Volume 8 - 2017 | https://doi.org/10.3389/fmicb.2017.01063 The emergence of new microbial pathogens can result in destructive outbreaks since their hosts have limited resistance and pathogens may be excessively aggressive Described as the major ecological incident of the twentieth century caused by ascomycete fungi from the Ophiostoma genus has caused a significant decline in elm tree populations (Ulmus sp.) in North America and Europe Genome sequencing of the two main causative agents of Dutch elm disease (Ophiostoma ulmi and Ophiostoma novo-ulmi) along with closely related species with different lifestyles allows for unique comparisons to be made to identify how pathogens and virulence determinants have emerged Among several established virulence determinants secondary metabolites (SMs) have been suggested to play significant roles during phytopathogen infection the secondary metabolism of Dutch elm pathogens remains almost unexplored and little is known about how SM biosynthetic genes are organized in these species To better understand the metabolic potential of O we performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in these species and assessed their conservation among eight species from the Ophiostomataceae family a fujikurin-like gene cluster (OpPKS8) was unique to Dutch elm pathogens Phylogenetic analysis revealed that orthologs for this gene cluster are widespread among phytopathogens and plant-associated fungi suggesting that OpPKS8 may have been horizontally acquired by the Ophiostoma genus the detailed identification of several BGCs paves the way for future in-depth research and supports the potential impact of secondary metabolism on Ophiostoma genus’ lifestyle This information allows for unique comparisons to be made between species to highlight putative virulence determinants in DED pathogens Dutch elm disease species are promising models to study the impact of invasive species and the evolution and adaptation to new niches in view of the success and persistence of these pathogens in the environment it is reasonable to propose that SMs may assist in the infection process of DED pathogens secondary metabolism and genes enrolled in SM production are virtually unexplored in the Ophiostoma genus and Ophiostomataceae family we assessed the conservation of BGCs and related genes identified within the eight available Ophiostomataceae family genomes a specific PKS BGC (OpPKS8) was conserved only in DED pathogens and was absent in other members of the Ophiostomataceae family Through phylogenetic inference and comparative genomic analyses we showed that this cluster may have been horizontally acquired by DED pathogens orthologs for this BGC were found in several plant-associated fungi which supports a putative role for the products of this gene cluster in phytopathogenic infection Corresponding amino acid sequences were aligned with the predicted OpPKS8 backbone gene products from O Genes that satisfied the previously fixed cutoffs were included in further analyses and a phylogenetic tree was constructed using PhyML 3.1 with 100 bootstrap replicates (alignment shown in Supplementary Data Sheet 2) A logical diagram describing the step-by-step process and the connections between methodologies applied in this section is shown in Supplementary Figure 2 based primarily on backbone gene conservation (e-value < 1 × 10-5 These results indicate that the SM potential is remarkably similar between Ophiostomataceae family members two BGCs were found only in the Ophiostoma genus: OpOTHER3 (OphioH327gp1586) was identified in O while OpPKS8 is exclusive to DED pathogens To further confirm the absence of OpPKS8 in O the vicinities of the OpPKS8 locus were explored The results confirmed the absence of the OpPKS8 gene cluster in O as several genes in the vicinity were conserved despite the absence of OpPKS8 (Supplementary Figure 4) Considering that no putative BGCs have been functionally characterized in DED pathogens to date we employed comparative genomics to determine the likely final products of these BGCs These comparisons revealed three interesting BGCs that exhibited previously characterized orthologous gene clusters in other species and which were putatively linked with the biosynthesis of a fujikurin-like compound (OpPKS8) crassa and the Ophiostomataceae family species are grouped in the same class which may indicate that proteins with similar functions to those of N crassa could represent the final product of the OpPKS10 backbone gene and that these similar proteins could act in the biosynthesis of similar metabolites Synteny comparison between the type III PKS locus in DED pathogens and in N crassa did not reveal extensive conservation (Supplementary Figure 4) suggesting that it might not be grouped in a biosynthetic cluster CASSIS was unable to discriminate homologous regulatory sequences between type III PKS of DED pathogens and neighboring genes This clustering of the ferricrocin ortholog backbone gene and the L-ornithine N5-oxygenase gene suggests that DED pathogens utilize a siderophore biosynthesis pathway that is similar to other model fungi we hypothesize that the products of OpNRPS1 BGC modulate iron dynamics in DED pathogens Putative fujikurin and fujikurin-like compound cluster (OpPKS8) conservation and synteny The OpPKS8 cluster from DED pathogens resembled the characterized fujikurin cluster from F fujikuroi IMI 58289 (36–68% identity in protein-by-protein comparisons of BGC constituent genes) and putative BGCs from Scedosporium apiospermum (66–84% identity in protein-by-protein comparisons of BGC constituent genes) Aureobasidium pullulans (58–80% in protein-by-protein comparisons of BGC constituent genes) and Setosphaeria turcica (66–70% identity in protein-by-protein comparisons of BGC constituent genes) The conservation between OpPKS8 and these putative BGCs from S turcica helped to define the boundaries from OpPKS8 Phylogenetic analyses were performed using Maximum-likelihood and Bayesian Inference and dehydrogenase domains of the fujikurin-like backbone gene of DED pathogens and orthologous sequences exhibited by several fungi The orthologous sequences were classified according to fungal lifestyle trait The Bayesian tree is displayed and branch support values (aLRT SH-like supports and Bayesian posterior probability) are associated with nodes The Bayesian inference ran for 35,000 generations fujikuroi IMI 58289 harboring the fujikurin and fujikurin-like gene clusters are highlighted in bold BGCs closely related to the OpPKS8 gene cluster are shaded gray species that harbor these BGCs closely related to the OpPKS8 gene cluster have a plant-associated trait the OpPKS8 gene cluster is the only BGC exclusively found in DED pathogens (among the evaluated species) raising the possibility that HGT events could have shaped the evolution of OpPKS8 orthologs an ancestor of the Sordariomycetes and Dothideomycetes classes may have contained a fujikurin-like compound gene cluster which remained in the genome of some species would have required the loss of constituent genes across several Sordariomycetes and Dothideomycetes species species from Leotiomycetes (Hymenoscyphus fraxineus and Phialocephala scopiformis) and Eurotiomycetes (Talaromyces cellulolyticus and Penicillium griseofulvum) classes also have orthologs making this vertical descent hypothesis unlikely several species that possess orthologous genes have a plant-associated lifestyle leading us to speculate that fujikurin-like SMs allow several fungal