The soil bacterial community regulates germination of Plasmodiophora brassicae resting spores rather than root exudates.

Clubroot, caused by Plasmodiophora brassicae, is a severe soil-borne disease that restricts the production of cruciferous crops worldwide. A better understanding of biotic and abiotic factors regulating germination of P. brassicae resting spores in the soil is significant for developing novel control methods. Previous studies reported that root exudates can trigger P. brassicae resting spore germination, thus enabling a targeted attack of P. brassicae on host plant roots. However, we found that native root exudates collected under sterile conditions from host or non-host plants cannot stimulate the germination of sterile spores, indicating that root exudates may not be direct stimulation factors. Instead, our studies demonstrate that soil bacteria are essential for triggering germination. Through 16s rRNA amplicon sequencing analysis, we found that certain carbon sources and nitrate can reshape the initial microbial community to an inducing community leading to the germination of P. brassicae resting spores. The stimulating communities significantly differed in composition and abundance of bacterial taxa compared to the non-stimulating ones. Several enriched bacterial taxa in stimulating community were significantly correlated with spore germination rates and may be involved as stimulation factors. Based on our findings, a multi-factorial 'pathobiome' model comprising abiotic and biotic factors is proposed to represent the putative plant-microbiome-pathogen interactions associated with breaking spore dormancy of P. brassicae in soil. This study presents novel views on P. brassicae pathogenicity and lays the foundation for novel sustainable control strategies of clubroot.

Chemotaxis and Motility of Spongospora subterranea Zoospores in Response to Potato Root Exudate Constituents and pH.

Spongospora subterranea f. sp. subterranea is an important pathogen of potato responsible for major losses in most potato growing regions of the world. Infection is initiated by biflagellated motile zoospores released from long-lived resting spores. Zoospore chemotaxis to the host plant root is widely believed to be stimulated by host root exudate compounds, although direct evidence is lacking. This study refined the traditional chemotaxis capillary assay, with which we provided the first empirical evidence of S. subterranea zoospore chemotaxis. Individual potato root exudate metabolites were either taxis neutral, inhibitory, or attractant to the zoospores. L-Glutamine was the strongest chemoattractant, while spermine was the most inhibitory. Zoospore motility and chemotaxis were constrained by strongly acidic or alkaline solutions of pH < 5.3 and >8.5, respectively. Beyond pH, ionic constituents of the test solution affected zoospore motility as Sorensen's phosphate buffer stalled zoospore motility, but HEPES buffer at the same concentration and pH had little or no negative motility effect. Zoospore motility, as characterized by several parameters, influenced chemotaxis. Among the parameters measured, total distance traveled was the best predictor of zoospore chemotaxis. The characterization of environmental and ecological effects on zoospore motility and chemotaxis highlights useful targets for S. subterranea disease control through manipulation of zoospore taxis or selection of host resistance traits.

The polyketide synthase StlA is involved in inducing aggregation in Polysphondylium violaceum.

In the social amoeba Dictyostelium discoideum, the polyketide MPBD (4-methyl-5-pentylbenzene-1,3-diol) regulates the gene expressions of cAMP signaling to make cells aggregation-competent and also induces spore maturation. The polyketide synthase StlA is responsible for MPBD biosynthesis in D. discoideum and appears to be conserved throughout the major groups of the social amoeba (Dictyostelia). In this study, we analyzed the function of StlA in Polysphondylium violaceum by identifying the gene sequence and creating the knockout mutants. We found that Pv-stlA- mutants had defects only in cell aggregation but not in spore maturation, indicating that the function of StlA in inducing spore maturation is species-specific. We also found that MPBD could rescue the aggregation defect in Pv-stlA- mutants whereas the mutants normally exhibited chemotaxis to their chemoattractant, glorin. Our data suggest that StlA is involved in inducing aggregation in P. violaceum by acting on signaling pathways other than chemotaxis in P. violaceum.

Plasmodiophora brassicae in Mexico: From Anecdote to Fact.

