Bacterial Leaf Nodules Research Articles

Keetia nodulosa sp. nov. (Rubiaceae - Vanguerieae) of West-Central Africa: bacterial leaf nodulation discovered in a fourth genus and tribe of Rubiaceae

Keetia nodulosa Cheek, a cloud forest climber nearly endemic to Cameroon, with a single record from Nigeria, is described and illustrated. It is remarkable as the first known species to be recorded with bacterial leaf nodules (BLN) in the genus Keetia, and also, in the tribe Vanguerieae. Other genera in Rubiaceae with BLN are Psychotria (Psychotrieae-Rubioideae), Sericanthe (Coffeeae) and Pavetta (Pavetteae), both Ixoroideae/Dialypetalanthoideae. The BLN in Keetia (Vanguerieae) are illustrated for the first time here. The characteristics and significance of bacterial leaf nodulation in Keetia nodulosa are discussed in the context of rapidly growing knowledge on the subject in flowering plants. Keetia nodulosa is provisionally assessed using the 2012 IUCN standard as Endangered (EN B2ab(iii)). The importance of its conservation, and options for achieving this are discussed in the context of recent extinctions of other plant species in Cameroon. This discovery of a new cloud forest species is discussed in relation to other cloud forest plant species described in the last twenty years which are also distributed over the highlands of the western half of Cameroon.

Is the bacterial leaf nodule symbiosis obligate for Psychotria umbellata? The development of a Burkholderia-free host plant.

Background & aimsThe bacterial leaf nodule symbiosis is an interaction where bacteria are housed in specialised structures in the leaves of their plant host. In the Rubiaceae plant family, host plants interact with Burkholderia bacteria. This interaction might play a role in the host plant defence system. It is unique due to its high specificity; the vertical transmission of the endophyte to the next generation of the host plant; and its supposedly obligatory character. Although previous attempts have been made to investigate this obligatory character by developing Burkholderia-free plants, none have succeeded and nodulating plants were still produced. In order to investigate the obligatory character of this endosymbiosis, our aims were to develop Burkholderia-free Psychotria umbellata plants and to investigate the effect of the absence of the endophytes on the host in a controlled environment.MethodsThe Burkholderia-free plants were obtained via embryo culture, a plant cultivation technique. In order to analyse the endophyte-free status, we screened the plants morphologically, microscopically and molecularly over a period of three years. To characterise the phenotype and growth of the in vitro aposymbiotic plants, we compared the growth of the Burkholderia-free plants to the nodulating plants under the same in vitro conditions.Key resultsAll the developed plants were Burkholderia-free and survived in a sterile in vitro environment. The growth analysis showed that plants without endophytes had a slower development.ConclusionsEmbryo culture is a cultivation technique with a high success rate for the development of Burkholderia-free plants of P. umbellata. The increased growth rate in vitro when the specific endophyte is present cannot be explained by possible benefits put forward in previous studies. This might indicate that the benefits of the endosymbiosis are not yet completely understood.

Detection of Burkholderia in the seeds of Psychotria punctata (Rubiaceae) - Microscopic evidence for vertical transmission in the leaf nodule symbiosis.

Background and aimsThe bacterial leaf nodule symbiosis is a close interaction between endophytes and their plant hosts, mainly within the coffee family. The interaction between Rubiaceae species and Burkholderia bacteria is unique due to its obligate nature, high specificity, and predominantly vertical transmission of the endophytes to the next generation of host plants. This vertical transmission is intriguing since it is the basis for the uniqueness of the symbiosis. However, unequivocal evidence of the location of the endophytes in the seeds is lacking. The aim of this paper is therefore to demonstrate the presence of the host specific endophyte in the seeds of Psychotria punctata and confirm its precise location. In addition, the suggested location of the endophyte in other parts of the host plant is investigated.MethodsTo identify and locate the endophyte in Psychotria punctata, a two-level approach was adopted using both a molecular screening method and fluorescent in situ hybridisation microscopy.Key resultsThe endophytes, molecularly identified as Candidatus Burkholderia kirkii, were detected in the leaves, vegetative and flower buds, anthers, gynoecium, embryos, and young twigs. In addition, they were in situ localised in leaves, flowers and shoot apical meristems, and, for the first time, in between the cotyledons of the embryos.ConclusionsBoth independent techniques detected the host specific endophyte in close proximity to the shoot apical meristem of the embryo, which confirms for the first time the exact location of the endophytes in the seeds. This study provides reliable proof that the endophytes are maintained throughout the growth and development of the host plant and are transmitted vertically to the offspring.

