A comprehensive analysis of the microbial communities of healthy and diseased marine macroalgae and the detection of known and potential bacterial pathogens

Microorganisms are increasingly being recognized as the causative agents in the diseases of marine higher organisms, such as corals, sponges and macroalgae. Delisea pulchra is a common, temperate red macroalga, which suffers from a bleaching disease. Two bacterial strains, Nautella italica R11 and Phaeobacter gallaeciensis LSS9, have been shown in vitro to cause bleaching symptoms, but previous work has failed to detect them during a natural bleaching event. To provide a link between in vitro observations and natural occurrences of the disease, we employ here deep-sequencing of the 16S rRNA gene to comprehensively analyze the community composition of healthy and diseased D. pulchra samples from two separate locations. We observed operational taxonomic units (OTUs) with 100% identity and coverage to the 16S RNA gene sequence of both in vitro pathogens, but only the OTU with similarity to strain LSS9 showed a statistically significant higher abundance in diseased samples. Our analysis also reveals the existence of other bacterial groups within the families Rhodobacteraceae and Flavobacteriaceae that strongly contribute to difference between diseased and healthy samples and thus these groups potentially contain novel macroalgal pathogens and/or saprophytes. Together our results provide evidence for the ecological relevance of one kind of in vitro pathogen, but also highlight the possibility that multiple opportunistic pathogens are involved in the bleaching disease of D. pulchra

Effects of temperature stress and aquarium conditions on the red macroalga Delisea pulchra and its associated microbial community

In recent years there has been an increase in the rate and severity of diseases affecting habitat-forming marine organisms, such as corals, sponges and macroalgae. Delisea pulchra is a temperate red macroalga that suffers from a bleaching disease that is more frequent during summer, when seawater temperatures are elevated and the alga’s chemical defense is weakened. A bacterial cause for the disease is implied by previous studies showing that some isolated strains can cause bleaching in vitro and that host-associated microbial communities are distinct between diseased and healthy individuals. However, nothing is known about the successional events in the microbial community that occur during the development of the disease. To study this aspect in the future, we aimed here to develop an experimental setup to study the bleaching disease in a controllable aquarium environment. Application of a temperature stress (up to 27° C) did not cause a clear and consistent pattern of bleaching, suggesting that temperature alone might not be the only or main factor to cause the disease. The results also showed that the aquarium conditions alone seem to be sufficient to produce bleaching symptoms. Microbial community analysis based on 16S rRNA gene fingerprinting and sequencing showed significant changes after 15 days in the aquarium, indicating that the native microbial associates of D. pulchra are not stably maintained. Microbial taxa that were enriched in the aquarium-held D. pulchra thalli, however did not match on a taxonomic level those that have been found to be enriched in natural bleaching events. Together our observations indicate that environmental factors, other than the ones investigated here, might drive the bleaching disease in D. pulchra and that the aquarium conditions have substantial impact on the algal-associated microbiome

Sample processing impacts the viability and cultivability of the sponge microbiome

Sponges host complex microbial communities of recognized ecological and biotechnological importance. Extensive cultivation efforts have been made to isolate sponge bacteria, but most still elude cultivation. To identify the bottlenecks of sponge bacterial cultivation, we combined high-throughput 16S rRNA gene sequencing with a variety of cultivation media and incubation conditions. We aimed to determine the extent to which sample processing and cultivation conditions can impact bacterial viability and recovery in culture.We isolated 325 sponge bacteria from 6 specimens of Cymbastela concentrica and 3 specimens of Scopalina sp.. These isolates were distributed over 37 different genera and 47 operational taxonomic units (defined at 97% 16S rRNA gene sequence identity). The cultivable bacterial community was highly specific to its sponge host and different media compositions yielded distinct microbial isolates. Around 97% of the isolates could be detected in the original sponge and represented a large but highly variable proportion (0.5% to 92% total abundance, depending on sponge species) of viable bacteria obtained after sample processing, as determined by propidium monoazide (PMA) selective DNA modification of compromised cells.Our results show that the most abundant viable bacteria are also the most predominant groups found in cultivation, reflecting, to some extent, the relative abundances of the viable bacterial community, rather than the overall community estimated by direct molecular approaches. Cultivation is therefore shaped not only by the growth conditions provided, but also by the different cell viabilities of the bacteria that constitute the cultivation inoculum. These observations highlight the need to perform experiments to assess each method of sample processing for its accurate representation of the actual in situ bacterial community and its yield of viable cells