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been stated as a fact. However, an intensive search of the scientific literature in English and Spanish, as well as gray literature including theses and government reports, did not reveal any information about the actual detection of the pathogen, affected hosts, or areas with clubroot presence, or any information about clubroot (hernia de la col in Mexico). We followed a multistep process to confirm whether P. brassicae was indeed in Mexico. First, we identified agricultural communities with a history of cruciferous crop cultivation. Second, we asked growers if they had seen clubroot on their crops, using pictures of the characteristic root galls. Third, we collected soil from the locations where clubroot was reported and looked for clubroot/P. brassicae in the soil using several cruciferous bait plants. For the first time we confirm the presence of the clubroot pathogen P. brassicae in Mexico, through a bioassay, the presence of resting spores, and a P. brassicae-specific PCR assay. The identification of P. brassicae in Mexico will contribute to our understanding of the genetic diversity of this elusive and devastating plant pathogen in future studies.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Hard to Kill: Inactivation of Plasmodiophora brassicae Resting Spores Using Chemical Disinfectants.

Biosafety practices, such as bioexclusion via sanitization, can prevent the spread of infectious soilborne threats such as the clubroot pathogen Plasmodiophora brassicae. Twenty-three chemical disinfectants were evaluated for efficacy against P. brassicae resting spores. Evans blue staining was used to directly measure the viability of P. brassicae resting spores after 20-min exposures to 10 concentrations of each of the 23 chemical disinfectants. Only nine disinfectants were capable of >95% inactivation, and only five were capable of inactivating >99% of resting spores. Bleach (sodium hypochlorite) and Spray Nine were the most effective disinfectants for inactivation of clubroot resting spores. AES 2500, SaniDate, and ethanol also inactivated >99% of resting spores but only at very high concentrations. A time course experiment showed that 10- to 12-min contact time was sufficient for ≥95% resting spore inactivation with Spray Nine and sodium hypochlorite, but ≥30-min contact was required for other disinfectants evaluated. These results will assist in guiding management recommendations for sanitization aimed at bioexclusion and biocontainment of P. brassicae.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

Transport and Retention of Free-Living Amoeba Spores in Porous Media: Effects of Operational Parameters and Extracellular Polymeric Substances.

Amoebas are protists that are widespread in water and soil environments. Some species are pathogenic, inducing potentially lethal effects on humans, making them a major threat to public health. Nonpathogenic amoebas are also of concern because they have the potential to carry a mini-microbiome of bacteria, either transiently or via more long-term stable transport. Due to their resistance to disinfection processes, the physical removal of amoeba by filtration is necessary to prevent their propagation throughout drinking water distribution networks and occurrence in tap water. In this study, a model amoeba species Dictyostelium discoideum was used to study the transport and retention behavior of amoeba spores in porous media. The key factors affecting the transport behavior of amoeba spores in fully saturated media were comprehensively evaluated, with experiments performed using a quartz crystal microbalance with dissipation monitoring (QCM-D) and parallel plate chamber system. The effects of ionic strength (IS) on the deposition of spores were found to be in contrast to the predicted Derjaguin-Landau-Verwey-Overbeek (DLVO) theory that more deposition is observed under lower-IS conditions. The presence of extracellular polymeric substances (EPS) was found to be the main contributor to deposition behavior. Overall, these results provide plausible evidence for the presence of amoeba in tap water. Furthermore, this is one of the first studies to examine the mechanisms affecting the fate of amoeba spores in porous media, providing a significant baseline for future research to minimize the safety risk presented by amoeba in drinking water systems.

Strategic investment explains patterns of cooperation and cheating in a microbe.

