Genetic diversity of symbiotic Bradyrhizobium elkanii populations recovered from inoculated and non-inoculated Acacia mangium field trials in Brazil

Acacia mangium is a legume tree native to Australasia. Since the eighties, it has been introduced into many tropical countries, especially in a context of industrial plantations. Many field trials have been set up to test the effects of controlled inoculation with selected symbiotic bacteria versus natural colonization with indigenous strains. In the introduction areas, A. mangium trees spontaneously nodulate with local and often ineffective bacteria. When inoculated, the persistence of inoculants and possible genetic recombination with local strains remain to be explored. The aim of this study was to describe the genetic diversity of bacteria spontaneously nodulating A. mangium in Brazil and to evaluate the persistence of selected strains used as inoculants. Three different sites, several hundred kilometers apart, were studied, with inoculated and non-inoculated plots in two of them. Seventy-nine strains were isolated from nodules and sequenced on three housekeeping genes (glnII, dnaK and recA) and one symbiotic gene (nodA). All but one of the strains belonged to the Bradyrhizobium elkanii species. A single case of housekeeping gene transfer was detected among the 79 strains, suggesting an extremely low rate of recombination within B. elkanii, whereas the nodulation gene nodA was found to be frequently transferred. The fate of the inoculant strains varied depending on the site, with a complete disappearance in one case, and persistence in another. We compared our results with the sister species Bradyrhizobium japonicum, both in terms of population genetics and inoculant strain destiny.     

Chytrid infecting the bloom-forming marine diatom Skeletonema sp.: Morphology,phylogeny and distribution of a novel species within the Rhizophydiales

Chytrids have long been recognised as important parasites of microalgae in freshwater systems, able to shape the dynamics of blooms, the gene pool of their host and phytoplankton succession. In the sea however, where the presence of these organisms is erratic and ephemeral, studies concerning chytrids are sparse and confined to metabarcoding surveys or microscopy observations. Despite the scarcity of data, chytrid epidemics are supposed to play an important role in marine biogeochemical cycles, being one of the drivers of phytoplankton dynamics. Here we combine microscopy observations and in silico mining of a single-cell whole genome to molecularly and morphologically characterise a novel chytrid parasite of the dominant diatom genus Skeletonema. Morphological observations highlight features of the thallus and ascertain the parasitic nature of the interaction whilst the genetic markers obtained allows for a phylogenetic reconstruction, placing the new species in the order Rhizophydiales. Thanks to the molecular data obtained we are also able to provide a first investigation of the global distribution of this organism by screening the Ocean Sampling Day (OSD) dataset, highlighting a northern transatlantic dissemination.     

Differing Courses of Genetic Evolution of Bradyrhizobium Inoculants as Revealed by Long-Term Molecular Tracing in Acacia mangium Plantations

Introducing nitrogen-fixing bacteria as an inoculum in association with legume crops is a common practice in agriculture. However, the question of the evolution of these introduced microorganisms remains crucial, both in terms of microbial ecology and agronomy. We explored this question by analyzing the genetic and symbiotic evolution of two Bradyrhizobium strains inoculated on Acacia mangium in Malaysia and Senegal 15 and 5 years, respectively, after their introduction. Based on typing of several loci, we showed that these two strains, although closely related and originally sampled in Australia, evolved differently. One strain was recovered in soil with the same five loci as the original isolate, whereas the symbiotic cluster of the other strain was detected with no trace of the three housekeeping genes of the original inoculum. Moreover, the nitrogen fixation efficiency was variable among these isolates (either recombinant or not), with significantly high, low, or similar efficiencies compared to the two original strains and no significant difference between recombinant and nonrecombinant isolates. These data suggested that 15 years after their introduction, nitrogen-fixing bacteria remain in the soil but that closely related inoculant strains may not evolve in the same way, either genetically or symbiotically. In a context of increasing agronomical use of microbial inoculants (for biological control, nitrogen fixation, or plant growth promotion), this result feeds the debate on the consequences associated with such practices.     

“Ectrogella” Parasitoids of the Diatom Licmophora sp. are Polyphyletic

The diatom genera Licmophora and Fragilaria are frequent epiphytes on marine macroalgae and can be infected by intracellular parasitoids traditionally assigned to the oomycete genus Ectrogella. Much debate and uncertainty remains about the taxonomy of these oomycetes, not least due to their morphological and developmental plasticity. Here, we used single‐cell techniques to obtain partial sequences of the parasitoids 18S and cox2 genes. The former falls into two recently identified clades of Pseudo‐nitzschia parasites temporarily named OOM_1_2 and OOM_2, closely related to the genera of brown and red algal pathogens Anisolpidium and Olpidiopsis. A third group of sequences falls at the base of the red algal parasites assigned to Olpidiopsis. In one instance, two oomycete parasitoids seemed to co‐exist in a single diatom cell; this co‐occurrence of distinct parasitoid taxa not only within a population of diatom epiphytes, but also within the same host cell, possibly explains the ongoing confusion in the taxonomy of these parasitoids. We demonstrate the polyphyly of Licmophora parasitoids previously assigned to Ectrogella (sensu Sparrow, 1960) and show that parasites of red algae assigned to the genus Olpidiopsis are most likely not monophyletic. We conclude that combining single‐cell microscopy and molecular methods is necessary for their full characterisation.     