A combination of biochar-mineral complexes and compost improves soil bacterial processes, soil quality and plant properties

Organic farming avoids the use of synthetic fertilizers and promises food production with minimal environmental impact, however this farming practice does not often result in the same productivity as conventional farming. In recent years, biochar has received increasing attention as an agricultural amendment and by coating it with minerals to form biochar-mineral complex (BMC) carbon retention and nutrient availability can be improved. However, little is known about the potential of BMC in improving organic farming. We therefore investigated here how soil, bacterial and plant properties respond to a combined treatment of BMC and an organic fertilizer, i.e. a compost based on poultry manure. In a pakchoi pot trial, BMC and compost showed synergistic effects on soil properties, and specifically by increasing nitrate content. Soil nitrate has been previously observed to increase leaf size and we correspondingly saw an increase in the surface area of pakchoi leaves under the combined treatment of BMC and chicken manure. The increase in soil nitrate was also correlated with an enrichment of bacterial nitrifiers due to BMC. Additionally, we observed that the bacteria present in the compost treatment had a high turnover, which likely facilitated organic matter degradation and a reduction of potential pathogens derived from the manure. Overall our results demonstrate that a combination of BMC and compost can stimulate microbial process in organic farming that result in better vegetable production and improved soil properties for sustainable farming

Expanding our understanding of the seaweed holobiont: RNA viruses of the red alga Delisea pulchra.

Marine seaweeds are holobionts comprised of the macroalgal hosts and their associated microbiota. While the composition of the bacterial component of seaweed microbiomes is increasingly studied, almost nothing is known about the presence, diversity and composition of viruses in macroalgae in situ. In this study, we characterise for the first time the viruses associated with a red macroalga, Delisea pulchra. Using transmission electron microscopy we identified diverse morphotypes of virus-like particles in D. pulchra ranging from icosahedral to bacilliform to coiled pleomorphic as well as bacteriophages. Virome sequencing revealed the presence of a diverse group of dsRNA viruses affiliated to the genus Totivirus, known to infect plant pathogenic fungi. We further identified a ssRNA virus belonging to the order Picornavirales with a close phylogenetic relationship to a pathogenic virus infecting marine diatoms. The results of this study shed light on a so far neglected part of the seaweed holobiont, and suggest that some of these may be possible pathogens for a host that is already known to be significantly impacted by bacterial infections

VarR controls colonization and virulence in the marine macroalgal pathogen Nautella italica R11

There is increasing evidence to suggest that macroalgae (seaweeds) are susceptible to infectious disease. However, to date, little is known about the mechanisms that facilitate the colonization and virulence of microbial seaweed pathogens. One well-described example of a seaweed disease is the bleaching of the red alga Delisea pulchra, which can be caused by the bacterium Nautella italica R11, a member of the Roseobacter clade. This pathogen contains a unique luxR-type gene, varR, which we hypothesize controls its colonization and virulence. We show here that a varR knock-out strain is deficient in its ability to cause disease in D. pulchra and is defective in biofilm formation and attachment to a common algal polysaccharide. Moreover complementation of the varR gene in trans can restore these functions to the wild type levels. Proteomic analysis of bacterial cells in planktonic and biofilm growth highlight the potential importance of nitrogen scavenging, mobilization of energy reserves, and stress resistance in the biofilm lifestyle of N. italica R11. Moreover, we show that VarR regulates the expression of a specific subset of biofilm-associated proteins. Taken together these data suggest that VarR controls colonization and persistence of N. italica R11 on the surface of a macroalgal host and that it is an important regulator of virulence