Evidence of horizontal gene transfer between obligate leaf nodule symbionts.

Bacteria of the genus Burkholderia establish an obligate symbiosis with plant species of the Rubiaceae and Primulaceae families. The bacteria, housed within the leaves, are transmitted hereditarily and have not yet been cultured. We have sequenced and compared the genomes of eight bacterial leaf nodule symbionts of the Rubiaceae plant family. All of the genomes exhibit features consistent with genome erosion. Genes potentially involved in the biosynthesis of kirkamide, an insecticidal C7N aminocyclitol, are conserved in most Rubiaceae symbionts. However, some have partially lost the kirkamide pathway due to genome erosion and are unable to synthesize the compound. Kirkamide synthesis is therefore not responsible for the obligate nature of the symbiosis. More importantly, we find evidence of intra-clade horizontal gene transfer (HGT) events affecting genes of the secondary metabolism. This indicates that substantial gene flow can occur at the early stages following host restriction in leaf nodule symbioses. We propose that host-switching events and plasmid conjugative transfers could have promoted these HGTs. This genomic analysis of leaf nodule symbionts gives, for the first time, new insights in the genome evolution of obligate symbionts in their early stages of the association with plants.

Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species.

• Premise of the study: The pantropical, species-rich Psychotrieae and Palicoureeae are sister tribes of mostly drupe-bearing and nonbacterial leaf-nodulating species with problematic generic limits. This problem is more complicated in Psychotrieae due to the paraphyly of the genus Psychotria, the lack of diagnostic characters for some major lineages, and the poor sampling from some biodiversity hotspots. Schizocarps and bacterial leaf nodules have been used for recognizing formal groups in Psychotrieae, but their evolution and taxonomic value have not been studied using a robust phylogeny of the tribe.• Methods: We analyzed 287 samples from the entire ranges of the tribes, with particular emphasis on the Western Indian Ocean region, with the Bayesian Markov chain Monte Carlo method.• Key results: All allied Psychotria genera investigated are nested within a paraphyletic Psychotria. Schizocarps evolved independently two times within Psychotria, and one reversal back to the drupaceous condition is inferred. The Malagasy leaf-nodulated Psychotrieae (except Apomuria bullata) and the Comorian non-leaf-nodulated Psychotria conocarpa are nested within the (African) leaf-nodulated clade. Within Palicoureeae, Chassalia is paraphyletic with respect to Geophila sensu stricto, and the Malagasy Geophila gerrardii and the African Hymenocoleus are closely related.• Conclusions: A widely circumscribed Psychotria encompassing the entire Psychotrieae is supported. Within Psychotria, two separate origins of schizocarps from drupes, one reversal back to the drupaceous condition, and two independent origins of the Malagasy leaf-nodulated species are inferred. A new genus Puffia is described to accommodate Geophila gerrardii, and a narrow circumscription of Chassalia is adopted. Thirty-two new combinations, two lectotypifications, and 25 new names are presented.

Screening for leaf-associated endophytes in the genus Psychotria (Rubiaceae)

Burkholderia endophytes were identified within the leaves of non-nodulated members of the genus Psychotria. In contrast to leaf-nodulated Psychotria species, which are known to accommodate their endosymbionts into specialized endosymbiont-housing structures, non-nodulated species lack bacterial leaf nodules and harbor endosymbionts intercellularly between mesophyll cells. Based on molecular data (rps16, trnG, and trnLF), the phylogenetic reconstruction of the host plants revealed a separate origin of leaf-nodulated and non-nodulated Psychotria species. Despite a distinct phylogenetic position of the two host clades, the endophytes of the non-nodulated plants were not placed into a single monophyletic group but were found to be closely related to the leaf-nodulated endosymbionts. The observation of genetically similar endophytes in both nodulated and non-nodulated Psychotria lineages suggests that the host plant is playing a crucial role in the induction of leaf nodule formation. Moreover, the concentration of endosymbionts into specialized leaf nodules may be considered as a more derived evolutionary adaptation of the host plant, serving as an interface structure to facilitate metabolic exchange between plant and endosymbiont.

