The greenbeard gene tgrB1 regulates altruism and cheating in Dictyostelium discoideum.

Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards have been described in various organisms but direct evidence for all the properties in one system is scarce. The tgrB1-tgrC1 allorecognition system of Dictyostelium discoideum encodes two polymorphic membrane proteins which protect cells from chimerism-associated perils. During development, TgrC1 functions as a ligand-signal and TgrB1 as its receptor, but evidence for altruism has been indirect. Here, we show that mixing wild-type and activated tgrB1 cells increases wild-type spore production and relegates the mutants to the altruistic stalk, whereas mixing wild-type and tgrB1-null cells increases mutant spore production and wild-type stalk production. The tgrB1-null cells cheat only on partners that carry the same tgrC1-allotype. Therefore, TgrB1 activation confers altruism whereas TgrB1 inactivation causes allotype-specific cheating, supporting the greenbeard concept and providing insight into the relationship between allorecognition, altruism, and exploitation.

In the social amoeba Dictyostelium discoideum, shortened stalks may limit obligate cheater success even when exploitable partners are available.

Cooperation is widespread across life, but its existence can be threatened by exploitation. The rise of obligate social cheaters that are incapable of contributing to a necessary cooperative function can lead to the loss of that function. In the social amoeba Dictyostelium discoideum, obligate social cheaters cannot form dead stalk cells and in chimeras instead form living spore cells. This gives them a competitive advantage within chimeras. However, obligate cheaters of this kind have thus far not been found in nature, probably because they are often enough in clonal populations that they need to retain the ability to produce stalks. In this study we discovered an additional cost to obligate cheaters. Even when there are wild-type cells to parasitize, the chimeric fruiting bodies that result have shorter stalks and these are disadvantaged in spore dispersal. The inability of obligate cheaters to form fruiting bodies when they are on their own combined with the lower functionality of fruiting bodies when they are not represent limits on obligate social cheating as a strategy.

Didymium arenosum, a myxomycete new to science from the confluence of deserts in northwestern China.

A new myxomycete species, Didymium arenosum, was described based on morphological evidence and phylogenetic analyses. The species was discovered in the arid region at the confluence of the Badain Jaran desert and Tengger desert on the leaves of Betula platyphylla and was cultivated in a moist chamber culture. Morphologically, the species is distinguished by the greenish-yellow calcium carbonate crystals on the surface and the spores covered with small warts, some of which are connected into a short line. A phylogenetic analysis of D. arenosum strongly supports its classification as a separate clade. The spore to spore agar culture of D. arenosum requires 23 days, and this study provides a detailed description of its life cycle.

Tasmaniomyxa umbilicata, a new genus and new species of myxomycete from Tasmania.

A new genus and species of myxomycete, Tasmaniomyxa umbilicata, is described based on numerous observations in Tasmania and additional records from southeastern Australia and New Zealand. The new taxon is characterized by an unusual combination of characters from two families: Lamprodermataceae and Didymiaceae. With Lamprodermataceae the species shares limeless sporocarps, a shining membranous peridium, an epihypothallic stalk, and a cylindrical columella. Like Didymiaceae, it has a soft, flaccid, sparsely branched capillitium, with rough tubular threads that contain fusiform nodes and are firmly connected to the peridium. Other characters of T. umbilicata that also occur in many Didymiaceae are the peridium dehiscing into petaloid lobes, the yellow, motile plasmodium, and the spores ornamented with larger, grouped and smaller, scattered warts. The transitional position of the new taxon is reflected by a three-gene phylogeny, which places T. umbilicata at the base of the branch of all lime-containing Physarales, thus justifying its description as a monotypic genus.

Efficient inactivation of amoeba spores and their intraspore bacteria by solar/chlorine: Kinetics and mechanisms.

Amoebae are widespread in water and serve as environment vectors for pathogens, which may threaten public health. This study evaluated the inactivation of amoeba spores and their intraspore bacteria by solar/chlorine. Dictyostelium discoideum and Burkholderia agricolaris B1qs70 were selected as model amoebae and intraspore bacteria, respectively. Compared to solar irradiation and chlorine, solar/chlorine enhanced the inactivation of amoeba spores and intraspore bacteria, with 5.1 and 5.2-log reduction at 20 min, respectively. The enhancement was similar in real drinking water by solar/chlorine under natural sunlight. However, the spore inactivation decreased to 2.97-log by 20 min solar/chlorine under oxygen-free condition, indicating that ozone played a crucial role in the spore inactivation, as also confirmed by the scavenging test using tert­butanol to scavenge the ground-state atomic oxygen (O(3P)) as a ozone precursor. Moreover, solar/chlorine induced the shape destruction and structural collapse of amoeba spores by scanning electron microscopy. As for intraspore bacteria, their inactivation was likely ascribed to endogenous reactive oxygen species. As pH increased from 5.0 to 9.0, the inactivation of amoeba spores decreased, whereas that of intraspore bacteria was similar at pH 5.0 and 6.5 during solar/chlorine treatment. This study first reports the efficient inactivation of amoeba spores and their intraspore pathogenic bacteria by solar/chlorine in drinking water.

