Metataxonomic analysis of endophytic bacteria of blackberry (Rubus ulmifolius Schott) across tissues and environmental conditions.

(1) Background: Endophytic bacteria represent an important component of plant wellness. They have been widely studied for their involvement in plant development and enhancement of stress tolerance. In this work, the endophytic communities of roots, stems, and leaves of blackberry (Rubus ulmifolius Schott) were studied in three different niches: natural, riverside, and human-impacted niches. (2) Results: The microbiome composition revealed that Sphingomonadaceae was the most abundant family in all samples, accounting for 9.4-45.8%. In contrast, other families seem to be linked to a specific tissue or niche. Families Microbacteriaceae and Hymenobacteraceae increased their presence in stem and leaf samples, while Burkholderiaceae abundance was important in riverside samples. Alpha and beta diversity analyses showed that root samples were the most diverse, and they gathered together in the same cluster, apart from the rest of the samples. (3) Conclusions: The analysis of the microbiome of R. ulmifolius plants revealed that the composition was essentially the same in different niches; the differences were primarily influenced by plant tissue factors with a core genome dominated by Sphingomonadaceae. Additionally, it was observed that R. ulmifolius can select its own microbiome, and this remains constant in all tissues evaluated regardless the niche of sampling.

Determining the hierarchical order by which intestinal tract, administered diet, and individual relay can shape the gut microbiome of fattening quails.

A bacterial metabarcoding approach was used to compare the microbiome composition of caecal and faecal samples from fattening Japanese quails (Coturnix coturnix japonica) fed three different diet regimes. The tested feedstuffs included (1) a commercial diet for fattening quails, (2) a commercial diet containing 12% full-fat silkworm (Bombyx mori) pupae meal, and (3) a commercial diet containing 12% defatted silkworm pupae meal. The aim of the experiment was to verify the relative effect of three variables (diet type, gut tract comparing caecum to rectum, and individual animal) in determining the level of bacterial community dissimilarity to rank the relevance of each of the three factors in affecting and shaping community composition. To infer such ranking, the communities resulting from the high-throughput sequencing from each sample were used to calculate the Bray-Curtis distances in all the pairwise combinations, whereby identical communities would score 0 and totally different ones would yield the maximum distance, equal to 1. The results indicated that the main driver of divergence was the gut tract, as distances between caecal and faecal samples were higher on average, irrespective of diet composition, which scored second in rank, and of whether they had been sampled from the same individual, which was the least effective factor. Simpson's species diversity indexes was not significantly different when comparing tracts or diets, while community evenness was reduced in full-fat silkworm diet-fed animals. The identities of the differentially displayed taxa that were statistically significant as a function of gut tract and diet regimen are discussed in light of their known physiological and functional traits.

The impact of milk storage temperatures on cheese quality and microbial communities at dairy processing plant scale.

Cheese production is an applied biotechnology whose proper outcome relies strictly on the complex interactive dynamics which unfold within defined microbial groups. These may start being active from the collection of milk and continue up to its final stages of maturation. One of the critical parameters playing a major role is the milk refrigeration temperature before pasteurization as it can affect the proportion of psychrotrophic taxa abundance in the total milk bacterial population. While a standard temperature of 4 °C is the common choice, due to its general growth control effect, it does have a potential drawback. This is due to the fact that some cold-tolerant genera present a proteolytic activity with uncompleted proliferation, which could negatively affect curd clotting and regular cheese maturation. Moreover, accidental thermal variations of milk before cheese-making, in a plus or minus direction, can occur both at farm collection sites and during transfer to dairy plant. This present research, directly commissioned by a major fresh cheese-producing company, includes an in-factory trial. In this trial, a gradient of temperatures from 4 °C to 13 °C, which were subsequently reversed, was purposely adopted to: (a) verify sensory alterations in the resulting product at different maturation stages, and, (b) analyze, in parallel, using DNA extraction and 16S-metabarcoding sequencing from the same samples, the presence, abundance and corresponding taxonomical identity of all the bacteria featured in communities found in milk and cheese samples. Overall, 1,714 different variants were detected and sorted into 394 identified taxa. Significant bacterial community shifts in cheese were observed in response to milk refrigeration temperature and subsequently associated with samples having altered scores in sensory panel tests. In particular, proteolytic psychrotrophes were outcompeted by Enterobacteriales and by other taxa at the peak temperature of 13 °C, but aggressively increased in the descent phases, upon the cooling down of milk to values of 7 °C. Relevant clues have been collected for better anticipation of thermal abuse effects or parameter variations allowing for improved handling of technical processing conditions by the cheese manufacturing industry.

Characterization of bacterial communities isolated from municipal waste compost and screening of their plant-interactive phenotypes.