species to interact with plants not only as phytopatogens or endophytes but also in a nematophagous/entomopathogenic or opportunistic manner Comparative genomics have provided important insights into the evolution of fungal pathogens Several fungal genomes have been sequenced in recent years including multiple Ophiostomataceae family members and the publication of this data enables deep comparisons to highlight putative virulence determinants in view of a singular characteristic of different pathogenic traits in close family members (e.g. phytopathogenic and Sporothrix mammalian pathogenic fungi) enabling the identification of virulence determinants linked with the phytopathogenic trait (i.e. as the Ophiostomataceae family harbors several phytopathogenic fungi a putative virulence determinant may be widespread in several species Secondary metabolites are important virulence determinants for microorganisms. These molecules are produced to circumvent host defenses and ensure the success of these organisms in the environment (Keller, 2015) We explored the SM potential of DED pathogens and identified three interesting BGCs: OpNRPS1 A mycelium phase is essential for invasion of uninfected xylem vessels and for posterior saprophytic growth within moribund trees and the products of OpPKS10 may be of significant importance during this growth phase since this BGC may generate functional transcripts and subsequent proteins which would help to catalyze the biosynthesis of natural products This genome exhibits an ortholog (obic_04723) for the OpPKS8 backbone gene (65% identity between proteins) indicating that other phytopathogenic species from the Ophiostoma genus may harbor OpPKS8 orthologs The SM potential of DED pathogens was meticulously analyzed and yielded insights into aspects of genome organization of BGCs and their possible role as virulence determinants and mainly OpPKS8 gene clusters during DED pathogenic infection requires further confirmation using wet lab techniques our findings are important for future research Fujikurins and fujikurin-like compounds can play significant roles in fungal-plant interactions in several models including several economically important phytopathogenic fungi in addition to presenting horizontally transferred origins we anticipate that other putative virulence determinants can be found in DED pathogens and Ophiostomaceae family members using comparative genomic approaches as this family is rich in pathogenic traits Other relevant genetic characteristics could be explored in both Sporothrix and Ophiostoma genera in addition to genetic singularities that may be important virulence determinants Conceived and designed the experiments: NS Contributed reagents/materials/analysis tools: MV All authors read and approved the final manuscript This study was supported by grants and fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Grant: Universal 2014 458160/2014-8] Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [Grant: Biocomputacional Processo 23038.010041/2013-13] Fundação de Amparo a Pesquisa do Estado do RS (FAPERGS) and Fundação de Amparo a Pesquisa do Estado do RJ (FAPERJ) and is part of the Advanced Network of Computational Biology (RABICÓ) The publication charges for this article were funded by CAPES (process no The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Patrícia Aline Gröhs Ferrareze and the staff of LNCC for their support Stefanie Hartmann for kindly providing the O The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2017.01063/full#supplementary-material Parallel metropolis coupled markov chain monte carlo for Bayesian phylogenetic inference CrossRef Full Text | 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) or licensor are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Augusto Schrank, YXNjaHJhbmtAY2Jpb3QudWZyZ3MuYnI= Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details A recent survey in Germany revealed the wide presence of ‘Candidatus Phytoplasma ulmi’ in native elm stands Accessions were studied for their genetic variability and phylogenetic relationship based on the conserved groEL and the variable imp gene While the groEL sequences revealed a high intraspecific homology of more than 99% the homology of the imp gene dropped to 71% between distantly related sequences Twenty-nine groEL and 74 imp genotypes were distinguished based on polymorphic sites Phylogenetic analysis of the groEL gene clustered all ‘Ca ulmi’ strains and separated them from related phytoplasmas of the 16SrV group The inferred phylogeny of the imp gene resulted in a different tree topology and separated the ‘Ca one closely related to the flavescence dorée phytoplasma strain FD-D (16SrV-D) the other affiliated with the flavescence dorée phytoplasma strains FD-C and FD70 and the alder yellows phytoplasma (16SrV-C) ulmi’ genotypes from Scots elm trees formed a coherent cluster while genotypes from European white elms and field elms grouped less strictly The regional distribution pattern was congruent for some of the groEL and imp genotypes but a strict linkage for all genotypes was not apparent phylogenetic linkages and geographical distribution the phylogenetic relationship and the regional distribution of ‘Ca ulmi’ accessions with a groEL gene fragment and the imp gene—two markers of different resolving powers adding specific information to survey results published recently by the authors The selected forward and reverse primers for the groEL- and imp genes amplified fragments of about 880 bp and 675 bp, respectively, from all 288 ‘Ca. P. ulmi’ accessions (Fig. 1). The primers also amplified groEL- and imp fragments from the phytoplasmas alder yellows strain ALY, the ‘Ca. P. ulmi’ strain ULW and the flavescence dorée strain FD70. Amplification of imp (upper panel) and groEL (lower panel) fragments from ‘Ca. P. ulmi’ accessions. Only examples are shown. Sample names are indicated on the top, including the host plant and Federal State. Ug_h, DNA from a healthy Scots elm accession. The gel was photographed with a VWR Imager 2 System, Software Version 1.5.6.0 (https://de.vwr.com) and cropped with Photoshop CS3 (www.adobe.com) FD70 (MT638098) and ‘Candidatus Phytoplasma ziziphi’ sequences ranged between 94% and 96.4% shared a lower sequence homology (95.7% to 96.4%) to all other ‘Ca ulmi’ sequences and was closer related to the ALY (99.1%) and FD70 (98.6%) sequences This accession was included in the phylogenetic analysis but not considered as a sensu stricto member of the species ‘Ca Phytoplasma mali’ groEL sequence was on average 69.7% The multiple alignment of the 790 bp groEL fragment from the ‘Ca. P. ulmi’ sequences, excluding sequence 4120_Ul_SN, revealed a total of 18 polymorphic sites, of which 12 caused a nonsynonymous substitution (Table 1) mutations altering the amino acid in a significant number of isolates were only present at positions 18 The remaining non-silent mutations occurred in genotype groups with a low number of isolates A comparison of the 288 groEL sequences, including accession 4120_Ul_SN, on the basis of complete sequence homology, differentiated 29 genotypes, of which 18 occurred more than once (Table 2) The genotype groups correlated in most cases with the elm species from which they were isolated ulmi’ genotype was associated with a different elm species more than one elm species grew at the sampling location if fruits were unavailable or leaf shapes difficult to interpret a misidentification of the elm species cannot be excluded While the majority of