Parasitism causes changes in caterpillar odours and associated bacterial communities with consequences for host-location by a hyperparasitoid.

Microorganisms living in and on macroorganisms may produce microbial volatile compounds (mVOCs) that characterise organismal odours. The mVOCs might thereby provide a reliable cue to carnivorous enemies in locating their host or prey. Parasitism by parasitoid wasps might alter the microbiome of their caterpillar host, affecting organismal odours and interactions with insects of higher trophic levels such as hyperparasitoids. Hyperparasitoids parasitise larvae or pupae of parasitoids, which are often concealed or inconspicuous. Odours of parasitised caterpillars aid them to locate their host, but the origin of these odours and its relationship to the caterpillar microbiome are unknown. Here, we analysed the odours and microbiome of the large cabbage white caterpillar Pieris brassicae in relation to parasitism by its endoparasitoid Cotesia glomerata. We identified how bacterial presence in and on the caterpillars is correlated with caterpillar odours and tested the attractiveness of parasitised and unparasitised caterpillars to the hyperparasitoid Baryscapus galactopus. We manipulated the presence of the external microbiome and the transient internal microbiome of caterpillars to identify the microbial origin of odours. We found that parasitism by C. glomerata led to the production of five characteristic volatile products and significantly affected the internal and external microbiome of the caterpillar, which were both found to have a significant correlation with caterpillar odours. The preference of the hyperparasitoid was correlated with the presence of the external microbiome. Likely, the changes in external microbiome and body odour after parasitism were driven by the resident internal microbiome of caterpillars, where the bacterium Wolbachia sp. was only present after parasitism. Micro-injection of Wolbachia in unparasitised caterpillars increased hyperparasitoid attraction to the caterpillars compared to untreated caterpillars, while no differences were found compared to parasitised caterpillars. In conclusion, our results indicate that host-parasite interactions can affect multi-trophic interactions and hyperparasitoid olfaction through alterations of the microbiome.

Genetic diversity and structure of the coffee leaf rust fungus Hemileia vastatrix across different coffee management systems in Ethiopia.

Although coffee leaf rust (CLR), caused by Hemileia vastatrix, poses an increasing threat to coffee production in Ethiopia, little is known regarding its genetic diversity and structure and how these are affected by coffee management. Here, we used genetic fingerprinting based on sequence-related amplified polymorphism (SRAP) markers to genotype H. vastatrix samples from different coffee shrubs, across 40 sites, covering four coffee production systems (forest coffee, semi plantation coffee, home garden coffee, and plantation coffee) and different altitudes in Ethiopia. In total, 96 H. vastatrix samples were successfully genotyped with three primer combinations, producing a total of 79 scorable bands. We found 35.44% of amplified bands to be polymorphic, and the polymorphic information content (PIC) was 0.45, suggesting high genetic diversity among our CLR isolates. We also found significant isolation-by-distance across the samples investigated and detected significant differences in fungal genetic composition among plantation coffee and home garden coffee and a marginally significant difference among plantation coffee and forest coffee. Furthermore, we found a significant effect of altitude on CLR genetic composition in the forest coffee and plantation systems. Our results suggest that both spore dispersal and different selection pressures in the different coffee management systems are likely responsible for the observed high genetic diversity and genetic structure of CLR isolates in Ethiopia. When selecting Ethiopian coffee genotypes for crop improvement, it is important that these genotypes carry some resistance against CLR. Because our study shows large variation in genetic composition across relatively short geographical distances, a broad selection of rust isolates must be used for coffee resistance screening.

Phylogenomic analysis of the genus Rosenbergiella and description of Rosenbergiella gaditana sp. nov., Rosenbergiella metrosideri sp. nov., Rosenbergiella epipactidis subsp. epipactidis subsp. nov., Rosenbergiella epipactidis subsp. californiensis subsp. nov., Rosenbergiella epipactidis subsp. japonicus subsp. nov., Rosenbergiella nectarea subsp. nectarea subsp. nov. and Rosenbergiella nectarea subsp. apis subsp. nov., isolated from floral nectar and insects.

