Frond transformation system mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> for <Emphasis Type="Italic">Lemna minor</Emphasis>

The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8–9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.     

The hidden dynamics of low coral cover communities

Our study presents a low-cost method (no expensive hardware platforms required) of quantified biomonitoring based on computer image analysis. The negative influence of toxins on surface waters was analysed. The method was verified on widespread freshwater macrophyte Lemna minor to test populations treated with non-ionic detergents. We showed that the proposed automated bioassay has a broad applicability in assessing the negative impacts of aquatic toxicants. This approach enabled fast and precise evaluation of the morphometric parameters of the duckweed test population. We observed that growth rate of L. minor reacts to non-ionic detergents, which is reflected by the change in the surface area. The decrease in the growth of L. minor was revealed at high doses of detergents. This test proved to be highly useful, because it is well repeatable and fast in its implementation. Unlike classical bioassays, the proposed test allows the elimination of measurement errors, resulting from observers’ subjectivity.     

Taxonomic relations of three flounder species of the subfamily pleuronectinae of the sea of Japan

The taxonomic relations of two flatfish genera, Pseudopleuronectes and Liopsetta were examined for the species Ps. yokohamae, L. obscura and L. pinnifasciata with the use of morphometry and lateral line system structure analysis. Ps. yokohamae and L. obscura were found to be very similar in the majority of plastic and meristic characters and in the arrangement of the lateral line sensory canals on the head. Our findings confirmed the recent suggestions of a close relationship between Ps. yokohamae and L. obscura and provided a solid argument for the transfer of L. obscura from the genus Liopsetta to the genus Pseudopleuronectes.     

Non-native fish species in Neotropical freshwaters: how did they arrive,and where did they come from?

The invasion of aquatic ecosystems by introduced invasive alien species (IAS) has become a worldwide phenomenon, and often leads to competitive interactions with native species. At high-nutrient levels, native species mostly are outcompeted by the introduced species. We performed an outdoor competition experiment between IAS free-floating Lemna minuta and native Lemna minor in a eutrophicated pond to examine whether the invasive species is the better competitor. We additionally performed an indoor experiment resembling mesotrophic phosphorus (P) conditions to investigate both species’ competitiveness in low P availability and compared with previous experiments at high-nutrient levels. Our results showed that in field conditions, the alien L. minuta was the better competitor. In the mesotrophic indoor condition, however, the native L. minor was the better competitor. Both species produced longer roots in the indoor experiment compared to field conditions. The species’ relative growth rates were also lower in the indoor experiment. A P reduction to mesotrophic condition in the water column thus might reduce invasive L. minuta growth and competitive performance. Additionally, introduction and recovery of L. minor could reduce L. minuta cover, but only following P reduction. Field experiments in mesotrophic ponds are needed to confirm these indoor findings.     

Shoot endophytic plant growth-promoting bacteria reduce cadmium toxicity and enhance switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis> L.) biomass

The aims of the study were to increase the biomass and to alleviate the deleterious effects of cadmium (Cd) in the switchgrass cultivars (Panicum virgatum L.) Alamo and Cave-in-Rock (CIR) under cadmium (Cd) stress using Cd-tolerant shoot endophytic plant growth-promoting bacteria (PGPB). Four shoot endophytic bacterial strains, viz. Bc09, So23, E02, and Oj24, were isolated from the above-ground parts of plants grown in a Cd-polluted soil and were successfully identified by 16S rRNA gene sequencing as Pseudomonas grimontii, Pantoea vagans, Pseudomonas veronii, and Pseudomonas fluorescens, respectively. These four strains were adapted to high CdCl₂ concentrations as they had higher Cd uptake capacities. In addition, they possessed a huge amount of growth regulatory activities e.g., indole acetic acid production, 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) activity, and phosphate solubilization. Growth particularly the height and biomass of both cultivars increased significantly in response to PGPB inoculation in the 20 µM CdCl₂ stress. The shoot biomass of the PGPB-inoculated Alamo was higher than the CIR under Cd stress. Interestingly, the level of Cd inside PGPB-inoculated plant tissues and the translocation factors were lower compared with the noninoculated Cd control plants. CIR plants exhibited higher Cd content than Alamo plants. Through confocal microscopy, green fluorescence was observed in roots and leaf tissues 2 days after the inoculation of green fluorescent protein (GFP)-labeled bacteria in Alamo, which confirmed the successful colonization of bacteria inside the plant tissues. These shoot endophytic PGPB and switchgrass interactions are useful for the sustainable biomass production of bioenergy crop in a Cd-contaminated environment.     

