Decoding the Arsenal: Protist Effectors and Their Impact on Photosynthetic Hosts.

Interactions between various microbial pathogens including viruses, bacteria, fungi, oomycetes and their plant hosts have traditionally been the focus of phytopathology. In recent years, a significant and growing interest in the study of eukaryotic microorganisms not classified among fungi or oomycetes has emerged. Many of these protists establish complex interactions with photosynthetic hosts, and understanding these interactions is crucial in understanding the dynamics of these parasites within traditional and emerging types of farming, including marine aquaculture. Many phytopathogenic protists are biotrophs with complex polyphasic life cycles, which makes them difficult or impossible to culture, a fact reflected in a wide gap in the availability of comprehensive genomic data when compared to fungal and oomycete plant pathogens. Furthermore, our ability to use available genomic resources for these protists is limited by the broad taxonomic distance that these organisms span, that makes comparisons with other genomic datasets difficult. The current rapid progress in genomics and computational tools for the prediction of protein functions and interactions is revolutionising the landscape in plant pathology. This is also opening novel possibilities, specifically for a deeper understanding of protist effectors. Tools such as Alphafold enable better and more targeted functional predictions of divergent protein sequences. In turn this allows to ask better biological questions and, coupled with innovative experimental strategies, will lead into a new era of effector research, especially for protists, to expand our knowledge on these elusive pathogens and their interactions with photosynthetic hosts.

Co-culture Wood Block Decay Test with Bacteria and Wood Rotting Fungi to Analyse Synergism/Antagonism during Wood Degradation.

Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany). Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments. Key features • Quantification of wood decay ability of mixed cultures. • Allows testing if fungi are more efficient in degrading wood when bacteria are present.

Prokaryote communities associated with different types of tissue formed and substrates inhabited by Serpula lacrymans.

The basidiomycete Serpula lacrymans is responsible for major timber devastation in houses. Basidiomycetes are known to harbour a diverse but poorly understood microbial community of bacteria, archaea, yeasts and filamentous fungi. In this study, we used amplicon-sequencing to analyse the abundance and composition of prokaryotic communities associated with fruiting bodies of S. lacrymans and compared them to communities of surrounding material to access the 'background' community structure. Our findings indicate that bacterial genera cluster depended on sample type and that the main driver for microbial diversity is specimen, followed by sample origin. The most abundant bacterial phylum identified in the fruiting bodies was Pseudomonadota, followed by Actinomycetota and Bacteroidota. The prokaryote community of the mycelium was dominated by Actinomycetota, Halobacterota and Pseudomonadota. Actinomycetota was the most abundant phylum in both environment samples (infested timber and underground scree), followed by Bacillota in wood and Pseudomonadota in underground samples. Nocardioides, Pseudomonas, Pseudonochardia, Streptomyces and Rubrobacter spp. were among others found to comprise the core microbiome of S. lacrymans basidiocarps. This research contributes to the understanding of the holobiont S. lacrymans and gives hints to potential bacterial phyla important for its development and lifestyle.

Phagocytosis underpins the biotrophic lifestyle of intracellular parasites in the class Phytomyxea (Rhizaria).

Phytomyxea are intracellular biotrophic parasites infecting plants and stramenopiles, including the agriculturally impactful Plasmodiophora brassicae and the brown seaweed pathogen Maullinia ectocarpii. They belong to the clade Rhizaria, where phagotrophy is the main mode of nutrition. Phagocytosis is a complex trait of eukaryotes, well documented for free-living unicellular eukaryotes and specific cellular types of animals. Data on phagocytosis in intracellular, biotrophic parasites are scant. Phagocytosis, where parts of the host cell are consumed at once, is seemingly at odds with intracellular biotrophy. Here we provide evidence that phagotrophy is part of the nutritional strategy of Phytomyxea, using morphological and genetic data (including a novel transcriptome of M. ectocarpii). We document intracellular phagocytosis in P. brassicae and M. ectocarpii by transmission electron microscopy and fluorescent in situ hybridization. Our investigations confirm molecular signatures of phagocytosis in Phytomyxea and hint at a small specialized subset of genes used for intracellular phagocytosis. Microscopic evidence confirms the existence of intracellular phagocytosis, which in Phytomyxea targets primarily host organelles. Phagocytosis seems to coexist with the manipulation of host physiology typical of biotrophic interactions. Our findings resolve long debated questions on the feeding behaviour of Phytomyxea, suggesting an unrecognized role for phagocytosis in biotrophic interactions.