The genus Rosenbergiella is one of the most frequent bacterial inhabitants of flowers and a usual member of the insect microbiota worldwide. To date, there is only one publicly available Rosenbergiella genome, corresponding to the type strain of Rosenbergiella nectarea (8N4T), which precludes a detailed analysis of intra-genus phylogenetic relationships. In this study, we obtained draft genomes of the type strains of the other Rosenbergiella species validly published to date (R. australiborealis, R. collisarenosi and R. epipactidis) and 23 additional isolates of flower and insect origin. Isolate S61T, retrieved from the nectar of an Antirrhinum sp. flower collected in southern Spain, displayed low average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values when compared with other Rosenbergiella members (≤86.5 and ≤29.8 %, respectively). Similarly, isolate JB07T, which was obtained from the floral nectar of Metrosideros polymorpha plants in Hawaii (USA) had ≤95.7 % ANI and ≤64.1 % isDDH with other Rosenbergiella isolates. Therefore, our results support the description of two new Rosenbergiella species for which we propose the names Rosenbergiella gaditana sp. nov. (type strain: S61T=NCCB 100789T=DSM 111181T) and Rosenbergiella metrosideri sp. nov. (JB07T=NCCB 100888T=LMG 32616T). Additionally, some R. epipactidis and R. nectarea isolates showed isDDH values<79 % with other conspecific isolates, which suggests that these species include subspecies for which we propose the names Rosenbergiella epipactidis subsp. epipactidis subsp. nov. (S256T=CECT 8502T=LMG 27956T), Rosenbergiella epipactidis subsp. californiensis subsp. nov. (FR72T=NCCB 100898T=LMG 32786T), Rosenbergiella epipactidis subsp. japonicus subsp. nov. (K24T=NCCB 100924T=LMG 32785T), Rosenbergiella nectarea subsp. nectarea subsp. nov. (8N4T = DSM 24150T = LMG 26121T) and Rosenbergiella nectarea subsp. apis subsp. nov. (B1AT=NCCB 100810T= DSM 111763T), respectively. Finally, we present the first phylogenomic analysis of the genus Rosenbergiella and update the formal description of the species R. australiborealis, R. collisarenosi, R. epipactidis and R. nectarea based on new genomic and phenotypic information.

Nectar-Inhabiting Bacteria Affect Olfactory Responses of an Insect Parasitoid by Altering Nectar Odors.

Floral nectar is ubiquitously colonized by a variety of microorganisms among which yeasts and bacteria are the most common. Microorganisms inhabiting floral nectar can alter several nectar traits, including nectar odor by producing microbial volatile organic compounds (mVOCs). Evidence showing that mVOCs can affect the foraging behavior of insect pollinators is increasing in the literature, whereas the role of mVOCs in altering the foraging behavior of third-trophic level organisms such as insect parasitoids is largely overlooked. Parasitoids are frequent visitors of flowers and are well known to feed on nectar. In this study, we isolated bacteria inhabiting floral nectar of buckwheat, Fagopyrum esculentum (Polygonales: Polygonaceae), to test the hypothesis that nectar bacteria affect the foraging behavior of the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae) via changes in odors of nectar. In behavioral assays, we found that T. basalis wasps are attracted toward nectar fermented by 4 out of the 14 bacterial strains isolated, which belong to Staphylococcus epidermidis, Terrabacillus saccharophilus (both Firmicutes), Pantoea sp. (Proteobacteria), and Curtobacterium sp. (Actinobacteria). Results of chemical investigations revealed significant differences in the volatile blend composition of nectars fermented by the bacterial isolates. Our results indicate that nectar-inhabiting bacteria play an important role in the interactions between flowering plants and foraging parasitoids. These results are also relevant from an applied perspective as flowering resources, such as buckwheat, are largely used in agriculture to promote conservation biological control of insect pests.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

Microbial profile during fermentation and aerobic stability of ensiled mixtures of maize stover and banana pseudostem in South Ethiopia.

