Pseudomonas fortuita sp. nov., isolated from the endosphere of a wild yam.

A Gram-negative, strictly aerobic bacterial strain was isolated from asymptomatic leaf tissue of a wild yam plant. Optimal growth was observed at 28 °C and pH 7, and catalase and oxidase activities were detected. Polyphasic taxonomic and comparative genomics revealed that strain LMG 33091T represents a novel species of Pseudomonas. The nearest phylogenetic neighbours of strain LMG 33091T were Pseudomonas putida NBRC 14164T (with 99.79 % 16S rRNA sequence identity), Pseudomonas alkylphenolica KL28T (99.28 %) and Pseudomonas asplenii (99.07 %) ATCC 23835T. MALDI-TOF MS analysis yielded distinct profiles for strain LMG 33091T and the nearest phylogenetic neighbours. Average nucleotide identity analyses between the whole genome sequence of strain LMG 33091T and of the type strains of its nearest-neighbour taxa yielded values below the species delineation threshold and thus confirmed that the strain represented a novel Pseudomonas species, for which we propose the name Pseudomonas fortuita sp. nov., with strain LMG 33091T (=GMI12077T= CFBP 9143T) as the type strain.

Induction of antibiotic specialized metabolism by co-culturing in a collection of phyllosphere bacteria.

A diverse set of bacteria live on the above-ground parts of plants, composing the phyllosphere, and play important roles for plant health. Phyllosphere microbial communities assemble in a predictable manner and diverge from communities colonizing other plant organs or the soil. However, how these communities differ functionally remains obscure. We assembled a collection of 258 bacterial isolates representative of the most abundant taxa of the phyllosphere of Arabidopsis and a shared soil inoculum. We screened the collection for the production of metabolites that inhibit the growth of Gram-positive and Gram-negative bacteria either in isolation or in co-culture. We found that isolates capable of constitutive antibiotic production in monoculture were significantly enriched in the soil fraction. In contrast, the proportion of binary cultures resulting in the production of growth inhibitory compounds differed only marginally between the phyllosphere and soil fractions. This shows that the phyllosphere may be a rich resource for potentially novel molecules with antibiotic activity, but that production or activity is dependent upon induction by external signals or cues. Finally, we describe the isolation of antimicrobial acyloin metabolites from a binary culture of Arabidopsis phyllosphere isolates, which inhibit the growth of clinically relevant Acinetobacter baumannii.

The association between Dioscorea sansibarensis and Orrella dioscoreae as a model for hereditary leaf symbiosis.

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.

Motility-Independent Vertical Transmission of Bacteria in Leaf Symbiosis.

Hereditary symbioses have the potential to drive transgenerational effects, yet the mechanisms responsible for transmission of heritable plant symbionts are still poorly understood. The leaf symbiosis between Dioscorea sansibarensis and the bacterium Orrella dioscoreae offers an appealing model system to study how heritable bacteria are transmitted to the next generation. Here, we demonstrate that inoculation of apical buds with a bacterial suspension is sufficient to colonize newly formed leaves and propagules, and to ensure transmission to the next plant generation. Flagellar motility is not required for movement inside the plant but is important for the colonization of new hosts. Further, tissue-specific regulation of putative symbiotic functions highlights the presence of two distinct subpopulations of bacteria in the leaf gland and at the shoot meristem. We propose that bacteria in the leaf gland dedicate resources to symbiotic functions, while dividing bacteria in the shoot tip ensure successful colonization of meristematic tissue, glands, and propagules. Compartmentalization of intrahost populations together with tissue-specific regulation may serve as a robust mechanism for the maintenance of mutualism in leaf symbiosis. IMPORTANCE Hereditary symbioses with bacteria are common in the animal kingdom, but relatively unexplored in plants. Several plant species form associations with bacteria in their leaves, which is called leaf symbiosis. These associations are highly specific, but the mechanisms responsible for symbiont transmission are poorly understood. Using the association between the yam species Dioscorea sansibarensis and Orrella dioscoreae as a model leaf symbiosis, we show that bacteria are distributed to specific leaf structures via association with shoot meristems. Flagellar motility is required for initial infection but does not contribute to spread within host tissue. We also provide evidence that bacterial subpopulations at the meristem or in the symbiotic leaf gland differentially express key symbiotic genes. We argue that this separation of functional symbiont populations, coupled with tight control over bacterial infection and transmission, explain the evolutionary robustness of leaf symbiosis. These findings may provide insights into how plants may recruit and maintain beneficial symbionts at the leaf surface.

Adaptations and evolution of a heritable leaf nodule symbiosis between Dioscorea sansibarensis and Orrella dioscoreae.

Various plant species establish intimate symbioses with bacteria within their aerial organs. The bacteria are contained within nodules or glands often present in distinctive patterns on the leaves in what is commonly referred to as leaf nodule symbiosis. We describe here a highly specific symbiosis between a wild yam species from Madagascar, Dioscorea sansibarensis and bacteria of the species Orrella dioscoreae. Using whole-genome sequencing of plastids and bacteria from wild-collected samples, we show phylogenetic patterns consistent with a dominant vertical mode of transmission of the symbionts. Unique so far among leaf nodule symbioses, the bacteria can be cultured and are amenable to comparative transcriptomics, revealing a potential role in complementing the host's arsenal of secondary metabolites. We propose a recent establishment of a vertical mode of transmission in this symbiosis which, together with a large effective population size explains the cultivability and apparent lack of genome reductive evolution in O. dioscoreae. We leverage these unique features to reveal pathways and functions under positive selection in these specialized endophytes, highlighting the candidate mechanisms enabling a permanent association in the phyllosphere.