isolates (N = 253) showed a gene length of 465 bases The imp gene of accession 4120_Ul_SN (MT668488) was shorter by six bases a feature shared with the phytoplasma strain ALY ulmi’ imp gene sequences ranged from 71 to 100% whilst homology to the imp gene of the 16SrV-D group phytoplasma FD-D ranged between 66 and 79% and homology to the 16SrV-C group sequences of ALY (MT668499) FD70 (MT6684500) and FD-C ranged between 67 and 71% Homology to the 16SrV-B subgroup member ‘Ca ziziphi’ was generally lower with a narrower bandwidth and ranged between 61 and 65% higher homology with 16SrV-C phytoplasmas (ALY ulmi’ strains to the distantly related species ‘Ca Alignment of imp sequences from 4319_Ug_SN (MT668492), 3486_Ug_ST (MT668480), 0371_Ul_BB (MT668429), ULW (MT418908) and the 16SrV-group phytoplasmas ALY (MT668499), FD70 (MT668500) and ‘Ca. P. ziziphi’ (CP025121) with Clustal W. The consensus sequence is given above the alignment, showing bases represented in more than 50% of sequences. Sequence alignments have been performed with Clustal W (www.ebi.ac.uk/clustalw/) Based on complete sequence identity, 74 genotypes were distinguished among the 288 ‘Ca. P. ulmi’ accessions. Twenty-eight genotypes were represented by more than one sequence (Table 3) and 46 genotypes were unique and their electropherograms re-assessed when a single nucleotide mismatch defined their group exclusion like the groEL genotypes according to the elm species from which they originated The imp genotype I comprised most of the accessions from Scots elm trees and the gene showed higher conservation compared to the linked groEL genes which associated the remaining 44 accessions to 14 groEL genotype groups the strain ULW and FD70 coded for a protein of 154 amino acids (aa) The imp gene of accession 4120_Ul_SN coded for a protein of 152 aa only Due to the high sequence heterogeneity between the ‘Ca homology between distantly related isolates dropped to 48% A protein-based reassessment of the genotype groups revealed that three sequences shared identical IMP sequences to others reducing the number of genotypes to 71 Homology to the 16SrV-C group phytoplasmas ALY and FD70 ranged from 42 to 52% to the 16SrV-D group phytoplasma FD-D from 45 to 61% and decreased below 39% in comparison to the 16SrV-B group phytoplasma ‘Ca The accession 4120_Ul_SN shared homology of 34% The ratio between non-synonymous over synonymous mutations was 1.53 (dN = 0.23; dS = 0.15) for imp and 1.00 (dN = 0.01; dS = 0.01) for the groEL-fragment The ratio of dN/dS > 1 for the imp gene indicates a positive selection which was not the case for the groEL-fragment The distribution of the imp genotypes I, II and V largely matched the respective groEL genotype spread (Fig. 5 A greater disparity of distribution was observed between the ‘Ca ulmi’ imp and groEL genotype groups III and IV The imp genotype III was absent in East Germany while the imp genotype IV did not occur in the south of the country The regional distribution of genotypes was largely congruent with the distribution of the elm species Genotype groups I and III were mostly associated with Scots elms genotype II with field elms and genotypes IV and V with European white elms there was some flexibility in the linkage of groEL- and imp genotypes was linked to 15 different groEL genotype groups of which the groEL genotype group I comprised the most with 57 sequences followed by group III with 16 sequences and group VI with six sequences The remaining 22 sequences were part of 12 other groEL genotype groups The same was true when considering the groEL group II in comparison with imp genotype group II All 26 sequences of the imp genotype group II were part of the corresponding groEL group but the remaining 35 groEL sequences were associated with a further 19 imp genotype groups Neither the amplification products nor the electropherograms of the groEL or imp sequences provided evidence that the amplimers consisted of polymorphic fragments The observed gene combinations were therefore considered as a true linkage and the result of selective evolutionary pressure and the variable imp gene was chosen to tap the full range of genetic polymorphism of a variable gene The primer sets for amplifying both genes were deduced from the database entries of the respective genes from the ‘Ca The oligonucleotides were compared to target genes of close relatives from the 16SrV-B Due to the high conservation of the groEL gene and the likelihood of an unspecific amplification of other bacterial groEL genes from crude plant extracts the reverse primer showed six mismatches to the ‘Ca the only other full-length groEL gene in the 16SrV group published Primers for the amplification of the imp-pyrG region were fully homologous to the respective sequences of ‘Ca ulmi’ strain ULW and the flavescence dorée strain FD-C Both primer sets enabled the amplification of the target fragments from all ‘Ca the sequencing results revealed no evidence of mixed infection of plants by more than one ‘Ca but they must have been present in small numbers so alternative base calls were below the threshold line of the electropherogram A closer examination of this matter would have exceeded the scope of this work samples were carefully selected based on an even geographical spread and on a representative number of samples from all three elm species The groEL and imp genotype groups comprised accessions originating from the same elm species although parallel genotype groups with the same host species existed Taking into consideration the many sequences of the larger genotype groups the distinct host–pathogen strain association seems not to be arbitrary It is unlikely that the groEL gene product is responsible for this specificity; however it is likely that groEL is correlated to one or more such genes specifying this feature Challenging the attachment of phytoplasmas to host cell membranes in the presence of recombinant AMP or opsonating anti-AMP antibodies reduced the transmission frequency significantly for two vector species the origin of the high polymorphism remains unclear One likely notion to explain the host-genotype association might involve monophagous vectors which specifically transmit the prevailing regional ‘Ca An occasional presence of this strain in other elm species might be explained by a rare probe feeding of the vector or by root anastomoses as no vector or vectors have been identified in Germany The phylogenetic analysis of the groEL fragment placed all on one root separated from members of the 16SrV-B and C subgroup The accessions on this root were arranged according to the elm species from which they were isolated This highly ordered elm species-related grouping broke up when shorter sequences (< 560 bp) were included in the analysis and although these sequences did not cluster with the ‘Ca the loss of discriminating characters changed the tree hierarchy This example demonstrates that although the groEL gene is a proven universal target for phylogenetic analysis a certain sequence length might be necessary to exploit the full information content of a gene biologically significant features might be obstructed resulting in a less meaningful phylogenetic tree all stamp sequences grouped according to established tuf clusters Why the immunodominant membrane protein genes of some phytoplasma groups seem to evolve independently from other genes might be explained by their interaction with host proteins but this does not seem to apply for the imp sequences of the flavescence