Alterations to unionid transformation during agricultural and urban contaminants of emerging concern exposures.

Highly imperiled unionids have a complex life cycle including the metamorphosis of an obligate parasite life stage, larval glochidia, to the juvenile stage. Despite the known vulnerabilities of both glochidia and juveniles to pollutants, little is known on how metamorphosis success may be affected by chemical stress. Disruption of the transformation process in which glochidia encyst on the gills of a host fish, could lead to lowered recruitment and population declines. Transformation rates of Lampsilis cardium on host fish Micropterus salmoides were empirically derived from experimental exposures to low, medium, or high concentrations of an agricultural or urban mixture of contaminants of emerging concern (CECs) over two exposure durations. Transformation was characterized by: (1) a zero-inflated Poisson general linear mixed effects model to compare difference in transformation between exposure durations and (2) time response curves to describe the transformation curve using long-term exposure data. Lampsilis cardium transformation was similar between exposure durations. When compared to controls, CEC stress significantly reduced juvenile production (p « 0.05) except for the agricultural medium treatment and tended to increased encapsulation duration which while statistically insignificant (p = 0.16) may have ecological relevancy. Combining the empirically derived reduction of transformation rates with parameters values from the literature, a Lefkovich stage-based population model predicted strong declines in population size of L. cardium for all treatments if these results hold in nature. Management focus on urban CECs may lead to best conservation efforts though agricultural CECs may also have a concentration dependent impact on transformation and therefore overall recruitment and conservation success.

Evaluation of the Characteristics and Infectivity of the Secondary Inoculum Produced by Plasmopara viticola on Grapevine Leaves by Means of Flow Cytometry and Fluorescence-Activated Cell Sorting.

Plasmopara viticola, the oomycete causing grapevine downy mildew, is one of the most important pathogens in viticulture. P. viticola is a polycyclic pathogen, able to carry out numerous secondary cycles of infection during a single vegetative grapevine season, by producing asexual spores (zoospores) within sporangia. The extent of these infections is strongly influenced by both the quantity (density) and quality (infectivity) of the inoculum produced by the pathogen. To date, the protocols for evaluating all these characteristics are quite limited and time-consuming and do not allow all the information to be obtained in a single run. In this study, a protocol combining flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) was developed to investigate the composition, the infection efficiency and the dynamics of the inoculum produced by P. viticola for secondary infection cycles. In our analyses, we identified different structures within the inoculum, including degenerated and intact sporangia. The latter have been sorted, and single sporangia were directly inoculated on grapevine leaf discs, thus allowing a thorough investigation of the infection dynamics and efficiency. In detail, we determined that, in our conditions, 8% of sporangia were able to infect the leaves and that on a susceptible variety, the time required by the pathogen to reach 50% of total infection is about 10 days. The analytical approach developed in this study could open a new perspective to shed light on the biology and epidemiology of this important pathogen. IMPORTANCE P. viticola secondary infections contribute significantly to the epidemiology of this important plant pathogen. However, the infection dynamics of asexual spores produced by this organism are still poorly investigated. The main challenges in dissecting the grapevine-P. viticola interaction in vitro are attributable to the biotrophic adaptation of the pathogen. This work provides new insights into the infection efficiency and dynamics imputable to P. viticola sporangia, contributing useful information on grapevine downy mildew epidemiology. Moreover, future applications of the sorting protocol developed in this work could yield a significant and positive impact in the study of P. viticola, providing unmatched resolution, precision, and accuracy compared with the traditional techniques.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on New York Aster (Symphyotrichum novi-belgii) in Korea.

