Australian Acacia Research Articles

Questioning the effectiveness of seed-reducing agents on invasive Acacia: Pod production relative to gall abundance of classical biological control agents

It has been widely accepted that the biological control agents released on invasive Australian Acacia in South Africa, have impacted on the population dynamics of their host plants. However, there are no quantitative data to support these claims and these invasive species remain at similar abundance levels prior to the release of their associated biological control agents and also show evidence of range expansion. The effect of biological control agents, two bud galling wasps (Trichilogaster acaciaelongifoliae and T. signiventris) and one gall-forming rust fungus (Uromycladium morrisii), on the pod production of their respective hosts (Acacia longifolia, A. pycnantha and A. saligna) was assessed by quantifying the reproductive and galled trees per hectare as well as the pods and galls produced per tree and per square meter over a one to two year period. It was shown that invasive Australian Acacia satiate their gall-forming biological control agents, with many pods surviving in their presence. Pod production and loss was placed into perspective using recent findings of seed rain and seed bank studies. It was concluded, based on pod production, seed rain and seed bank data that despite T. acaciaelongifoliae, T. signiventris and U. morrisii reducing pod production, they have had no impact on the population dynamics of their invasive Australian Acacia hosts in South Africa.

The identification of psychedelic compounds within Australian Acacia species

ABSTRACT N,N-dimethyltryptamine (DMT) is a psychedelic substance produced within various Acacia species; however, the range of species that contain it and the quantity of the compound found within each species is relatively unknown. This work focused on the identification of DMT in 104 plant samples, representing 79 Acacia species, utilizing a reverse phase liquid chromatographic separation prior to mass spectral characterization with a robust Q-TOF-MS method. The findings of this work determined 51 DMT-containing species, 41 of which were previously unknown to contain the psychedelic.

A new hydrocolloid to rival gum Arabic: Characterisation of a traditional food gum from Australian Acacia cambagei.

This study investigated a new gum sourced from Acacia cambagei, Gidyea gum, by comparative characterisation with gum Arabic. The polysaccharide compositional analysis of Gidyea gum via Fourier transfer infrared and nuclear magnetic resonance spectroscopies was conducted, showing common structural features to gum Arabic, with minor differences in polymer branching. Microstructural imaging using transmission electron microscopy revealed the typical globular arabinogalactan glycoprotein structures of ∼10 nm diameter for both samples. Size exclusion chromatography also confirmed a similar molecular weight with 28 × 103 g mol−1 and 25 × 103 g mol−1 for Gidyea gum and gum Arabic, respectively. Gum rheological analysis determined a significantly lower apparent viscosity in the Gidyea gum. Modelling of the temporal evolution of dynamic surface and interfacial tensions revealed that Gidyea gum had a significantly lower rate of diffusion to the interfaces but similar adsorption and rearrangement rates. The dilatational viscoelastic response of Gidyea gum at the oil-water interface was consistent with gum Arabic, indicating similarities in functional performance. Indeed, Gidyea gum could stabilise orange oil-water emulsions for 15 days, exhibiting a comparable performance to those stabilised with gum Arabic. This study revealed that Gidyea gum is a functional emulsifier with the potential to be a gum Arabic alternative within the food and pharmaceutical industries.

Host-specificity of biocontrol agents in the field – The significance of sampling sympatric relatives of target weeds (Acacia holosericea versus A. auriculiformis)

Acacia auriculiformis is an invasive weed that causes significant environmental damage across Florida, USA. Previous native range surveys in Australia identified a number of herbivores that are commonly associated with this plant, including several species of leaf-tying moths (Macrobathra spp.) and a chrysomelid beetle (Calomela intemerata). These herbivores are considered strong candidates as biological control agents, but the range of host species they use has not been determined. We therefore conducted a transect survey of herbivorous insects on A. holosericea, a closely-related sympatric relative of A. auriculiformis. These species share areas of sympatry and the latter was sampled simultaneously (in sympatry and allopatry). DNA barcoding approach identified an array of herbivorous insects feeding on A. holosericea, but few were found on both the Acacia species sampled. A literature survey suggested this pattern of host specificity may be consistent across Australian acacias, to the extent that each Acacia species may largely host its own suite of herbivores. These results do not negate the need for host testing procedures in biological control, but they do increase our confidence substantially that several promising potential biological control agents of A. auriculiformis (Macrobathra moths and Calomela beetles) have a restricted host species range. Our study illustrates how exploring the herbivore diversity of plant species closely related to the target weed can provide crucial information for biological control researchers.

