Metrics details Manipulating the rhizosphere microbiome to enhance plant stress tolerance is an environmentally friendly technology and a renewable resource to restore degraded environments Here we suggest a sustainable bioremediation strategy on the example of Stebnyk mine tailings storage We consider Salicornia europaea rhizosphere community and the ability of the phytoremediation plant Salix viminalis to recruit its beneficial microbiome to mediate the pollution stress at the Stebnyk mine tailings storage The tailings contain large amounts of brine salts and heavy metals that contaminate the ground water and surrounding areas changing soil biogeochemistry and causing increased erosion The species richness of the endophytic bacterial community of S viminalis roots was assessed based on observed OTUs Our results obtained using the plant-based enrichment strategy show that biodiversity was decreased across the contamination zones and that S europaea supplementation significantly increased the species richness Our results also indicate that the number of dominating bacteria was not changed across zones in both S europaea-treated and untreated bacterial populations and that the decrease in richness was mainly caused by the low abundant bacterial OTUs The importance of selecting the bioremediation strains that are likely to harbor a reservoir of genetic traits that aid in bioremediation function from the target environment is discussed Here we considered the potential of the basket willow (Salix viminalis) for the tailing storage bioremediation Here we hypothesized that bacteria that have been living under HM and salt pollution stress over a long period of time have gained the potential to mitigate plant pollution stress Hence as a first step we determined the pattern of endophytic bacterial colonization of S viminalis across the Stebnyk contamination gradient europaea treatments would increase the abundance and species richness of the endophytic community We tested these hypotheses by conducting DNA metabarcoding along replicated pollution zones was collected on Stebnyk tailing under field conditions and transferred to sterile bags All soils were stored at 4 °C until potting the next day 5 rooted cuttings were grown using a sand culture After three months of growth the willow cuttings were harvested and DNA extracted from the below-ground part of the plants DNA was extracted from the roots grown in the pots with soil from the zones 1 Z2 and Z3) and from the ones where the growth substrate contained soil from zones enriched with the S Microbial DNA was obtained from 200 mg of S viminalis roots using the Nucleo Spin® Soil kit (Macherey–Nagel Quantitative determination of the concentration (ng/μl) and purity of DNA (A260/280) was performed spectrophotometrically using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific) DNA concentration was determined fluorometrically using Qubit 2.0 (Invitrogen) The quality of the DNA was checked via 1% agarose gel electrophoresis DNA samples were stored at − 20 °C for further analysis Prokaryotic 16S rRNA gene fragments were amplified by a two-step PCR procedure where the first step consisted of 2.5 ng extracted DNA 2× Phusion PCR Mastermix (Thermo Scientific US) and 10 μM of the primers pro341F/pro805R in 15 μl reactions Two independent PCRs were run under the following conditions: 3 min at 98 °C 55 °C for 30 s and 72 °C for 30 s and a final extension step of 10 min at 72 °C The PCR products were then pooled and checked by 1% agarose gel electrophoresis A single 30 μl reaction was performed for the second PCR using 2 μM of primers with Nextera adaptor and index sequences and 3 μl of the pooled PCR product from the first PCR Conditions were the same as the first step except for an annealing temperature of 55 °C and an extension time of 45 s The final PCR products were purified using an E.Z.N.A.