Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.     

Infection of an Insect Vector with a Bacterial Plant Pathogen Increases Its Propensity for Dispersal

The spread of vector-transmitted pathogens relies on complex interactions between host, vector and pathogen. In sessile plant pathosystems, the spread of a pathogen highly depends on the movement and mobility of the vector. However, questions remain as to whether and how pathogen-induced vector manipulations may affect the spread of a plant pathogen. Here we report for the first time that infection with a bacterial plant pathogen increases the probability of vector dispersal, and that such movement of vectors is likely manipulated by a bacterial plant pathogen. We investigated how Candidatus Liberibacter asiaticus (CLas) affects dispersal behavior, flight capacity, and the sexual attraction of its vector, the Asian citrus psyllid (Diaphorina citri Kuwayama). CLas is the putative causal agent of huanglongbing (HLB), which is a disease that threatens the viability of commercial citrus production worldwide. When D. citri developed on CLas-infected plants, short distance dispersal of male D. citri was greater compared to counterparts reared on uninfected plants. Flight by CLas-infected D. citri was initiated earlier and long flight events were more common than by uninfected psyllids, as measured by a flight mill apparatus. Additionally, CLas titers were higher among psyllids that performed long flights than psyllid that performed short flights. Finally, attractiveness of female D. citri that developed on infected plants to male conspecifics increased proportionally with increasing CLas bacterial titers measured within female psyllids. Our study indicates that the phytopathogen, CLas, may manipulate movement and mate selection behavior of their vectors, which is a possible evolved mechanism to promote their own spread. These results have global implications for both current HLB models of disease spread and control strategies.     

Impact of Vector Dispersal and Host-Plant Fidelity on the Dissemination of an Emerging Plant Pathogen

Dissemination of vector-transmitted pathogens depend on the survival and dispersal of the vector and the vector''s ability to transmit the pathogen, while the host range of vector and pathogen determine the breath of transmission possibilities. In this study, we address how the interaction between dispersal and plant fidelities of a pathogen (stolbur phytoplasma tuf-a) and its vector (Hyalesthes obsoletus: Cixiidae) affect the emergence of the pathogen. Using genetic markers, we analysed the geographic origin and range expansion of both organisms in Western Europe and, specifically, whether the pathogen''s dissemination in the northern range is caused by resident vectors widening their host-plant use from field bindweed to stinging nettle, and subsequent host specialisation. We found evidence for common origins of pathogen and vector south of the European Alps. Genetic patterns in vector populations show signals of secondary range expansion in Western Europe leading to dissemination of tuf-a pathogens, which might be newly acquired and of hybrid origin. Hence, the emergence of stolbur tuf-a in the northern range was explained by secondary immigration of vectors carrying stinging nettle-specialised tuf-a, not by widening the host-plant spectrum of resident vectors with pathogen transmission from field bindweed to stinging nettle nor by primary co-migration from the resident vector''s historical area of origin. The introduction of tuf-a to stinging nettle in the northern range was therefore independent of vector''s host-plant specialisation but the rapid pathogen dissemination depended on the vector''s host shift, whereas the general dissemination elsewhere was linked to plant specialisation of the pathogen but not of the vector.     

Long-Distance Dispersal of a Female Cougar in a Basin and Range Landscape

Abstract: We used Global Positioning System technology to document distance, movement path, vegetation, and elevations used by a dispersing subadult female cougar (Puma concolor) through the fragmented habitat of the Intermountain West, USA. Over the course of 1 year, female number 31 moved 357 linear km, but an actual distance of 1,341 km from the Oquirrh Mountains, Utah to the White River Plateau, Colorado, constituting the farthest dispersal yet documented for a female cougar. This cougar successfully negotiated 4 major rivers and one interstate highway while traversing portions of 3 states. Our data suggest that transient survival, and therefore total distance moved, may be enhanced when dispersal occurs during the snow-free season due to low hunting pressure and greater access to high elevation habitats. Long-distance movements by both sexes will be required for the recolonization of vacant habitats, and thus inter-state management may be warranted where state boundaries do not coincide with effective dispersal barriers.     