Fifteen new species from the myxomycete genus Lycogala.

Based on a study of 255 collections from four continents and four floristic kingdoms, we describe 15 new species of the genus Lycogala. The new species, all morphologically close to L. epidendrum, L. exiguum, and L. confusum, differ from each other by the structure of the peridium and, in some cases, also by the color of the fresh spore mass and the ornamentation of the capillitium and spores. Species delimitation is confirmed by two independently inherited molecular markers, as well as previously performed tests of reproductive isolation and genetic distances. We studied authentic material of L. exiguum and L. confusum and found fresh specimens of these species, which allowed us to obtain molecular barcodes and substantiate the separation of new species from these taxa. We propose to retain the name L. epidendrum for the globally most abundant species, for which we provide a more precise description and a neotypification. Two formerly described species, L. leiosporum and L. fuscoviolaceum, we consider to be dubious. We do not recognize the species L. terrestre.

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.

Vegetation and climate change at the southern margin of the Neo-Tethys during the Cenomanian (Late Cretaceous): Evidence from Egypt.

Changes in terrestrial vegetation during the mid-Cretaceous and their link to climate and environmental change are poorly understood. In this study, we use plant macrofossils and analysis of fossil pollen and spores from the Western Desert, Egypt, to assess temporal changes in plant communities during the Cenomanian. The investigated strata have relatively diverse sporomorph assemblages, which reflect the nature of parent vegetation. Specifically, the palynofloras represent ferns, conifers, monosulcate pollen producers, Gnetales, and a diverse group of angiosperms. Comparisons of both, dispersed palynoflora and plant macrofossils reveal different characteristics of the palaeoflora owing to a plethora of taphonomical and ecological biases including the depositional environment, production levels, and discrepancies between different plant organs. A combination of detailed records of sporomorphs, leaves, and charcoal from the studied successions provide new understandings of the palaeoclimate and palaeogeography during the Cenomanian and Albian-Cenomanian transition in Egypt. The mixed composition of the palynofloral assemblages reflects the presence of different depositional situations with a weak marine influence, as evidenced by a minor dinoflagellate cysts component. The local vegetation comprised various categories including herbaceous groups including ferns and eudicots, fluvial, open environments, and xeric arboreal communities dominated by Cheirolepidiaceae and perhaps including drought- and/or salt-tolerating ferns (Anemiaceae) and other gymnosperms (Araucariaceae, Ginkgoales, Cycadales, and Gnetales) as well as angiosperms. The presence of riparian and freshwater wetland communities favouring aquatic and/or hygrophilous ferns (of Salviniaceae and Marsileaceae), is noted. The wide variation of depositional settings derived from the palynological data may be attributed to a prevalent occurrence of producers in local vegetation during the early Cenomanian of Egypt. For the purpose of this work on the studied Bahariya Formation and its equivalent rock units, where iconic dinosaurs and other fossil fauna roamed, we attempt to improve the understanding of Egypt's Cenomanian climate, which is reconstructed as generally warm and humid punctuated by phases of considerably drier conditions of varying duration.

High Environmentally Induced Plasticity in Spore Size and Numbers of Nuclei per Spore in Physarum albescens (Myxomycetes).

Spore size enables dispersal in plasmodial slime molds (Myxomycetes) and is an important taxonomic character. We recorded size and the number of nuclei per spore for 39 specimens (colonies of 50-1000 sporocarps) of the nivicolous myxomycete Physarum albescens, a morphologically defined taxon with several biological species. For each colony, three sporocarps were analyzed from the same spore mount under brightfield and DAPI-fluorescence, recording ca. 14,000 spores per item. Diagrams for spore size distribution showed narrow peaks of mostly uninucleate spores. Size was highly variable within morphospecies (10.6-13.5 µm, 11-13%), biospecies (3-13%), even within spatially separated colonies of one clone (ca. 8%); but fairly constant for a colony (mean variation 0.4 µm, ca. 1.5%). ANOVA explains most of this variation by the factor locality (within all colonies: 32.7%; within a region: 21.4%), less by biospecies (13.5%), whereas the contribution of intra-colony variation was negligible (<0.1%). Two rare aberrations occur: 1) multinucleate spores and 2) oversized spores with a double or triple volume of normal spores. Both are not related to each other or limited to certain biospecies. Spore size shows high phenotypic plasticity, but the low variation within a colony points to a strong genetic background.

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.