Contribution of glutamate decarboxylase in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> to acid resistance and persistence in sourdough fermentation

Background

Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri.

Results

A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ?gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ?gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ?gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ?gadB after 5 cycles of fermentation in back-slopped sourdough fermentations.

Conclusions

The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.

     

Diversity of Culturable Plant Growth-Promoting Bacterial Endophytes Associated with Sugarcane Roots and Their Effect of Growth by Co-Inoculation of Diazotrophs and Actinomycetes

Application of environmentally friendly agents to reduce the use of chemicals and to enhance growth of plants is an ultimate goal of sustainable agriculture. The use of plant growth-promoting endophytes has become of great interest as a way to enhance plant growth and additionally protect plants from phytopathogens. In this study, 135 isolates of endophytic bacteria including actinomycetes were isolated from roots of commercial sugarcane plants cultivated in Thailand and were characterized for plant growth-promoting (PGP) traits. Based on morphological and 16S rRNA sequence analysis, the endophytes were distributed into 14 genera of which the most dominant species belong to Bacillus, Enterobacter, Microbispora, and Streptomyces. Two strains of endophytic diazotrophs, Bacillus sp. EN-24 and Enterobacter sp. EN-21; and two strains of actinomycetes, Microbispora sp. GKU 823 and Streptomyces sp. GKU 895, were selected based on their PGP traits including 1-aminocyclopropane-1-decarboxylate deaminase, indole-3-acetic acid, nitrogen fixation, phosphate solubilization, and siderophore production for evaluation of sugarcane growth enhancement by individual and co-inoculation. Sixty days after co-inoculation by endophytic diazotrophs and actinomycetes, the growth parameters of sugarcane plants were significantly greater than that of individual and un-inoculated plants. The results indicated that these endophytes have high potential as PGP agents that could be applied to promote sugarcane growth and could be developed as active added value biofertilizers in the future.     

Protective Effects of <Emphasis Type="Italic">Mekabu</Emphasis> Aqueous Solution Fermented by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Sanriku-SU7 on Human Enterocyte-Like HT-29-luc Cells and DSS-Induced Murine IBD Model

Most wakame Undaria pinnatifida, a brown algae, products are made from the frond portion. In this study, the polysaccharide content and antioxidant property of aqueous extract solutions (AESs) of the four parts (frond: wakame, stem of the frond: kuki-wakame, sporophyll: mekabu, and kuki-mekabu) of wakame were investigated. Polysaccharide content was high in both the wakame and mekabu. Superoxide anion (O₂ ^?) radical-scavenging capacities were high in the mekabu. These AESs could be fermented by Lactobacillus plantarum Sanriku-SU7. The O₂ ^? radical-scavenging activity of the kuki-wakame, mekabu, and kuki-mekabu were increased by the fermentation. Fermented mekabu clearly showed a protective effect on human enterocyte-like HT-29-luc cells and in a mouse model of dextran sodium sulphate-induced inflammatory bowel disease (IBD). These results suggest that the mekabu fermented by L. plantarum Sanriku-SU7 has anti-IBD effect related to O₂ ^? radical-scavenging.     

<Emphasis Type="Italic">Azospirillum</Emphasis> spp. from native forage grasses in Brazilian Pantanal floodplain: biodiversity and plant growth promotion potential

A sustainable alternative to improve yield and the nutritive value of forage is the use of plant growth-promoting bacteria (PGPB) that release nutrients, synthesize plant hormones and protect against phytopathogens (among other mechanisms). Azospirillum genus is considered an important PGPB, due to the beneficial effects observed when inoculated in several plants. The aim of this study was to evaluate the diversity of new Azospirillum isolates and select bacteria according to the plant growth promotion ability in three forage species from the Brazilian Pantanal floodplain: Axonopus purpusii, Hymenachne amplexicaulis and Mesosetum chaseae. The identification of bacterial isolates was performed using specific primers for Azospirillum in PCR reactions and partial sequencing of the 16S rRNA and nifH genes. The isolates were evaluated in vitro considering biological nitrogen fixation (BNF) and indole-3-acetic acid (IAA) production. Based on the results of BNF and IAA, selected isolates and two reference strains were tested by inoculation. At 31 days after planting the plant height, shoot dry matter, shoot protein content and root volume were evaluated. All isolates were able to fix nitrogen and produce IAA, with values ranging from 25.86 to 51.26 mg N mL^?1 and 107–1038 µmol L^?1, respectively. The inoculation of H. amplexicaulis and A. purpusii increased root volume and shoot dry matter. There were positive effects of Azospirillum inoculation on Mesosetum chaseae regarding plant height, shoot dry matter and root volume. Isolates MAY1, MAY3 and MAY12 were considered promising for subsequent inoculation studies in field conditions.     