AIMS: This study evaluated pH reduction and microbial growth during fermentation of maize stover (MS) mixed with banana pseudostem (BPS) under South Ethiopian conditions. MATERIALS AND RESULTS: The MS and BPS were chopped and mixed into six treatments (T): 80% BPS plus 20% DMS (T1), 70% BPS plus 30% DMS (T2), 40% BPS plus 60% FMS (fresh MS) (T3), 20% BPS plus 80% FMS (T4), 100% FMS (T5), and 95% BPS plus 5% molasses (T6). At 0, 7, 14, 30, 60, and 90 days, pH and dry matter were determined. Microbiological quality was assessed using plate counts and Illumina MiSeq sequencing. On day 60 and 90, aerobic stability was investigated. The results showed a significant reduction in pH in all mixtures, except in T1 and T2. Lactic acid bacteria counts reached a maximum in all treatments within 14 days. Sequencing showed marked changes in dominant bacteria, such as Buttiauxella and Acinetobacter to Lactobacillus and Bifidobacterium. CONCLUSIONS: The fresh MS and BPS mixtures and fresh maize showed significant pH reduction and dominance of desirable microbial groups. SIGNIFICANCE AND IMPACT OF THE STUDY: The study enables year-round livestock feed supplementation to boost milk and meat production in South Ethiopia.

Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov., isolated from floral nectar and honey bees.

A detailed evaluation of eight bacterial isolates from floral nectar and animal visitors to flowers shows evidence that they represent three novel species in the genus Acinetobacter. Phylogenomic analysis shows the closest relatives of these new isolates are Acinetobacter apis, Acinetobacter boissieri and Acinetobacter nectaris, previously described species associated with floral nectar and bees, but high genome-wide sequence divergence defines these isolates as novel species. Pairwise comparisons of the average nucleotide identity of the new isolates compared to known species is extremely low (<83 %), thus confirming that these samples are representative of three novel Acinetobacter species, for which the names Acinetobacter pollinis sp. nov., Acinetobacter baretiae sp. nov. and Acinetobacter rathckeae sp. nov. are proposed. The respective type strains are SCC477T (=TSD-214T=LMG 31655T), B10AT (=TSD-213T=LMG 31702T) and EC24T (=TSD-215T=LMG 31703T=DSM 111781T).

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

The Pupal Parasitoid Trichopria drosophilae Is Attracted to the Same Yeast Volatiles as Its Adult Host.

There is increasing evidence that microorganisms, particularly fungi and bacteria, emit volatile compounds that mediate the foraging behaviour of insects and therefore have the potential to affect key ecological relationships. However, to what extent microbial volatiles affect the olfactory response of insects across different trophic levels remains unclear. Adult parasitoids use a variety of chemical stimuli to locate potential hosts, including those emitted by the host's habitat, the host itself, and microorganisms associated with the host. Given the great capacity of parasitoids to utilize and learn odours to increase foraging success, parasitoids of eggs, larvae, or pupae may respond to the same volatiles the adult stage of their hosts use when locating their resources, but compelling evidence is still scarce. In this study, using Saccharomyces cerevisiae we show that Trichopria drosophilae, a pupal parasitoid of Drosophila species, is attracted to the same yeast volatiles as their hosts in the adult stage, i.e. acetate esters. Parasitoids significantly preferred the odour of S. cerevisiae over the blank medium in a Y-tube olfactometer. Deletion of the yeast ATF1 gene, encoding a key acetate ester synthase, decreased attraction of T. drosophilae, while the addition of synthetic acetate esters to the fermentation medium restored parasitoid attraction. Bioassays with individual compounds revealed that the esters alone were not as attractive as the volatile blend of S. cerevisiae, suggesting that other volatile compounds also contribute to the attraction of T. drosophilae. Altogether, our results indicate that pupal parasitoids respond to the same volatiles as the adult stage of their hosts, which may aid them in locating oviposition sites.

