Filamentous fungi asymptomatically colonize the inner tissues of macroalgae, yet their ecological roles remain largely underexplored. Here, we tested if metabolites produced by fungal endophytes might protect their host against a phylogenetically broad spectrum of protistan pathogens. Accordingly, the cultivable fungal endophytes of four brown algal species were isolated and identified based on LSU and SSU sequencing. The fungal metabolomes were tested for their ability to reduce the infection by protistan pathogens in the algal model Ectocarpus siliculosus. The most active metabolomes effective against the oomycetes Eurychasma dicksonii and Anisolpidium ectocarpii, and the phytomixid Maullinia ectocarpii were further characterized chemically. Several pyrenocines isolated from Phaeosphaeria sp. AN596H efficiently inhibited the infection by all abovementioned pathogens. Strikingly, these compounds also inhibited the infection of nori (Pyropia yezoensis) against its two most devastating oomycete pathogens, Olpidiopsis pyropiae, and Pythium porphyrae. We thus demonstrate that fungal endophytes associated with brown algae produce bioactive metabolites which might confer protection against pathogen infection. These results highlight the potential of metabolites to finely-tune the outcome of molecular interactions between algae, their endophytes, and protistan pathogens. This also provide proof-of-concept toward the applicability of such metabolites in marine aquaculture to control otherwise untreatable diseases.

The bloom and bust patterns of microalgae in aquatic systems contribute massively to global biogeochemical cycles. The decline of algal blooms is mainly caused by nutrient limitation resulting in cell death, the arrest of cell division and the aging of surviving cells. Nutrient intake can re-initiate proliferation, but the processes involved are poorly understood. Here we characterize how the bloom-forming diatom Coscinodiscus radiatus recovers from starvation after nutrient influx. Rejuvenation is mediated by extracellular vesicles that shuttle reactive oxygen species, oxylipins and other harmful metabolites out of the old cells, thereby re-enabling their proliferation. By administering nutrient pulses to aged cells and metabolomic monitoring of the response, we show that regulated pathways are centred around the methionine cycle in C. radiatus. Co-incubation experiments show that bacteria mediate aging processes and trigger vesicle production using chemical signalling. This work opens new perspectives on cellular aging and rejuvenation in complex microbial communities.

Unicellular phototrophic algae can form massive blooms with up to millions of individual cells per milliliter in freshwater and marine ecosystems. Despite the temporal dominance of bloom formers many algal species can co-exist and compete for nutrients and space, creating a complex and diverse community. While microscopy and single cell genomics can address the taxonomic inventory, the cellular metabolome has yet to be thoroughly explored to determine the physiological status of microalgae. This might, however, provide a key to understand the observed species diversity in the homogeneous environment. Here, we introduce an effective, rapid and versatile method to analyze living single cells from aqueous substrata with laser-desorption/ionization mass spectrometry (LDI-MS) using a simple and inexpensive matrix-free support. The cells deposited on a cultivation-medium wetted support are analyzed with minimal disturbance as they remain in their natural viable state until their disruption during LDI-MS. Metabolites desorbed from single cells are analyzed on High-Resolution Mass Spectrometry (HR-MS) using the Orbitrap FT-MS technology to fingerprint cellular chemistry. This live single-cell mass spectrometry (LSC-MS) allows assessing the physiological status and strain-specifics of different microalgae, including marine diatoms and freshwater chlorophytes, at the single-cell level. We further report a reliable and robust data treatment pipeline to perform multivariate statistics on the replicated LSC-MS data. Comparing single cell MS spectra from natural phytoplankton samples and from laboratory strains allows the identification and discrimination of inter and intra-specific metabolic variability and thereby has promising applications in addressing highly complex phytoplankton communities. Notably, the herein described matrix-free live-single-cell LDI-HR-MS approach enables monitoring dynamics of the plankton and might explain why key-players survive, thrive, avoid selective feeding or pathogenic virus and bacteria, while others are overcome and die.

Mulberry (Morus) is an economically important woody tree that is suitable for use in sericulture as forage and in medicine. However, this broad-leaved tree is facing multiple threats ranging from phytopathogens to insect pests. Here, a Gram-positive, endospore-forming bacterium (ZJU1) was frequently isolated from healthy mulberry plants by screening for foliar endophytes showing antagonism against pathogens and pests. Whole-genome sequencing and annotation resulted in a genome size of 4.06 Mb and classified the bacterium as a novel strain of Bacillus amyloliquefaciens that has rarely been identified from tree leaves. An integrative approach combining traditional natural product chemistry, activity bioassays, and high-resolution mass spectrometry confirmed that strain ZJU1 uses a blend of antimicrobials including peptides and volatile organic compounds to oppose Botrytis cinerea, a major phytopathogenic fungus causing mulberry gray mold disease. We showed that the inoculation of endophyte-free plants with ZJU1 significantly decreased both leaf necrosis and mortality under field conditions. In addition to the direct interactions of endophytes with foliar pathogens, in planta studies suggested that the inoculation of endophytes also induced plant systemic defense, including high expression levels of mulberry disease resistance genes. Moreover, when applied to the generalist herbivore Spodoptera litura, ZJU1 was sufficient to reduce the pest survival rate below 50%. A previously undiscovered crystal toxin (Cry10Aa) could contribute to this insecticidal effect against notorious lepidopteran pests. These unique traits clearly demonstrate that B. amyloliquefaciens ZJU1 is promising for the development of successful strategies for biocontrol applications. The search for new plant-beneficial microbes and engineering microbiomes is therefore of great significance for sustainably improving plant performance.