® Cycle-Pure Kit (Omega Bio-tek Georgia) following the manufacturer’s instructions The amplicon size was checked by gel electrophoresis and the quality control was performed on a BioAnalyzer (Agilent After quantification using a Qubit fluorometer (Invitrogen Sequencing was performed on an Illumina MiSeq instrument using the 2,250 bp chemistry Sequences classified as belonging to taxa “Chloroplast-Mitochondria-unknown-Archaea-Eukaryota” were removed from further analysis Sequences were clustered to OTU-s using the cluster.split command with parameters taxlevel = 4 and cutoff = 0.03 OTU counts were obtained using command make.shared and the consensus taxonomy of each OTU was obtained using the classify.otu command The mothurs’ make.biom command was used with inputs from make.shared and classify.otu to generate a biom v1.0 file for downstream analyses We ran minimally 2000 iterations and four chains to fit models Stan NUTS control parameter adapt_delta was increased to 0.95–0.99 and max tree depth to 12–15 Hypotheses were tested using the alpha = 0.05 level specifying the Bayesian confidence interval (credible interval) containing 1 − alpha = 0.95 (95%) of the posterior values material identification and deposition was performed complying with national and international guidelines and legislation of the Department of Plant Physiology and Ecology of Ivan Franko National University of Lviv and ChML West-Plast (certifcate of GOE “Lvivstandardmetrologiya” No Relationships between OTU sets identified from S viminalis roots grown in unsupplemented soils or in S europaea rhizosphere-supplemented (rhiz) soils from three contamination zones viminalis cuttings grown in unsupplemented soil samples from three zones europaea rhizosphere-supplemented soil from three zones viminalis cuttings grown in unsupplemented vs S viminalis plants grown in unsupplemented or in S europaea rhizosphere-supplemented soils from Z1 (D) (G) Proportion of common OTUs relative to total from S Points denote the best fit of the aggregated binomial model [common|trials(total) ~ rhiz + zone + rhiz:zone] Thick and thin lines denote 67% and 95% credible intervals individual replicates were first rarefied and then merged by treatment groups Alpha diversity measures of OTUs identified from S viminalis roots grown in soils from three contamination zones with or without S (A) Conditional effects of the contamination zone and S europaea rhizosphere supplementation to the observed OTU richness estimated from negative binomial model (B) Conditional effects of the contamination zone and S europaea rhizosphere supplementation to the Shannon diversity index estimated from the robust linear model (Student’s t likelihood) (C) Conditional effects of the contamination zone and S europaea rhizosphere supplementation to the Inverse Simpson’s diversity index estimated from the robust linear model Non-rarefied OUT sets were used for diversity index calculations Thick- and thin lines denote 67% and 95% credible intervals Small points denote individual observations Asterisks denote non-zero effect size in diversity measures (probability > 0.95) of S viminalis cuttings grown in unsupplemented versus S (A) Non-metric multidimensional scaling (NMDS) using Bray–Curtis distances Ellipses are 95% confidence regions of t distributions (B) Distances to centroid calculated for NMDS Bray–Curtis distances Taxonomic analysis suggests a higher abundance of genera Halomonas, Marinobacter, Idiomarina, and Marinimicrobium in S. viminalis cuttings grown in Z3 soils (Fig. 5). Relative abundance (Abd) of the top twenty genera Samples were ordered by the contamination zone and S By a similar token here we hypothesise that the bacteria that have lived for long periods under HM and salt pollution stress have acquired the ability to tolerate the pollution and are suitable for supporting bioremediation processes While the Stebnyk tailings storage represents a threat to plant and microbial communities putting ecosystem integrity at risk europaea plants grown under the pollution over four decades represent a reservoir of genetic traits to aid bioremediation of S Our data representing the contamination gradient clearly showed that the alfa diversity of the bacterial community decreased suggesting a homogenization of the communities as the pollution appeared The sequencing of the 16S rDNA library revealed the presence of a relatively rich endophytic community indicated by the number of identified OTUs The biodiversity and the richness of the bacterial endophytic community were assessed based on the observed number of OTUs Modelling showed that the observed number of OTUs decreased from zone 1 to zone 3 europaea supplementation significantly increased the number of OTUs in zones 1 and 3 The biodiversity was significantly decreased across the contamination in un-supplemented plants as indicated by the Shannon index europaea supplementation