Acacia longifolia is one of the most aggressive invaders worldwide whose invasion is potentiated after a fire, a common perturbation in Mediterranean climates. As a legume, this species establishes symbioses with nitrogen-fixing bacteria inside root nodules; however, the overall microbial diversity is still unclear. In this study, we addressed root nodules’ structure and biodiversity through histology and Next-Generation Sequencing, targeting 16S and 25S-28S rDNA genes for bacteria and fungi, respectively. We wanted to evaluate the effect of fire in root nodules from 1-year-old saplings, by comparing unburnt and burnt sites. We found that although having the same general structure, after a fire event, nodules had a higher number of infected cells and greater starch accumulation. Starch accumulated in uninfected cells can be a possible carbon source for the microbiota. Regarding diversity, Bradyrhizobium was dominant in both sites (ca. 77%), suggesting it is the preferential partner, followed by Tardiphaga (ca. 9%), a non-rhizobial Alphaproteobacteria, and Synechococcus, a cyanobacteria (ca. 5%). However, at the burnt site, additional N-fixing bacteria were included in the top 10 genera, highlighting the importance of this process. Major differences were found in the mycobiome, which was diverse in both sites and included genera mostly described as plant endophytes. Coniochaeta was dominant in nodules from the burnt site (69%), suggesting its role as a facilitator of symbiotic associations. We highlight the presence of a large bacterial and fungal community in nodules, suggesting nodulation is not restricted to nitrogen fixation. Thus, this microbiome can be involved in facilitating A. longifolia invasive success.

Acacia longifolia is a worldwide invader that cause damage in ecosystems, expanding largely after wildfires, which promote germination of a massive seed bank. As a legume, symbiosis is determinant for adaptation. Our study aims to isolate a wider consortium of bacteria harboured in nodules, including both nitrogen and non-nitrogen fixers. Furthermore, we aim to evaluate the effects of fire in nodulation and bacterial diversity on young acacias growing in unburnt and burnt zones, one year after the fire. For this, we used molecular approaches, M13 fingerprinting and 16S rRNA partial sequencing, to identify species/genera involved and δ¹⁵N isotopic composition in leaves and plant nodules. Nitrogen isotopic analyses in leaves suggest that in unburnt zones, nitrogen fixation contributes more to plant nitrogen content. Overall, A. longifolia seems to be promiscuous and despite Bradyrhizobium spp. dominance, Paraburkholderia spp. followed by Pseudomonas spp. was also found. Several species not previously reported as nitrogen-fixers were identified, proposing other functions besides ammonia acquisition. Our study shows that bacterial communities are different in nodules after fire. Fire seems to potentiate nodulation and drives symbiosis towards nitrogen-fixers. Taken together, a multifunctional community inside nodules is pointed out which potentiate A. longifolia invasiveness and adaptation.

Woody invasive species pose a big threat to ecosystems worldwide. Among them, Acacia longifolia is especially aggressive, fundamentally changing ecosystem structure through massive biomass input. This biomass is rarely harvested for usage; thus, these plants constitute a nuisance for stakeholders who invest time and money for control without monetary return. Simultaneously, there is an increased effort to valorise its biomass, e.g., for compost, growth substrate or as biofuel. However, to incentivise A. longifolia harvest and usage, stakeholders need to be able to estimate what can be obtained from management actions. Thus, the total biomass and its quality (C/N ratio) need to be predicted to perform cost–benefit analyses for usage and determine the level of invasion that has already occurred. Here, we report allometric biomass models for major biomass pools, as well as give an overview of biomass quality. Subsequently, we derive a simplified volume-based model (BM ~ 6.297 + 0.982 × Vol; BM = total dry biomass and Vol = plant volume), which can be applied to remote sensing data or with in situ manual measurements. This toolkit will help local stakeholders, forest managers or municipalities to predict the impact and valorisation potential of this invasive species and could ultimately encourage its management.