The global distribution and environmental drivers of the soil antibiotic resistome.

BACKGROUND: Little is known about the global distribution and environmental drivers of key microbial functional traits such as antibiotic resistance genes (ARGs). Soils are one of Earth's largest reservoirs of ARGs, which are integral for soil microbial competition, and have potential implications for plant and human health. Yet, their diversity and global patterns remain poorly described. Here, we analyzed 285 ARGs in soils from 1012 sites across all continents and created the first global atlas with the distributions of topsoil ARGs. RESULTS: We show that ARGs peaked in high latitude cold and boreal forests. Climatic seasonality and mobile genetic elements, associated with the transmission of antibiotic resistance, were also key drivers of their global distribution. Dominant ARGs were mainly related to multidrug resistance genes and efflux pump machineries. We further pinpointed the global hotspots of the diversity and proportions of soil ARGs. CONCLUSIONS: Together, our work provides the foundation for a better understanding of the ecology and global distribution of the environmental soil antibiotic resistome. Video Abstract.

Molecular data reallocates Sorosphaerula radicalis (Plasmodiophorida, Phytomyxea, Rhizaria) to the genus Hillenburgia.

This study reports the first record of Sorosphaerula radicalis (Phytomyxea, Rhizaria) in continental Europe (Tirol, Austria) and provides first molecular data for this species. An 18S rRNA phylogeny placed S. radicalis into the Plasmodiophorida, although distant from other members of the genus Sorosphaerula and close to the parasite of water cress Hillenburgia nasturtii. To resolve this polyphyly, we compare morphological data and life cycles of Sorosphaerula veronicae (the type species of the genus Sorosphaerula), Hillenburgia nasturtii, and Sorosphaerula radicalis. We conclude that Sorosphaerula radicalis belongs to the recently established genus Hillenburgia.

Microbiota Associated with Different Developmental Stages of the Dry Rot Fungus Serpula lacrymans.

The dry rot fungus Serpula lacrymans causes significant structural damage by decaying construction timber, resulting in costly restoration procedures. Dry rot fungi decompose cellulose and hemicellulose and are often accompanied by a succession of bacteria and other fungi. Bacterial-fungal interactions (BFI) have a considerable impact on all the partners, ranging from antagonistic to beneficial relationships. Using a cultivation-based approach, we show that S. lacrymans has many co-existing, mainly Gram-positive, bacteria and demonstrate differences in the communities associated with distinct fungal parts. Bacteria isolated from the fruiting bodies and mycelia were dominated by Firmicutes, while bacteria isolated from rhizomorphs were dominated by Proteobacteria. Actinobacteria and Bacteroidetes were less abundant. Fluorescence in situ hybridization (FISH) analysis revealed that bacteria were not present biofilm-like, but occurred as independent cells scattered across and within tissues, sometimes also attached to fungal spores. In co-culture, some bacterial isolates caused growth inhibition of S. lacrymans, and vice versa, and some induced fungal pigment production. It was found that 25% of the isolates could degrade pectin, 43% xylan, 17% carboxymethylcellulose, and 66% were able to depolymerize starch. Our results provide first insights for a better understanding of the holobiont S. lacrymans and give hints that bacteria influence the behavior of S. lacrymans in culture.

The influence of soil age on ecosystem structure and function across biomes.