The Impact of Recombination Hotspots on Genome Evolution of a Fungal Plant Pathogen

Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host.     

Evolution of Pathogen Specialisation in a Host Metapopulation: Joint Effects of Host and Pathogen Dispersal

Metapopulation processes are important determinants of epidemiological and evolutionary dynamics in host-pathogen systems, and are therefore central to explaining observed patterns of disease or genetic diversity. In particular, the spatial scale of interactions between pathogens and their hosts is of primary importance because migration rates of one species can affect both spatial and temporal heterogeneity of selection on the other. In this study we developed a stochastic and discrete time simulation model to specifically examine the joint effects of host and pathogen dispersal on the evolution of pathogen specialisation in a spatially explicit metapopulation. We consider a plant-pathogen system in which the host metapopulation is composed of two plant genotypes. The pathogen is dispersed by air-borne spores on the host metapopulation. The pathogen population is characterised by a single life-history trait under selection, the infection efficacy. We found that restricted host dispersal can lead to high amount of pathogen diversity and that the extent of pathogen specialisation varied according to the spatial scale of host-pathogen dispersal. We also discuss the role of population asynchrony in determining pathogen evolutionary outcomes.     

TmpL,a Transmembrane Protein Required for Intracellular Redox Homeostasis and Virulence in a Plant and an Animal Fungal Pathogen

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus ΔtmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola ΔtmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus ΔtmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola ΔtmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the ΔtmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola ΔtmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.     

Monoclonal Antibodies to Glycoprotein Antigens of a Fungal Plant Pathogen, Phytophthora megasperma f. sp. glycinea

Genetic studies of plants and their pathogens indicate that dominant alleles for resistance in hosts are complemented by corresponding dominant alleles for avirulence in pathogens. Products of these genes have not yet been identified. We have produced murine monoclonal antibodies (mAbs) to extracellular antigens of the fungal soybean pathogen Phytophthora megasperma f. sp. glycinea (Pmg, race 1) as part of a larger effort to identify antigenic determinants associated with particular avirulence genes. Thirty-six independent mAbs have been characterized by binding to Western blots of Pmg extracellular glycoproteins and by enzyme-linked immunosorbent assay with glycoproteins modified by treatment with periodate, α-mannosidase, and endo-β-N-acetylglucosaminidase H. The mAbs are predominantly carbohydrate-specific and can be placed in six groups based on interactions with Pmg glycoproteins. Binding patterns of various mAbs to Western blots indicate that Pmg proteins may have single or multiple types of attached carbohydrate antigens. Races of Pmg with differing avirulence genes exhibit more characteristic differences by Western blot analysis than by protein staining of glycoprotein profiles. Several of the mAbs show much higher reaction levels to glycoproteins from race 1 than from two other races. All of the glycoprotein-specific mAbs cross-react with purified mycelial walls.     

Taxol Determination from Pestalotiopsis pauciseta, a Fungal Endophyte of a Medicinal Plant

Taxol is the most effective antitumor agent developed in the past three decades. It has been used for effective treatment of a variety of cancers. A taxol-producing endophytic fungus Pestalotiopsis pauciseta (strain CHP-11) was isolated from the leaves of Cardiospermum helicacabum and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores and screened for taxol production. The amount of taxol produced by this endophytic fungus was quantified by HPLC and it produced 113.3 mg/L, thus the fungus can serve as a potential material for fungus engineering to improve taxol production. This fungal taxol also had strong anticancer activity against some cancer cells viz., BT 220, H116, Int 407, HL 251 and HLK 210 tested by Apoptotic assay and it is indicated that with the increase of taxol concentration from 0.005–0.05 mmol/L, taxol induced increased cell death through apoptosis.     