Intraspecific morphological variation in myxosporeans: high pleomorphic myxospores in the same plasmodium of Myxobolus drjagini (Akhmerov, 1954).

The taxonomy of myxosporeans was traditionally dependent solely upon the spore morphological and morphometric data. Intensive reports of intraspecific morphological variation, however, are increasingly challenging the taxonomic approaches for myxosporeans. In the present work, the morphological pleomorphism of myxospores of Myxobolus drjagini (Akhmerov, 1954) was observed. More interestingly, all of these pleomorphic myxospores occurred in the same plasmodium of M. drjagini, which refutes the previous hypothesis that morphological variation of M. drjagini was derived from its responses to differences in nutrition and immunological responses associated with different host tissues. Bearing the intraspecific morphometric and morphotype variation in mind, the combination of morphological, ecological and molecular data should be applied to the species identification and delimitation for myxosporeans. This is the first reported myxobolid species with high pleomorphic myxospores which are present in the same plasmodium.

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.

Morphological and Phylogenetic Features of Ceratomyxa macapaensis n. sp. (Myxozoa: Ceratomyxidae) in Mesonauta festivus Heckel, 1840 (Cichliformes: Cichlidae) from the Eastern Amazon Region.

PURPOSE: The class Myxozoa consists of microscopic spores that typically present uniform morphological simplifications, containing one or two sporoplasms surrounded by valve cells. The present study analysed the morphological and phylogenetic characteristics of what proved to be a new species of Ceratomyxa found in the gallbladder of Mesonauta festivus Heckel, 1840 captured on the Piririm River in the municipality of Macapá, in Amapá state, Brazil. METHODS: The fish were collected in gillnets, and were transported alive to Amapá State University in Macapá for the analysis of the organs. Crescent- or arch-shaped free spores were observed under light microscopy, containing two polar capsules and a suture line, which is typical of the morphology of the genus Ceratomyxa, contained within plasmodia that had snake-like movements. The gallbladder was conserved in 80% alcohol for the analysis of the 18S rDNA gene, and in Davidson solution for standardhistology. RESULTS: Ceratomyxa spores (n = 20) were observed in 11 of the 50 samplesanalysed, and thus had a prevalence of 22%, and had a mean length of 4.2 ± 0.5 µm, mean thickness of 22.75 ± 0.3 µm, and two polar capsules, 1.86 ± 0.3 µm long and 1.63 ± 0.1 µm thick. CONCLUSIONS: The phylogenetic analysis indicated that the new species forms a cluster with other freshwater Amazonian Ceratomyxa species, described previously. The new species represents the first description of a taxon of the genus Ceratomyxain fish from the Brazilian state of Amapá.

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.

Efficient inactivation of intracellular bacteria in dormant amoeba spores by FeP.

Waterborne pathogens and related diseases are a severe public health threat worldwide. Recent studies suggest that microbial interactions among infectious agents can significantly disrupt the disinfection processes, and current disinfection methods cannot inactivate intracellular pathogens effectively, posing an emerging threat to the safety of drinking water. This study developed a novel strategy, the FeP/persulfate (PS) system, to effectively inactivate intracellular bacteria within the amoeba spore. We found that the sulfate radical (SO4•-) produced by the FeP/PS system can be quickly converted into hydroxyl radicals (•OH), and •OH can penetrate the amoeba spores and inactivate the bacteria hidden inside amoeba spores. Therefore, this study proposes a novel technique to overcome the protective effects of microbial interactions and provides a new direction to inactivate intracellular pathogens efficiently.

Spore ultrastructural features and significance of their diverse ornamental elements in the evolutionary history of the order Trichiales (Myxomycetes, Amoebozoa).

Spores are the dispersal and reproductive units in Myxomycetes, and their ornamentation, usually at the light microscopy resolution limit, is taxonomically meaningful. Here, we analyze with scanning and transmission electron microscopy the spore ultrastructural features in Trichiales, one of the most morphologically diverse orders. In Trichiales, the spore wall consists of two layers, an outer one, including the ornamentation, and an inner layer subdivided into two sections. The diversity of ornamental elements includes verrucae, bacula, pila, muri, and cristae, the two latter creating reticulate patterns. Each of these elements defines a broadly recognized ornamentation type except cristae, from which we differentiate for the first time the cristate reticulate and patched subtypes. Besides, our results point out a previous incorrect classification of the spores of the species Trichia decipiens and T. scabra. Advances in Trichiales phylogeny showed that the capillitium ornamental elements, such as the spirals, do not reflect phylogenetic relationships among the species. The same seems to apply to some of spore ornamentation types, e.g., baculate. Consequently, we may consider the need to combine multiple characters, like the spore and capillitium ornamentation, so that Trichiales systematics better reflect the still cryptic phylogenetic affinities.

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.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.