dorée strains FD-C and FD-D which differ but share the same plant and insect hosts Both strains were closely related to alder yellows to accession 4120_Ul_SN and to a subset of ‘Ca ulmi’ accessions mainly isolated from European white elm This situation demonstrates the range of genetic diversity within the 16SrV group and the complex phylogenetic relationship between its members is more related between otherwise more remote strains highlights its biological importance a number of polyphagous phloem-feeders such as Allygidius atomarius or Empoasca vitis have been described but for many Auchenorryhncha species found on elm trees the host specificity is not completely clear ulmi’ vectors might provide important new information on how the spatial distribution of ‘Ca The nucleic acid pellets were resuspended in 200 µl of ddH2O PCR fragment amplification was carried out in 20 µl reactions using 20 pmol of forward and reverse primer 10 µl of 2 × primaQuant PCR master mix (Steinbrenner The amplification was performed in a qTower (Analytik Jena AG Jena) with the following cycling conditions: three minutes at 95 °C for the initial denaturation and polymerase activation followed by 35 cycles consisting of 10 s at 95 °C The final elongation step was set to 5 min at 72 °C 2 µl of the PCR reaction mix were used to verify product yield and size by electrophoresis in a GelRed-stained 2% agarose gel The PCR fragments of the remaining 18 µl were purified by a PCR purification kit (Qiagen Hilden) and quantified using a Qubit fluorometer (Thermo Fisher Scientific Sequencing was performed with the forward and reverse primers used for amplification Coordinates of individual trees were recorded in WGS84 format and imported to the software program BaseCamp version 4.7.1 (Garmin Ltd For a graphical display of spatial distribution groEL- and imp genotypes were depicted as differently coloured dots on maps GroEL- and imp sequences obtained from the same DNA extract were considered as linked when sequencing electropherograms gave no indication of polymorphic PCR products it was assumed that primer sets amplified the genes of the prevalent ‘Ca ulmi’ strain and that both genes were present in single copy Accession codes: DNA sequences determined in the course of this study have been deposited in GenBank (ncbi.nlm.nih.gov) under accession numbers MT638069 to MT638098 for the groEL fragments and MT668426 to MT668500 for the imp sequences In Molecular Biology and Pathology of Mycoplasmas (eds Razin R.) 91–116 (Kluwer Academic/Plenum Publishers Diverse targets of 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infecting grapevine and alder in Europe Macropsis mendax as a vector of elm yellows phytoplasma of Ulmus species The Leafhoppers and Planthoppers of Germany (Hemiptera Auchenorrhyncha): Patterns and Strategies in a Highly Diverse Group of Phytophagous Insects (Pensoft Publishers Detection of DNA of plant pathogenic mycoplasma-like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene Transmission de la flavescence dorée de la vigne aux plantes herbacées par l’allongement du temps d’utilisation de la cicadelle Scaphoideus littoralis Ball et l’étude de sa survie sur un grand nombre d’espèces végétales Multiple sequence alignment using ClustalW and ClustalX MEGA X: molecular evolutionary genetics analysis across computing platforms Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions Download references 22026316) was funded by the “Fachagentur Nachwachsende Rohstoffe e.V.” (FNR) a promotor of the German Federal Ministry for Food and Agriculture Open Access funding enabled and organized by Projekt DEAL Max Planck Institute for Plant Breeding Research Department of Integrative Infection Biology Crops-Livestock performed all experiments and drafted the manuscript designed the study and contributed to manuscript preparation 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/s41598-020-78745-w 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: Microbiology newsletter — what matters in microbiology research Romania ranked among global pioneers in sustainability the global household appliances manufacturer and a subsidiary of Koç Holding Romania has been awarded Sustainability Lighthouse status by the World Economic Forum (WEF) in recognition of its effective on-site sustainability measures This follows Arçelik's existing status as a Global Lighthouse granted by WEF for its Ulmi plant in 2019 and Eskişehir plant in 2021 for the successful adoption of Fourth Industrial Revolution (4IR) technologies to enhance productivity and performance The World Economic Forum's Global Lighthouse Network (GLN) seeks to promote 4IR technologies to transform production facilities the GLN introduced Sustainability Lighthouse as a new designation to recognize manufacturers applying innovative 4IR transformations to drive productivity while safeguarding the environment Arçelik's Ulmi washing machine plant is leading the way in efficient and sustainable manufacturing and uses cutting edge technologies for enhanced energy and water efficiency The Ulmi plant is now designated one of the ten Sustainability Lighthouse sites in the Global Lighthouse Network Arçelik CEO Hakan Bulgurlu commented on the honor "We are proud to be acknowledged as a pioneer for sustainability by the World Economic Forum It's a reflection of our deep commitment to sustainability across the organization." "We have ambitious environmental goals and believe that innovation is key in accomplishing them We aim to hit net-zero emissions in all operations by 2050 through green investments in renewable energy as well as energy efficiency in products and production Our Ulmi factory serves as a laboratory and sets an example for the entire Arçelik ecosystem We set out to build not only a production-efficient Industry 4.0 unit but also one that reflects our commitment to contribute to a better future thanks to investments in cutting-edge technologies and green manufacturing applications I'm proud of all my colleagues who worked hard and with drive to make this happen." the Arçelik Ulmi Washing Machine Plant stands out with the following achievements: With 45,000 employees throughout the world Arçelik's global operations include subsidiaries in 53 countries and 30 production facilities in 9 countries and 12 brands (Arçelik which is among the three largest white goods companies in Europe with its market share (based on volumes) reached a consolidated turnover of 6.5 billion Euros in 2021 Arçelik's 29 R&D and Design Centers & Offices across the globe are home to over 2,300 researchers and hold up to 3,000 international registered patent applications to date Arçelik achieved the highest score in the DHP Household Durables category for the 3rd year in a row (based on the results dated November 2021) in the Dow Jones Sustainability Index of the S&P Global Corporate Sustainability Assessment Through its leadership position in sustainability and credible decarbonization roadmap for achieving net zero Arçelik became the first and only company from its industry to receive the Terra Carta Seal by His Majesty King Charles III  www.arcelikglobal.com/en  Do not sell or share my personal information: Microbe and Virus Interactions with Plants Volume 12 - 2021 | https://doi.org/10.3389/fmicb.2021.699783 The dimorphic fungus Ophiostoma novo-ulmi is the highly aggressive pathogen responsible for the current Genome and transcriptome analyses of this pathogen previously revealed that a large set of genes expressed during