Symphyotrichum novi-belgii (L.) G.L. Nesom (syn. Aster novi-belgii L.), known as New York aster, is a perennial herb used in gardens and as a potted plant. The plant is native to North America but has been developed into various horticultural varieties. In Korea, it is one of the most common plants used for autumn bloom. In September 2011, New York asters (variety unknown) showing typical signs of powdery mildew were observed in a public garden in Seoul, Korea. Since then, the disease on New York asters has been continuously found in parks and flower markets in different regions of Korea. Voucher specimens (n=3) were deposited in the Korea University Herbarium (KUS-F 30752, 31865, and 32103). On leaves, circular to irregular white patches appeared which subsequently showed abundant hyphal growth on both sides of the leaves and on young stems and inflorescences, reducing the aesthetic value and vigor of the plants affected. Hyphae were septate, branched, and 4 to 8 µm wide. Appressoria on the mycelium were nipple-shaped. Conidiophores measured 110 to 200 × 9 to 11.5 µm, were simple, and produced 2 to 5 immature conidia in chains with a sinuate outline, followed by 2 to 3 cells. Foot-cells of conidiophores were straight, cylindric, and 55 to 125 µm long. Conidia were hyaline, ellipsoid to barrel-shaped, measured 22 to 52 × 15 to 20 µm (length/width ratio = 1.5-2.5), lacked distinct fibrosin bodies, and produced germ tubes on the subterminal position, with reticulate wrinkling of the outer walls. No chasmothecia were observed. The structures described above were typical of the Oidium subgenus Euoidium anamorph of the genus Golovinomyces, and the fungus measurements were consistent with those of G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA were amplified with primers PM10/ITS4 for ITS and PM3/TW14 for LSU (Mori et al. 2000, Bradshaw and Tobin 2020). The resulting sequences were deposited in GenBank (Accession No. OP028065-7 for ITS and OP028053-5 for LSU). A GenBank BLAST search of these sequences revealed 100% identity with sequences of G. ambrosiae on many asteraceous plants, including S. novi-belgii from China (MK452575-9 for ITS and MK452648-52 for LSU). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto leaves of five healthy potted New York aster plants. Five non-inoculated plants served as controls. Plants were maintained in an incubator at 24°C. Inoculated plants developed signs and symptoms after three weeks, whereas the control plants remained symptomless. The fungus present on the inoculated plants was morphologically identical to that observed initially on diseased plants, fulfilling Koch's postulates. The powdery mildew infections of S. novi-belgii associated with G. ambrosiae have been widely known in Europe and North America but only recently in China (Qiu et al. 2020, Farr and Rossman 2022). In Japan, Podosphaera fuliginea was known to be associated with powdery mildew of S. novi-belgii (Farr and Rossman 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on S. novi-belgii in Korea. The powdery mildew on this ornamental plant can be considered a severe threat.

Pneumocystis Mating-Type Locus and Sexual Cycle during Infection.

Pneumocystis species colonize mammalian lungs and cause deadly pneumonia if the immune system of the host weakens. Each species presents a specificity for a single mammalian host species. Pneumocystis jirovecii infects humans and provokes pneumonia, which is among the most frequent invasive fungal infections. The lack of in vitro culture methods for these fungi complicates their study. Recently, high-throughput sequencing technologies followed by comparative genomics have allowed a better understanding of the mechanisms involved in the sexuality of Pneumocystis organisms. The structure of their mating-type locus corresponding to a fusion of two loci, Plus and Minus, and the concomitant expression of the three mating-type genes revealed that their mode of sexual reproduction is primarily homothallism. This mode is favored by microbial pathogens and involves a single self-compatible mating type that can enter into the sexual cycle on its own. Pneumocystis sexuality is obligatory within the host's lungs during pneumonia in adults, primary infection in children, and possibly colonization. This sexuality participates in cell proliferation, airborne transmission to new hosts, and probably antigenic variation, processes that are crucial to ensure the survival of the fungus. Thus, sexuality is central in the Pneumocystis life cycle. The obligate biotrophic parasitism with obligate sexuality of Pneumocystis is unique among fungi pathogenic to humans. Pneumocystis organisms are similar to the plant fungal obligate biotrophs that complete their entire life cycle within their hosts, including sex, and that are also difficult to grow in vitro.

RNAi of a Putative Grapevine Susceptibility Gene as a Possible Downy Mildew Control Strategy.

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the diseases causing the most severe economic losses to grapevine (Vitis vinifera) production. To date, the application of fungicides is the most efficient method to control the pathogen and the implementation of novel and sustainable disease control methods is a major challenge. RNA interference (RNAi) represents a novel biotechnological tool with a great potential for controlling fungal pathogens. Recently, a candidate susceptibility gene (VviLBDIf7) to downy mildew has been identified in V. vinifera. In this work, the efficacy of RNAi triggered by exogenous double-stranded RNA (dsRNA) in controlling P. viticola infections has been assessed in a highly susceptible grapevine cultivar (Pinot noir) by knocking down VviLBDIf7 gene. The effects of dsRNA treatment on this target gene were assessed by evaluating gene expression, disease severity, and development of vegetative and reproductive structures of P. viticola in the leaf tissues. Furthermore, the effects of dsRNA treatment on off-target (EF1α, GAPDH, PEPC, and PEPCK) and jasmonic acid metabolism (COI1) genes have been evaluated. Exogenous application of dsRNA led to significant reductions both in VviLBDIf7 gene expression, 5 days after the treatment, and in the disease severity when artificial inoculation was carried out 7 days after dsRNA treatments. The pathogen showed clear alterations to both vegetative (hyphae and haustoria) and reproductive structures (sporangiophores) that resulted in stunted growth and reduced sporulation. Treatment with dsRNA showed signatures of systemic activity and no deleterious off-target effects. These results demonstrated the potential of RNAi for silencing susceptibility factors in grapevine as a sustainable strategy for pathogen control, underlying the possibility to adopt this promising biotechnological tool in disease management strategies.