Coléteres dendróides em Alibertia sessilis (Vell.) K. Schum., uma espécie não-nodulada de Rubiaceae

This paper describes the distribution, structure and histochemistry of the colleters in vegetative and reproductive apices of Alibertia sessilis (Vell.) K. Schum., a native Rubiaceae species of the Brazilian cerrado. In this family, dendroid colleters have been associated with the presence of bacterial nodules in the leaves. Samples were prepared according to common light and scanning electron microscopy techniques, and histochemical assays involved freshly collected material. Colleters are dendroid and occur on the adaxial surface of stipules, bracts and sepals; they consist of a multicellular and multiseriate central axis of parenchymatous nature, covered by digitiform or sharply pointed epidermal cells of irregular sizes, joined together only in the proximal portion and separated from each other in the distal portion. The columnar cells are axially elongated, with thick cell walls, prominent nucleus and reduced cytoplasm, developed vacuole with lipid inclusions and phenolic compounds. The epidermal cells have thin walls, a conspicuous nucleus, abundant cytoplasm and a poorly developed vacuome. The colleters are devoid of cuticle. The secretion is more abundant in the vegetative apex, impregnating the leaf buds and the stipules, forming a spherical cap that is hard, bright, and hydrophobic. In all analyzed samples, the histochemical assays detected polysaccharides, proteins and lipids in the protoplast of both epidermal and columnar cells; phenolic compounds were detected solely in the columnar cells. The resinous nature of the exudate was confirmed with the use of the diethylic ether solvent. Bacterial leaf nodules were not found.

Structural, Functional and Phylogenetic Aspects of the Colleter

This article surveys the structural, functional and phylogenetic significance of colleters in different dicotyledonous families. Colleters are multicellular secretory structures attached to the stipule, petiole, lamina, bract, bracteole, calyx and corolla. Colleters are grouped into standard (S), dendroid (D) and brush-like (B) types on the basis of their morphology and structure. D and B-type colleters occur in certain members of Rubiaceae that also have bacterial leaf nodules. Besides the normal structure, epithelial hairs, thin-walled subepidermal cells, laticifers and vasculature are present in many colleters of Apocynaceae. It is probable that the colleter functions to protect the developing meristem by secreting a viscous fluid. Exudate of D-type colleters are mucilaginous, providing the substrate necessary for the nutrition of endophytic bacteria.

Location and distribution of symbiotic bacteria during floral development in Ardisia crispa

Abstract. The location and distribution of symbiotic bacteria during floral development in Ardisia crispa (Thunb.) A.DC., a species characterized by bacterial leaf nodules, has been studied using light, scanning and transmission electron microscopy. During early floral development, bacteria in mucilage derived from host plant trichomes, become enclosed in a small conical chamber on top of the placenta, as a result of the closure and fusion of the carpel initials. The placental epidermal cells, which appear to be secretory in nature, become detached apically in places forming a network of grooves which traverse the placental surface. The symbiotic bacteria are preferentially located in these grooves. As growth and development of the placenta proceed, the grooves widen and deepen to form channels. The cells lining these channels secrete a mucilaginous material. The network of channels covers the entire placental surface and terminates at the placental margins surrounding the ovules. Bacteria are found within the channels, at the ends of the channels near the margin of the placenta, on the surface of the ovules and in the micropyle. It is suggested that these mucilage‐filled channels are responsible for, and a prerequisite of, ensuring that the bacterial partner is efficiently transmitted from one host generation to the next by providing a mechanism by which the bacteria arc accurately placed within the developing seed.