lessened the decline based on observed OTUs and the Shannon index The inverse Simpson index was not significantly affected by the S This suggests that the number of dominating OTUs is not changing across zones in both S whereas the decrease in richness is mainly caused by low-abundance OTUs The basic biodiversity principle is that different organisms enhance the ecosystem functions under stress conditions The observed increase in species richness can differentially influence the functioning of the ecosystem If all species contribute approximately equally to the functioning of the ecosystem the species effect may be additively decelerating if some of the species are to some extent functionally redundant If the pool of species contains few species that can mediate handling pollutants efficiently Any effective plant beneficial microbial application requires effective colonization Although several endophytes have shown bioremediation activities microorganisms under field conditions face competition with a myriad of microbes naturally adapted to the environment on the example of Stebnyk tailing we present a sustainable bioremediation strategy using plant-based enrichment We identified native endophytic strains of S viminalis (the bioremediation species) and S europaea (local species adapted to pollution) that would be capable of colonizing and have potential efficiently supporting phyto-remediation at the Stebnyk mine How the microbial isolates behave under specific environmental conditions and what are the most influential consortia needs further metagenomic study in order to predict functional traits under the conditions The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB73979 (https://www.ebi.ac.uk/ena/browser/view/PRJEB73979) Timmusk, S., Behers, L., Muthoni, J., Muraya, A. & Aronsson, A. C. Perspectives and challenges of microbial application for crop improvement. Front. Plant Sci. 8, 49. https://doi.org/10.3389/fpls.2017.00049 (2017) Hartmann, M., Frey, B., Mayer, J., Mäder, P. & Widmer, F. Distinct soil microbial diversity under long-term organic and conventional farming. ISME J. 9(5), 1177–1194. https://doi.org/10.1038/ismej.2014.210 (2015) Averill, C. et al. Defending Earth’s terrestrial microbiome. Nat. Microbiol. 7(11), 1717–1725. https://doi.org/10.1038/s41564-022-01228-3 (2022) Nevo E (2023) The potential for plant growth-promoting bacteria to impact crop productivity in future agricultural systems is linked to understanding the principles of microbial ecology Batista, B. D. & Singh, B. K. Realities and hopes in the application of microbial tools in agriculture. Microb. Biotechnol. 14(4), 1258–1268. https://doi.org/10.1111/1751-7915.13866 (2021) Uber neuere Erfahrungen und Probleme auf dem Gebiete der Bodenbakteriologie unter besonderden berucksichtigung und Brache Ray, P., Lakshmanan, V., Labbe, J. L. & Craven, K. D. Microbe to microbiome: A paradigm shift in the application of microorganisms for sustainable agriculture. Front. Microbiol. 11, 622926. https://doi.org/10.3389/fmicb.2020.622926 (2020) Inoculants of plant growth promoting bacteria for use in agriculture Glick, B. R. Plant growth-promoting bacteria: Mechanisms and applications. Scientifica. 2012, 963–401. https://doi.org/10.6064/2012/963401 (2012) de-Bashan L, Hernandez J, Bashan Y. The potential contribution of plant growth-promoting bacteria to reduce environmental degradation—A comprehensive evaluation. Appl. Soil Ecol. https://doi.org/10.1016/japsoil201109003 Timmusk, S. et al. Drought-tolerance of wheat improved by rhizosphere bacteria from harsh environments: Enhanced biomass production and reduced emissions of stress volatiles. PLoS One. 9(5), e96086. https://doi.org/10.1371/journal.pone.0096086 (2014) Sonawane, J. M., Rai, A. K., Sharma, M., Tripathi, M. & Prasad, R. Microbial biofilms: Recent advances and progress in environmental bioremediation. Sci. Total Environ. 824, 153843. https://doi.org/10.1016/j.scitotenv.2022.153843 (2022) Tufail, M. A. et al. Recent advances in bioremediation of heavy metals and persistent organic pollutants: A review. Sci. Total Environ. 