Contributing to cooperation is typically costly, while its rewards are often available to all members of a social group. So why should individuals be willing to pay these costs, especially if they could cheat by exploiting the investments of others? Kin selection theory broadly predicts that individuals should invest more into cooperation if their relatedness to group members is high (assuming they can discriminate kin from nonkin). To better understand how relatedness affects cooperation, we derived the ?Collective Investment" game, which provides quantitative predictions for patterns of strategic investment depending on the level of relatedness. We then tested these predictions by experimentally manipulating relatedness (genotype frequencies) in mixed cooperative aggregations of the social amoeba Dictyostelium discoideum, which builds a stalk to facilitate spore dispersal. Measurements of stalk investment by natural strains correspond to the predicted patterns of relatedness-dependent strategic investment, wherein investment by a strain increases with its relatedness to the group. Furthermore, if overall group relatedness is relatively low (i.e., no strain is at high frequency in a group) strains face a scenario akin to the "Prisoner's Dilemma" and suffer from insufficient collective investment. We find that strains employ relatedness-dependent segregation to avoid these pernicious conditions. These findings demonstrate that simple organisms like D. discoideum are not restricted to being ?cheaters" or ?cooperators" but instead measure their relatedness to their group and strategically modulate their investment into cooperation accordingly. Consequently, all individuals will sometimes appear to cooperate and sometimes cheat due to the dynamics of strategic investing.

Sporulation rate and viability of Eimeria tenella oocysts stored in potassium sorbate solution.

In order to find a new preservation solution for avian coccidial oocysts that can replace potassium dichromate (K2Cr2O7) solution, Eimeria tenella oocysts were preserved in 0.1 to 10% potassium sorbate (C6H7KO2) solution in this study. The results showed that there was no significant difference between the sporulation rate of E. tenella oocysts preserved in 0.1 to 10% C6H7KO2 solution and in 2.5% K2Cr2O7 solution (p > 0.05). The 0.5 to 10% C6H7KO2 solution could also effectively inhibit the growth of bacterial microorganisms. E. tenella oocysts preserved in 1% C6H7KO2 solution at 4 °C for 3, 6, 9, and 12 months, with the oocyst production of E. tenella oocysts being 1.3-, 1.2-, 1.6-, and 1.3-fold higher than that of oocysts stored in 2.5% K2Cr2O7 solution (p < 0.05). In conclusion, C6H7KO2 could replace K2Cr2O7 as the preservation solution of avian coccidial oocysts.

An Improved Technique for Isolation and Characterization of Single-Spore Isolates of Plasmodiophora brassicae.

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease that occurs in cruciferous crops worldwide. P. brassicae usually exists as a mixture of several pathotypes, which has hampered the research on resistance mechanisms of cruciferous crops against P. brassicae. In this study, clubroot galls were collected from a field in Shenyang, China, as a pathogen source to develop an efficient protocol for a single-spore isolation system of P. brassicae by optimizing the seedling age for inoculation, host inoculation method, and plant culture method. The operational steps of the single-spore isolation method were optimized as follows: the use of 2-day-old seedlings for inoculation, substituting a cryobox (100 × 2.0-ml vials) for culture dishes, the addition of nutrient solution culture, and microscopic observations of single spores. The rate of infection success was substantially improved, and single-spore isolates of four pathotypes (4, 8, 9, and 11) were acquired in this system. Subsequently, the optimized system was used to isolate and characterize the pathotypes of single-spore isolates of P. brassicae collected from five fields in regions in China. Approximately four to nine pathotypes were isolated from each region. Among these, pathotype 4 was the most prevalent. This study provides a source of valuable information that can eventually be used for the genetic analysis of host-P. brassicae interaction.

Spatiotemporal Quantification of Plasmodiophora brassicae Inoculum in Relation to Clubroot Development Under Inoculated and Naturally Infested Field Conditions.

Clubroot caused by Plasmodiophora brassicae is a destructive disease of cruciferous plants worldwide. A quantitative PCR (qPCR) system specific to P. brassicae was developed. Analysis of the qPCR sensitivity indicated that the lower limit of detection was 1 × 101 resting spores/ml, 1 × 102 spores/g of soil, and 1 × 103 spores/g of roots and seeds. The regression curves generated from the qPCR data of different samples had a parallel relationship. The difference between the theoretical and actual concentrations was lowest at 1 × 105 spores/g of sample, compared with other concentrations. The P. brassicae biomass in soil and plant root tissues after inoculated with different spore concentrations was correlated. A correlation analysis confirmed that the clubroot incidence and disease index at 6 weeks after inoculation increased as the spore concentration increased. Under field conditions, the natural inoculum density of the P. brassicae population decreased at the early stage and then increased, with P. brassicae mainly being detected at a soil depth of 0 to 50 cm. The horizontal distribution of P. brassicae varied in the field with occurrences of hot spots. This study established a qPCR-based method for quantitative detection of clubroot. The developed assay is useful for monitoring the spatiotemporal dynamics of P. brassicae in the field. It may also be applicable for clubroot forecasting as a part of proactive disease management.