Acacia longifolia is an aggressive invader in Mediterranean-type ecosystems severely impacting biodiversity and ecosystem functions. The species’ invasiveness has been linked to its ability to thrive in nutrient poor soils, high seed production, and quick establishment after fire. In this study, we identify and compare the bacterial endophytes of A. longifolia seeds collected from populations in the species’ native (Australia) and invasive (Portugal) ranges. For this, we characterised the morphology (length, width, and weight) of seeds from two sites in each range and isolated and cultivated bacteria from seeds. DNA fingerprinting and cluster analyses revealed slightly higher, and distinct, bacterial diversity associated with seeds collected from native range populations in comparison to those collected from invasive populations. Sequencing of the 16S rDNA gene identified 119 bacterial isolates from 15 genera, with Curtobacterium strains being common in both ranges. Several differences in bacterial genera were found among ranges and sites: Dermacoccus, Frigoribacterium, Kocuria, Pantoea and Phyllobacterium taxa were each unique to seeds from the native populations, while Brevundimonas, Microbacterium, Rhizobium and Sphingomonas taxa were only found in the invasive seeds. The genus Paraburkholderia occurred in all invasive-range seeds but was not isolated from the native-range. Bacillus and Paenibacillus co-occurred in seeds collected from all invaded sites, but the simultaneous presence of both taxa was not found in native-range seeds. We propose that the bacterial endophytes present in invasive-range seeds may be important players for the invasiveness of A. longifolia, due to their role as plant growth promoters, providing extra capabilities helping acacia expansion.

Acacia longifolia, a species native to Australia, is an aggressive invasive in Mediterranean-type ecosystems worldwide. Its success in diverse habitats, expanding from coastal dunes to forests, is often attributed to its ability to establish interactions with a variety of microbes, including bacteria and fungi. This study investigates the seed and root-nodule microbiomes of A. longifolia to understand the roles these microbial communities play in its adaptation and invasive behaviour. Using high-throughput sequencing, we characterized the bacteriome and mycobiome associated with the plant, considering nodules and seeds, and the surrounding soil in three different locations in Portugal with different climate conditions (North, Center and South), and a comparison between two different habitats (Dune versus Forest). Our results reveal a dynamic interaction between A. longifolia and its microbial partners, supporting the importance of these plant-microbe interactions in nutrient acquisition and stress tolerance for A. longifolia, ultimately leading to their impact in an invaded ecosystem. The seed microbiome of A. longifolia was less diverse than for nodules but with more functions assigned, while nodules showed a broader diversity, assigned to more specific functions. Here we provide evidence for the role of seed microbiota in germination and seed-to-seedling transition along with the beneficial role of nodulation in development and seedling-to-sapling switch. We also propose a local signature for microbial communities as we found a dissimilarity in microbial partners when considering habitat, with dune communities showing a functional plasticity, aiding A. longifolia to cope in such nutrient-limiting environment. For forests, functions more related with plant and microbe associations are evidenced, possibly facilitating interspecific competition. These findings contribute to an understanding of the plant-microbe interactions and dynamics that underpin A. longifolia ecological success as an invasive plant.

Os fogos florestais provocam perturbações nos ecossistemas, sendo cada vez mais frequentes nos climas Mediterrânicos. A introdução de espécies exóticas, que podem tornar-se invasoras, como o género Acacia, tem-se revelado impactante em regiões do nosso país, diminuindo a biodiversidade e alterando a composição dos solos. Atualmente, Acacia longifolia é uma das invasoras mais agressivas, e como leguminosa, é capaz de fixar azoto atmosférico por associação simbiótica com bactérias no interior de nódulos radiculares, com outras funções ainda desconhecidas. Neste trabalho foram amostradas áreas não ardidas e ardidas, onde se estudou (1) o impacto do fogo nas propriedades químicas dos solos e (2) a diversidade de bactérias cultiváveis isoladas do interior de nódulos radiculares de A. longifolia, por técnicas de microbiologia clássica, DNA fingerprinting e identificação por sequenciação de genes. Em zonas ardidas, verificou-se um aumento da matéria orgânica, fósforo e azoto nos solos e uma alteração do bacterioma de A. longifolia com Bradyrhizobium sp. mantendo-se como principal mutualista. Encontrou-se uma menor diversidade, associada a uma maior especificidade para os fixadores de azoto. Estas alterações podem potenciar a invasão de A. longifolia após o fogo.