Antagonistic Changes in Sensitivity to Antifungal Drugs by Mutations of an Important ABC Transporter Gene in a Fungal Pathogen

Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS and recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital for dealing with this emerging issue. In this study, we show that dramatic sequence changes in PDR5, an ABC (ATP-binding cassette) efflux transporter protein gene in an opportunistic fungal pathogen, caused the organism to become hypersensitive to azole, a widely used antifungal drug. Surprisingly, the same mutations conferred growth advantages to the organism on polyenes, which are also commonly used antimycotics. Our results indicate that Pdr5p might be important for ergosterol homeostasis. The observed remarkable sequence divergence in the PDR5 gene in yeast strain YJM789 may represent an interesting case of adaptive loss of gene function with significant clinical implications.     

Host-Pathogen Interactions : XXXIII. A Plant Protein Converts a Fungal Pathogenesis Factor into an Elicitor of Plant Defense Responses

This paper describes the effect of a plant-derived polygalacturonase-inhibiting protein (PGIP) on the activity of endopolygalacturonases isolated from fungi. PGIP's effect on endopolygalacturonases is to enhance the production of oligogalacturonides that are active as elicitors of phytoalexin (antibiotic) accumulation and other defense reactions in plants. Only oligogalacturonides with a degree of polymerization higher than nine are able to elicit phytoalexin synthesis in soybean cotyledons. In the absence of PGIP, a 1-minute exposure of polygalacturonic acid to endopolygalacturonase resulted in the production of elicitor-active oligogalacturonides. However, the enzyme depolymerized essentially all of the polygalacturonic acid substrate to elicitor-inactive oligogalacturonides within 15 minutes. When the digestion of polygalacturonic acid was carried out with the same amount of enzyme but in the presence of excess PGIP, the rate of production of elicitor-active oligogalacturonides was dramatically altered. The amount of elicitor-active oligogalacturonide steadily increased for 24 hours. It was only after about 48 hours that the enzyme converted the polygalacturonic acid into short, elicitor-inactive oligomers. PGIP is a specific, reversible, saturable, high-affinity receptor for endopolygalacturonase. Formation of the PGIP-endopolygalacturonase complex results in increased concentrations of oligogalacturonides that activate plant defense responses. The interaction of the plant-derived PGIP with fungal endopolygalacturonases may be a mechanism by which plants convert endopolygalacturonase, a factor important for the virulence of pathogens, into a factor that elicits plant defense mechanisms.     

Laboratory and Field Trials of Bait Formulations of the Fungal Pathogen, Metarhizium flavoviride, Against a Tropical Grasshopper and Locust

Extrusion technology was used to produce maize-starch 'contact bait' carriers, and vegetable-oil suspensions of Metarhizium flavoviride were incorporated into the baits after extrusion. Laboratory bioassays using the locust, Schistocerca gregaria, demonstrated mortality and reduction of 8 1 feeding after exposure to the baits, and gave an LC 50 of 1.5 10 spores l slope 0.63 for fifth instar nymphs. In laboratory trials, maize-starch contact baits showed improved efficacy relative to wheat-bran edible bait formulations of M. flavoviride. Field cage experiments, undertaken in Mali, using fourth and fifth instar nymphs of the grasshopper, Hieroglyphus dagenensis, demonstrated significant mortality with both fresh and weathered maize-starch contact bait formulations of M. flavoviride, relative to untreated controls.     

Molecular Characterization of Legionella Populations Present within Slow Sand Filters Used for Fungal Plant Pathogen Suppression in Horticultural Crops

The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products. One dominant band had sequence homology to Legionella species, indicating that these bacteria were a large component of the SSF bacterial community. Populations within experimental and commercial SSF units were studied by using Legionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses. In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a single Legionella strain was predominant in each sample. Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample. Sequence analysis of these PCR products showed that several Legionella species were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples. Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number of Legionella cells decreased, resulting in a lower number of cells in the outflow water. In the commercial SSF, legionellae were also detected in the sand column samples. Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth of Legionella and should be avoided. Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation.     