Plant and Bacterial Cell Wall Deformations in the Functional Part of the Bacterial Leaf Nodule of Spearflower [Ardisia crispa (Thunb.) A.DC., Myrsinaceae

Plant and Bacterial Cell Wall Deformations in the Functional Part of the Bacterial Leaf Nodule of Spearflower [<i>Ardisia crispa</i> (Thunb.) A.DC., Myrsinaceae

Trichome forms in Ardisia (Myrsinaceae) in relation to the bacterial leaf nodule symbiosis

Symbiotic leaf-nodule bacteria in nodulated members of Rubiaceae live in mucilage secreted by colleters located on stipules within buds. These differ from colleters on most nodule-free species. This study was undertaken to examine buds of Ardisia and the related monotypic Amblyanthus of Myrsinaceae to see if nodulated species had secretory structures dissimilar from those of nodule-free species. Buds removed from herbarium specimens (61 species) and live plants (3 species) were paraffin-sectioned. Diverse trichome forms occur, including dimorphism between adaxial and abaxial trichomes in some species. Species within each subgenus were arranged according to trichome form: peltate scale, irregularly capitate, capitate, sessile capitate, bicellular capitate, and uniseriate. Only seven Ardisia species (all in subgenus Crispardisia, widely assumed to have bacteria in marginal leaf nodules of all 30 species) have short-lived trichomes bearing one or more elongate, swollen, distal cells that appear to be secretory cells. These trichomes are analogous to the dendroid or brushlike colleters of nodulated Rubiaceae. Druses occur in most subgenera and this appears to be by far the most predominant crystal type m Ardisia.

Bacterial leaf nodule symbiosis in angiosperms with emphasis on Rubiaceae and Myrsinaceae

Over 400 species of three genera of Rubiaceae and one genus of Myrsinaceae reportedly have bacterial leaf nodules. Light and/or electron microscope studies of a few species have shown that bacteria exist in spaces within buds filled with mucilage secreted by glands. These bacteria enter substomatal chambers (Rubiaceae) or marginal hydathodes (Myrsinaceae) and establish short-lived colonies, in intercellular spaces, that die out almost before full leaf expansion. Bacteria occur in seeds between endosperm and embryo, but only two studies have followed bacteria into flowers and ovules. Previous work on the physical relations of bacteria and host plants is discussed critically. Reviewing work done on isolation and identification of presumed endophytes leads to the conclusion that there is no agreement whether one or several bacterial taxa are the endophyte, and no unambiguous identifications, although four genera are suggested as possibilities. Nitrogen fixation was considered as the bacterial contribution until quite recently, but a review of such studies reveals that fixation has been detected almost exclusively in isolated presumed endophytes, whereas almost all studies involving the bacterium in intact leaves have failed to detect nitrogen fixation. Studies of particular substances (besides combined nitrogen) contributed by the endophyte have been inconclusive, although the most recent works suggest that cytokinins are involved. Host plants lacking the endophyte have been reportedly produced many times, either spontaneously or by seed treatment. Such “cripples”, used for several aspects of symbiosis study, frequently revert to a nodulated condition, and a more reliable method of producing them is needed. Tissue culture may offer the best potential, but this approach has not yet produced whole bacteria-free plants. A proposed scheme for the evolution of the symbiosis suggests that a variety of bacteria entered buds first, and only in rare instances were compatible with the host bud mucilage. In a few of these cases, specific bacteria, compatible with the microenvironment, contributed a useful substance to the host, and bud mucilage and those bacteria co-evolved until large numbers of bacteria thrived in the buds. Nodules may have resulted from accidental entry of bacteria into leaves, with the possibility that some host plant nodules are merely pathogenic responses, whereas in others the bacteria are beneficial and further selection has resulted in numerous, regularly produced nodules. This review deals with taxonomy of host plants and endophytes, morphology of the symbiosis, its physiology, and speculation on the evolution of the symbiosis.