dimorphic transition were also potentially related to plant infection processes which seem to be regulated by molecular mechanisms different from those described in other dimorphic pathogens novo-ulmi can be used as a representative species to study the lifestyle of dimorphic pathogenic fungi that are not shared by the “model species” Candida albicans and Ustilago maydis In order to gain better knowledge of molecular aspects underlying infection process and symptom induction by dimorphic fungi that cause vascular wilt disease we developed a high-throughput gene deletion protocol for O The protocol is based on transforming a Δmus52 O novo-ulmi mutant impaired for non-homologous end joining (NHEJ) as the recipient strain and transforming this strain with the latest version of OSCAR plasmids The latter are used for generating deletion constructs containing the toxin-coding Herpes simplex virus thymidine kinase (HSVtk) gene which prevents ectopic integration of the T-DNA in Ophiostoma DNA The frequency of gene deletion by homologous recombination (HR) at the ade1 locus associated with purine nucleotide biosynthesis was up to 77.8% in the Δmus52 mutant compared to 2% in the wild-type (WT) To validate the high efficiency of our deletion gene methodology we deleted ade7 which also belongs to the purine nucleotide pathway and opf2 which encode fungal binuclear transcription factors (TFs) The frequency of gene replacement by HR for these genes reached up to 94% We expect that our methodology combining the use of NHEJ deficient strains and OSCAR plasmids will function with similar high efficiencies for other O novo-ulmi genes and other filamentous fungi these works suggest that dimorphic growth could be closely related to DED infection development but regulated by other molecular mechanisms in Ophiostoma species which offers a new perspective for understanding the relationship between pathogenesis novo-ulmi did not show any alteration in vegetative development and pathogenicity while showing increased gene replacement by HR at the ppo1 locus encoding a putative cyclooxygenase In spite of enhancement of HR events in NHEJ-defective strains extensive screening is still required to ensure replacement at the desired locus This step is overall quite laborious and time-consuming and thus limits systematic gene disruption and genomic functional analysis A high efficiency deletion gene protocol based on HR also requires a high-throughput approach to generate deletion constructs and simplify the identification of deletion mutants upon fungal transformation we used the OSCAR-based counterselection system in combination with a Δmus52 mutant defective in NHEJ to develop an efficient strategy for systematic gene deletion in the highly aggressive DED fungus O Although these two methods can be used separately the NHEJ mutant exhibited a significant increase in the frequency of HR [over 75% compared to 2% in wild-type (WT)] when using the counterselection marker approach We first targeted genes ade1 and ade7 since their deletion resulted in the production of characteristic pink mutants on non-selective (rich) media thereby facilitating the phenotypic identification of knockout mutants we validated our strategy by successfully deleting genes bct2 and opf2 which encode TFs belonging to the Zn(II)2Cys6 family These new efficient tools for targeted gene deletion together with existing resources for the study and manipulation of DED fungi open the way to a new level of functional analysis and understanding the relationship between morphogenesis and pathogenesis in vascular tree pathogens yeast cells of each parental strain were harvested by filtration through one layer of Miracloth (Calbiochem #475855) and centrifugation at 5500 g for 10 min Yeast cells were then suspended in sterile distilled water to a final concentration of 5 × 107 cells/mL Transformants expressing either of the hygromycin B phosphotransferase (hph) or nourseothricin acetyltransferase (nat1) resistance genes were cultivated on 2PDA supplemented with 300 μg/L hygromycin B (hygromycin #10687010) or 50 μg/L nourseothricin dihydrogen sulfate (nourseothricin To counterselect transformants expressing HSVtk 50 μg/L 5FU (Thermo Fisher Scientific The ccdB SurvivalTM 2 T1R Escherichia coli strain (Thermo Fisher Scientific, #A10460) was used to propagate the vector pOSCAR-HSVtk (Sarmiento-Villamil et al., 2020) which harbors the ccdB gene lethal for most E coli One shot® OmniMAXTM 2 T1R competent cells (Thermo Fisher Scientific #C854003) were used to produce the deletion construct coli strain DH5α (Bethesda Research Laboratories coli strains were grown in or on Luria Bertani (LB) media containing 100 μg/mL ampicillin sodium salt (ampicillin #A9518) or 100 μg/mL spectinomycin dihydrochloride pentahydrate (spectinomycin Fungal parental strains were transformed with A tumefaciens which was grown on LB or in minimal medium (MM) supplemented with 50 μg/mL spectinomycin PCRs were performed according to the manufacturer’s recommendations in a total volume of 50 μL and under the following PCR cycling conditions: an initial denaturation of 1 min at 94°C and completed with a final extension of 5 min at 68°C All PCR products were gel-purified using EZ-10 spin column DNA gel extraction miniprep kit (Bio Basic #BS367) according to the manufacturer’s instructions a 5 μL BP clonase reaction was set up to include 1 μL BP clonase II enzyme mix (Thermo Fisher Scientific 95 ng of pOSCAR-HSVtk and 100 ng of pA-Hyg-GFP-OSCAR or 60 ng of marker vector pA-NTC-OSCAR The BP reaction was incubated at 25°C for 20 h in a PCR thermocycler the reaction mixture was used to transform One Shot OmniMAXTM 2 T1R Chemically Competent E coli according to the manufacturer’s recommendations Bacterial colonies were recovered on LB plates supplemented with 100 μg/mL spectinomycin following overnight incubation at 37°C Correct structure of each deletion construct was verified by restriction enzyme digestion Constructs pade1_hyg_1kb and pade1_hyg_2kb were developed using pA-Hyg-GFP-OSCAR Agrobacterium tumefaciens strain GV3101:pMP90 (Koncz and Schell, 1986) was used for transferring T-DNA harboring dual molecular selection markers The deletion constructs were transformed into A tumefaciens strain GV3101:pMP90 using the heat shock method spread onto LB plates supplemented with 100 μg/mL spectinomycin and incubated at 25°C for 2 days For A. tumefaciens-Mediated Transformation (ATMT) of O. novo-ulmi, the method described by Mullins et al. (2001) was used with some variations tumefaciens strains containing a deletion construct were grown at 28°C for 2 days in MM supplemented with 100 μg/mL spectinomycin tumefaciens cultures were diluted to optical density (OD600) of 0.2 in 6 mL induction medium (IM) which contained MM supplemented with 0.5% (W/V) glycerol 40 mM 2-(N-morpholino) ethanesulfonic acid (MES #M5287) at pH 5.3 and 100 μg/mL spectinomycin Bacterial cells were grown for an additional length of time until the OD600 value reached 0.6–0.9 tumefaciens culture and 100 μL of a yeast cell suspension (5 × 108 cells/mL) from each fungal parental strain were mixed and spread on a sterile 0.45 μm pore nitrocellulose membrane (Sartorius Stedim Biotech GmbH #1140647ACN) overlaid on top of 20 mL of co-culture medium plate which contained IM plus 200 μM 3′,5′-dimethoxy-3′hydroxyacetophenone acetosyringone (AS Following incubation at 25°C for 2 days the filters were cut in strips and transferred to selection medium which contained 2PDA supplemented