Apibacter muscae sp. nov., a novel bacterial species isolated from house flies.

We describe the isolation and characterization of three bacterial isolates from the common house fly, Musca domestica, caught in Londerzeel, Belgium and Huye District, Rwanda. Although isolated from distinct geographical locations, the strains show >99 % identical 16S rRNA gene sequences and are <95 % identical to type strains of Apibacter species. Whole-genome sequences were obtained for all three strains. The genomes are 2.4-2.5 Mb with a G+C content of ~30.3 mol%. Bacteriological and biochemical analysis of the strains demonstrate distinctly different characteristics compared to known Apibacter species. Particularly, the three strains investigated in this study can be distinguished from the known Apibacter species (Apibacter mensalisand Apibacter adventoris) through urease and ß-glucosidase activities. Whole-cell fatty acid methyl ester analysis shows that the fatty acid composition of the novel strains is also unique. On the basis of phylogenetic, genotypic and phenotypic data, we propose to classify these isolates as representatives of a novel species of the genus Apibacter, Apibacter muscae sp. nov., in reference to its prevalence in house flies, with strain G8T (=LMG 30898T=DSM 107922T) as the type strain.

Microbial characterisation of the edible grasshopper Ruspolia differens in raw condition after wild-harvesting in Uganda.

This research aimed at establishing the chemical intrinsic properties and the microbial quality of an edible grasshopper Ruspolia differens and the effect of its source (geographical area) in Uganda, trading point, swarming season and plucking on these parameters. The intrinsic properties of the grasshopper can support the growth of a wide variety of microorganisms. High counts of total aerobic microbes, Enterobacteriaceae, lactic acid bacteria, total aerobic spores, and yeasts and moulds were obtained. Metagenetic analyses yielded 1793 Operational Taxonomic Units (OTUs) belonging to 24 phyla. Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were the most abundant phyla, while members of the genera Acinetobacter, Buttiauxella, Lactococcus, Staphylococcus and Undibacterium were the most abundant OTUs. A number of genera harbouring potential pathogens (Acinetobacter, Bacillus, Buttiauxella, Campylobacter, Clostridium, Staphylococcus, Pseudomonas and Neisseria) were identified. The geographical area, trading point, swarming season and plucking significantly influenced microbial counts and bacterial diversity. The high microbial counts predispose R. differens to fast microbial spoilage, while the presence of Clostridium and Campylobacter makes this grasshopper a potential source of food borne diseases. Further research should identify the specific spoilage microorganisms of R. differens and assess the characteristics of this grasshopper that support growth of food pathogens.

A comparative hierarchical analysis of bacterioplankton and biofilm metacommunity structure in an interconnected pond system.

It is unknown whether bacterioplankton and biofilm communities are structured by the same ecological processes, and whether they influence each other through continuous dispersal (known as mass effects). Using a hierarchical sampling approach we compared the relative importance of ecological processes structuring the dominant fraction (relative abundance ≥0.1%) of bacterioplankton and biofilm communities from three microhabitats (open water, Nuphar and Phragmites sites) at within- and among-pond scale in a set of 14 interconnected shallow ponds. Our results demonstrate that while bacterioplankton and biofilm communities are highly distinct, a similar hierarchy of ecological processes is acting on them. For both community types, most variation in community composition was determined by pond identity and environmental variables, with no effect of space. The highest ß-diversity within each community type was observed among ponds, while microhabitat type (Nuphar, Phragmites, open water) significantly influenced biofilm communities but not bacterioplankton. Mass effects among bacterioplankton and biofilm communities were not detected, as suggested by the absence of within-site covariation of biofilm and bacterioplankton communities. Both biofilm and plankton communities were thus highly structured by environmental factors (i.e., species sorting), with among-lake variation being more important than within-lake variation, whereas dispersal limitation and mass effects were not observed.

