Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry

White-nose syndrome (WNS) caused by the pathogenic fungus Pseudogymnoascus destructans is decimating the populations of several hibernating North American bat species. Little is known about the molecular interplay between pathogen and host in this disease. Fluorescence microscopy ambient ionization mass spectrometry was used to generate metabolic profiles from the wings of both healthy and diseased bats of the genus Myotis. Fungal siderophores, molecules that scavenge iron from the environment, were detected on the wings of bats with WNS, but not on healthy bats. This work is among the first examples in which microbial molecules are directly detected from an infected host and highlights the ability of atmospheric ionization methodologies to provide direct molecular insight into infection.     

Coinfection with a virus constrains within‐host infection load but increases transmission potential of a highly virulent fungal plant pathogen

The trade‐off between within‐host infection rate and transmission to new hosts is predicted to constrain pathogen evolution, and to maintain polymorphism in pathogen populations. Pathogen life‐history stages and their correlations that underpin infection development may change under coinfection with other parasites as they compete for the same limited host resources. Cross‐kingdom interactions are common among pathogens in both natural and cultivated systems, yet their impacts on disease ecology and evolution are rarely studied. The host plant Plantago lanceolata is naturally infected by both Phomopsis subordinaria, a seed killing fungus, as well as Plantago lanceolata latent virus (PlLV) in the Åland Islands, SW Finland. We performed an inoculation assay to test whether coinfection with PlLV affects performance of two P. subordinaria strains, and the correlation between within‐host infection rate and transmission potential. The strains differed in the measured life‐history traits and their correlations. Moreover, we found that under virus coinfection, within‐host infection rate of P. subordinaria was smaller but transmission potential was higher compared to strains under single infection. The negative correlation between within‐host infection rate and transmission potential detected under single infection became positive under coinfection with PlLV. To understand whether within‐host and between‐host dynamics are correlated in wild populations, we surveyed 260 natural populations of P. lanceolata for P. subordinaria infection occurrence. When infections were found, we estimated between‐hosts dynamics by determining pathogen population size as the proportion of infected individuals, and within‐host dynamics by counting the proportion of infected flower stalks in 10 infected plants. In wild populations, the proportion of infected flower stalks was positively associated with pathogen population size. Jointly, our results suggest that the trade‐off between within‐host infection load and transmission may be strain specific, and that the pathogen life‐history that underpin epidemics may change depending on the diversity of infection, generating variation in disease dynamics.     

The natural occurrence of Pandora heteropterae (Zygomycetes: Entomophthorales) infecting Lygus lineolaris (Hemiptera: Miridae)

An unknown fungal pathogen was recovered from Lygus lineolaris (Palisot de Beauvois) during a survey of parasitic and pathogenic natural enemies conducted in Franklin County, Arkansas. The pathogen was identified as Pandora heteropterae (Ba?azy) Keller based on characteristics of the morphology, as well as growth and sporulation on hosts. The fungus infected 11 of the 3405 (0.32%) wild L. lineolaris collected. In a laboratory host-range bioassay, five of seven hemipteran species from the families Miridae, Coreidae, Lygaeidae, and Pentatomidae were successfully infected. P. heteropterae was previously reported only once, from an unidentified host species in Poland. Here we describe the morphology and growth of P. heteropterae and discuss its potential impact on L. lineolaris in the field.     

A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides

Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization.     

A Lipoxygenase Pathway Is Activated in Rice after Infection with the Rice Blast Fungus Magnaporthe grisea

Lipoxygenase (LOX) and lipid hydroperoxide-decomposing activity (LHDA) markedly increased in the fifth leaves of rice (Oryza sativa cv Aichiasahi) after infection with the rice blast fungus, Magnaporthe grisea. The increases in the enzyme activities were significantly higher in response to infection with an incompatible strain (race 131) compared with infection with a compatible strain (race 007) of the fungus. Using ion-exchange chromatography, we isolated three LOX activities (leaf LOX-1, -2, -3) from both uninoculated and infected leaves. The activity of leaf LOX-3, in particular, increased in the incompatible race-infected leaves. The leaf LOX-3 had a pH optimum of 5.0 and produced preferentially 13-l-hydroperoxy-9,11 (Z,E)-octadecadienoic acid (13-HPODD) from linoleic acid. 13-HPODD and 13-l-hydroxy-9,11 (Z,E)-octadecadienoic acid, one of the reaction products from 13-HPODD by LHDA, were highly inhibitory to the germination of conidia of the fungus. The present study provides correlative evidence for important roles of LOX and LHDA in the resistance response of rice against the blast fungus.     

Use of quantitative PCR to understand within-host competition between two entomopathogenic fungi

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.     