850, 157961. https://doi.org/10.1016/j.scitotenv.2022.157961 (2022) Orozco-Mosqueda, M. D. C., Fadiji, A. E., Babalola, O. O., Glick, B. R. & Santoyo, G. Rhizobiome engineering: Unveiling complex rhizosphere interactions to enhance plant growth and health. Microbiol. Res. 263, 127137. https://doi.org/10.1016/j.micres.2022.127137 (2022) Santoyo, G., Urtis-Flores, C. A., Loeza-Lara, P. D., Orozco-Mosqueda, M. D. C. & Glick, B. R. Rhizosphere colonization determinants by plant growth-promoting rhizobacteria (PGPR). Biology (Basel). https://doi.org/10.3390/biology10060475 (2021) Bar-On, Y. M., Phillips, R. & Milo, R. The biomass distribution on Earth. Proc. Natl. Acad. Sci. USA. 115(25), 6506–6511. https://doi.org/10.1073/pnas.1711842115 (2018) Kaminsky, L. M., Trexler, R. V., Malik, R. J., Hockett, K. L. & Bell, T. H. The inherent conflicts in developing soil microbial inoculants. Trends Biotechnol. 37(2), 140–151. https://doi.org/10.1016/j.tibtech.2018.11.011 (2019) Timmusk S, de-Bashan L. Microbiome: A tool for plant stress management in future production systems. Stresses. (2022). https://doi.org/10.3390/stresses2020014 Timmusk, S., Seisenbaeva, G. A. & Behers, L. Titania (TiO2) nanoparticles enhance the performance of growth-promoting rhizobacteria. Nat. Sci. Rep. https://doi.org/10.1038/s41598-017-18939-x (2018) Oyserman, B. O., Medema, M. H. & Raaijmakers, J. M. Road MAPs to engineer host microbiomes. Curr. Opin. Microbiol. 43, 46–54. https://doi.org/10.1016/j.mib.2017.11.023 (2018) Dini-Andreote, F. & Raaijmakers, J. M. Embracing community ecology in plant microbiome research. Trends Plant Sci. 23(6), 467–469. https://doi.org/10.1016/j.tplants.2018.03.013 (2018) Trivedi, P., Mattupalli, C., Eversole, K. & Leach, J. E. Enabling sustainable agriculture through understanding and enhancement of microbiomes. New Phytol. 230(6), 2129–2147. https://doi.org/10.1111/nph.17319 (2021) Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T. & Singh, B. K. Plant–microbiome interactions: From community assembly to plant health. Nat. Rev. Microbiol. 18(11), 607–621. https://doi.org/10.1038/s41579-020-0412-1 (2020) Wagg, C., Bender, S. F., Widmer, F. & van der Heijden, M. G. Soil biodiversity and soil community composition determine ecosystem multifunctionality. Proc. Natl. Acad. Sci. USA. 111(14), 5266–5270. https://doi.org/10.1073/pnas.1320054111 (2014) Abbas, M. et al. Vibrio fischeri bioluminescence inhibition assay for ecotoxicity assessment: A review. Sci. Total Environ. 626, 1295–1309. https://doi.org/10.1016/j.scitotenv.2018.01.066 (2018) Baldassarre, L., Ying, H., Reitzel, A. M., Franzenburg, S. & Fraune, S. Microbiota mediated plasticity promotes thermal adaptation in the sea anemone Nematostella vectensis. Nat. Commun. 13(1), 3804. https://doi.org/10.1038/s41467-022-31350-z (2022) Delannoy-Bruno, O. et al. An approach for evaluating the effects of dietary fiber polysaccharides on the human gut microbiome and plasma proteome. Proc. Natl. Acad. Sci. USA. 119(20), e2123411119. https://doi.org/10.1073/pnas.2123411119 (2022) Content of enzymatic and nonenzymatic antioxidants in Salix viminalis L Salamon I (2023) Information supply on hydrotechnical reconstruction concept of Stebnyk tailing storage (Ukraine) Timmusk, S. et al. Bacterial distribution in the rhizosphere of wild barley under contrasting microclimates. PLoS One. 6(3), e17968. https://doi.org/10.1371/journal.pone.0017968 (2011) Abdollahzadeh, T. et al. Phytoremediation of petroleum-contaminated soil by Salicornia: From PSY activity to physiological and morphological communications. Environ. Technol. 40(21), 2789–2801. https://doi.org/10.1080/09593330.2018.1453551 (2019) Hrynkiewicz, K., Patz, S. & Ruppel, S. Salicornia europaea L. as an underutilized saline-tolerant plant inhabited by endophytic diazotrophs. J. Adv. Res. 19, 49–56. https://doi.org/10.1016/j.jare.2019.05.002 (2019) Fetsiukh A, Buno L, Patsula O, Terek O. Accumulation of heavy metals by Salix viminalis plants under growing at the substrate from Stebnyk tailings. Series Biology; Visnyk of the Lviv University. 2019;(81):96–110. https://doi.org/10.30970/vbi.2019.81.11 Burkner, P. C., Doebler, P. & Holling, H. Optimal design of the Wilcoxon–Mann–Whitney-test. Biom. J. 59(1), 25–40. https://doi.org/10.1002/bimj.201600022 (2017) Timmusk, S. & Zucca, C. The plant microbiome as a resource to increase crop productivity and soil resilience: A systems approach. J. Cameroon Acad. Sci. https://doi.org/10.4314/jcasv14i32 (2019) Desale, P., Patel, B., Singh, S., Malhotra, A. & Nawani, N. Plant growth promoting properties of Halobacillus sp. and Halomonas sp. in presence of salinity and heavy metals. J. Basic Microbiol. 