Four batches of commercial compost obtained from the organic fraction of municipal solid waste were analyzed from chemical and microbiological standpoints. The working hypothesis was that, being this type of compost derived partly from plant waste, it could contain plant-growth promoting bacterial endophytes, prone to be active again upon its usual delivery as fertilizer. Culturable bacteria were isolated at different temperatures, quantified by colony morphology, identified taxonomically by 16S sequencing and screened for plant-growth promoting phenotypes including auxin and siderophore production, phosphate solubilization and peptide mineralization to ammonia. In parallel, the total community was assessed by culture independent DNA metabarcoding. The capability of plants to select, uptake and internally multiply bacteria from these compost samples was analyzed using grapevine in-vitro rooting cuttings from which acquired bacteria were reisolated, quantified and their identities determined as above. Major differences in compost bacterial composition were observed as function of the season, with the winter sample being rather distinct from the summer ones. Bacillales and Actinomycetales dominated the culturable communities while Alteromonadales, Oceanospirillales and Flavobacteriales prevailed in the total community. In spite of the challenging composting cycle conditions, the plant nature of the main input substrates appeared determinant in guaranteeing that 82% of the culturable bacteria were found endowed with one or more of the plant growth-promoting phenotypes tested. Beside its fertilization role, compost proved to be also a potential inoculant carrier for the in-soil delivery of plant beneficial microorganisms. Furthermore, upon an in vitro passage through grapevine plants under axenic conditions, the subsequently recoverable endophyte community yielded also members of the Rhizobiales order which had not been detectable when culturing directly from compost. This observation further suggests that compost-borne plant-interacting taxa could be also rescued from non-culturable states and/or enriched above detectability levels by a contact with their potential host plants.

Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera.

BACKGROUND: Bacillus licheniformis GL174 is a culturable endophytic strain isolated from Vitis vinifera cultivar Glera, the grapevine mainly cultivated for the Prosecco wine production. This strain was previously demonstrated to possess some specific plant growth promoting traits but its endophytic attitude and its role in biocontrol was only partially explored. In this study, the potential biocontrol action of the strain was investigated in vitro and in vivo and, by genome sequence analyses, putative functions involved in biocontrol and plant-bacteria interaction were assessed. RESULTS: Firstly, to confirm the endophytic behavior of the strain, its ability to colonize grapevine tissues was demonstrated and its biocontrol properties were analyzed. Antagonism test results showed that the strain could reduce and inhibit the mycelium growth of diverse plant pathogens in vitro and in vivo. The strain was demonstrated to produce different molecules of the lipopeptide class; moreover, its genome was sequenced, and analysis of the sequences revealed the presence of many protein-coding genes involved in the biocontrol process, such as transporters, plant-cell lytic enzymes, siderophores and other secondary metabolites. CONCLUSIONS: This step-by-step analysis shows that Bacillus licheniformis GL174 may be a good biocontrol agent candidate, and describes some distinguished traits and possible key elements involved in this process. The use of this strain could potentially help grapevine plants to cope with pathogen attacks and reduce the amount of chemicals used in the vineyard.

Quorum vs. diffusion sensing: a quantitative analysis of the relevance of absorbing or reflecting boundaries.

The consequences of the boundary conditions (signal reflecting vs. signal adsorbing) on bacterial intercellular communication were addressed by a combined physics and microbiology approach. A predictive biophysical model was devised that considered system size, diffusion from given points, signal molecule decay and boundary properties. The theoretical predictions were tested with two experimental agarose-gel-based set-ups for reflecting or absorbing boundaries. N-acyl homoserine lactone (AHL) concentration profiles were measured using the Agrobacterium tumefaciens NTL4 bioassay and found to agree with model predictions. The half-life of AHL was estimated to be 7 days. The absorbing vs. reflecting nature of the boundaries drastically changed AHL concentration profiles. The effect of a single nonreflecting boundary side was equivalent to a 100-fold lower cell concentration. Results suggest that the kinetics of signal accumulation vs. signal removal and their threshold-mediated phenotypic consequences are directly linked to the properties of biofilm boundaries, stressing the relevance of the diffusion sensing component in bacterial communication.

A unique midgut-associated bacterial community hosted by the cave beetle Cansiliella servadeii (Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors.

BACKGROUND: Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches. RESULTS: Abundant microbial cell masses were observed on the external appendages. Cansiliella's midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals' digestive traits. CONCLUSIONS: Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.

Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules.

In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants.

Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall.

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group.

Identification of two fungal endophytes associated with the endangered orchid Orchis militaris L.

A survey of the endangered orchid Orchis militaris populations was carried out in north-eastern Italy. The occurrence of fungal root endophytes was investigated by light and electron microscopies and molecular techniques. Two main sites of presence were individuated in the Euganean Hills, differing as to the percentage of flowering individuals and of capsules completing maturity. Fluorescence microscopy revealed an intracellular cortical colonization by hyphal pelotons. Two ITS PCR products co-amplified. Sequencing revealed for the former an identity and a high similarity (99%) with a Tulasnellaceae (Basidiomycota) fungus found within tissues of the same host in independent studies in Hungary and Estonia, suggesting an interesting case of tight specificity throughout the Eurosiberian home range. The second amplicon had 99% similarity with Tetracladium species (Ascomycota) recently demonstrated as potential endophytes. TEM revealed two different hyphal structures. Double fungal colonization appears to occur in Orchis militaris and the possible requirement of a specific fungal partner throws light on the causes of this plant's rarity and threatened status.

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes.

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.