with 300 μg/mL hygromycin or 50 μg/L nourseothricin to select the transformants with or without 50 μg/mL 5FU as a counterselecting agent of ectopic transformants and 100 μg/mL cefotaxime sodium salt (cefotaxime #C7039) and 100 μg/L moxalactam sodium salt (Sigma-Aldrich single colonies began to appear on the selection medium Three independent ATMTs of each parental fungal strain were performed with each combination of A Phenotype analysis based on the recovery of pink colonies from individual transformants was used for distinguishing visually Ade– from Ade+ survivors. To confirm gene replacement, randomly selected pink colonies were analyzed by PCR with primer combinations ade1F_809 and ade1R_1221, and ade7F_368 and ade7R_809 (Supplementary Table 1) which amplified roughly 441 and 594 bp of ade1 and ade7 The HR frequencies for each gene in each strain treatment and replicate experiment were estimated by the pink/white transformant colony ratio and analyzed by the application of generalized linear models (GzLMs) using Logit as the link function and Binomial as the underlying distribution An additional analysis to obtain orthogonal contrasts was also conducted among treatments with Bonferroni adjustments to the p-values americana saplings was analyzed by application of GzLMs using identity as the link function and Gaussian as the underlying distribution Orthogonal contrasts among the treatment medians with Bonferroni adjustments to the p-values were also performed All statistical analyses were conducted using Lme4 and emmeans packages under R environment (R Project software Each PCR resulted in three fragments of roughly 1.9 Kb of the entire HVStk cassette 1.1 Kb of the HVStk promoter proximal to the left border of the T-DNA and 0.3 Kb of the HVStk terminator (data not shown) Eight transformants had truncations of their HVStk cassettes while two transformants had intact HSVtk cassettes (data not shown) Effect of ade1 deletion on colony morphology of Ophiostoma novo-ulmi after 7 days incubation on potato dextrose agar (PDA) at 21°C The plate on the left is the parental strain and the plate on the right is a Δade1 mutant HR frequency was significantly higher in transformants obtained with construction containing 2 Kb of flanking homologous region thereby showing a correlation between the length of homologous region and the efficiency of homologous integration Bar graphs showing the effects of using 1 Kb or 2 Kb of homologous sequence flanking targeted loci and applying counterselection with 5-Fluoro-2′-deoxyuridine on the frequency of homologous recombination (HR) in Ophiostoma novo-ulmi strain 174_68Δmus52 Standard error bars are displayed at the top of each bar Different letters above each bar indicate statistically significant differences (p < 0.05) according to the sequential Bonferroni method for error correction There was no statistically significant difference between both HR rates comparison of HR rates between transformants isolated from supplemented (5FU) versus non-supplemented medium revealed a statistically significant difference caused by a three-fold increase in the number of gene replacement mutants as well as a stark reduction (94%) in the number of ectopic transformants in the presence of 5FU This result showed that HSVtk worked well in preventing random integration of the deletion construct T-DNA in the NHEJ-impaired mutant of O thus greatly simplifying the selection of null mutants These slight variations in HR frequencies between ade1 and ade7 may be closely related to locus-specific properties including differences in chromatin structure Recovery data for ade7 mutants also corroborated the effect of 5FU in enrichment of gene replacement mutants as well as in decreasing the number of ectopic transformants Gel images showing PCR confirmation of ade1 or ade7 deletion in Ophiostoma novo-ulmi transformants (A) Total genomic DNA samples from twelve pink colony transformants and one white colony transformant (t13) were amplified with ade1 specific primer pair ade1F-809 and ade1R-1221 (top panel) and nat1 marker primers ntcF_562 and ntc_975_R (bottom panel) 1 kb plus (FroggaBio); t1 through t6 and t7 through 13 174_68Δmus52 transformants obtained using pade1_NCT_2kb and pade1_NTC_1kb (B) Total genomic DNA samples from twelve pink colony transformants and one white colony transformant (t26) were amplified with ade7 specific primer pair ade7F-368 and ade7R-809 (top panel) and nat1 marker primers ntcF_562 and ntc_975_R (bottom panel) 1kb plus (FroggaBio); t14 through t19 and t20 through 26 174_68Δmus52 transformants obtained using pade7_NTC_2kb and pade1_NTC_1kb Disease severity of Ulmus americana saplings inoculated with Ophiostoma novo-ulmi H327 wild-type NHEJ-defective mutant strain 174_68Δmus52 and two adenine auxotrophs (ade1-6 and ade7-13) derived from it by targeted deletion of genes ade1 and ade7 novo-ulmi strain 174_68Δmus52 transformed with OSCAR constructs containing 1 Kb of homologous arm length for genes encoding fungal binuclear TFs bct2 ogf1 and opf2 (located on chromosomes 1 or 2) were grown on selection medium with nourseothricin and 5FU PCR analysis of 17 transformants from each fungal transformation confirmed deletion of bct2 These results confirmed that null mutants could be recovered at high frequency in O novo-ulmi through an approach combining OSCAR-based counterselection with a Δmus52 mutant defective in NHEJ In order to fully exploit the new knowledge obtained from NGS approaches and gain insight into molecular aspects underlying infection process and symptom induction by DED pathogens we developed a high-efficiency gene deletion methodology by HR for O Truncation of T-DNA could therefore explain why counterselection by HSVtk failed in WT O Our results confirmed that NHEJ plays a major role in HR and that efficacy depends on the length of homologous arms that flank the gene targeted for deletion in spite of the significant increase in HR frequency resulting from mus52 deletion in O a high rate of random integration of the T-DNA still occurred in strain 174_68Δmus52 thereby suggesting the preferential use of the NHEJ over the HR pathway in O we suspected that counterselection by HSVtk could work in O A comparison of 174_68Δmus52 ectopic transformants recovered from selective medium showed a drastic reduction (up to 94%) of transformants in the presence of 5FU These results suggest that Ku80 intervenes in the deletion of foreign DNA segments during the genomic integration process coupling of ATMT with HSVtk-based counterselection worked efficiently for enrichment of gene replacement mutants and prevention of ectopic transformants in strain 174_68Δmus52 successful targeted deletion of five genes suggests that the ATMT-OSCAR-HSVtk strategy is not restricted to certain loci The virulence of knockout mutants for genes bct2 ogf1 and opf2 will be evaluated when elm saplings are available for pathogenicity tests novo-ulmi was limited by the absence of an efficient gene deletion tool as we were able to recover from 64 to 94% of targeted deletion mutants among transformants derived from NHEJ-defective strain 174_68Δmus52 subjected to a dual-selection system in OSCAR systematic gene function studies can now be envisioned for elucidating the molecular bases of pathogenicity and virulence in the DED fungi The original contributions presented in the study are included in the article/Supplementary Material further inquiries can be directed to the corresponding authors ESN constructed the Ophiostoma novo-ulmi