54(8), 781–791. https://doi.org/10.1002/jobm.201200778 (2014) Mukherjee, P., Mitra, A. & Roy, M. Halomonas rhizobacteria of avicennia marina of indian sundarbans promote rice growth under saline and heavy metal stresses through exopolysaccharide production. Front. Microbiol. 10, 1207. https://doi.org/10.3389/fmicb.2019.01207 (2019) Saha, L., Tiwari, J., Bauddh, K. & Ma, Y. Recent developments in microbe-plant-based bioremediation for tackling heavy metal-polluted soils. Front. Microbiol. 12, 731723. https://doi.org/10.3389/fmicb.2021.731723 (2021) Haque, S. et al. Functional microbiome strategies for the bioremediation of petroleum-hydrocarbon and heavy metal contaminated soils: A review. Sci. Total Environ. 833, 155222. https://doi.org/10.1016/j.scitotenv.2022.155222 (2022) Raklami, A., Meddich, A., Oufdou, K. & Baslam, M. Plants-microorganisms-based bioremediation for heavy metal cleanup: Recent developments, phytoremediation techniques, regulation mechanisms, and molecular responses. Int. J. Mol. Sci. https://doi.org/10.3390/ijms23095031 (2022) Sharma, P., Singh, S. P., Iqbal, H. M. N. & Tong, Y. W. Omics approaches in bioremediation of environmental contaminants: An integrated approach for environmental safety and sustainability. Environ. Res. 211, 113102. https://doi.org/10.1016/j.envres.2022.113102 (2022) Prosser, J. I. et al. The role of ecological theory in microbial ecology. Nat. Rev. Microbiol. 5(5), 384–392. https://doi.org/10.1038/nrmicro1643 (2007) Download references David Clapham is gratefully acknowledged for critically reading the manuscript This study was supported by the Swedish Research Council VR 2017-05524 and VR2021-05471 to ST Sequencing was performed by the SNP&SEQ Technology Platform in Uppsala The facility is part of the National Genomics Infrastructure (NGI) TheSNP&SEQ Platform is supported by the Swedish Research Council and the Knut and Alice Wallenberg Foundation Open access funding provided by Swedish University of Agricultural Sciences These authors contributed equally: Anastasiia Fetsiukh and Taavi Pall Department of Forest Mycology and Pathology Swedish University of Agricultural Sciences (SLU) Institute of Biomedicine and Translational Medicine ST conceived and designed the study; AF Stebnyk sampling library preparation; TP processing the reads statistical analysis; ST and TP wrote the manuscript; All authors contributed to the article and approved the submitted version The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-024-68123-1 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Ukraine (AP) — Coronavirus infections in Ukraine began surging in late summer and the ripples are now hitting towns like Stebnyk in the western part of the country Natalia Stetsik is watching the rising number of patients with alarm and anguish We are catastrophically short of doctors,” says Stetsik the chief doctor at the only hospital in the town of 20,000 people “It’s very hard for a doctor to even see all the patients.” The hospital is supposed to accommodate 100 patients Ukraine’s ailing health care system struggled with the outbreak and authorities introduced a tight lockdown in March to prevent hospitals from getting overwhelmed The number of cases slowed during the summer but began to rise again quickly prompting the government at the end of August to close Ukraine's borders for a month the number of positive tests reported in the country continued climbing quickly and reached a new daily peak of 5,397 on Thursday COVID-19 infections in Ukraine have nearly doubled in the past month and an increasing share of them are in grave condition,” Stetsik told The Associated Press of the situation in Stebnyk “The virus is becoming more aggressive and more difficult to deal with.” She said many of those doing poorly are in their 30s adding that an increasing number of them need expensive medication “There is a similar situation across entire Ukraine,” she said adding that hospitals have run out of funds to provide drugs forcing patients in some areas to buy their own The World Health Organization warns that the number of infections in Ukraine could continue to grow and reach 7,000-9,000 a day The government wants to avoid imposing a new lockdown but officials acknowledge that the rising number of infections could make it necessary It has sought to introduce a more flexible approach to minimize the economic