The importance of soil age as an ecosystem driver across biomes remains largely unresolved. By combining a cross-biome global field survey, including data for 32 soil, plant, and microbial properties in 16 soil chronosequences, with a global meta-analysis, we show that soil age is a significant ecosystem driver, but only accounts for a relatively small proportion of the cross-biome variation in multiple ecosystem properties. Parent material, climate, vegetation and topography predict, collectively, 24 times more variation in ecosystem properties than soil age alone. Soil age is an important local-scale ecosystem driver; however, environmental context, rather than soil age, determines the rates and trajectories of ecosystem development in structure and function across biomes. Our work provides insights into the natural history of terrestrial ecosystems. We propose that, regardless of soil age, changes in the environmental context, such as those associated with global climatic and land-use changes, will have important long-term impacts on the structure and function of terrestrial ecosystems across biomes.

Demystifying biotrophs: FISHing for mRNAs to decipher plant and algal pathogen-host interaction at the single cell level.

Plant-pathogen interactions follow spatial and temporal developmental dynamics where gene expression in pathogen and host undergo crucial changes. Therefore, it is of great interest to detect, quantify and localise where and when key genes are active to understand these processes. Many pathosystems are not accessible for genetic amendments or other spatially-resolved gene expression monitoring methods. Here, we adapt single molecule FISH techniques to demonstrate the presence and activity of mRNAs at the single-cell level using phytomyxids in their plant and algal host in lab and field material. This allowed us to monitor and quantify the expression of genes from the clubroot pathogen Plasmodiophora brassicae, several species of its Brassica hosts, and of several brown algae, including the genome model Ectocarpus siliculosus, infected with the phytomyxid Maullinia ectocarpii. We show that mRNAs are localised along a spatiotemporal gradient, thus providing a proof-of-concept of the usefulness of single-molecule FISH to increase knowledge about the interactions between plants, algae and phytomyxids. The methods used are easily applicable to any interaction between microbes and their algal or plant host, and have therefore the potential to rapidly increase our understanding of key, spatially- and temporally-resolved processes underpinning complex plant-microbe interactions.

Revised Taxonomy and Expanded Biodiversity of the Phytomyxea (Rhizaria, Endomyxa).

Phytomyxea (phytomyxids) is a group of obligate biotrophic pathogens belonging to the Rhizaria. Some phytomyxids are well studied and include known plant pathogens such as Plasmodiophora brassicae, the causal agent of clubroot disease. Despite this economic importance, the taxonomy and biodiversity of this group are largely cryptic, with many species described in the premolecular area. Some of these species were key for establishing the morphotaxonomic concepts that define most genera to this day, but systematic efforts to include and integrate those species into molecular studies are still lacking. The aim of this study was to expand our understanding of phytomyxid biodiversity in terrestrial environments. Thirty-eight environmental samples from habitats in which novel and known diversity of Phytomyxea was expected were analysed. We were able to generate 18S rRNA sequences from Ligniera verrucosa, a species which is well defined based on ultrastructure. Phylogenetic analyses of the collected sequences rendered the genera Lignera, Plasmodiophora and Spongospora polyphyletic, and identified two novel and apparently diverse lineages (clade 17, clade 18). Based on these findings and on data from previous studies, we formally establish the new genera Pseudoligniera n. gen. for L. verrucosa,Hillenburgia n. gen. for Spongospora nasturtii and revert Plasmodiophora diplantherae to its original name Ostenfeldiella diplantherae.

Phyllobilins from Senescence-Associated Chlorophyll Breakdown in the Leaves of Basil (Ocimum basilicum) Show Increased Abundance upon Herbivore Attack.

In view of the common use of the herb basil (Ocimum basilicum) in nutrition and in phytomedicine, the contents of its leaves are of obvious interest. In extracts of fresh yellowish-green basil leaves, phyllobilins (PBs), which are bilin-type catabolites of chlorophyll (Chl), were detected using high-performance liquid chromatography (HPLC). Two such PBs, provisionally named Ob-nonfluorescent chlorophyll catabolite (NCC)-40 and Ob-YCC-45, exhibited previously unknown structures that were delineated by a thorough spectroscopic characterization. When basil leaves were infested with aphids or thrips or underwent fungal infections, areas with chlorosis were observed. HPLC analyses of the infested parts of leaves compared to those of the healthy parts showed a significant accumulation of PBs in the infested areas, demonstrating that the senescence-associated pheophorbide a oxygenase/phyllobilin (PAO/PB) pathway is activated by herbivore feeding and fungal infection.