Flower palate structure of the aquatic bladderworts <Emphasis Type="Italic">Utricularia bremii</Emphasis> Heer and <Emphasis Type="Italic">U. minor</Emphasis> L. from section <Emphasis Type="Italic">Utricularia</Emphasis> (Lentibulariaceae)

There is an enormous diversity in the structure of the flower palate of the carnivorous rootless genus Utricularia. This study aims to examine the structure of the palates in Utricularia bremii Heer and U. minor L of the Utricularia sect. Utricularia, which have a glandular palate type. In both species, the palate has only one type of glandular trichomes. Because of the occurrence of cell wall ingrowths in its glandular cells, any exudation may be transported via eccrinous secretion. It was proposed that the palate trichomes of the examined species act as scent glands and that the palate may play a role as an unguentarium. Both U. bremii and U. minor are of an open flower type. Thus, U. bremii and U. minor flowers can be penetrated by small, weak insects, which then easily have access to their generative structure. Small Hymenoptera (member of families Mymaridae and Braconidae) were observed as flower visitors of the male-sterile species Utricularia bremii.     

Promotion of Peanut Growth by Co-inoculation with Selected Strains of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis>

The ability of inoculated rhizobial strains to increase root nodulation of host legumes often depends on their competitiveness with existing native soil strains. Results of studies to date on rhizobial inoculation for improvement of peanut (Arachis hypogaea L.) production in Argentina have been inconsistent and controversial. In many cases, nodulation and yield of peanut crops have been increased by inoculation of specific rhizobial strains. Native peanut-nodulating strains are generally present in soils of agricultural areas, but their growth-promoting effect is often lower than that of inoculated strains. Many species of the genus Bradyrhizobium interact in a host-specific manner with legume species and form nitrogen-fixing root nodules. Other free-living rhizobacteria such as species of the genus Azospirillum are facultatively capable of interacting with legume roots and promoting plant growth. We evaluated and compared the effects of various single inoculation and co-inoculation treatments on peanut growth parameters in greenhouse and field experiments. In the greenhouse studies, co-inoculation with various Bradyrhizobium strains (native 15A and PC34, and recommended peanut inoculant C145), and Azospirillum brasilense strain Az39 generally resulted in increases in the measured parameters. The growth-promoting effect of 15A was similar to or higher than that of C145. In the field studies, 15A-Az39 co-inoculation had a greater promoting effect on measured growth parameters than did C145-Az39 co-inoculation. Our findings indicate that careful selection of native rhizobacterial strains adapted to peanut soils is useful in strategies for growth promotion, and that 15A in particular is a promising candidate for future inoculant formulation.     

Enhanced expression of ginsenoside biosynthetic genes and in vitro ginsenoside production in elicited <Emphasis Type="Italic">Panax sikkimensis</Emphasis> (Ban) cell suspensions

Dual metabolite, i.e., ginsenoside and anthocyanin, co-accumulating cell suspensions of Panax sikkimensis were subjected to elicitation with culture filtrates of Serratia marcescens (SD 21), Bacillus subtilis (FL11), Trichoderma atroviridae (TA), and T. harzianum (TH) at 1.25% and 2.5% v/v for 1- and 3-week duration. The fungal-derived elicitors (TA and TH) did not significantly affect biomass accumulation; however, bacterial elicitors (SD 21 and FL11), especially SD 21, led to comparable loss in biomass growth. In terms of ginsenoside content, differential responses were observed. A maximum of 3.2-fold increase (222.2 mg/L) in total ginsenoside content was observed with the use of 2.5% v/v TH culture filtrate for 1 week. Similar ginsenoside accumulation was observed with the use of 1-week treatment with 2.5% v/v SD 21 culture filtrate (189.3 mg/L) with a 10-fold increase in intracellular Rg2 biosynthesis (31 mg/L). Real-time PCR analysis of key ginsenoside biosynthesis genes, i.e., FPS, SQS, DDS, PPDS, and PPTS, revealed prominent upregulation of particularly PPTS expression (20–23-fold), accounting for the observed enhancement in protopanaxatriol ginsenosides. However, none of the elicitors led to successful enhancement in in vitro anthocyanin accumulation as compared to control values.     

