populations of the pathogen from Alberta, Ontario, and British Columbia, was characterized on the differentials of Williams and Somé et al. Symptoms were rated 6 weeks after inoculation and Fisher's least significant difference (P < 0.05) was used to differentiate resistant from susceptible host reactions. The pathotype composition of P. brassicae in Canada appeared more diverse when single-spore isolates were examined rather than populations of the pathogen. In Alberta, at least three and possibly four pathotypes were identified among the 14 isolates tested, whereas a maximum of only two pathotypes had been reported previously when populations of the pathogen were examined. Pathotype 3 or P2, as classified on the differentials of Williams and Somé et al., respectively, was found to be predominant in the province. The occurrence of other pathotypes at lower frequencies suggests that caution should be used in any breeding strategy, because rare pathotypes of P. brassicae may quickly become predominant if susceptible host genotypes are continuously grown.

Susceptibility in Viburnum to Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death on oak and tanoak and Ramorum blight or Ramorum shoot dieback on ornamentals, is a recently emerged pathogen. Viburnum is a genus of commonly grown ornamental shrubs known to be susceptible to P. ramorum. The entire genus has been placed on the P. ramorum host list. The range of susceptibility of genotypes in the genus is currently not understood. We evaluated whether or not cultivars in the genus Viburnum differ in susceptibility to P. ramorum in controlled, detached leaf inoculations using two isolates belonging to the two clonal lineages found in Oregon. The genus Viburnum shows remarkable differences in susceptibility to infection by P. ramorum. Viburnum genotypes differed significantly in susceptibility to P. ramorum in detached leaf inoculations. V. × burkwoodii was consistently the most susceptible host genotype, followed by V. plicatum 'Mariesii' and V. lentago. Most cultivars evaluated for resistance to P. ramorum were not susceptible or developed only small lesions as determined with a detached leaf assay. Our work only provides information on leaf based resistance, and inferences on epidemic development in whole canopies and under field conditions cannot currently be made.

Transcriptional Adaptation of Mycosphaerella graminicola to Programmed Cell Death (PCD) of Its Susceptible Wheat Host

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.