Preventing Adipogenesis and Preserving Mitochondria and GLUT-4 Functions by Extracts and Isolated Compounds of Australian Acacia saligna.

Acacia saligna's secondary metabolites show promise in treating type 2 diabetes mellitus and its related conditions. We previously discovered that methanolic extracts, isolated flavonoids, and cyclitols effectively preserve mitochondria in 3T3-L1 adipocytes. In this current work, quantification of lipid droplet levels with Oil Red O assay showed a noticeable decrease in lipogenesis in 3T3-L1 cells. Methanolic leaf and bark extracts and isolated compounds, (-)-epicatechin 6 and myricitrin 8, reduced cellular lipid levels by 21.15% to 25.28%, respectively. mRNA levels of key regulators of mitochondrial biogenesis, such as adiponectin, PGC-1α, and mtTFA, were increased. Methanolic flower extract (FL-MeOH) and its chemical components, naringenin 1 and D-(+)-pinitol 5a, increased these gene levels from 10% to 29% at the higher dose. Our study found that FL-MeOH slightly reduced pro-inflammatory cytokines TNF-α and IL-6, attributed to two phytochemicals, naringenin-7-O-α-L-arabinofuranoside 2 and D-(+)-pinitol 5a. Western blot analysis also showed that adipocytes treated with MeOH extracts had higher GLUT-4 expression levels than untreated adipocytes. Overall, A. saligna extracts and their isolated compounds demonstrated anti-lipogenesis activity during 3T3-L1 cell differentiation, modulation of transcriptional levels of adiponectin, PGC-1α, and mtTFA, reducing TNF-α and IL-6 mRNA levels, promoting mitochondrial biogenesis, and enhancing GLUT-4 expression.

Bradyrhizobium xenonodulans sp. nov. isolated from nodules of Australian Acacia species invasive to South Africa

A genealogical concordance approach was used to delineate strains isolated from Acacia dealbata and Acacia mearnsii root nodules in South Africa. These isolates form part of Bradyrhizobium based on 16S rRNA sequence similarity. Phylogenetic analysis of six housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) confirmed that these isolates represent a novel species, while pairwise average nucleotide identity (ANIb) calculations with the closest type strains (B. cosmicum 58S1T, B. betae PL7HG1T, B. ganzhouense CCBAU 51670 T, B. cytisi CTAW11T and B. rifense CTAW71T) resulted in values well below 95–96%. We further performed phenotypic tests which revealed that there are high levels of intraspecies variation, while an additional analysis of the nodA and nifD loci indicated that the symbiotic loci of the strains are closely related to those of Bradyrhizobium isolates with an Australian origin. Strain 14ABT (=LMG 31415 T = SARCC-753 T) is designated as the type strain of the novel species for which we propose the name Bradyrhizobium xenonodulans sp. nov.

Domesticated Australian wattle seeds (Acacia species): nutritional values, techno‐functional properties and toxicological assessments after roasting

SummaryThis study evaluated the nutritional composition, anti‐nutritional factors, djenkolic acid (DKA) contents and techno‐functional properties of commercial raw, light and medium roasted wattle seed (WS) flour from 10 domesticated Australian Acacia species. The results showed that the species are a good source of dietary fibre, protein, fat and minerals (K, Ca, Mg, P and Fe). Moreover, the species have substantial amounts of linolenic acid and essential amino acids (EAAs), except for methionine and phenylalanine, indicating the high fat and protein quality of the species. Low‐trypsin inhibitor activity (TIA) and DKA contents were found in the species, except for the high TIA in A. longifolia spp. sophorae (18.3 mg TUI/g), A. longifolia spp. longifolia (12.3 mg TUI/g) and A. provincialis (12.2 mg TUI/g). In contrast, light and medium roasting effectively reduced the TIA and DKA levels as well as foaming and emulsifying properties. The greatest reduction in these properties was noticed in medium‐roasted samples. However, roasting appears to retain most of the nutritive value in the species and, the species exhibited high‐water absorption capacity, making them suitable for use as ingredients in bakery applications.

Antihyperglycemic Properties of Extracts and Isolated Compounds from Australian Acacia saligna on 3T3-L1 Adipocytes.