Inhibition of neutrophil adhesion by pectic galacturonans

The inhibition of the adhesion of neutrophils to fibronectin by the fragments of the main galacturonan chain of the following pectins was demonstrated: comaruman from the marsh cinquefoil Comarum polustre, bergenan from the Siberian tea Bergenia crassifolia), lemnan from the duckweed Lemna minor), zosteran from the eelgrass Zostera marina), and citrus pectin. The parent pectins, except for comaruman, did not affect the cell adhesion. Galacturonans prepared from the starting pectins by acidic hydrolysis were shown to reduce the neutrophil adhesion stimulated by phorbol 12-myristate 13-acetate (1.625 μM) and dithiothreitol (0.5 mM) at a concentration of 50–200 μg/ml. The presence of carbohydrate chains with molecular masses higher than 300, from 100 to 300, and from 50 to 100 kDa in the galacturonan fractions was proved by membrane ultrafiltration.     

Habitat uncertainty explains variation in offspring provisioning strategies in a temporary pond crustacean

Along with the development of nanotechnology, an increase in production and application of nanosized magnetite (Fe₃O₄) is expected. Though magnetite is considered relatively safe, information concerning potential hazards of synthetic magnetite nanoparticles with unique physico-chemical characteristics to aquatic organisms is still limited. In this study, we evaluated the toxicity of nanosized (27.2 ± 9.8 nm) and bulk (144.2 ± 67.7 nm) magnetite particles to different life stages of the aquatic crustacean Daphnia magna. In addition, phytotoxicity of the magnetite was evaluated using duckweed Lemna minor. The study did not reveal any statistically significant differences between the biological effects of nanosized and bulk magnetite particles. Both forms of magnetite induced very low toxicity (EC50 > 100 ppm) to D. magna and L. minor in the standard acute assays. However, it was demonstrated that at acutely subtoxic magnetite concentrations (10 and 100 ppm), the number of neonates hatched from D. magna ephippia was decreased. Moreover, short-term (48 h) exposure of neonate daphnids to these concentrations may significantly affect the long-term survival and reproductive potential of daphnids. These results indicate that substantial contamination of aquatic ecosystems by magnetite may disrupt the stability of cladoceran populations.     

Comparative Growth Behaviour and Biofunctionality of Lactic Acid Bacteria During Fermentation of Soy Milk and Bovine Milk

The study reports the growth, acidification and proteolysis of eight selected lactic acid bacteria in skim and soy milk. Angiotensin-converting enzyme inhibition and antimicrobial profiles of skim and soy milk fermented by the lactic acid bacteria were also determined. Among eight lactic cultures (S. thermophilus MD2, L. helveticus V3, L. rhamnosus NS6, L. rhamnosus NS4, L. bulgaricus NCDC 09, L. acidophilus NCDC 15, L. acidophilus NCDC 298 and L. helveticus NCDC 292) studied, L. bulgaricus NCDC 09 and S. thermophilus MD2 decreased the pH of skim and soy milk in greater extent. Acid production (i.e. titratable acidity) by L. bulgaricus NCDC 09 and L. helveticus V3 was higher than other strains. Higher viable counts were observed in S. thermophilus MD2 and L. helveticus V3. Higher proteolysis was exhibited by S. thermophilus MD2 and L. rhamnosus NS6 in both skim and soy milk. Milk fermented by S. thermophilus (MD2) exhibited highest angiotensin-converting enzyme inhibition. Antimicrobial activities of cell-free supernatant of milk fermented by S. thermophilus MD2 and L. helveticus V3 were higher. All the tested lactic acid bacteria performed better in skim milk as compared to soy milk.     

Physiological responses and antioxidant enzyme changes in <Emphasis Type="Italic">Sulla coronaria</Emphasis> inoculated by cadmium resistant bacteria

Plant growth promoting bacteria (PGPB) may help to reduce the toxicity of heavy metals on plants growing in polluted soils. In this work, Sulla coronaria inoculated with four Cd resistant bacteria (two Pseudomonas spp. and two Rhizobium sullae) were cultivated in hydroponic conditions treated by Cd; long time treatment 50 µM CdCl₂ for 30 days and short time treatment; 100 µM CdCl₂ for 7 days. Results showed that inoculation with Cd resistant PGPB enhanced plant biomass, thus shoot and root dry weights of control plants were enhanced by 148 and 35% respectively after 7 days. Co-inoculation of plants treated with 50 and 100 µM Cd increased plant biomasses as compared to Cd-treated and uninoculated plants. Cadmium treatment induced lipid peroxidation in plant tissues measured through MDA content in short 7 days 100 µM treatment. Antioxidant enzyme studies showed that inoculation of control plants enhanced APX, SOD and CAT activities after 30 days in shoots and SOD, APX, SOD, GPOX in roots. Application of 50 µM CdCl₂ stimulated all enzymes in shoots and decreased SOD and CAT activities in roots. Moreover, 100 µM of CdCl₂ increased SOD, APX, CAT and GPOX activities in shoots and increased significantly CAT activity in roots. Metal accumulation depended on Cd concentration, plant organ and time of treatment. Furthermore, the inoculation enhanced Cd uptake in roots by 20% in all treatments. The cultivation of this symbiosis in Cd contaminated soil or in heavy metal hydroponically treated medium, showed that inoculation improved plant biomass and increased Cd uptake especially in roots. Therefore, the present study established that co-inoculation of S. coronaria by a specific consortium of heavy metal resistant PGPB formed a symbiotic system useful for soil phytostabilization.     

Co-inoculation with <Emphasis Type="Italic">Enterobacter</Emphasis> and Rhizobacteria on Yield and Nutrient Uptake by Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) in the Alluvial Soil Under Indo-Gangetic Plain of India

The aim of this work was to evaluate the effects of co-inoculation with phosphate-solubilizing and nitrogen-fixing rhizobacteria on growth promotion, yield, and nutrient uptake by wheat. Out of twenty-five bacteria isolated from the rhizosphere soils of cereal, vegetable, and agro-forestry plants in eastern Uttar Pradesh, three superior most plant growth-promoting (PGP) isolates were characterized as Serratia marcescens, Microbacterium arborescens, and Enterobacter sp. based on their biochemical and 16S rDNA gene sequencing data and selected them for evaluating their PGP effects on growth and yield of wheat. Among them, Enterobacter sp. and M. arborescens fixed significantly higher amounts (9.32?±?0.57 and 8.89?±?0.58 mg Ng^?1 carbon oxidized, respectively) of atmospheric nitrogen and produced higher amounts (27.06?±?1.70 and 26.82?±?1.63 TP 100 µg mL^?1, respectively) of IAA in vitro compared to S. marcescens (8.32?±?0.39 mg Ng^?1 carbon oxidized and 21.29?±?0.99 TP 100 µg mL^?1). Although both M. arborescens and S. marcescens solubilized remarkable amounts of phosphate from tricalcium phosphate likely through production of organic acids, however, Enterobacter sp. was inactive. The effects of these three rhizobacteria were evaluated on wheat in alluvial soils of the Indo-Gangetic Plain by inoculation of plants with bacterial isolates either alone or in combinations in both pot and field conditions for two successive years. Rhizobacterial inoculation either alone or in consortium of varying combinations significantly (P?≤?0.05) increased growth and yield of wheat compared to mock inoculated controls. A consortium of two or three rhizobacterial isolates also significantly increased plant height, straw yield, grain yield, and test weight of wheat in both pot and field trials compared to single application of any of these isolates. Among the rhizobacterial treatment, co-inoculation of three rhizobacteria (Enterobacter, M. arborescens and S. marcescens) performed best in promotion of growth, yield, and nutrient (N, P, Cu, Zn, Mn, and Fe) uptake by wheat. Taken together, our results suggest that co-inoculation of Enterobacter with S. marcescens and M. arborescens could be used for preparation of an effective formulation of PGP consortium for eco-friendly and sustainable production of wheat.     

A nodule endophytic plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> and its effects on growth,nodulation and metal uptake in <Emphasis Type="Italic">Medicago lupulina</Emphasis> under copper stress

The aim of this study was to determine the plant growth-promoting potential of the nodule endophytic Pseudomonas brassicacearum strain Zy-2-1 when used as a co-inoculant of Medicago lupulina with Sinorhizobium meliloti under copper (Cu) stress conditions. Strain Zy-2-1 was capable of producing ACC deaminase activity, IAA and siderophores, and was able to grow in the presence of Cu²⁺ up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 enhanced M. lupulina root fresh weight, total plant dry weight, number of nodules, nodule fresh weight and nitrogen content in the presence of 100 or 300 mg/kg Cu²⁺. In the presence of 500 mg/kg Cu²⁺, co-inoculation with S. meliloti and strain Zy-2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoculation alone. Furthermore, a higher amount of Cu accumulation in both shoots and roots and a higher level of Cu translocation to shoots were observed in co-inoculated plants. These results demonstrate that co-inoculation of M. lupulina with S. meliloti and P. brassicacearum Zy-2-1 improves plant growth, nitrogen nutrition and metal extraction potential. This can be of practical importance in the remediation of heavy metal-contaminated soils.