damage At a meeting Monday with officials in Kyiv President Volodymyr Zelenskiy chastised them for failing to do enough to slow the spread and taking too long to provide necessary supplies “We spend weeks on doing things that must be done within days,” he said Zelenskiy specifically urged them to move faster on ensuring that hospitals have enough supplementary oxygen noting that only about 40% of beds for COVID-19 patients have access to it Ukraine’s corruption-ridden economy has been drained by a six-year conflict with Russia-backed separatists in the eastern part of the country and Zelenskiy's administration inherited health care reforms from his predecessor that slashed government subsidies leaving hospital workers underpaid and poorly equipped Zelenskiy ordered the government to increase wages for medical workers Official statistics show that 132 medical workers have died from the coronavirus although the figure doesn't include those who tested negative but had symptoms typical for COVID-19 a 51-year-old therapist from the western town of Pochaiv who described the challenges of dealing with the outbreak in an interview with the AP in May Venzhynovych died last week of double pneumonia which his colleagues believed was caused by the coronavirus “He certainly had COVID-19,” said Venzhynovych's widow “There are many infections among medical workers The government pays the equivalent to $56,000 to families of medical workers who die from the coronavirus But Venzhynovych's widow can't receive the payment because he tested negative many lawmakers and top officials are testing positive including former President Petro Poroshenko who was hospitalized in serious condition with virus-induced pneumonia Medical professionals want the government to bring back a sweeping lockdown pointing to the scarce resources for the health care system “It’s possible that Ukraine would need to return to a tight quarantine like in the spring The number of patients is really big,” said Dr He blamed the new infections on public negligence and I feel sorry that they aren’t impressed by numbers,” he said Gloshovskiy said he had to switch to treating COVID-19 patients because of the personnel shortage “I had to change my specialty because my colleagues simply wouldn’t be able to cope with it without me,” he said Health Minister Maxim Stepanov acknowledged that the shortage of doctors and nurses is a big problem “We may increase the hospital capacity and improve oxygen supply but we could just be simply short of doctors,” he said A tight lockdown would be a severe blow to the already weakened economy warning that authorities could be forced to do it anyway the Health Ministry would propose to return to tough quarantine measures,” he said some patients said they only realized the coronavirus threat after falling ill “I didn’t believe in its existence until I became infected,” said 43-year-old Natalia Bobyak “When I got here I saw that people get sick en masse.” Follow AP’s pandemic coverage at http://apnews.com/VirusOutbreak and https://apnews.com/UnderstandingtheOutbreak rewritten or redistributed without permission Kyiv official says he thinks Istanbul proposals could lead to summit but Kremlin says there is ‘a lot of work to be done’ Vladimir Putin and Volodymyr Zelenskiy could meet “soon”, a senior aide to Ukraine’s president has claimed, even as the Kremlin downplayed hopes of an early breakthrough in the peace talks. The head of Ukraine’s negotiating team, Mykhailo Podolyak, a key adviser to Zelenskiy, said on Wednesday that Moscow was scrutinising proposals submitted by Kyiv in Istanbul which he believed could lead to a presidential peace summit. Read more“We can expect a presidential meeting to be held some time soon,” Podolyak said said it was “positive” that Ukraine had set out its position but he talked down expectations of a summit “We cannot state that there was anything too promising or any breakthroughs,” he said adding that there was “a lot of work to be done” 1:42Full peace is needed to agree treaty with Russia says Ukrainian negotiator – videoVladimir Medinsky the head of Russia’s delegation in the talks this week by claiming that Zelenskiy’s proposals indicated a readiness by Ukraine to reach a deal “for the first time in years” adding that “if it fulfils the obligations the threat of creating a Nato bridgehead on the Ukrainian territory will be