Virulence Spectrum of Single-Spore and Field Isolates of Plasmodiophora brassicae Able to Overcome Resistance in Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important disease of canola (Brassica napus L.) that is managed mainly by planting clubroot-resistant (CR) cultivars. Field isolates of P. brassicae can be heterogeneous mixtures of various pathotypes, making assessments of the genetics of host-pathogen interactions challenging. Thirty-four single-spore isolates were obtained from nine field isolates of the pathogen collected from CR canola cultivars. The virulence patterns of the single-spore and field isolates were assessed on the 13 host genotypes of the Canadian Clubroot Differential (CCD) set, which includes the differentials of Williams and Somé et al. Indices of disease (IDs) severity of 25, 33, and 50% (±95% confidence interval) were compared as potential thresholds to distinguish between resistant and susceptible reactions, with an ID of 50% giving the most consistent responses for pathotype classification purposes. With this threshold, 13 pathotypes could be distinguished based on the CCD system, 7 on the differentials of Williams, and 3 on the hosts of Somé et al. The highest correlations were observed among virulence matrices generated using the three threshold IDs on the CCD set. Genetically homogeneous single-spore isolates gave a clearer profile of the P. brassicae pathotype structure. Novel pathotypes, not reported in Canada previously, were identified among the isolates. This large collection of single-spore isolates can serve as a reference in screening and breeding for clubroot resistance.

Scanning electron microscopic observation of the in vitro cultured protozoan, Perkinsus olseni, isolated from the Manila clam, Ruditapes philippinarum.

BACKGROUND: Perkinsosis is a major disease affecting the commercially important marine mollusk Ruditapes philippinarum (Manila clam) in Asian waters. In this study, we investigated the morphological characteristics of Perkinsus olseni, the causative agent of perkinsosis, cultured under laboratory conditions at different stages of its life cycle using a scanning electron microscope (SEM). RESULTS: The prezoosporangia formed after induction with Ray's fluid thioglycollate medium (RFTM) developed into zoosporangia. During this process, a discharge tube formed a porous sponge-like structure that detached before the zoospores were released; thus, this organelle operated as a bung. Liberated zoospores gradually transformed into immature trophozoites, during which detachment of the anterior flagella occurred, but the loss of the posterior flagella was not clearly observed in the present study. Mature trophozoites underwent schizogony by cleaving the cell forming some merozoites in schizonts, which were released by the rupturing of the cellular membrane of the schizont within a few days. CONCLUSIONS: Our morphological and ultrastructural studies contribute new information on the life cycle and propagation of P. olseni.

Expression and Identification of a Novel Spore Wall Protein in Microsporidian Nosema bombycis.

Microsporidia are a group of obligate intracellular parasites causing significant disease in human beings and economically important animals. Though a few spore wall proteins (SWPs) have now been identified in these intriguing species, the information on SWPs remains too little to elucidate the spore wall formation mechanisms of microsporidia. It has been well described that numerous proteins with tandem repeats tend to be localized on the cell wall of fungi and parasites. Previously, by scanning the proteins with tandem repeats in microsporidian Nosema bombycis, we obtained 83 candidate SWPs based on whether those proteins possess a signal peptide and/or transmembrane domain. Here, we further characterized a candidate protein (EOB13250) with three tandem repeats in the N-terminal region and a transmembrane domain in C-terminus of N. bombycis. Sequence analysis showed that the tandem repeat domain of EOB13250 was species-specific for this parasite. RT-PCR indicated that the expression of the gene encoding this protein started on the fourth day postinfection. After cloned and expressed in Escherichia coli, a polyclone antibody against the recombinant EOB13250 protein was prepared. Western blotting demonstrated this protein exist in N. bombycis. Immunofluorescence analysis (IFA) and immunoelectron microscopy analysis (IEM) further provided evidence that EOB13250 was an endospore wall protein. These results together suggested that EOB13250 was a novel spore wall protein of N. bombycis. This study provides a further enrichment of the number of identified spore wall proteins in microsporidia and advances our understanding of the spore wall formation mechanism in these obligate unicellular parasites.

