Spatio-temporal analysis of an invasive plant pathogen (Phytophthora ramorum) in England and Wales

Phytophthora ramorum is a damaging invasive plant pathogen and was first discovered in the UK in 2002. Spatial point analyses were applied to the occurrence of this disease in England and Wales during the period of 2003-2006 in order to assess its spatio-temporal spread. Out of the 4301 garden centres and nurseries (GCN) surveyed, there were 164, 105, 123 and 41 sites with P. ramorum in 2003, 2004, 2005 and 2006, respectively. Spatial analysis of the observed point patterns of GCN outbreaks suggested that these sites were significantly clumped within a radius of ca 60 km in 2003, but not in later years. Further analyses were conducted to determine the relationship of GCN outbreak sites over two consecutive years and thus to infer possible disease spread over time. This analysis suggested that disease spread among GCN sites was most likely to have occurred within a distance of 60 km for 2003-2004, but not for the later years. There were 35, 63, 81 and 58 sites with P. ramorum in the semi-natural environment (SNE). Analyses were carried out to assess whether infected GCN sites could act as an inoculum source of infected SNE plants or vice versa. In all years, there was a significant spatial closeness among GCN and SNE outbreak sites within a distance of 1 km. But a significant relationship over a longer distance (within 60 km) was only observed between cases in 2003 and 2004. These analyses suggest that statutory actions taken so far appear to have reduced the extent of long-distance spread of P. ramorum among garden centres and nurseries, but not the disease spread at a shorter distance between GCN and SNE sites.

Apical leaf necrosis as a defence mechanism against pathogen attack: effects of high nutrient availability on onset and rate of necrosis

An outdoor experiment was conducted to increase understanding of apical leaf necrosis in the presence of pathogen infection. Holcus lanatus seeds and Puccinia coronata spores were collected from two adjacent and otherwise similar habitats with differing long-term N fertilization levels. After inoculation, disease and necrosis dynamics were observed during the plant growing seasons of 2003 and 2006. In both years high nutrient availability resulted in earlier disease onset, a higher pathogen population growth rate, earlier physiological apical leaf necrosis onset and a reduced time between disease onset and apical leaf necrosis onset. Necrosis rate was shown to be independent of nutrient availability. The results showed that in these nutrient-rich habitats H. lanatus plants adopted necrosis mechanisms which wasted more nutrients. There was some indication that these necrosis mechanisms were subject to local selection pressures, but these results were not conclusive. The findings of this study are consistent with apical leaf necrosis being an evolved defence mechanism.

A qualitative host-pathogen interaction in the Theobroma cacao-Moniliophthora perniciosa pathosystem

The aim of this study was to test whether resistance of clones of Theobroma cacao ( cocoa) varied between isolates of Moniliophthora (formerly Crinipellis) perniciosa, the cause of witches' broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 x Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.

The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

Evaluation of cacao (Theobroma cacao L.) clones against seven Colombian isolates of Moniliophthora roreri (Cif.) Evans et al., representing four major genetic groupings of the pathogen in cacao

Artificial pod inoculation was used to compare the relative aggressiveness of seven Colombian isolates of Moniliophthora roreri (the causal agent of moniliasis or frosty pod disease), representing four major genetic groupings of the pathogen in cacao (cocoa), when applied to five diverse cacao genotypes (ICS-1, ICS-95, TSH-565, SCC-61 and CAP-34) at La Suiza Experimental Farm, Santander Department, Colombia. The following variables were evaluated 9 weeks after inoculation of 2- to 3-month-old pods with spore suspensions (1.2 x 10(5) spores mL(-1)): (i) disease incidence (DI); (ii) external severity (ES); and (iii) internal severity (IS). IS was found to be of greatest value in classifying the reaction of the host genotype against M. roreri. Genetic variation reported between isolates and cacao genotypes was not matched by similar diversity in their aggressiveness. All isolates were generally highly aggressive against most cacao genotypes, with only two isolates showing reduced IS and ES reactions. There was considerable variation between clones in the IS and ES scores, but one cultivated clone (ICS-95) displayed a significant level of resistance against all seven isolates. This clone may be useful in cacao breeding initiatives for resistance to moniliasis of cacao.

