Andrea Campisano

The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes

SUMMARY All plants are inhabited internally by diverse microbial communities comprising bacterial, archaeal, fungal, and protistic taxa. These microorganisms showing endophytic lifestyles play crucial roles in plant development, growth, fitness, and diversification. The increasing awareness of and information on endophytes provide insight into the complexity of the plant microbiome. The nature of plant-endophyte interactions ranges from mutualism to pathogenicity. This depends on a set of abiotic and biotic factors, including the genotypes of plants and microbes, environmental conditions, and the dynamic network of interactions within the plant biome. In this review, we address the concept of endophytism, considering the latest insights into evolution, plant ecosystem functioning, and multipartite interactions.

Human Host Defense Peptide LL-37 Prevents Bacterial Biofilm Formation

ABSTRACT The ability to form biofilms is a critical factor in chronic infections by Pseudomonas aeruginosa and has made this bacterium a model organism with respect to biofilm formation. This study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37. In addition to its key role in modulating the innate immune response and weak antimicrobial activity, LL-37 potently inhibited the formation of bacterial biofilms in vitro. This occurred at the very low and physiologically meaningful concentration of 0.5 μg/ml, far below that required to kill or inhibit growth (MIC = 64 μg/ml). LL-37 also affected existing, pregrown P. aeruginosa biofilms. Similar results were obtained using the bovine neutrophil peptide indolicidin, but no inhibitory effect on biofilm formation was detected using subinhibitory concentrations of the mouse peptide CRAMP, which shares 67% identity with LL-37, polymyxin B, or the bovine bactenecin homolog Bac2A. Using microarrays and follow-up studies, we were able to demonstrate that LL-37 affected biofilm formation by decreasing the attachment of bacterial cells, stimulating twitching motility, and influencing two major quorum sensing systems (Las and Rhl), leading to the downregulation of genes essential for biofilm development.

Fungal Endophytic Communities in Grapevines (Vitis vinifera L.) Respond to Crop Management

ABSTRACT We studied the distribution of fungal endophytes of grapevine (Vitis vinifera L.) plants in a subalpine area of northern Italy, where viticulture is of high economic relevance. We adopted both cultivation-based and cultivation-independent approaches to address how various anthropic and nonanthropic factors shape microbial communities. Grapevine stems were harvested from several locations considering organic and integrated pest management (IPM) and from the cultivars Merlot and Chardonnay. Cultivable fungi were isolated and identified by internal-transcribed-spacer sequence analysis, using a novel colony-PCR method, to amplify DNA from fungal specimens. The composition of fungal communities was assessed using a cultivation-independent approach, automated ribosomal intergenic spacer analysis (ARISA). Multivariate statistical analysis of both culture-dependent and culture-independent data sets was convergent and indicated that fungal endophytic communities in grapevines from organically managed farms were different from those from farms utilizing IPM. Fungal communities in plants of cv. Merlot and cv. Chardonnay overlapped when analyzed using culture-dependent approaches but could be partially resolved using ARISA fingerprinting.

Bacterial endophytic communities in the grapevine depend on pest management.

Microbial plant endophytes are receiving ever-increasing attention as a result of compelling evidence regarding functional interaction with the host plant. Microbial communities in plants were recently reported to be influenced by numerous environmental and anthropogenic factors, including soil and pest management. In this study we used automated ribosomal intergenic spacer analysis (ARISA) fingerprinting and pyrosequencing of 16S rDNA to assess the effect of organic production and integrated pest management (IPM) on bacterial endophytic communities in two widespread grapevines cultivars (Merlot and Chardonnay). High levels of the dominant Ralstonia, Burkholderia and Pseudomonas genera were detected in all the samples We found differences in the composition of endophytic communities in grapevines cultivated using organic production and IPM. Operational taxonomic units (OTUs) assigned to the Mesorhizobium, Caulobacter and Staphylococcus genera were relatively more abundant in plants from organic vineyards, while Ralstonia, Burkholderia and Stenotrophomonas were more abundant in grapevines from IPM vineyards. Minor differences in bacterial endophytic communities were also found in the grapevines of the two cultivars.

Impact of Alginate Overproduction on Attachment and Biofilm Architecture of a Supermucoid Pseudomonas aeruginosa Strain

ABSTRACT The supermucoid Pseudomonas aeruginosa strain PDO300Δalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.

Interkingdom transfer of the acne causing agent, Propionibacterium acnes, from human to grapevine

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa.

ABSTRACT The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.