Microbial Dynamics during Industrial Rearing, Processing, and Storage of Tropical House Crickets (Gryllodes sigillatus) for Human Consumption.

In this study, the microbiota during industrial rearing, processing, and storage of the edible tropical house cricket, Gryllodessigillatus, was investigated. To this end, we analyzed samples from the cricket feed, obtained before feeding as well as from the cages, and from the crickets during rearing, after harvest, and after processing into frozen, oven-dried, and smoked and oven-dried (smoked/dried) end products. Although the feed contained lower microbial numbers than the crickets, both were dominated by the same species-level operational taxonomic units, as determined by Illumina MiSeq sequencing. They corresponded, among others, to members of Porphyromonadaceae, Fusobacterium, Parabacteroides, and Erwinia The harvested crickets contained high microbial numbers, but none of the investigated food pathogens Salmonella spp., Listeria monocytogenes, Bacillus cereus, or coagulase-positive staphylococci. However, some possible mycotoxin-producing fungi were isolated from the crickets. A postharvest heat treatment, shortly boiling the crickets, reduced microbial numbers, but an endospore load of 2.4 log CFU/g remained. After processing, an increase in microbial counts was observed for the dried and smoked/dried crickets. Additionally, in the smoked/dried crickets, a high abundance of a Bacillus sp. was observed. Considering the possible occurrence of food-pathogenic species from this genus, it is advised to apply a heat treatment which is sufficient to eliminate spores. Nevertheless, the microbial numbers remained constant over a 6-month storage period, whether frozen (frozen end product) or at ambient temperature (oven-dried and smoked/dried end products).IMPORTANCE The need for sustainable protein sources has led to the emergence of a new food sector, producing and processing edible insects into foods. However, insight into the microbial quality of this new food and into the microbial dynamics during rearing, processing, and storage of edible insects is still limited. Samples monitored for their microbiota were obtained in this study from an industrial rearing and processing cycle. The results lead first to the identification of process steps which are critical for microbial food safety. Second, they can be used in the construction of a Hazard Analysis and Critical Control Points (HACCP) plan and of a Novel Food dossier, which is required in Europe for edible insects. Finally, they confirm the shelf-life period which was determined by the rearer.

Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.

Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae, but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity.IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination.

Bioflavoring by non-conventional yeasts in sequential beer fermentations.

Non-conventional yeast species have great capacity for producing diverse flavor profiles in production of alcoholic beverages, but their potential for beer brewing, in particular in consecutive fermentations with Saccharomyces cerevisiae, has only poorly been explored. We have screened 17 non-conventional yeast species for production of an appealing profile of flavor esters and phenolics in the first phase of alcoholic fermentation, followed by inoculation with S. cerevisiae to complete the fermentation. For measurement of phenolic compounds and their precursors we developed an improved and highly sensitive methodology. The results show that non-conventional yeast species possess promising potential for enhancement of desirable flavors in beer production. Notable examples are increasing isoamyl acetate (fruity, banana flavor) by application of P. kluyverii, augmenting ethyl phenolic compounds (spicy notes) with Brettanomyces species and enhancing 4-vinyl guaiacol (clove-like aroma) with T. delbrueckii. All Pichia strains also produced high levels of ethyl acetate (solvent-like flavor). This might be selectively counteracted by selection of an appropriate S. cerevisiae strain for the second fermentation phase, which lowers total ester profile. Hence, optimization of the process conditions and/or proper strain selection in sequentially inoculated fermentations are required to unlock the full potential for aroma improvement by the non-conventional yeast species.

Mycorrhizal specificity does not limit the distribution of an endangered orchid species.