strain 174_68Δmus52 used in the improved OSCAR-based protocol JLS-V and TCO constructed the gene delete OSCAR plasmids and performed transformation experiments LB carried out the inoculations on elm saplings and supervised the research JLS-V wrote the manuscript with input from all authors All authors contributed to the article and approved the submitted version Genome British Columbia and Génome Québec within the framework of project bioSAFE (Biosurveillance of Alien Forest Enemies; project number 10106) and Philippe Silar at the Université Paris-Diderot for their help in producing Ophiostoma novo-ulmi strain 174_68Δmus52 impaired for Non-Homologous End Joining André Gagné and Jean-Guy Catford and Camille Bédard at the Université Laval for technical assistance María Dolores García Pedrajas at Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM La Mayora-CSIC-UMA) for providing OSCAR plasmids and Philippe Tanguay at the Natural Resources 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited *Correspondence: Jorge Luis Sarmiento-Villamil, anNhcm1pZW50b0BlZWxtLmNzaWMuZXM= one of the largest home appliances producers in Europe will make a Greenfield type investment to Romania for the construction of a factory that will produce washing machines in Ulmi Dambovita County.The new washing machine factory will be the company’s first production unit to integrate 4.0 standards The factory will introduce Cyber Physical Systems fleet and inter-connected network) and analysis systems The total value of the project investment announced in Dambovita county totals RON 471 million will invest EUR 70 million from the company’s profit in the past years to build the new factory Arctic transforms Romania into a production hub for Europe investments in Romania have always been important and this new investment will allow us to transform Romania into a production hub for the whole Europe,” Arçelik CEO Hakan Bulgurlu said our project will create 480 work places in Dambovita County in a region with the highest unemployment in Romania this investment will contribute to improving the quality of life at a local level through the professional development of the work force but also by attracting new investors in the area,” Nihat Bayiz “The new factory will integrate up-to-date technologies, as well as the wireless communication of production lines with those in another countries that are part of the Arçelik group, but also smart tools,” Nihai Bayiz said. We use cookies for keeping our website reliable and secure providing social media features and to analyse how our website is used Please enable JS and disable any ad blocker the leader of the Romanian home appliance market and one of the main contributors to the local economy marks a milestone of one million washing machines produced at its Ulmi manufacturing plant in Dambovita county the first Industry 4.0 factory in Romania and one of the most modern production facilities in Europe The one million Arctic washing machines were produced with minimal impact on the environment Thanks to technologies based on Industry 4.0 principles the energy consumption per unit of product has decreased by 28% the factory is partly powered by renewable energy the photovoltaic system having produced over 1.7 million kWh so far thus reducing carbon dioxide emission by around 600 tons The water used during the production process is also almost 100% recycled and reused. Owing to its advanced wastewater treatment and rainwater collection systems, 26.000 cubic meters of water have been recycled and reused – the equivalent of 10 Olympic pools. The Ulmi unit received an impressive number of accreditations for being a green factory and it is the only one in Romania certified as LEED Platinum This certification acknowledges its sustainable production performance In Romania Arctic also owns and operates the largest household appliance manufacturing factory in continental Europe, the Gaesti unit having produced so far over 38 million refrigerators which have been exported to more than 70 countries. Turkish group Arcelik will hire 4,000 people at its new factory in Ulmi The group will invest around EUR 200 million in the factory The factory will be built on a plot of land of 70 hectares which was purchased by the company from a local Ulmi has a population of around 4,000 inhabitants According to Oprea, the company hopes to start production 10 months after receiving the building permit. “For me, as county authority, is an extremely important moment and we welcome the initiative of the Arcelik group to make this factory in Dambovita county,” Oprea told Mediafax the leader of the Romanian home appliances market and one of the main contributors to the local economy marks the production of 1,000,000 washing machines at the factory in Ulmi the first factory in Romania operating according to the principles of Industry 4.0 and one of the the most modern production facilities in Europe following a total investment of 153 million euros the Arctic factory in Ulmi integrates the latest machine learning technologies and automation processes which allow employees to work side by side with robots generating an increase of productivity by up to 30% More than 200 robots were involved in the production process of the one million washing machines the production center in Ulmi incorporates automatic systems for handling and storage of components which are transported from the warehouse to the assembly line with the help of autonomous machines Over 70% of production operations are based on self-determined and self-managed systems more than 407,000 hours of testing have been conducted in the Research and Development departments Arctic also owns and operates in Romania the largest household appliances factory in Continental Europe the place where over 38 million refrigerators have been produced so far which have been exported to over 70 countries the leader in Romania’s home appliance market has reached a significant milestone by producing its four millionth washing machine at the Ulmi factory Romania’s first Industry 4.0 unit and one of the few in Europe certified by the World Economic Forum (WEF) as a Sustainability Lighthouse acknowledging its outstanding sustainability performance the factory stands as a benchmark facility for energy efficiency equipped with cutting-edge automation and machine learning technologies the factory’s two production lines have the capability to produce 407 models of washing machines destined to both domestic and international markets 82 percent of Ulmi’s production is destined towards exports Appliances reach consumers in 42 countries yet over 94 percent of total exports are destined for European markets ”The inauguration of our Ulmi facility in 2019 marked a significant leap into the next generation of manufacturing aligning with the principles of Industry 4.0 positioning us as a benchmark in terms of energy efficiency The production of the four-millionth washing machine is the result of a collective effort of the entire team We focused all our energy on finding the best solutions so that each product that goes out of the factory leaves the smallest possible carbon footprint Arctic is not just a company that manufactures appliances but also one that focuses on a greener future for us and the generations to come Finding a balance between sustainability and efficiency is a proof that excellence in production can be achieved keeping in our hearts the care for the environment and we intend to surpass the milestone of 5 million