Editorial special issue: soil, plants and endophytes

Despite the inception of the endophyte research dates back to the nineteenth century, studies on this topic have only recently bloomed, producing a fast-growing body of literature (Compant et al. 2012; Hardoim et al. 2015; Saikkonen et al. 2016). Several definitions of endophyte have been furthermore proposed; while some have been criticized, others have been suggested to better define this particular lifestyle. We currently describe endophytes as any microbe that can be isolated from asymptomatic plant tissue (Chanway 1995; Wilson 1995; Hardoim et al. 2015). This definition includes neutral, commensal and/or beneficial microorganisms as well as dormant saprobes and pathogens during their latent phase of their life cycle. These microorganisms engage an intimate association within their host plants, establishing a microbial community in the plant endosphere and could be found in various plant parts (Hardoim et al. 2015). Although endophytes have been previously extensively studied, the interplay between different endophytes and their hosts is however yet poorly understood. The mechanisms underlying these interactions, the driving forces determining specific microbial community assemblages found in different plant organs, the influence that a range of factors (e.g. environmental, genetic, phenological, etc.) have on shaping the endophytic microbiomes, or how to exploit/harness the benefits these inner microbial consortia provide to their hosts are just a few exciting matters currently under study. Nevertheless, novel and powerful methodological approaches already available must be implemented to obtain a better picture of this complex and fascinating ecological niche (i.e. the host plant and its inner microbiome). Despite descriptive and comprehensive research is done, important gaps in our knowledge are evident, and thorough insights are still needed to fully understand these particular plant-microbe interactions. We need, for instance, to move beyond cataloguing microorganism identities in different plant organs/tissues, or from plants growing in different soils and/or under diverse environmental conditions. Indeed, providing more hypothesis-driven studies will help answering Plant Soil (2016) 405:1–11 DOI 10.1007/s11104-016-2927-9

Diversity in Endophyte Populations Reveals Functional and Taxonomic Diversity between Wild and Domesticated Grapevines

Endophytes hold great potential for implementing a more sustainable, bio-based agriculture. Here we investigated the differences among populations of endophytic bacteria isolated from wild and domesticated grapevines to improve our understanding of how agriculture and agro-environment affect endophytic communities. Bacterial isolates from 88 wild and domesticated grapevines that shared the same climate for at least four years were identified and a representative set of 155 strains was characterized for 30 features, including quorum sensing-related, enzyme production, antibiotic resistance, plant growth promotion (PGP), and biocontrol (BiCo) traits. Microbial diversity was greater in wild grapevines (25 genera) than in domesticated grapevines (six genera). Molecular fingerprinting by automated ribosomal intergenic spacer analysis also indicated a greater diversity in wild grapevines. Multivariate analysis of phenotypic traits indicated that strains isolated from wild and cultivated plants often formed distinct clusters, even when they belonged to the same genus. Interestingly, endophytes from domesticated grapevines were more likely to perform well in PGP and BiCo tests than endophytes from wild grapevines, suggesting that the decreased taxonomic diversity in domesticated grapevine microbiota did not correspond to a loss of agriculturally relevant traits.

The polyhydroxyalkanoate biosynthesis genes are differentially regulated in planktonic- and biofilm-grown Pseudomonas aeruginosa

The planktonic and biofilm growth mode, the presence of polyhydroxyalkanoate (PHA) granules and the nitrogen availability were considered as parameters to study the regulation of genes involved in PHA biosynthesis in Pseudomonas aeruginosa. The transcriptional start point of the phaIF gene cluster, encoding PHA granule-associated proteins with proposed regulatory function, was experimentally verified. Gfp and lacZ transcriptional reporter fusions, respectively, were used to analyse promoter activities. In planktonic growth under nitrogen limitation, the phaC promoter (PphaC) showed increased induction, while in the PHA-negative mutant the activity of PphaC was reduced to 25% of the wild type and was independent of nitrogen availability. Promoter activity in biofilms was assessed using 2, 0.05 or 0 g/l of NH(4)Cl as nitrogen source, respectively. PphaC activity was increased during early biofilm growth, whereas in mature biofilms PphaC activity was preferentially localised to stalks of microcolonies. Nitrogen starvation led to an increased PphaC activity at the biofilm surface. PHA granule formation was confirmed by electron microscopy in planktonic and in biofilm cells. PphaI activity in planktonic cultures was less dependent on the conditions assayed and presented an oscillatory behaviour. In biofilms, PphaI was strongly activated during early stages of biofilm development, but was inactive in mature biofilms. Under nitrogen starvation PphaI activation resembled that of PphaC. These data suggested a differential regulation of PHA biosynthesis genes in planktonic and biofilm cells, as well as an important regulatory function of PhaF, when considering nitrogen availability. Interestingly, in biofilms PHA biosynthesis gene regulation showed a spatial distribution similar to rhamnolipid biosynthesis genes.