Phylogeny of Physarida (Amoebozoa, Myxogastria) Based on the Small-Subunit Ribosomal RNA Gene, Redefinition of Physarum pusillum s. str. and Reinstatement of P. gravidum Morgan.

Myxomycetes (also called Myxogastria or colloquially, slime molds) are worldwide occurring soil amoeboflagellates. Among Amoebozoa, they have the notable characteristic to form, during their life cycle, macroscopic fruiting bodies, that will ultimately release spores. Some 1,000 species have been described, based on the macroscopic and microscopic characteristics of their fruiting bodies. We were interested in Physarum pusillum (Berk. & M.A. Curtis) G. Lister, a very common species described with two variants, each bearing such morphological differences that they could represent two distinct species. In order to test this, we observed key characters in a large selection of specimens attributed to P.  pusillum, to its synonyms (in particular Physarum gravidum), and to related species. In addition, the small-subunit ribosomal RNA gene was obtained from seven of these specimens. Based on these data, we provide a comprehensive phylogeny of the order Physarida (Eukaryota: Amoebozoa: Conosa: Macromycetozoa: Fuscisporidia). Morphology and phylogeny together support the reinstatement of P. gravidum Morgan 1896 with a neotype here designated, distinct from P. pusillum, here redefined.

Morphological and molecular characterization of Plasmodium cathemerium (lineage PADOM02) from the sparrow Passer domesticus with complete sporogony in Culex pipiens complex.

Avian malaria is a mosquito-borne disease caused by Plasmodium spp. protozoa. Although these parasites have been extensively studied in North America and Eurasia, knowledge on the diversity of Plasmodium, its vectors and avian hosts in Africa is scarce. In this study, we report on natural malarial infections in free-ranging sparrows (Passer domesticus) sampled at Giza Governorate, Egypt. Parasites were morphologically characterized as Plasmodium cathemerium based on the examination of thin blood smears from the avian host. Sequencing a fragment of the mitochondrial cytochrome b gene showed that the parasite corresponded to lineage PADOM02. Phylogenetic analysis showed that this parasite is closely related to the lineages SERAU01 and PADOM09, both of which are attributed to P. cathemerium. Experimental infection of Culex pipiens complex was successful, with ookinetes first detected at 1-day post infection (dpi), oocysts at 4 dpi and sporozoites at 6 dpi. The massive infection of the salivary glands by sporozoites corroborates that Cx. pipiens complex is a competent vector of PADOM02. Our findings confirm that Plasmodium lineage PADOM02 infects sparrows in urban areas along the Nile River, Egypt, and corroborate that Cx. pipiens complex is a highly competent vector for these parasites. Furthermore, our results demonstrate that this lineage corresponds to the morphospecies P. cathemerium and not P. relictum as previously believed.

Refining the Life Cycle of Plasmodiophora brassicae.