Multiple elements of soil biodiversity drive ecosystem functions across biomes.

The role of soil biodiversity in regulating multiple ecosystem functions is poorly understood, limiting our ability to predict how soil biodiversity loss might affect human wellbeing and ecosystem sustainability. Here, combining a global observational study with an experimental microcosm study, we provide evidence that soil biodiversity (bacteria, fungi, protists and invertebrates) is significantly and positively associated with multiple ecosystem functions. These functions include nutrient cycling, decomposition, plant production, and reduced potential for pathogenicity and belowground biological warfare. Our findings also reveal the context dependency of such relationships and the importance of the connectedness, biodiversity and nature of the globally distributed dominant phylotypes within the soil network in maintaining multiple functions. Moreover, our results suggest that the positive association between plant diversity and multifunctionality across biomes is indirectly driven by soil biodiversity. Together, our results provide insights into the importance of soil biodiversity for maintaining soil functionality locally and across biomes, as well as providing strong support for the inclusion of soil biodiversity in conservation and management programmes.

Global ecological predictors of the soil priming effect.

Identifying the global drivers of soil priming is essential to understanding C cycling in terrestrial ecosystems. We conducted a survey of soils across 86 globally-distributed locations, spanning a wide range of climates, biotic communities, and soil conditions, and evaluated the apparent soil priming effect using 13C-glucose labeling. Here we show that the magnitude of the positive apparent priming effect (increase in CO2 release through accelerated microbial biomass turnover) was negatively associated with SOC content and microbial respiration rates. Our statistical modeling suggests that apparent priming effects tend to be negative in more mesic sites associated with higher SOC contents. In contrast, a single-input of labile C causes positive apparent priming effects in more arid locations with low SOC contents. Our results provide solid evidence that SOC content plays a critical role in regulating apparent priming effects, with important implications for the improvement of C cycling models under global change scenarios.

Transcriptomic response in symptomless roots of clubroot infected kohlrabi (Brassica oleracea var. gongylodes) mirrors resistant plants.

BACKGROUND: Clubroot disease caused by Plasmodiophora brassicae (Phytomyxea, Rhizaria) is one of the economically most important diseases of Brassica crops. The formation of hypertrophied roots accompanied by altered metabolism and hormone homeostasis is typical for infected plants. Not all roots of infected plants show the same phenotypic changes. While some roots remain uninfected, others develop galls of diverse size. The aim of this study was to analyse and compare the intra-plant heterogeneity of P. brassicae root galls and symptomless roots of the same host plants (Brassica oleracea var. gongylodes) collected from a commercial field in Austria using transcriptome analyses. RESULTS: Transcriptomes were markedly different between symptomless roots and gall tissue. Symptomless roots showed transcriptomic traits previously described for resistant plants. Genes involved in host cell wall synthesis and reinforcement were up-regulated in symptomless roots indicating elevated tolerance against P. brassicae. By contrast, genes involved in cell wall degradation and modification processes like expansion were up-regulated in root galls. Hormone metabolism differed between symptomless roots and galls. Brassinosteroid-synthesis was down-regulated in root galls, whereas jasmonic acid synthesis was down-regulated in symptomless roots. Cytokinin metabolism and signalling were up-regulated in symptomless roots with the exception of one CKX6 homolog, which was strongly down-regulated. Salicylic acid (SA) mediated defence response was up-regulated in symptomless roots, compared with root gall tissue. This is probably caused by a secreted benzoic acid/salicylic acid methyl transferase from the pathogen (PbBSMT), which was one of the highest expressed pathogen genes in gall tissue. The PbBSMT derived Methyl-SA potentially leads to increased pathogen tolerance in uninfected roots. CONCLUSIONS: Infected and uninfected roots of clubroot infected plants showed transcriptomic differences similar to those previously described between clubroot resistant and susceptible hosts. The here described intra-plant heterogeneity suggests, that for a better understanding of clubroot disease targeted, spatial analyses of clubroot infected plants will be vital in understanding this economically important disease.