Interactions between a braconid,Microplitis croceipes,and a fungus,Nomuraea rileyi,in laboratory-reared bollworm larvae

The parasite Microplitis croceipes required 1.1 days longer at 26°C to complete development in Heliothis zea larvae than was required for the fungus Nomuraea rileyi to kill the host larvae and sporulate. Host larvae parasitized by M. croceipes or infected with N. rileyi failed to complete a fifth larval molt or pupate. Of the remaining healthy larvae, one-half completed six larval stadia before popation. Larvae parasitized by M. croceipes were predisposed to infection by N. rileyi, but the fungus inhibited development of M. croceipes if host larvae were infected with N. rileyi within 1 day after parasitization.     

Invasive North American bullfrogs transmit lethal fungus <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> infections to native amphibian host species

Invasive species can be a threat to native species in several ways, including transmitting lethal infections caused by the parasites they carry. However, invasive species may also be plagued by novel and lethal infections they acquire when invading, making inferences regarding the ability of an invasive host to vector disease difficult from field observations of infection and disease. This is the case for the pathogenic fungus Batrachochytrium dendrobatidis (Bd) in Europe and one invasive host species, the North American bullfrog Lithobates catesbeianus, hypothesized to be responsible for vectoring lethal infection to European native amphibians. We tested this hypothesis experimentally using the alpine newt Ichthyosaura alpestris as our model native host. Our results show that infected bullfrog tadpoles are effective vectors of Bd. Native adult newts co-housed with experimentally infected bullfrog tadpoles became Bd infected (molecular and histological tests). Moreover, the exposed adult newts suffered mortality while the majority of infected bullfrog tadpoles survived until metamorphosis. These results cannot resolve the historical role of alien species in establishing the distribution of Bd across Europe or other regions in the world where this species was introduced, but they show its potential role as a Bd reservoir capable of transmitting lethal infections to native amphibians. Finally, our results also suggest that the removal of infected bullfrogs from aquatic environments may serve to reduce the availability of Bd in European amphibian communities, offering another justification for bullfrog eradication programmes that are currently underway or may be considered.     

Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.     

Cell Surface Interactions between Bean Leaf Cells and Colletotrichum lindemuthianum: Cytochemical Aspects of Pectin Breakdown and Fungal Endopolygalacturonase Accumulation

After a brief period of biotrophic growth, the anthracnose fungus Colletotrichum lindemuthianum (Sacc. et Mgn.) Bri et Cav. develops extensively in bean leaf cells, causing severe wall alterations and death of the host protoplast. Aplysia gonad lectin, a polygalacturonic acid-binding agglutinin, was complexed to gold and used to study the extent of pectin breakdown during the necrotrophic phase of the infection process. In view of its specific binding properties for the endopolygalacturonase produced by C. lindemuthianum, a polygalacturonase-inhibiting protein isolated from bean cell walls was successfully tagged with gold particles and used for localizing the sites of enzyme accumulation in infected host tissues. The basal level of endopolygalacturonase produced by C. lindemuthianum grown in culture was found to increase severalfold when the fungus developed in host plant tissues. The enzyme was able to diffuse freely in the host cell wall, causing drastic degradation of the pectic material of primary walls and middle lamella matrices. The enzymatic alteration of plant cell walls was accompanied by the release of pectic fragments and by the accumulation of pectic molecules at specific sites, such as intercellular spaces and aggregated cytoplasm of infected host cells. The occurrence of pectic molecules at those sites where fungal growth is likely to be restricted is discussed in relation to their origin and their implication in the plant's defense system.     

Proteins in intercellular washing fluid from noninoculated and rust-affected leaves of wheat and barley

Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19°C, four infection-related CBB-stainable proteins were detected in IWF.

To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25°C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley.

IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust-affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19°C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium.

     

Influenza A Virus Migration and Persistence in North American Wild Birds

Wild birds have been implicated in the emergence of human and livestock influenza. The successful prediction of viral spread and disease emergence, as well as formulation of preparedness plans have been hampered by a critical lack of knowledge of viral movements between different host populations. The patterns of viral spread and subsequent risk posed by wild bird viruses therefore remain unpredictable. Here we analyze genomic data, including 287 newly sequenced avian influenza A virus (AIV) samples isolated over a 34-year period of continuous systematic surveillance of North American migratory birds. We use a Bayesian statistical framework to test hypotheses of viral migration, population structure and patterns of genetic reassortment. Our results reveal that despite the high prevalence of Charadriiformes infected in Delaware Bay this host population does not appear to significantly contribute to the North American AIV diversity sampled in Anseriformes. In contrast, influenza viruses sampled from Anseriformes in Alberta are representative of the AIV diversity circulating in North American Anseriformes. While AIV may be restricted to specific migratory flyways over short time frames, our large-scale analysis showed that the long-term persistence of AIV was independent of bird flyways with migration between populations throughout North America. Analysis of long-term surveillance data provides vital insights to develop appropriately informed predictive models critical for pandemic preparedness and livestock protection.     