Our early work indicated that methanolic extracts from the flowers, leaves, bark, and isolated compounds of Acacia saligna exhibited significant antioxidant activities in vitro. The overproduction of reactive oxygen species (ROS) in the mitochondria (mt-ROS) interfered with glucose uptake, metabolism, and its AMPK-dependent pathway, contributing to hyperglycemia and diabetes. This study aimed to screen the ability of these extracts and isolated compounds to attenuate the production of ROS and maintain mitochondrial function via the restoration of mitochondrial membrane potential (MMP) in 3T3-L1 adipocytes. Downstream effects were investigated via an immunoblot analysis of the AMPK signalling pathway and glucose uptake assays. All methanolic extracts effectively reduced cellular ROS and mt-ROS levels, restored the MMP, activated AMPK-α, and enhanced cellular glucose uptake. At 10 µM, (-)-epicatechin-6 (from methanolic leaf and bark extracts) markedly reduced ROS and mt-ROS levels by almost 30% and 50%, respectively, with an MMP potential ratio 2.2-fold higher compared to the vehicle control. (-)-Epicatechin 6 increased the phosphorylation of AMPK-α by 43%, with an 88% higher glucose uptake than the control. Other isolated compounds include naringenin 1, naringenin-7-O-α-L-arabinopyranoside 2, isosalipurposide 3, D-(+)-pinitol 5a, and (-)-pinitol 5b, which also performed relatively well across all assays. Australian A. saligna active extracts and compounds can reduce ROS oxidative stress, improve mitochondrial function, and enhance glucose uptake through AMPK-α activation in adipocytes, supporting its potential antidiabetic application.

Metabolic groups of plants in neotropical hyperseasonal savannas threatened by Australian Acacia invasion

Metabolic groups of plants in neotropical hyperseasonal savannas threatened by Australian Acacia invasion

Feeding Australian Acacia Gums and Gum Arabic Leads to Non-Starch Polysaccharide Accumulation in the Cecum of Rats

Exudative gums from two Australian Acacia species (A. pycnantha and A. baileyana) and gum arabic (from A. senegal) were fed to rats at graded levels (0, 20, 40, 80 g/kg), replacing cellulose in purified diets containing cholesterol plus cholic acid. Compared with consumption of the control diet containing cellulose only, consumption of the gums had no significant effects on concentrations of plasma or liver cholesterol. Plasma triacylglycerol concentrations were higher in rats fed gum arabic, whereas liver triacylglycerols were lower in rats fed the gums. The gums did not affect the total pool of volatile fatty acids in the ceca, as compared with results in controls, but did promote the relative contribution of propionate at the expense of acetate. In rats fed the diet containing cellulose (80 g/kg) the proportions of cecal acetate:propionate:butyrate were 76:15:9, whereas in the rats fed A. pycnantha gum, gum arabic and A. baileyana gum (80 g/kg) the ratios were 42:54:4, 35:46:19 and 43:53:4, respectively. The low apparent fermentability of the gums was confirmed by the accumulation of non-starch polysaccharides in cecal digesta. In rats fed 80 g/kg A. pycnantha gum, 3.44 g of soluble non-starch polysaccharides was measured in the ceca, which was 58% of the dry weight of the cecal contents. We conclude that the biological activities of the Australian Acacia gums were similar to those of gum arabic and that these gums may have potential value as human food ingredients.

Structure – Function relationship of Australian Acacia seed protein concentrates: Amino acid composition, in vitro protein digestibility and molecular properties

The nutritional, functional, in vitro protein digestibility (PD), structural, and molecular properties of Acacia seed protein concentrates (ASPC) from three Acacia species (Acacia victoriae, A. coriacea and A. cowleana) were investigated and compared with a commercial soy protein isolate (SPI). The essential amino acid profile in ASPC, except for methionine, exceeded the minimum daily requirements for adults recommended by the FAO/WHO. The ASPC PD, particularly from A. victoriae and A. coriacea, was considerably high (64.2–79.3%), but lower than SPI (89.5%). ASPC proteins had higher molecular weights ranging from 50 to 65 kDa (associated with 7S vicilin subunit), better protein solubility and oil absorption capacity than SPI. Emulsifying properties were comparable between ASPC and SPI. Among the Acacia species, the proteins from A. victoriae and A. cowleana exhibited higher protein quality, foaming capacity and solubility compared to A. coriacea. Overall, ASPC from A. victoriae and A. cowleana showed potential as ingredients in food processing.