Colleter types in Rubiaceae, especially in relation to the bacterial leaf nodule symbiosis

Colleter types in Rubiaceae, especially in relation to the bacterial leaf nodule symbiosis

Morphology and Distribution of Colleters and Crystals in Relation to the Taxonomy and Bacterial Leaf Nodule Symbiosis of Psychotria (Rubiaceae)

Earlier investigators established that a finger-like colleter with elongate axial cells and a palisade epidermis is the standard type in the Rubiaceae. This type, with some variation, also prevails in Psychotria, based on a worldwide anatomical survey of vegetative buds from herbarium specimens of 296 species (about 50 % of total). It is virtually the only type found outside of continental Africa. Among African species, it is most common in subgenus Psychotria, with mostly nodule-free species. In subgenus Tetramerae, with only leaf-nodulated species, there is a strong tendency toward brushlike and dendroid colleters in which epidermal cells are extremely elongate and separated from each other. It is speculated that this change in colleter morphology associated with presence of nodule bacteria may be correlated with a change in secretion product more suitable for support of bacteria. Three morphological forms of crystal occur: raphides, styloids, clustered crystals. They may occur singly or in combination. Several patterns and trends were noted in crystal distribution that could be of taxonomic significance.

Colleter morphology in Pavetta, Neorosea and Tricalysia (Rubiaceae) and its relationship to the bacterial leaf nodule symbiosis

Buds removed from herbarium specimens were processed for light microscopic examination of colleter form and anatomy. Most Pavetta species have dendroid colleters and bacterial leaf nodules, a correlation also found in an earlier study of Psychotria. Colleters of Neorosea, another genus with leaf-nodulated species, are more like the standard rubiaceous type that predominates in the family except that they have irregular, bulging epidermal cells. Tricalysia, a nodule-free genus closely related to Neorosea, shows a range of colleter form from standard to dendroid, and some species have the Neorosea type of colleter. Such morphological correlation between the bacterial leaf nodule symbiosis in Pavetta and Psychotria and dendroid colleters may indicate a chemical change in colleter secretion. Apart from their involvement with the bacterial symbiosis, rubiaceous colleters have now been shown to vary sufficiently in certain taxa to be considered as additional useful taxonomic characters.

Developmental Aspects of Bacterial Leaf Nodules in Psychotria bacteriophila Val. (Rubiaceae)

The ontogeny and biological activity of bacterial leaf nodules in Psychotria bacteriophila were studied. The acridine-orange fluorescence method coupled with ribonuclease extractions showed that young and developing nodules were rich in ribonucleic acid (RNA), whereas nodules in older leaves displayed less RNA fluorescence, and nodules in leaves near abscission did not fluoresce. Fluorescence was attributed to normal and dividing ribosome-rich bacteria. As nodules age, the bacterial population becomes crowded, and degenerating bacteria appear. The latter become a membrane mass which fills the nodule network, replacing the bacteria. The loss of fluorescence is therefore correlated with bacterial cell degeneration. These results suggest that the nodule activity is high in young and maturing leaves and decreases in mature leaves and in leaves nearing abscission. Therefore, the presumed beneficial aspects of this symbiosis appear to occur early in nodule development.

Development and structure of bacterial leaf nodules in Psychotria bacteriophila Val. (Rubiaceae).

The development and mature structure of bacterial leaf nodules in Psychotria bacteriophila were studied by using light and electron microscopy. Bacteria in mucilage surrounding the shoot apex pass through certain stomates in leaf primordia into the substomatal chamber. These chambers enlarge and become nodules as the yound leaves grow out of the apical region. Surrounding mesophyll cells grow into each nodule and form a cellular reticulum whose interstices are occupied by bacteria. Each intrusive mesophyll cell wall is unusually thick and continually supplemented by vesicles originating from dictyosomes. The gram-negative bacteria are often surrounded by capsules. Nodule bacteria contain several crystal-like dense bodies. A population of normal, dividing, and degenerating bacteria is found in each nodule. Extensive membranes occur between the bacteria. A hypothesis is proposed to explain certain aspects of this obligate symbiotic relationship.