Ochropsora staphyleae,a new rust pathogen of Japanese bladdernut,found in central Japan

Japanese bladdernut shrub, Staphylea bumalda (Staphyleaceae), was found infected by a fungus in Prefectures of Ibaraki and Tochigi, central Japan. The fungus produced telia beneath the host epidermis and basidiospores on metabasidia emerging from the sori on the abaxial leaf surface. Probasidia were single-celled, sessile, short-cylindrical, thin-walled, and laterally free. A four-celled metabasidium arose from a probasidium by apical elongation. Apparent biotrophic nature and the basidium morphology of the Staphylea fungus suggested its taxonomic affinity to the rust genus Ochropsora. Molecular phylogenetic analyses of D1/D2 region of LSU rDNA showed that two collections of the fungus were grouped with Ochropsora ariae (type species of the genus) and O. nambuana but formed an independent clade. The fungus under consideration was, therefore, classified in Ochropsora, and a new name, O. staphyleae, was proposed for it.     

First record of the termite ectoparasite Laboulbeniopsis termitarius thaxter in Japan

Laboulbeniopsis termitarius, an ectoparasitic fungus of termites, was harvested from the body surface of Reticulitermes speratus (Isoptera: Rhinotermitidae), collected in Uji, Kyoto Prefecture, Japan. This is the first record of this fungus from Japan. Three to eighteen termite workers from 500 individuals were infected among the eight colonies investigated. From our results, several hundred termite are required for the survey of this ectoparasitic fungi in Japan. The temperature effect on the relationship between the infection rate and the host activity was discussed.     

The Colletotrichum orbiculare ssd1 Mutant Enhances Nicotiana benthamiana Basal Resistance by Activating a Mitogen-Activated Protein Kinase Pathway

Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance.     

Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection

Downy mildew, caused by the oomycete Plasmopara viticola, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.). The class 10 of pathogenesis-related (PR) genes in grapevine leaves was reported to be accumulated at mRNA level in response to P. viticola infection. To elucidate the functional roles of PR10 genes during plant–pathogen interactions, a PR10 gene from a fungal-resistant accession of Chinese wild Vitis pseudoreticulata (designated VpPR10.2) was isolated and showed high homology to PR10.2 from susceptible V. vinifera (designated VvPR10.2). Comparative analysis displayed that there were significant differences in the patterns of gene expression between the PR10 genes from the two host species. VpPR10.2 was induced with high level in leaves infected by P. viticola, while VvPR10.2 showed a low response to this inoculation. Recombinant VpPR10.2 protein showed DNase activity against host genomic DNA and RNase activity against yeast total RNA in vitro. Meanwhile, recombinant VpPR10.2 protein inhibited the growth of tobacco fungus Alternaria alternata and over-expression of VpPR10.2 in susceptible V. vinifera enhanced the host resistance to P. viticola. The results from subcellular localization analysis showed that VpPR10.2 proteins were distributed dynamically inside or outside of host cell. Moreover, they were found in haustorium of P. viticola and nucleus of host cell which was associated with a nucleus collapse at 10 days post-inoculation. Taken together, these results suggested that VpPR10.2 might play an important role in host plant defense against P. viticola infection.     

Variation in phenotype and virulence among enzootic and panzootic amphibian chytrid lineages

The Global Panzootic Lineage of the fungus Batrachochytrium dendrobatidis (Bd-GPL) is threatening amphibians worldwide. In contrast, four lineages (Bd-Brazil, Bd-CH, Bd-Cape, and Bd-Korea) that diverged early in the history of Bd have not yet been directly linked to amphibian declines. Bd likely evolves in response to strong selective pressure imposed by hosts and the environment, leading to differences among pathogen phenotypes and genotypes that may directly affect virulence. Here, we report on variation in phenotype, genotype, and virulence of Bd-Brazil and Bd-GPL. Specifically, we (i) used a controlled infection experiment to compare virulence between one Bd-Brazil and three Bd-GPL isolates on a North American amphibian host (Lithobates sylvaticus), (ii) tested for relative phenotypic and genotypic differentiation among Bd isolates from Brazil, and (iii) tested for possible correlations between environmental variables and Bd phenotypes. We found substantial variation in virulence among Bd-GPL isolates and found that our Bd-Brazil isolate showed virulence comparable to an average North American Bd-GPL. North American hosts infected with a Bd-GPL isolate from Panama did not show significant mortality. Bd phenotypes varied significantly across sampling locations; these phenotypes were neither spatially clustered nor correlated with any environmental variables. Additionally, we found a surprising lack of correlation between genotypic divergence and zoospore and zoosporangium sizes in our sample. Although Bd-Brazil was less virulent infecting L. sylvaticus than one Bd-GPL isolate, this endemic lineage still caused ∼50% mortality in our experimental North American hosts. This indicates that Bd-Brazil has the potential to kill amphibians if introduced to naïve wild populations. Our findings underscore that characterizing virulence of multiple Bd isolates and lineages is important for understanding the evolutionary history and diversity of Bd.