What factors determine the distribution of a species is a central question in ecology and conservation biology. In general, the distribution of plant species is assumed to be controlled by dispersal or environmentally controlled recruitment. For plant species which are critically dependent on mycorrhizal symbionts for germination and seedling establishment, specificity in mycorrhizal associations and availability of suitable mycorrhizal fungi can be expected to have a major impact on successful colonization and establishment and thus ultimately on a species distribution. We combined seed germination experiments with soil analyses and fungal assessments using 454 amplicon pyrosequencing to test the relative importance of dispersal limitation, mycorrhizal availability and local growth conditions on the distribution of the orchid species Liparis loeselii, which, despite being widely distributed, is rare and endangered in Europe. We compared local soil conditions, seed germination and mycorrhizal availability in the soil between locations in northern Belgium and France where L. loeselii occurs naturally and locations where conditions appear suitable, but where adults of the species are absent. Our results indicated that mycorrhizal communities associating with L. loeselii varied among sites and plant life cycle stages, but the observed variations did not affect seed germination, which occurred regardless of current L. loeselii presence and was significantly affected by soil moisture content. These results indicate that L. loeselii is a mycorrhizal generalist capable of opportunistically associating with a variety of fungal partners to induce seed germination. They also indicate that availability of fungal associates is not necessarily the determining factor driving the distribution of mycorrhizal plant species.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Phylogenetic signal in phenotypic traits related to carbon source assimilation and chemical sensitivity in Acinetobacter species.

A common belief is that the phylogeny of bacteria may reflect molecular functions and phenotypic characteristics, pointing towards phylogenetic conservatism of traits. Here, we tested this hypothesis for a large set of Acinetobacter strains. Members of the genus Acinetobacter are widespread in nature, demonstrate a high metabolic diversity and are resistant to several environmental stressors. Notably, some species are known to cause opportunistic human infections. A total of 133 strains belonging to 33 species with validly published names, two genomic species and species of an as-yet unknown taxonomic status were analyzed using the GENIII technology of Biolog, which allows high-throughput phenotyping. We estimated the strength and significance of the phylogenetic signal of each trait across phylogenetic reconstructions based on partial RNA polymerase subunit B (rpoB) and core genome sequences. Secondly, we tested whether phylogenetic distance was a good predictor of trait differentiation by Mantel test analysis. And finally, evolutionary model fitting was used to determine if the data for each phenotypic character was consistent with a phylogenetic or an essentially random model of trait distribution. Our data revealed that some key phenotypic traits related to substrate assimilation and chemical sensitivity are linked to the phylogenetic placement of Acinetobacter species. The strongest phylogenetic signals found were for utilization of different carbon sources such as some organic acids, amino acids and sugars, thus suggesting that in the diversification of Acinetobacter carbon source assimilation has had a relevant role. Future work should be aimed to clarify how such traits have shaped the remarkable ability of this bacterial group to dominate in a wide variety of habitats.

Species coexistence in simple microbial communities: unravelling the phenotypic landscape of co-occurring Metschnikowia species in floral nectar.

Identifying the ecological processes that underlie the distribution and abundance of species in microbial communities is a central issue in microbial ecology and evolution. Classical trade-off based niche theories of resource competition predict that co-occurrence in microbial communities is more likely when the residing species show trait divergence and complementary resource use. We tested the prediction that niche differentiation explained the co-occurrence of two yeast species (Metschnikowia reukaufii and M. gruessii) in floral nectar. Assessment of the phenotypic landscape showed that both species displayed a significantly different physiological profile. Comparison of utilization profiles in single versus mixed cultures indicated that these two species did not compete for most carbon and nitrogen sources. In mixed cultures, M. reukaufii grew better in sucrose solutions and in the presence of the antimicrobial compound digitonin than when grown as pure culture. M. gruessii, on the other hand, grew better in mixed cultures in glucose and fructose solutions. Overall, these results provide clear evidence that M. reukaufii and M. gruessii frequently co-occur in nectar and that they differ in their phenotypic response to variation in environmental conditions, suggesting that niche differentiation and resource partitioning are important mechanisms contributing to species co-occurrence in nectar yeast communities.