The Ecological Component of Ecological Risk Assessment: Lessons from a Field Experiment

To improve ecological relevance, regulatory agencies are promoting assessments of effects at higher levels of organization, an objective that requires an understanding of current ecological theories. One such theory, hierarchy theory, contends that the effects of a disturbance acting at one level of organization (e.g., population) are not, as a rule, transmitted to higher levels of organization (e.g., community). Conversely, effects at higher levels of organization only occur if lower level variables have been affected. Further, responses to disturbance depend on disturbance history. In this study, I determined the effects of a disturbance treatment at the population, guild, and community levels of organization for vegetation in five wetlands with a disturbance history ranging from highly to rarely disturbed. The 2-year field experiment revealed that the effects of the disturbance treatment were most strongly felt at the population level of organization in wetlands without a history of disturbance. These observed impacts took place against a backdrop of constant change. Thus, the eventual disappearance of treatment effects was not due to a return to the pre-treatment state, but rather a return to a trajectory similar to that exhibited by the control plots. The implications of these results for ecological risk assessment are: (1) the observed effects of a stressor in a system cannot be extrapolated to other systems unless they have similar disturbance histories, (2) detecting effects before they become serious requires monitoring at lower levels of organization, (3) recovery to a naturally innate state is not a viable concept, and (4) the traditional approach of using one post-treatment measurement to determine if reference and impact sites differ is of very questionable value.     

Eye Images Increase Charitable Donations: Evidence From an Opportunistic Field Experiment in a Supermarket

A number of studies have shown that the presence of simple images of eyes in the environment increases prosocial behaviour in humans. However, questions remain about the robustness of the effect, its explanation and the factors promoting it. In particular, it is not yet clear whether this effect is restricted to contexts where there is a normative requirement to behave prosocially and thus where punishment is a likely consequence of failing to do so. In an 11‐wk field experiment in a supermarket, we displayed either eye images or control images on charity collection buckets. There was no normative requirement to donate in this setting, and most people did not do so. However, the presence of eye images increased donations by 48% relative to control images. The effect of eye images was significantly stronger at times when the supermarket was quiet rather than busy. Results are consistent with models of the evolution of prosociality through reputation‐based partner choice and have potential practical benefits for those involved in charitable fundraising.     

A Field Experiment on Rain Splash Dispersal of Infective Larvae of Ostertagia Ostertagi (Trichostrongylidae) from Cow Pats to Surrounding Grass

Ostertagia ostertagi is an important tricho-strongyle nematode parasite of cattle in several countries, including Denmark. It leaves its host as eggs with faeces. After hatching it develops in the cow pat up to the third and infective stage, which is then transmitted to the surrounding grass.     

Horizontal Transfer of a Subtilisin Gene from Plants into an Ancestor of the Plant Pathogenic Fungal Genus Colletotrichum

The genus Colletotrichum contains a large number of phytopathogenic fungi that produce enormous economic losses around the world. The effect of horizontal gene transfer (HGT) has not been studied yet in these organisms. Inter-Kingdom HGT into fungal genomes has been reported in the past but knowledge about the HGT between plants and fungi is particularly limited. We describe a gene in the genome of several species of the genus Colletotrichum with a strong resemblance to subtilisins typically found in plant genomes. Subtilisins are an important group of serine proteases, widely distributed in all of the kingdoms of life. Our hypothesis is that the gene was acquired by Colletotrichum spp. through (HGT) from plants to a Colletotrichum ancestor. We provide evidence to support this hypothesis in the form of phylogenetic analyses as well as a characterization of the similarity of the subtilisin at the primary, secondary and tertiary structural levels. The remarkable level of structural conservation of Colletotrichum plant-like subtilisin (CPLS) with plant subtilisins and the differences with the rest of Colletotrichum subtilisins suggests the possibility of molecular mimicry. Our phylogenetic analysis indicates that the HGT event would have occurred approximately 150–155 million years ago, after the divergence of the Colletotrichum lineage from other fungi. Gene expression analysis shows that the gene is modulated during the infection of maize by C. graminicola suggesting that it has a role in plant disease. Furthermore, the upregulation of the CPLS coincides with the downregulation of several plant genes encoding subtilisins. Based on the known roles of subtilisins in plant pathogenic fungi and the gene expression pattern that we observed, we postulate that the CPLSs have an important role in plant infection.