removed” the Chechen leader whose forces are fighting in Ukraine said that Moscow would make no concessions And if you think that he (Putin) will quit what he started just the way it is presented to us today The claims and counter-claims from the two capitals highlight the negotiating games being played by both sides as the war continues to rage in hotspots across Ukraine The United Nations refugee agency said on Wednesday that more than 4 million people had fled Russia’s “utterly senseless” war 4,019,287 people have fled abroad since the start of Russia’s invasion on 24 February exceeding its initial estimate that the war would create up to 4 million refugees 14:42’Humans are all the same’: the Hungarian border village helping Ukraine's refugee families - video The UN high commissioner for refugees said on Twitter he had just arrived in Ukraine and was beginning discussions with authorities the UN and other partners on “ways to increase our support to people affected and displaced by this senseless war” The agency has said the speed and scale of the displacement is unprecedented in Europe since the second world war The UN’s International Organization for Migration (IOM) also said that as of mid-March 6.48 million people were internally displaced life-saving aid,” the IOM said on Wednesday Ukraine had a population of 37 million in the regions under government control excluding Crimea and the Russian-controlled regions in the east At the talks in Istanbul on Tuesday, Ukraine proposed a framework for peace under which it would remain neutral with its security guaranteed by third-party countries through a treaty similar to Nato’s article 5 mutual defence commitment intended to come into force only in the event of a complete ceasefire included a 15-year consultation period on the status of the Crimean peninsula which Moscow seized from Ukraine and annexed in 2014 Medinsky said Ukraine had appeared ready to commit to a nuclear-free status and drop its aspirations contained within the country’s constitution He said Ukraine had also signalled that it could agree not to host foreign military bases and that it would only hold joint drills with foreign militaries in consultation with countries serving as guarantors of a peace deal including the five permanent UN security council members Podolyak said any deal would need to be put to a referendum which could only happen once Russian troops had withdrawn but in what appeared to be a further concession he added that this could be only to “the positions as of 23 February 2022” in an apparent recognition that soldiers will remain at least in Crimea Key to Kyiv is that the four other security council members – the US France and China – commit to intervening should there be any future invasion of Ukrainian territory – which he claimed Moscow had accepted who said the Chelsea owner Roman Abramovich had been playing an effective role as a moderator of the talks said: “We have submitted our suggestions to the Russian party saying that in principle this is time for the presidential-level meeting When that is going to happen is rather a logistical question “The Russians need to go over our suggestions and give some preliminary response Now we are working at the working group level online clarifying different points so that package of documents is ready.” He added: “We came to Istanbul to define the intermediate positions in the key documents we see a possibility to sign preliminary agreements and to launch the presidential meeting process so in two or three or four days we will have a final version that will be confirmed by the Russians with amendments and then we can reach the next round of negotiations.” Moscow described the talks as “meaningful” and “constructive” and subsequently pledged to “radically reduce” its military activity around Kyiv and Chernihiv a heavily shelled city 100 miles north of the capital as a goodwill gesture to “to increase mutual trust” in the peace negotiations spokesperson for the Ukrainian ministry of defence said they had seen “some partial movement of certain units of the enemy from Kyiv and Chernihiv areas” “At the same time there is no mass-scale withdrawal from those areas,” he said “The enemy has been withdrawing units which suffered the highest losses in order to replenish them As far as we see the enemy has not abandoned its attempts to take or at least siege the capital