Changes in belowground biodiversity during ecosystem development.

Belowground organisms play critical roles in maintaining multiple ecosystem processes, including plant productivity, decomposition, and nutrient cycling. Despite their importance, however, we have a limited understanding of how and why belowground biodiversity (bacteria, fungi, protists, and invertebrates) may change as soils develop over centuries to millennia (pedogenesis). Moreover, it is unclear whether belowground biodiversity changes during pedogenesis are similar to the patterns observed for aboveground plant diversity. Here we evaluated the roles of resource availability, nutrient stoichiometry, and soil abiotic factors in driving belowground biodiversity across 16 soil chronosequences (from centuries to millennia) spanning a wide range of globally distributed ecosystem types. Changes in belowground biodiversity during pedogenesis followed two main patterns. In lower-productivity ecosystems (i.e., drier and colder), increases in belowground biodiversity tracked increases in plant cover. In more productive ecosystems (i.e., wetter and warmer), increased acidification during pedogenesis was associated with declines in belowground biodiversity. Changes in the diversity of bacteria, fungi, protists, and invertebrates with pedogenesis were strongly and positively correlated worldwide, highlighting that belowground biodiversity shares similar ecological drivers as soils and ecosystems develop. In general, temporal changes in aboveground plant diversity and belowground biodiversity were not correlated, challenging the common perception that belowground biodiversity should follow similar patterns to those of plant diversity during ecosystem development. Taken together, our findings provide evidence that ecological patterns in belowground biodiversity are predictable across major globally distributed ecosystem types and suggest that shifts in plant cover and soil acidification during ecosystem development are associated with changes in belowground biodiversity over centuries to millennia.

Draft Genome Resource for the Potato Powdery Scab Pathogen Spongospora subterranea.

The Plasmodiophorida (Phytomyxea, Rhizaria) are a group of protists that infect plants. Of this group, Spongospora subterranea causes major problems for the potato industry by causing powdery scab and root galling of potatoes and as vector for the Potato mop-top virus (PMTV) (genus Pomovirus, family Virgaviridae). A single tuber isolate (SSUBK13) of this uncultivable protist was used to generate DNA for Illumina sequencing. The data were assembled to a draft genome of 28.08 Mb consisting of 2,340 contigs and an L50 of 280. A total of 10,778 genes were predicted and 93% of the BUSCO genes were detected. The presented genome assembly is only the second genome of a plasmodiophorid. The data will accelerate functional genomics to study poorly understood interaction of plasmodiophorids and their hosts.

Rhizarian 'Novel Clade 10' Revealed as Abundant and Diverse Planktonic and Terrestrial Flagellates, including Aquavolon n. gen.

Rhizarian 'Novel Clade 10' (NC10) is frequently detected by 18S rRNA gene sequencing studies in freshwater planktonic samples. We describe a new genus and two species of eukaryovorous biflagellate protists, Aquavolon hoantrani n. gen. n. sp. and A. dientrani n. gen. n. sp., which represent the first morphologically characterized members of NC10, here named Aquavolonida ord. nov. The slightly metabolic cells possess naked heterodynamic flagella, whose kinetosomes lie at a right angle to each other and are connected by at least one fibril. Unlike their closest known relative Tremula longifila, they rotate around their longitudinal axis when swimming and only very rarely glide on surfaces. Screening of a wide range of environmental DNA extractions with lineage-specific PCR primers reveals that Aquavolonida consists of a large radiation of protists, which are most diversified in freshwater planktonic habitats and as yet undetected in marine environments. Earlier-branching lineages in Aquavolonida include less frequently detected organisms from soils and freshwater sediments. The 18S rRNA gene phylogeny suggests that Aquavolonida forms a common evolutionary lineage with tremulids and uncharacterized 'Novel Clade 12', which likely represents one of the deepest lineages in the Rhizaria, separate from Cercozoa (Filosa), Endomyxa, and Retaria.

