A comprehensive dataset on spatiotemporal variation of microbial plankton communities in the Baltic Sea.

The Baltic Sea is one of the largest brackish water environments on earth and is characterised by pronounced physicochemical gradients and seasonal dynamics. Although the Baltic Sea has a long history of microscopy-based plankton monitoring, DNA-based metabarcoding has so far mainly been limited to individual transect cruises or time-series of single stations. Here we report a dataset covering spatiotemporal variation in prokaryotic and eukaryotic microbial communities and physicochemical parameters. Within 13-months between January 2019 and February 2020, 341 water samples were collected at 22 stations during monthly cruises along the salinity gradient. Both salinity and seasonality are strongly reflected in the data. Since the dataset was generated with both metabarcoding and microscopy-based methods, it provides unique opportunities for both technical and ecological analyses, and is a valuable biodiversity reference for future studies, in the prospect of climate change.

Towards high-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton.

Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we applied a microarray single-cell sequencing (MASC-seq) technology, that generates microscope images of cells in parallel with capturing their transcriptomes, on three species representing important plankton groups with different cell structures; the ciliate Tetrahymena thermophila, the diatom Phaeodactylum tricornutum, and the dinoflagellate Heterocapsa sp. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating "microbial dark matter", although further optimizations are necessary to increase the signal-to-noise ratio.

The Fungal Endophyte Penicillium olsonii ML37 Reduces Fusarium Head Blight by Local Induced Resistance in Wheat Spikes.

The fungal endophyte Penicillium olsonii ML37 is a biocontrol agent of Fusarium head blight in wheat (caused by Fusarium graminearum), which has shown a limited direct inhibition of fungal growth in vitro. We used RNA-seq and LC-MS/MS analyses to elucidate metabolic interactions of the three-way system Penicillium-wheat-Fusarium in greenhouse experiments. We demonstrated that P. olsonii ML37 colonises wheat spikes and transiently activates plant defence mechanisms, as pretreated spikes show a faster and stronger expression of the defence metabolism during the first 24 h after pathogen inoculation. This effect was transient and the expression of the same genes was lower in the pathogen-infected spikes than in those infected by P. olsonii alone. This response to the endophyte includes the transcriptional activation of several WRKY transcription factors. This early activation is associated with a reduction in FHB symptoms and significantly lower levels of the F. graminearum metabolites 15-acetyl-DON and culmorin. An increase in the Penicillium-associated metabolite asperphanamate confirms colonisation by the endophyte. Our results suggest that the mode of action used by P. olsonii ML37 is via a local defence activation in wheat spikes, and that this fungus has potential as a novel biological alternative in wheat disease control.

Short- and long-read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

Succession of the fungal endophytic microbiome of wheat is dependent on tissue-specific interactions between host genotype and environment.

Fungi living inside plants affect many aspects of plant health, but little is known about how plant genotype influences the fungal endophytic microbiome. However, a deeper understanding of interactions between plant genotype and biotic and abiotic environment in shaping the plant microbiome is of significance for modern agriculture, with implications for disease management, breeding and the development of biocontrol agents. For this purpose, we analysed the fungal wheat microbiome from seed to plant to seeds and studied how different potential sources of inoculum contributed to shaping of the microbiome. We conducted a large-scale pot experiment with related wheat cultivars over one growth-season in two environments (indoors and outdoors) to disentangle the effects of host genotype, abiotic environment (temperature, humidity, precipitation) and fungi present in the seed stock, air and soil on the succession of the endophytic fungal communities in roots, flag leaves and seeds at harvest. The communities were studied with ITS1 metabarcoding and environmental climate factors were monitored during the experimental period. Host genotype, tissue type and abiotic factors influenced fungal communities significantly. The effect of host genotype was mostly limited to leaves and roots, and was location-independent. While there was a clear effect of plant genotype, the relatedness between cultivars was not reflected in the microbiome. For the phyllosphere microbiome, location-dependent weather conditions factors largely explained differences in abundance, diversity, and presence of genera containing pathogens, whereas the root communities were less affected by abiotic factors. Our findings suggest that airborne fungi are the primary inoculum source for fungal communities in aerial plant parts whereas vertical transmission is likely to be insignificant. In summary, our study demonstrates that host genotype, environment and presence of fungi in the environment shape the endophytic fungal community in wheat over a growing season.