city and Chernihiv.” Western analysts and diplomats noted that Russia’s offer to partially pull back came after its advance thwarted by stiff resistance and supply problems and that Moscow had already said it was refocusing its military goals on expanding the territory held by pro-Russia separatists in the eastern Donbas regions Officials in both Kyiv and Chernihiv said that both cities were still facing heavy shelling Log in for full access to stabroeknews.com Only one active session is allowed per subscriber (Reuters) – Ukrainian forces are preparing for new Russian attacks in the east of the country as Moscow builds up its troops there after suffering setbacks near the capital Kyiv President Volodymyr Zelenskiy said yesterday has driven around a quarter of Ukrainians from their homes and brought Russian-Western tensions to their worst point since the Cold War Tough resistance by Ukrainian forces has prevented Russia from capturing any major city where a Russian armed column was held back for weeks Russia said it would curtail operations near Kyiv and the northern city of Chernihiv to build trust dismissed Russia’s pledge as a ploy to stem its losses and prepare for other attacks The head of Britain’s GCHQ spy service said new intelligence showed some Russian soldiers had refused to carry out orders sabotaged their own equipment and accidentally shot down one of their own aircraft Russia says its forces are regrouping to focus on “liberating” the breakaway eastern Donbas region Zelenskiy referred to Russian troop movements away from Kyiv and Chernihiv and said that was not a withdrawal but rather “the consequence of our defenders’ work.” Zelenskiy added that Ukraine is seeing “a build-up of Russian forces for new strikes on the Donbas and we are preparing for that.” The Donbas region encompasses two self-proclaimed “people’s republics” that Russia says it is helping to free from Ukrainian control The leader of the Donetsk People’s Republic said yesterday that offensive operations were intensifying Donetsk includes the besieged port city of Mariupol which has seen some of the war’s heaviest fighting and bombardment and where about 170,000 people are trapped with scarce food and water some beetroot,” said former steel worker Viktor from Mariupol They cook using a rudimentary barbecue and sleep in a basement Russian forces have taken half of the strategic port city Russia’s defence ministry said it was prepared to observe a ceasefire in Mariupol on Thursday Kyiv has accused Russia of not fully respecting earlier such commitments Russia says it is carrying out a “special operation” to disarm and “denazify” its neighbour the biggest assault on a European country since World War Two The fate of Donbas was discussed at peace talks on Tuesday in Istanbul A senior Ukrainian negotiator said the talks will resume online on Friday Ukraine has sought a ceasefire without compromising on territory or sovereignty though it has proposed adopting a neutral status in exchange for security guarantees Russia opposes Ukraine joining the U.S.-led NATO military alliance and has cited its potential membership as a reason for the invasion Western sanctions imposed on Russia as punishment for its invasion have largely isolated its economy from world trade but Moscow is still the biggest supplier of oil and gas to Europe Russia demanded oil and gas payments be made in roubles by Friday raising fears of energy shortages and boosting recessionary risks in Europe Germany has warned of a possible emergency if Russia cuts supplies Russian President Vladimir Putin told German Chancellor Olaf Scholz that payments could be made in euros and sent to Gazprombank Scholz asked for written information to “better understand the procedure,” the spokesperson said one of the main channels for payments for Russian oil and gas Global restrictions on exports to Russia over its invasion have shut down a car maker and cut off a Russian computer maker’s access to circuits used in communications equipment “Necessity brought together this unprecedented collaboration on export controls and other measures that are having a meaningful impact on Putin’s war,” said Thea Kendler Russia says the West has effectively declared economic war on Russia and so it will now turn eastwards away from Europe to build a partnership with China A FREE roundup of top news from Guyana you might otherwise miss