As a soilborne protist pathogen, Plasmodiophora brassicae causes the devastating clubroot disease on Brassicaeae crops worldwide. Due to its intracellular obligate biotrophic nature, the life cycle of P. brassicae is still not fully understood. Here, we used fluorescent probe-based confocal microscopy and transmission electron microscopy (TEM) to investigate the infection process of P. brassicae on the susceptible host Arabidopsis under controlled conditions. We found that P. brassicae can initiate the primary infection in both root hairs and epidermal cells, producing the uninucleate primary plasmodium at 1 day postinoculation (dpi). After that, the developed multinucleate primary plasmodium underwent condensing and cytoplasm cleavage into uninucleate zoosporangia from 1 to 4 dpi. This was subsequently followed by the formation of multinucleate zoosporangia and the production of secondary zoospores within zoosporangium. Importantly, the secondary zoospores performed a conjugation in the root epidermal cells after their release. TEM revealed extensive uninucleate secondary plasmodium in cortical cells at 8 dpi, indicating the establishment of the secondary infection. The P. brassicae subsequently developed into binucleate, quadrinucleate, and multinucleate secondary plasmodia from 10 to 15 dpi, during which the clubroot symptoms appeared. The uninucleate resting spores were first observed in the cortical cells at 24 dpi, marking the completion of a life cycle. We also provided evidence that the secondary infection of P. brassicae may represent the diploid sexual life stage. From these findings, we propose a refined life cycle of P. brassicae which will contribute to understanding of the complicated infection biology of P. brassicae.

In vitro and in vivo activity of cinnamaldehyde against Eimeria kofoidi in chukar partridge (Alectoris chukar).

One hundred and twenty one-day-old chukar partridges were randomly divided into eight groups which received diets with different supplementations. There were four unchallenged groups. One group received salinomycin (50 ppm), two groups received cinnamaldehyde (CINN) (100 and 200 mg/kg of diet), and another one received only the basal diet from the 1st to the 31st day. There were also four corresponding groups orally challenged by 3 × 105Eimeria kofoidi sporulated oocysts at the 21st day. Three samplings were done at the 24th, 26th, and 31st days of rearing for pathological and biochemical assessments. Fecal samples were daily taken to check the pattern of oocyst shedding from the 26th to 31st day. The body weight of birds was measured at 21st and 31st days. Along with the in vivo experiment, an in vitro sporulation inhibition test was carried out. The in vitro results showed that CINN decreased sporulation rate at 1 and 0.5 mg/ml. In vivo, it was found that CINN did not prevent the oocyst shedding. Furthermore, the histopathological findings revealed that CINN and salinomycin had no effect on infection establishment. However, our findings showed that CINN (200 mg/kg of diet) could enhance the body weight and improve antioxidant status. Although our results did not support the in vivo anticoccidial activity of CINN, it had a promising potential to improve antioxidant status and body weight in the chukar partridge.

Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer.

Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca- structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP-induced cAMP synthesis as well as c-di-GMP-induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca- mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

Myxobolus spp. (Cnidaria: Myxozoa) in introduced yellow perch Perca flavescens (Mitchill, 1814).

We surveyed introduced yellow perch Perca flavescens (Mitchill, 1814) from the Willamette River, OR, USA, to determine if these fish have co-introduced myxosporean parasites. Mature parasite myxospores were observed in brains of 3/19 fish, and were morphologically and molecularly consistent with Myxobolus neurophilus (Guilford 1963), a parasite known from yellow perch in their native range. We identified another Myxobolus species from the gill filaments of 1/22 fish. The spores from the gill filaments were oval-shaped, 11.7 (10.7-12.3) µm long × 8.6 (7.7-9.0) µm wide × 5.2 (4.6-5.6) µm thick, with two oval-shaped polar capsules 5.7 (5.1-6.5) µm × 2.7 (2.4-3.2) µm, each containing a polar tubule with 8-9 turns. Small-subunit ribosomal DNA sequences from each of four plasmodia were identical, and 4.0% different (over 1800 nucleotides) from the closest known myxosporeans. Interestingly, these sequences had overlapping peaks in their chromatograms, which suggested that DNA from multiple species was present. Hence, we isolated and sequenced three individual myxospores and found that they too had mixed chromatograms, which indicated presence of at least two sequence types of small-subunit ribosomal DNA in each spore (GenBank accession MK592012, MK592013), a rare character among described myxosporeans. The spore morphology, morphometry, tissue tropism, and DNA sequence supported a diagnosis of a novel species, Myxobolus doubleae n. sp. This parasite is unknown from yellow perch in its native range, despite extensive historical surveys, which suggests that introduced yellow perch might have acquired an endemic Myxobolus species via spillback from another fish host.