Nutritional and techno-functional properties of Australian Acacia seed flour: Effects of roasting on chemical composition, physicochemical properties, and in vitro digestibility and intestinal iron absorption

Acacia seed (AS) is rich in protein and iron but contains protease inhibitors that can reduce protein digestibility (PD). The seeds are generally roasted prior to consumption, although no information on the PD of roasted AS is available. This study investigated the effect of roasting time (5, 7 and 9 min at 180 °C) on the chemical composition, physicochemical properties, and in vitro PD and intestinal iron absorption of three wild harvested Australian AS species, Acacia victoriae, A. coriacea and A. cowleana. Roasting A. victoriae and A. coriacea seeds for 7 min significantly increased PD in the seeds by 36 and 61 %, respectively. A 9-min roasting time was required to achieve 75 % reduction in trypsin inhibitor activity in A. coriacea seed, while a shorter roasting time (RT) was sufficient to achieve similar reduction rates in the other two Acacia species. Among the functional properties, water and oil absorption capacities were significantly enhanced as RT increased. The starch granules in 7- and 9-min roasted A. victoriae seed flour detached from the protein matrix while random coil increased in 7-min roasted A. victoriae and 9-min roasted A. coriacea and A. cowleana, thus, contributing to enhanced PD. Although the SDS-PAGE in 7- and 9-min roasted A. cowleana samples showed reductions in the intensity of bands for high molecular weight proteins, PD was not affected by RT. However, intestinal iron absorption was not significantly affected by roasting as compared to raw digesta samples. Compared to commercial roasted Acacia seed, the considerably shorter RT used in this study improved PD in the AS flour with less adverse effects on techno-functional properties.

Rumors of Psychedelics, Psychotropics and Related Derivatives in Vachellia and Senegalia in Contrast with Verified Records in Australian Acacia

There are almost 1000 species of Acacia sensu stricto in Australia, while the 44 species and 4 subspecies in southern Africa were taxonomically revised in the year 2011 to Senegalia and Vachellia. There are rumors of a chemical similarity between the Australian Acacia and their southern African sister genera. Chemical analysis has unequivocally demonstrated the presence of tryptamines (i.e., DMT), β-carbolines, histamines, and phenethylamines in Australian species. However, reliable published data were not found in support of similar alkaloids in southern African (or even African) species, indicating the need for exploratory phytochemical analysis. Interestingly, the Australian species are more like the Vachellia and Senegalia from the Americas. While many reliable chemical studies have been found, there are several more that report only tentative results. Tentative data and anecdotal accounts are included in the current review to guide researchers to areas where further work can be done. For example, the current review encourages further phytochemical work to confirm if the two metabolite families, tryptamine and β-carboline alkaloids, occur together in a single specimen. Tryptamines and β-carbolines are the prerequisite ingredients of the South American psychotropic drink ayahuasca, which utilizes two different species to create this synergistic combination. These observations and others are discussed in light of geochemical variability, the potential ethnobotanical implications, and the need for further research to confirm or nullify anecdotal reports and tentative chromatographic/spectroscopic data in southern African species.

Invader-induced soil abiotic and biotic conditions have little impact on the competitive interactions between a native and an invasive legume

• No competition occurred between native Psoralea pinnata and invasive A. saligna . • Each legume species associated with different rhizobia and used divergent nitrogen sources. • Australian rhizobia were naturally present in pristine Fynbos soils. • Acacia invasion and spread may not be limited by rhizobium availability in Fynbos soils. Invasive Australian acacias cause substantial biodiversity impacts in South Africa's Core Cape Subregion (CCR) via positive plant-soil feedbacks, driven by high growth rates and leaf litter input by these legumes and their associations with nitrogen-fixing rhizobia. However, it is unclear whether acacia-induced soil abiotic conditions, or the association between acacias and different types of rhizobia, or both, are driving the invasive performance of acacias. Here we aim to determine how familiar (i.e., Australian) vs. novel (i.e., South African) rhizobium associations and acacia leaf litter affect competition between the invasive Acacia saligna (Labill.) H.L.Wendl. and native Psoralea pinnata L.. We grew these two legumes alone and together in pots containing Psoralea -conditioned soils. Seedlings grown alone were subjected to Australian bradyrhizobia inoculum treatments, while seedlings grown in mixture received a combination of inoculum and topsoil conditioned by acacia leaf litter in a fully factorial design. We found that neither the addition of Australian rhizobia, nor acacia-conditioned topsoil, enhanced the performance of A. saligna . Surprisingly, these treatments also did not hinder P. pinnata ’s performance relative to that of A. saligna . Next generation sequencing (NGS) data revealed that each legume species associated with distinct (and familiar) rhizobia, regardless of inoculum treatment. Acacia saligna primarily met its nitrogen requirements by utilising soil nitrogen, while P. pinnata obtained much of its nitrogen via biological fixation. This apparent differentiation in nitrogen acquisition, coupled with the unique rhizobial associations of the two species, may explain the absence of strong asymmetric competition between them. This may also explain why P. pinnata is able to persist under Acacia invasion in the field and suggests that this native legume is a good candidate for restoring acacia-invaded habitats.