units produced at Ulmi by the end of the year.” Benefitting from advanced technologies based on Industry 4.0 concepts over 80 percent of the factory’s logistic processes are automated due to the high level of technological integration at the production points the facility records significant improvements in traceability capacity ensuring a high standardization of quality are involved in the production of washing machines leading to a 30 percent increase in productivity resource consumption within the appliance manufacturing process at the Ulmi facility has witnessed a significant decline since its opening with a 56 percent reduction in energy usage a 60 percent decrease in water consumption and a 26 percent decrease in production waste The facility utilizes energy from renewable sources with 20 percent being generated through the owned photovoltaic park at least half of the total energy used in the production of the 4 million washing machines originated from suppliers with certified origin in the manufacturing process of these appliances and nearly 5 tons of fishing net threads were utilized Metal and plastic materials are the primary elements required in appliance production has set out to increase the proportion of recycled plastic to 40 percent and the content of biomass-based materials to 5 percent by 2030 have come out to explain reasons behind the recently sponsored soccer tournament and honouring of deserving teachers in Uromi Explaining in details to Daily Independent Executive Chairman of the Uromi Like Minds Initiative Sylvester Abumere Ekpen said that the gesture was to help people loosen up while also encouraging scholarship and hard work ULMI had previously sponsored scholarship schemes for students of Uromi descent in from Edo State Made up principally of high flying successful sons and daughter of Uromi nation the group has become a strong organisation promoting well being of indigens from the town in wherever they find themselves Ekpan said: “all work and no play makes Jack a dull boy “We understand that for all-round development of the mind and body a mix of academic and social activities are required.” The soccer tournament was amongst Secondary Schools and also featured honoring deserving school teachers who had distinguished themselves within the last twenty-five years and still counting between Ebhoiyi and Obeidu secondary schools which began at 1.15 pm in the main bowl of the LGA Council Sports arena Ebhoiyi defeated Obeidu Secondary School 4-3 in a penalty shootout after the normal time scores were one apiece The goals were scored by Ikekhwa Dennis and Okoduwa George of Ebhoiyi and Obeidu secondary schools respectively The final proper saw a popular private school in Uromi pip the star-studded Agba grammar school to take home the inaugural gold cup against all odds The game was watched by many sons and daughters of Uromi who went to the commercial town for the yuletide celebration A first-half goal scored by Iyoha Friday of Buddie Group of Schools was all that made the difference between the school and Agba Grammar School Azeez Ugbodaga of BGS won the Tournament`s MVP award The top scorers’ award went to Iyoha Godspower of Ebhoiyi secondary school with 4 goals while Ugbodaga Miracle of BGS was crowned the best goalkeeper of the championship The highpoint of the fiesta was the awards of excellence given to about nine teachers who have served for about twenty-five years the group appreciated the teachers for their selfless services to the students and community at large and promised that the association would make it an annual event in Uromi “We have recently considered and passed a budget for 2022 that is almost twice the 2021 budget While that shows commitment on the part of existing members who shares a similar vision with the Association to team up and join hands with ULMI,” Ekpan said TV Independent on YouTube and IndependentNgr (Facebook Uromi Like Minds Initiative (ULMI) with members home and abroad has commenced the renovation of the Edo State Library situated in Uromi the administrative headquarters of Esan North-East Local Government Area of the state in a telephone conversation with selected journalists from the UK said that the project is part of its corporate social responsibility The group has in the last two years since its establishment been involved in some community and human capacity development projects it supplied some orphanage and old people’s homes relief materials and food items to celebrate the season While speaking to newsmen on the matter recently Chairman of welfare committee and Vice Chairman of the organisation Sebastine Osita described the library project as the biggest project the organisation has embarked on since its formation two years ago and harped on the need for students in the community to rekindle their interest in learning “ This particular project is dear to my heart because in my earlier years in the town I had the privilege of visiting the library to study and having unrestricted access to various textbooks “The serenity of the library provided a perfect environment for study It was also a place to meet and share ideas with students from other schools “Having enjoyed all these privileges in the past it was heartwarming when we as a group decided to take on the effort to revive the library” our strategy is to first provide a modern library with ICT facilities and then follow with an awareness campaign aimed at encouraging full utilization,” Osita said Picking the Uromi library was not a difficult decision to take according to the ULMI vice-chairman we met towards the end of the year to select projects/programs that will be executed in the following year once approved by the Exco team and blessed by the entire members at the General Meeting the executive chairman of the organisation which has its members across the globe recalled how the state-owned library had in the past been a source for learning and getting academic material and harped on the need to bring back the past glory of the institution through this renovation works and equipping it with books The renovation work on the building has cost ULMI N4.6m which is being handled by an indigenous firm Melvin Global LTD and the completion is estimated to be within five weeks according to Prince Kenneth Edenojie who is the association`s director of Projects the administrative headquarters of Esnortheastast LGA of Edo State will come alive with the Second Edition of the Uromi Like Minds Initiative Football Competition as it enters the Finals on Monday The games will see Agba Grammar school Uromi lock horns with Arue Secondary School both from Uromi by 3 pm The competition which began in October with about 20 Schools participating is expected to welcome dignitaries from Uromi and Esan land both at home and in the diaspora the third-place match would be played between Ulinlin Secondary School an NGO made up of sons and friends of Uromi at home and in the diaspora has been engaging in a secondary schools football competition in ENE LGA since 2021 and this is the second edition the Director of Projects and member of the organizing committee Prince Ken Edenojie express readiness to host a beautiful final as he said that everything is already in place to have a successful final the 26th and the people of Uromi are ready to treat themselves to an exciting moment on Sunday” we have made adequate preparations to ensure that we have a memorable final with the young boys who are already looking up to the games,” he said