Effects of Medicago truncatula Genetic Diversity, Rhizobial Competition, and Strain Effectiveness on the Diversity of a Natural Sinorhizobium Species Community

We investigated the genetic diversity and symbiotic efficiency of 223 Sinorhizobium sp. isolates sampled from a single Mediterranean soil and trapped with four Medicago truncatula lines. DNA molecular polymorphism was estimated by capillary electrophoresis-single-stranded conformation polymorphism and restriction fragment length polymorphism on five loci (IGS_NOD, typA, virB11, avhB11, and the 16S rRNA gene). More than 90% of the rhizobia isolated belonged to the Sinorhizobium medicae species (others belonged to Sinorhizobium meliloti), with different proportions of the two species among the four M. truncatula lines. The S. meliloti population was more diverse than that of S. medicae, and significant genetic differentiation among bacterial populations was detected. Single inoculations performed in tubes with each bacterial genotype and each plant line showed significant bacterium-plant line interactions for nodulation and N₂ fixation levels. Competition experiments within each species highlighted either strong or weak competition among genotypes within S. medicae and S. meliloti, respectively. Interspecies competition experiments showed S. meliloti to be more competitive than S. medicae for nodulation. Although not highly divergent at a nucleotide level, isolates collected from this single soil sample displayed wide polymorphism for both nodulation and N₂ fixation. Each M. truncatula line might influence Sinorhizobium soil population diversity differently via its symbiotic preferences. Our data suggested that the two species did not evolve similarly, with S. meliloti showing polymorphism and variable selective pressures and S. medicae showing traces of a recent demographic expansion. Strain effectiveness might have played a role in the species and genotype proportions, but in conjunction with strain adaptation to environmental factors.     

Exhaustive reanalysis of barcode sequences from public repositories highlights ongoing misidentifications and impacts taxa diversity and distribution

Accurate species identification often relies on public repositories to compare the barcode sequences of the investigated individual(s) with taxonomically assigned sequences. However, the accuracy of identifications in public repositories is often questionable, and the names originally given are rarely updated. For instance, species of the Sea Lettuce (Ulva spp.; Ulvophyceae, Ulvales, Ulvaceae) are frequently misidentified in public repositories, including herbaria and gene banks, making species identification based on traditional barcoding unreliable. We DNA barcoded 295 individual distromatic foliose strains of Ulva from the North-East Atlantic for three loci (rbcL, tufA, ITS1). Seven distinct species were found, and we compared our results with all worldwide Ulva spp. sequences present in the NCBI database for the three barcodes rbcL, tufA and the ITS1. Our results demonstrate a large degree of species misidentification, where we estimate that 24%–32% of the entries pertaining to foliose species are misannotated and provide an exhaustive list of NCBI sequences reannotations. An analysis of the global distribution of registered samples from foliose species also indicates possible geographical isolation for some species, and the absence of U. lactuca from Northern Europe. We extended our analytical framework to three other genera, Fucus, Porphyra and Pyropia and also identified erroneously labelled accessions and possibly new synonymies, albeit less than for Ulva spp. Altogether, exhaustive taxonomic clarification by aggregation of a library of barcode sequences highlights misannotations and delivers an improved representation of species diversity and distribution.     

Molecular phylogeny of <Emphasis Type="Italic">Pisolithus</Emphasis> species from Moroccan forest woodlands

The phylogenetic relationships among 200 Pisolithus basidiomata collected from pine, oak, and eucalypt forests and rockrose scrubs in Morocco were investigated. Using PCR-RFLP analysis of the internal transcribed spacer (ITS) of ribosomal DNA, this collection could be divided into 5 groups, by using PCR-RFLP analysis of the internal transcribed spacer (ITS) of ribosomal DNA. The ITS of a representative basidioma of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggests that at least five Pisolithus species are present in Morocco. Pisolithus basidiomata collected in the Pinus and Quercus forests correspond to Pisolithus arrhizus and P. species 4 as previously described by Martin and colleagues in 2002. Those collected from the eucalyptus forests, under E. gomphocephala and E. camaldulensis, correspond to P. albus and P. microcarpus. Basidiomata collected from the rockrose scrubs, under Cistus crispus, C. monspeliensis or C. salviifolius, are all identified as Pisolithus species 3. Phylogenetic analyses showed that our different Pisolithus grouped well with Pisolithus specimens from other geographical origins.     

Monoxenic nodulation process of Acacia mangium (Mimosoideae, Phyllodineae) by Bradyrhizobium sp

Acacia mangium Willd., a native tree in Australia and Papua New Guinea, has been introduced to countries in Asia and South America where plantations have been established that cover several hundred thousand ha. The present study investigated the early stages of the nodulation process in A. mangium using an homologous Australian Bradyrhizobium strain. After optimizing the axenic nodulation, histological and cytological studies were conducted using light and electron microscopy. These documented the proliferation of Bradyrhizobium, the lysis of mucilage at the root surface, root hair deformation and initiation, as well as the development and growth of multiple infection threads. A belt of tannin-filled cells was shown to surround the central nodular fixation zone. The nodules were of the indeterminate type and the bacteroids had a rod shape, without size modification and with few polyhydroxybutyrate (PHB) granules. Several bacteroids can share the same symbiosome. A. mangium exhibits both classical and novel features in its nodulation.