Provenance of rhizobial symbionts is similar for invasive and noninvasive acacias introduced to California.

Plant-soil interactions can be important drivers of biological invasions. In particular, the symbiotic relationship between legumes and nitrogen-fixing soil bacteria (i.e. rhizobia) may be influential in invasion success. Legumes, including Australian acacias, have been introduced into novel ranges around the world. Our goal was to examine the acacia-rhizobia symbiosis to determine whether cointroduction of non-native mutualists plays a role in invasiveness of introduced legumes. To determine whether acacias were introduced abroad concurrently with native symbionts, we selected four species introduced to California (two invasive and two noninvasive in the region) and identified rhizobial strains associating with each species in their native and novel ranges. We amplified three genes to examine phylogenetic placement (16S rRNA) and provenance (nifD and nodC) of rhizobia associating with acacias in California and Australia. We found that all Acacia species, regardless of invasive status, are associating with rhizobia of Australian origin in their introduced ranges, indicating that concurrent acacia-rhizobia introductions have occurred for all species tested. Our results suggest that cointroduction of rhizobial symbionts may be involved in the establishment of non-native acacias in their introduced ranges, but do not contribute to the differential invasiveness of Acacia species introduced abroad.

Active restoration in South African fynbos – A long-term perspective from the Agulhas Plain

In 2019 we re-surveyed a site on the Agulhas Plain, South Africa, which had been part of a restoration project conducted in 2008. The site was invaded by invasive trees, mainly Australian Acacia species. In the original restoration program, the site had been burned and various treatments (i.e. sowing of competitive, fast-growing native species) were conducted to determine the effectiveness of different restoration strategies. The sowing of native species to assist the recovery process is defined as an active restoration method, whereas passive restoration only removes stressors for the ecosystem (e.g. removal of the invader) and leaves it to recover by itself. For this study, we conducted a vegetation survey at the previously restored sites and compared them with a nearby reference site where no intervention had taken place and which is characterised by near-pristine native fynbos vegetation. Secondly, we compared the new data from 2019 with older data from 2010. However, a decade after the initial restoration effort, both active and passively treated plots had not achieved the same condition (species richness, diversity, evenness) as the reference site. The results also show that active restoration is most likely unnecessary in fynbos restoration projects where there are nearby patches with native vegetation or the existing native seed bank is of sufficient size. It seems that it is more important to prevent the reinvasion of invasive alien plants so that the vegetation can recover over time without further disruption.

SUSTAINABLE PLANTATION FOR RECLAMATION OF MUNICIPAL SOLID WASTE DISPOSAL SITE: A CASE STUDY

Performance of selected trees on municipal dumpsite at Mumbai has been studied for 30 months and results indicate that the tree survival rate was found to be 50% to 85 % respectively. Amongst the selected plant species, Rain Tree, Gulmohar, Australian Acacia and Neem were observed to be the fastest growing trees. Study shows how a dumpsite could be transformed from a highly unacceptable zone to ecological and environmentally sustainable. Study also attempts to evaluate the trees suitable for urban environment for dumpsite based on their carbon sequestration potential and conforming Sustainable Development Goals

A Review of Research and Developments with Insect Agents Used for Biological Control of Australian Acacia Species (Caesalpinioideae) in South Africa

There are 14 species of Australian Acacia now known to be invasive in South Africa, ten of which are under some form of biological control (biocontrol). The biocontrol agents introduced against this group include a fungal pathogen, Uromycladium morrisii Doungsa-ard, McTaggart, Geering & R.G. Shivas (Pucciniales: Raveneliaceae) for Acacia saligna (Labill.) H.L.Wendl., and ten insect species, including two bud-galling wasps, five seed-feeding weevils and three flower-galling fly species, all of which supress reproductive output of their hosts. There are also two native fungal pathogens that have become associated with the introduced acacias, and which have been developed for potential mycoherbicide use. Screening and importation of new agents has seen limited activity in the ten-year period reviewed here (i.e., 2011–2020). Most attention has been focussed on efforts to evaluate the effectiveness of the established agents and, importantly, to gain an understanding of the role of seeds in the population dynamics of the host plants.