The Coevolution Effect as a Driver of Spillover.

Global habitat fragmentation is associated with the emergence of infectious diseases of wildlife origins in human populations. Despite this well-accepted narrative, the underlying mechanisms driving this association remain unclear. We introduce a nuanced hypothesis, the 'coevolution effect'. The central concept is that the subdivision of host populations which occurs with habitat fragmentation causes localized coevolution of hosts, obligate parasites, and pathogens which act as 'coevolutionary engines' within each fragment, accelerating pathogen diversification, and increasing pathogen diversity across the landscape. When combined with a mechanism to exit a fragment (e.g., mosquitoes), pathogen variants will spill over into human communities. Through this combined ecoevolutionary approach we may be able to understand the fine-scale mechanisms that drive disease emergence in the Anthropocene.

Expression profiling and functional analyses of BghPTR2, a peptide transporter from Blumeria graminis f. sp. hordei.

The obligate ascomycete parasitic fungus Blumeria graminis f. sp. hordei (Bgh) has a unique lifestyle as it is completely dependent on living barley leaves as substrate for growth. Genes involved in inorganic nitrogen utilization are notably lacking, and the fungus relies on uptake of host-derived peptides and amino acids. The PTR2 transporter family takes up di- and tri- peptides in a proton coupled process and filamentous fungi typically have two or more di/tri peptide transporters. Here we show that Bgh appear to have one PTR2 that can restore dipeptide uptake in a Saccharomyces cerevisiae PTR2 deletion strain. The Bgh PTR2 gene is expressed in conidia and germinating conidia. During Bgh infection of barley the expression level of the BghPTR2 gene is high in the appressorial germ tube, low in the haustoria and high again during conidiation and secondary infection in the compatible and intermediate resistant interactions. BghPTR2 appears to be important for the initial establishment of fungal infection but not for uptake of di-tri-peptides at the haustorial interface. Based on the expression profile we suggest that BghPTR2 is active in internal transport of nutrient reserves and/or uptake of break down products from the plant surface during the early infection stages.

Comparative genomics suggests that the human pathogenic fungus Pneumocystis jirovecii acquired obligate biotrophy through gene loss.

Pneumocystis jirovecii is a fungal parasite that colonizes specifically humans and turns into an opportunistic pathogen in immunodeficient individuals. The fungus is able to reproduce extracellularly in host lungs without eliciting massive cellular death. The molecular mechanisms that govern this process are poorly understood, in part because of the lack of an in vitro culture system for Pneumocystis spp. In this study, we explored the origin and evolution of the putative biotrophy of P. jirovecii through comparative genomics and reconstruction of ancestral gene repertoires. We used the maximum parsimony method and genomes of related fungi of the Taphrinomycotina subphylum. Our results suggest that the last common ancestor of Pneumocystis spp. lost 2,324 genes in relation to the acquisition of obligate biotrophy. These losses may result from neutral drift and affect the biosyntheses of amino acids and thiamine, the assimilation of inorganic nitrogen and sulfur, and the catabolism of purines. In addition, P. jirovecii shows a reduced panel of lytic proteases and has lost the RNA interference machinery, which might contribute to its genome plasticity. Together with other characteristics, that is, a sex life cycle within the host, the absence of massive destruction of host cells, difficult culturing, and the lack of virulence factors, these gene losses constitute a unique combination of characteristics which are hallmarks of both obligate biotrophs and animal parasites. These findings suggest that Pneumocystis spp. should be considered as the first described obligate biotrophs of animals, whose evolution has been marked by gene losses.

Complete genome sequence of Arcanobacterium haemolyticum type strain (11018).

Arcanobacterium haemolyticum (ex MacLean et al. 1946) Collins et al. 1983 is the type species of the genus Arcanobacterium, which belongs to the family Actinomycetaceae. The strain is of interest because it is an obligate parasite of the pharynx of humans and farm animal; occasionally, it causes pharyngeal or skin lesions. It is a Gram-positive, nonmotile and non-sporulating bacterium. The strain described in this study was isolated from infections amongst American soldiers of certain islands of the North and West Pacific. This is the first completed sequence of a member of the genus Arcanobacterium and the ninth type strain genome from the family Actinomycetaceae. The 1,986,154 bp long genome with its 1,821 protein-coding and 64 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.