Plant Rhizosphere Selection of Plasmodiophorid Lineages from Bulk Soil: The Importance of "Hidden" Diversity.

Microbial communities closely associated with the rhizosphere can have strong positive and negative impacts on plant health and growth. We used a group-specific amplicon approach to investigate local scale drivers in the diversity and distribution of plasmodiophorids in rhizosphere/root and bulk soil samples from oilseed rape (OSR) and wheat agri-systems. Plasmodiophorids are plant- and stramenopile-associated protists including well known plant pathogens as well as symptomless endobiotic species. We detected 28 plasmodiophorid lineages (OTUs), many of them novel, and showed that plasmodiophorid communities were highly dissimilar and significantly divergent between wheat and OSR rhizospheres and between rhizosphere and bulk soil samples. Bulk soil communities were not significantly different between OSR and wheat systems. Wheat and OSR rhizospheres selected for different plasmodiophorid lineages. An OTU corresponding to Spongospora nasturtii was positively selected in the OSR rhizosphere, as were two genetically distinct OTUs. Two novel lineages related to Sorosphaerula veronicae were significantly associated with wheat rhizosphere samples, indicating unknown plant-protist relationships. We show that group-targeted eDNA approaches to microbial symbiont-host ecology reveal significant novel diversity and enable inference of differential activity and potential interactions between sequence types, as well as their presence.

Environmental Sequencing Fills the Gap Between Parasitic Haplosporidians and Free-living Giant Amoebae.

Class Ascetosporea (Rhizaria; Endomyxa) comprises many parasites of invertebrates. Within this group, recent group-specific environmental DNA (eDNA) studies have contributed to the establishment of the new order Mikrocytida, a new phylogeny and characterization of Paramyxida, and illuminated the diversity and distribution of haplosporidians. Here, we use general and lineage-specific PCR primers to investigate the phylogenetic "gap" between haplosporidians and their closest known free-living relatives, the testate amoeba Gromia and reticulate amoeba Filoreta. Within this gap are Paradinium spp. parasites of copepods, which we show to be highly diverse and widely distributed in planktonic and benthic samples. We reveal a robustly supported radiation of parasites, ENDO-3, comprised of Paradinium and three further clades (ENDO-3a, ENDO-3b and SPP). A further environmental group, ENDO-2, perhaps comprising several clades, branches between this radiation and the free-living amoebae. Early diverging haplosporidians were also amplified, often associated with bivalves or deep-sea samples. The general primer approach amplified an overlapping set of novel lineages within ENDO-3 and Haplosporida, whereas the group-specific primer strategy, targeted to amplify from the earliest known divergent haplosporidians to Gromia, generated greater sequence diversity across part of this phylogenetic range.

Review: Host-pathogen dynamics of seagrass diseases under future global change.

Human-induced global change is expected to amplify the disease risk for marine biota. However, the role of disease in the rapid global decline of seagrass is largely unknown. Global change may enhance seagrass susceptibility to disease through enhanced physiological stress, while simultaneously promoting pathogen development. This review outlines the characteristics of disease-forming organisms and potential impacts of global change on three groups of known seagrass pathogens: labyrinthulids, oomycetes and Phytomyxea. We propose that hypersalinity, climate warming and eutrophication pose the greatest risk for increasing frequency of disease outbreaks in seagrasses by increasing seagrass stress and lowering seagrass resilience. In some instances, global change may also promote pathogen development. However, there is currently a paucity of information on these seagrass pathosystems. We emphasise the need to expand current research to better understand the seagrass-pathogen relationships, serving to inform predicative modelling and management of seagrass disease under future global change scenarios.