Soil nitrogen availability favours the growth but not germination of secondary invaders after clearing invasive Acacia saligna

Invasive alien nitrogen-fixing species, such as Australian acacias, often leave a legacy of elevated available soil nitrogen after their removal. This legacy effect can facilitate secondary invasion by other alien species, thereby preventing natural restoration of areas being managed. To restore viable native plant communities in ecosystems where secondary invasion is a barrier to restoration, it is important to understand the soil legacy effects of invasions. Using Acacia saligna (Labill.) H. L. Wendl. (Fabaceae) invasions in the South African fynbos as case study, we determined (1) the extent to which levels of available soil nitrogen influence the germination and growth of secondary invaders; and (2) how this differs between secondary invader species. We chose five of the most common species that have been identified as secondary invaders after clearing invasive A. saligna in the Cape Flats Sand Fynbos: (i) Avena fatua (L.) (Poaceae), (ii) Briza maxima (L.) (Poaceae), (iii) Bromus diandrus (Roth.) (Poaceae), (iv) Hypochaeris radicata (L.) (Asteraceae), and (v) Raphanus raphanistrum (L.) (Brassicaceae). Using proportional fertigation, we created soil nitrate levels similar to those found in non-invaded areas (1mg/kg), and the lowest (3 mg/kg), median (7.5 mg/kg) and highest (12 mg/kg) levels typically found in areas previously invaded by A. saligna up to ten years after clearing. For each soil nitrate level, we germinated secondary invader seeds (five seeds per petri dish) in an incubator (five species × four soil nitrate levels × five replicates = 100 petri dishes). Furthermore, for each soil nitrate level, we grew secondary invaders (one plant per pot) in a greenhouse tunnel for five months (five species × four soil nitrate levels × five replicates = 100 pots). There was no significant relationship between germination success and soil nitrate level for any species. However, root and shoot dry mass were significantly positively correlated to soil nitrate level for all species. The relationship was nonetheless only linear for Bromus diandrus, Hypochaeris radicata, and Raphanus raphanistrum. These results indicate that the legacy of elevated available soil nitrogen does not have an effect on the germination of secondary invaders, but favours their growth. Therefore, secondary invasion can be managed through a reduction in soil nitrogen levels. However, secondary invader growth does not decrease at the same rate for all species in response to a decrease in available soil nitrogen. Thus, the use of soil nitrogen reduction as a tool for the management of secondary invasions should take into account the species being managed. However, given that multiple secondary invader species often dominate a restoration site, restoration efforts should reduce soil nitrogen to non-invaded levels to account for the different response rates to available soil nitrogen, and simply management efforts.

Culture-independent assessment of the diazotrophic Bradyrhizobium communities in the Pampa and Atlantic Forest Biomes localities in southern Brazil

The isolation of rhizobial strains from the root and stem nodules remains a commonly used method despite its limitations as it enables the identification of mainly dominant symbiotic groups within rhizobial communities. To overcome these limitations, we used genus-specific nifD primers in a culture-independent assessment of Bradyrhizobium communities inhabiting soils in southern Brazil. The majority of nifD sequences were generated from DNA isolated from tropical-lowland pasture soils, although some soil samples originated from the Campos de Cima da Serra volcanic plateau. In the nifD tree, all the bradyrhizobial sequences comprised 38 clades, including 18 new clades. The sequences generated in this study were resolved into 22 clades and 21 singletons. The nifD bradyrhizobial assemblage contained Azorhizobium and α-proteobacterial methylotrophic genera, suggesting that these genera may have acquired their nif loci from Bradyrhizobium donors. The most common in the lowland pasture soils subclade III.3D branch comprises the isolates of mainly an American origin. On the other hand, subclade III.4, which was earlier detected in Brazil among Bradyrhizobium isolates nodulating native lupins, appears more common in the Campos de Cima da Serra soils. The second-largest group, Clade XXXVIII, has not yet been reported in culture-dependent studies, while another common group called Clade I represents a symbiovar predominating in Australia. The identification of the diverse nifD Clade I haplotypes in the tropical-lowland pastures infested by Australian Acacia spp implies that the introduction of these legumes to southern Brazil has resulted in the dissemination of their bradyrhizobial symbionts.