Phytohormones and plant-herbivore-pathogen interactions: integrating the molecular with the ecological

Current research into indirect phytopathogen–herbivore interactions (i.e., interactions mediated by the host plant) is carried out in two largely independent directions: ecological/mechanistic and molecular. We investigate the origin of these approaches and their strengths and weaknesses. Ecological studies have determined the effect of herbivores and phytopathogens on their host plants and are often correlative: the need for long-term manipulative experiments is pressing. Molecular/cellular studies have concentrated on the role of signaling pathways for systemic induced resistance, mainly involving salicylic acid and jasmonic acid, and more recently the cross-talk between these pathways. This cross-talk demonstrates how interactions between signaling mechanisms and phytohormones could mediate plant–herbivore–pathogen interactions. A bridge between these approaches may be provided by field studies using chemical induction of defense, or investigating whole-organism mechanisms of interactions among the three species. To determine the role of phytohormones in induced resistance in the field, researchers must combine ecological and molecular methods. We discuss how these methods can be integrated and present the concept of “kaleidoscopic defense.” Our recent molecular-level investigations of interactions between the herbivore Gastrophysa viridula and the rust fungus Uromyces rumicis on Rumex obtusifolius, which were well studied at the mechanistic and ecological levels, illustrate the difficulty in combining these different approaches. We suggest that the choice of the right study system (possibly wild relatives of model species) is important, and that molecular studies must consider the environmental conditions under which experiments are performed. The generalization of molecular predictions to ecologically realistic settings will be facilitated by “middle-ground studies” concentrating on the outcomes of the interactions.

A model for the evolution of pathogen-induced leaf shedding

A size-structured plant population model is developed to study the evolution of pathogen-induced leaf shedding under various environmental conditions. The evolutionary stable strategy (ESS) of the leaf shedding rate is determined for two scenarios: i) a constant leaf shedding strategy and ii) an infection load driven leaf shedding strategy. The model predicts that ESS leaf shedding rates increase with nutrient availability. No effect of plant density on the ESS leaf shedding rate is found even though disease severity increases with plant density. When auto-infection, that is increased infection due to spores produced on the plant itself, plays a key role in further disease increase on the plant, shedding leaves removes disease that would otherwise contribute to disease increase on the plant itself. Consequently leaf shedding responses to infections may evolve. When external infection, that is infection due to immigrant spores, is the key determinant, shedding a leaf does not reduce the force of infection on the leaf shedding plant. In this case leaf shedding will not evolve. Under a low external disease pressure adopting an infection driven leaf shedding strategy is more efficient than adopting a constant leaf shedding strategy, since a plant adopting an infection driven leaf shedding strategy does not shed any leaves in the absence of infection, even when leaf shedding rates are high. A plant adopting a constant leaf shedding rate sheds the same amount of leaves regardless of the presence of infection. Based on the results we develop two hypotheses that can be tested if the appropriate plant material is available.

Mapping CD55 function: the structure of two pathogen-binding domains at 1.7 A

Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells. It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection. We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement. We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

Bacterial pathogen evolution: breaking news

The immense social and economic impact of bacterial pathogens, from drug-resistant infections in hospitals to the devastation of agricultural resources, has resulted in major investment to understand the causes and conse- quences of pathogen evolution. Recent genome se- quencing projects have provided insight into the evolution of bacterial genome structures; revealing the impact of mobile DNA on genome restructuring and pathogenicity. Sequencing of multiple genomes of relat- ed strains has enabled the delineation of pathogen evo- lution and facilitated the tracking of bacterial pathogens globally. Other recent theoretical and empirical studies have shown that pathogen evolution is significantly influenced by ecological factors, such as the distribution of hosts within the environment and the effects of co- infection. We suggest that the time is ripe for experi- mentalists to use genomics in conjunction with evolu- tionary ecology experiments to further understanding of how bacterial pathogens evolve.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria f.sp. hordei

Whilst there is increasing evidence tht the outcome of the interation between a pathogen and a host is dependent on protein-protein interactions, very little information is available on in planta proteomics of biotrophic plant pathogens. Here a proteogenomic approach has been employed to supplement the annotation of the recently sequenced genome and to cast light on the biology of the infection process of the economically important barley powdery mildew pathogen, Blumeria graminis f.sp hordei

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC30 supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC30 supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC30 supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC30-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC30, suggesting that a bi-modal lifecycle of GanedenBC30 in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution.