Effects of Heavy Metal from Polluted Soils on the Rhizobium Diversity

Heavy metals adversely influence microorganisms, affecting their growth, abundance, genetic diversity, nodulation ability and efficacy. The aim of this study was to isolate and characterize free-leaving Rhizobium from soil which were artificially polluted with Cu (100, 250, and 500 mg kg-1 soil), Zn (300, 700, and 1500 mg kg-1 soil) and Pb (50, 250, and 1000 mg kg-1 soil), but also with a mixture of all these metals, and cultivated with red clover (Trifolium pratense L.), and to compare them with bacteria isolated from similar type of soil, but unpolluted. Rhizobia from soil were isolated on YMA medium with or without bromothymol blue (0.00125%) as a pH-change indicator and the morpho-physiological characteristics of the colonies were examined. The number of Rhizobium was estimated using the most probable number method. Compared to the control, a decrease of rhizobia number and an increase of the metal concentration were observed. Several decameric primers (Operon Technology type) were used and a reduced polymorphism among isolated bacteria was observed. Moreover, significant differences were observed among these strains and the collection strains used as reference. Also, when primers nodCF/nodCI for detection of nod genes were used, several amplicons were obtained, different from the results obtained with similar strains isolated from unpolluted soil. These results suggest that the survival „price†of the Rhizobium in such polluted area was the alteration of some genes, including those involved in symbiosis and, probably, in nitrogen fixation

Root nodule bacteria isolated from Lotus uliginosus for future use in phytostabilization of arsenic contaminated soils

Funding Information: This work was supported by the cooperation project between Portugal and Argentina: 'The genus Lotus and their utilization for the restoration of soils contaminated with heavy metals. The biochemistry and their symbionts', FCT/DREBM 00264, Proc. 4.1.3 and also by the European project: 'Raising the bio-based industrial feedstock capacity of marginal Lands (Margin Up)', nº101082089. Publisher Copyright: © 2023 by the author(s).In recent decades there has been growing concern around heavy metals and metalloid contamination in soil. Arsenic (As) is a ubiquitous trace metalloid. The high levels of this metalloid in soils are a consequence of human activities and also from natural inputs. In general, the biodiversity of microorganisms and plants decreases drastically in contaminated soils. The knowledge that some leguminous plants, mainly certain species of Lotus, are growing well in such soils has attracted our attention for studying symbioses that are well adapted to harsh conditions. In this work we studied the rhizobial population existing in the root nodules of native Lotus uliginosus Sch. growing in a central region of Portugal. This legume grows in soils particularly affected by As due the discharge of industrial liquid effluents from fertilizer and chemical facilities. Diversity and tolerance to different concentrations of As of root nodule bacteria were studied. Our results showed that the symbioses between L. uliginosus and As tolerant Bradyrhizobium isolates were efficient when a nutrient medium containing high As concentrations was used. The present work highlights the capacity of L. uliginosus to grow and establish nitrogen-fixing symbioses in soils strongly contaminated with As and its potential for future use to promote vegetation cover to stabilize As contaminated soils.publishersversionpublishe

Plant growth promoting rhizobia: Challenges and opportunities

Modern agriculture faces challenges, such as loss of soil fertility, fluctuating climatic factors and increasing pathogen and pest attacks. Sustainability and environmental safety of agricultural production relies on eco-friendly approaches like biofertilizers, biopesticides and crop residue return. The multiplicity of beneficial effects of microbial inoculants, particularly plant growth promoters (PGP), emphasizes the need for further strengthening the research and their use in modern agriculture. PGP inhabit the rhizosphere for nutrients from plant root exudates. By reaction, they help in (1) increased plant growth through soil nutrient enrichment by nitrogen fixation, phosphate solubilization, siderophore production and phytohormones production (2) increased plant protection by influencing cellulase, protease, lipase and β-1,3 glucanase productions and enhance plant defense by triggering induced systemic resistance through lipopolysaccharides, flagella, homoserine lactones, acetoin and butanediol against pests and pathogens. In addition, the PGP microbes contain useful variation for tolerating abiotic stresses like extremes of temperature, pH, salinity and drought; heavy metal and pesticide pollution. Seeking such tolerant PGP microbes is expected to offer enhanced plant growth and yield even under a combination of stresses. This review summarizes the PGP related research and its benefits, and highlights the benefits of PGP rhizobia belonging to the family Rhizobiaceae, Phyllobacteriaceae and Bradyrhizobiaceae

Harnessing rhizobia to improve heavy-metal phytoremediation by legumes

Rhizobia are bacteria that can form symbiotic associations with plants of the Fabaceae family, during which they reduce atmospheric di-nitrogen to ammonia. The symbiosis between rhizobia and leguminous plants is a fundamental contributor to nitrogen cycling in natural and agricultural ecosystems. Rhizobial microsymbionts are a major reason why legumes can colonize marginal lands and nitrogen-deficient soils. Several leguminous species have been found in metal-contaminated areas, and they often harbor metal-tolerant rhizobia. In recent years, there have been numerous efforts and discoveries related to the genetic determinants of metal resistance by rhizobia, and on the effectiveness of such rhizobia to increase the metal tolerance of host plants. Here, we review the main findings on the metal resistance of rhizobia: the physiological role, evolution, and genetic determinants, and the potential to use native and genetically-manipulated rhizobia as inoculants for legumes in phytoremediation practices

Extent of cadmium stress on plant growth promoting microorganisms at the rhizosphere layers of S. stenocarpa and V. unguiculata accessions

The importance of role microbes in nodulation of leguminous crops helps in the nutritional diet of the Nigerian populace. However, heavy metal residues from heavy fertilization is a major cause of concern to soil and crop production. The study aimed to isolate and characterize free living microbe (bacteria and fungi) from the soil polluted with cadmium at different ecological screening value (0ESV, 2.5ESV and 5ESV) cultivated with Sphenostylis stenocarpa and Vigna unguiculata compared with similar soil but without the plants. The microbial count was estimated using the Most Probable Number (MPN) method. Compared to the control, a decrease of rhizobia number and an increase of the metal concentration were observed. From the results, cadmium toxicity had little to no effect on the bacteria diversity of the bulk soil has the increased bacterial and fungi diversity was recorded in the 2.5 ESV and 5 ESV respectively. The rhizosphere layer of Tss93 in the Cd-2.5 ESV had a significantly increased microbial diversity compared to the other accessions with the lowest total heterotrophic bacteria count recorded in Tss92 irrespective of metal concentration. Cd toxicity resulted in an insignificant difference (p>0.05) in total heterotrophic fungi count irrespective of plant accession or metal concentration, however, the fungi diversity was heightened in the Tss93 (2.5 ESV) and Tss95 (2.5 ESV) respectively. Cd toxicity increased the rhizosphere THB and THF counts of V. unguiculata with the highest microbial diversity recorded in TVu91 and TVu95 sown in the Cd-2.5ESV and Cd-5ESV respectively. The presence of heavy metal degradable bacteria – Pseudomonas aeruginosa and Bacillus subtilis and fungi – Aspergillus niger and penicillum sp. indicates bioremediation capacities of both accessions. This suggest that the survival microbes in polluted soil reveal adaptation of some traits especially those involved in symbiosis. Though cadmium had a significant effect on the soil productive capabilities, the THBC and THFC of Tss91 and Tss95 (S. stenocarpa) and TVu93 and TVu94 (V.unguiculata) were significantly increased as the presence of plant growth promoting bacteria and fungi were reported

Role of Trichoderma and Sinorhizobium Strains for Improving Growth and Nutritional Status of Alfalfa under Cd Stress

The plant rhizosphere is a major soil ecological environment for plant- microbe interactions involving colonization of different microorganisms in and around the roots of the growing plant. Plants can be used in the remediation of soils contaminated with heavy metals. The objective of this study was determine the relationship between the effect of Cd on the symbiotic model of Sinorhizobium meliloti – Medicago sativa and the application of Trichoderma sp. on the nutritional status as well as biochemical characterization of the sandy brown forest soil. The effects of biofertilizer Sinorhizobium and coinoculants Trichoderma strains on growth, chlorophyll and N, P and K content of alfalfa growing in soil polluted by cadmium were investigated. The results indicate that the presence of the saprobe fungi Trichoderma harzianum further enhanced shoot dry weight, N, P and K content of Sinorhizobium meliloti-alfalfa symbiotic model. The co-inoculation of alfalfa with T. harzianum was more effective for Cd uptake. The effects of the bio-multiple inoculants on the growth of alfalfa were stimulated the colonization of Sinorhizobium strains in the rhizosphere, promoted the nodulation potential and increased the dry organic matter. Sinorhizobium meliloti interacts with alfalfa as a model for rhizobioremediation and Trichoderma strains interact with this model as nodule promotors as well as a partner in the process of cleaning the plant rhizosphere from cadmium metal

Isolation of polysaccharide producer and heavy metal tolerant local rhizobial isolates

Background: Rhizobial bacteria is an important species among the soil bacteria that inter a relationship with leguminous plant and fix nitrogen symbiotically. Importance of this relation not only for soil as soil fertilizer but also to keep our environment without pollution.  Methods: Survey was conducted to collect different strains of rhizobial from different area in Nineveh Governate in Iraq. Isolation and biochemical tests were done under laboratory conditions. Determination of exopolysaccharide and tolerance of heavy metal was conducted also. Data obtained was recorded after cultivation and incubation of rhizobial strains.Results: The rhizobial bacteria were isolated from the following leguminous plants: Vigna unguiculata L., Trifolium alexandrinum, Trigonella foenum-graecum L., Leucaena leucocephala L., Medicago sativa L., Phaseolus vulgaris L., Tribulus terrestris L. and Vicia faba L. Maximum exopolysaccharide production were reached to 3.70 gm/L by the isolate R. leguminosarum bv. Viciae  RM25,after two days of incubation. The maximum cell dry weight was 2.90 gm/L. by the isolate E. meliloti RM14, after two days of incubation. Maximum reduction in pH were 4.30 by strain E. meliloti RM5, after two days incubation. All the local isolated rhizobia were tolerated to nickel chloride for the studied concentrations: 100, 500, 1000 and 5000 µg/ ml. Also were tolerance to 100 and 500 µg/ ml of zinc sulfate and copper sulfate. 1000 µg/ ml concentration of zinc sulfate were also tolerated by all rhizobial isolates.Conclusion: Rhizobium bacteria possess several mechanisms that allow them to tolerate heavy metal exposure. These mechanisms include the expression of efflux pumps, the presence of metal-resistance plasmids, the production of EPS, and the ability to adapt to environmental factors. Further research is needed to fully understand the mechanisms behind the heavy metal tolerance in Rhizobium and to explore the potential applications of these bacteria in bioremediation of heavy metal polluted soils

Plant growth-promoting rhizobacteria enhance the growth and Cd uptake of Sedum plumbizincicola in a Cd-contaminated soil

This study aimed to isolate plant growth-promoting rhizobacteria (PGPR) that exhibit heavy metal resistance to examine their influence on Cd uptake and soil microbial community structure during phytoremediation. Heavy metal-tolerant PGPR were isolated from the roots of possible hyperaccumulators using plates with 1-aminocyclopropane-1-carboxylate (ACC) as sole nitrogen source. Minimal inhibitory concentrations (MICs) of each isolate were determined by the plate dilution method. The impacts of isolated PGPR on the growth and Cd accumulation of Sedium plumbizincicola were conducted in a pot experiment. In addition, the effect of PGPR inoculation on the microbial community during phytoextraction by S. plumbizincicola was studied by 454 pyrosequencing. A total of nine Cd-resistant strains were isolated from the roots of Cd accumulators, and their plant growth-promoting activities were characterized. Isolates were able to produce indole-3-acetic acid (IAA) (28-133 mg L-1) and solubilize phosphate (65-148 mg L-1). In a pot experiment, the inoculation of isolates NSX2 and LCR1 significantly enhanced the growth of and uptake of Cd by the Cd hyperaccumulator S. plumbizincicola. 454 pyrosequencing revealed that the inoculation of the PGPR lead to a decrease in microbial community diversity in the rhizopshere during phytoextraction. Specifically, indigenous heavy metal-tolerant PGPR such as Actinospica, Bradyrhizobium, Rhizobium, Mesorhizobium, and Mycobacterium were selectively enriched in the treatments in which PGPR were added. It is suggested that a unique constitution of microbial communities in inoculated treatments plays a key role in enhancing Cd phytoremediation. Inoculation of strains Rhodococcus erythropolis NSX2 and Cedecea davisae LCR1 could promote S. plumbizincicola growth and enhance the remediation efficiency. The introduced PGPR could also affect the indigenous microbial community structure and the diversity in Cd-contaminated soil during phytoremediation.This study aimed to isolate plant growth-promoting rhizobacteria (PGPR) that exhibit heavy metal resistance to examine their influence on Cd uptake and soil microbial community structure during phytoremediation