Genetic and Metabolic Features in Host Specificity and Stress Tolerance of Potential Inoculant Rhizobia

Nitrogen is an essential nutrient for plants. Biological and industrial nitrogen fixation are the major sources of soil nitrogen. This study focuses on rhizobia that are the bacterial partners in the biologically nitrogen fixing symbiosis between leguminous plants and bacteria. In symbiosis, the plant and the rhizobia form a specified organ called nodule. In the nodule rhizobia differentiate into nitrogen fixing forms. Phenolic molecules secreted by the plant roots induce the rhizobia to produce lipochitooligosaccharide signalling molecules (LCOs). LCOs have an amino sugar backbone consisting of N-acetylglucosamine residues (GlcNAc), an acyl group on the non-reducing terminal GlcNAc residue and commonly other side-groups. When growing legumes in the field, inoculating the plants with compatible rhizobia usually results in better growth. The soil may not contain rhizobial strains that nodulate selective legumes. The promiscuous legumes are easily nodulated, yet most of the nodulating strains may be ineffective in nitrogen fixation. Environmental conditions, e.g. soil salinity, affect nodulation. The general aim of this study was to gain knowledge on the features of potential inoculant rhizobia strains. The specific aims were to assess host specific characteristics and the effect of salt stress on rhizobial signalling molecules, LCOs; to find potential inoculant strains for a promiscuous legume tree; and to characterize potential inoculant strains isolated from promiscuous legume trees and forage legumes to assess genetic features behind the characteristics of the strains. To reach the aims, LCOs of rhizobia nodulating Prosopis chilensis and Acacia senegal were analyzed using mass spectrometry; rhizobial strains were isolated from Leucaena leucocephala growing in China, and characterized with phenotypic and genotypic methods; and the genetic features behind the desirable characteristics of S. arboris and wild-type S. meliloti strains were analyzed by comparative genomic hybridization (CGH) using a model rhizobium S. meliloti Rm1021 microarray. The LCOs from prosopis- and acacia-nodulating strains were similar to those of other Acacia-nodulating rhizobia. The L. leucocephala nodulating Ensifer strains, representing at least three species, shared a common nodC, implying that their LCOs might be similar. Similarly, no divergence was detected between the S. meliloti model strain and the S. meliloti wild type strains nodulation genes. Even though rhizobia having a shared host range produced similar LCOs and carried similar nod genes, comparison of major LCOs between rhizobia with different host ranges suggested that LCOs have only a limited role in host specificity. L. leucocephala hosted eleven rhizobial strains that were efficient in nitrogen fixation making them good candidates as inoculants. Ten isolates had a growth slowing effect on the host. The variation in effectiveness between the isolates implied that the ability to compete with other strains is an important characteristic for the inoculant of a promiscuous host; the inoculant is of no use if nodules are mainly occupied by less efficient strains. The species distribution was different from those isolated from L. leucocephala in other locations, suggesting that strains adapted to local soil conditions would be preferred when choosing inoculants. The genotypic and phenotypic characteristics of Leucaena isolates did not distinguish the efficient from the inefficient and parasitic isolates. The results of the CGH analysis suggested that the osmotic shock response of the S. arboris and wild-type S. meliloti strains is considerably different to that of S. meliloti Rm1021, and that the stress tolerance of S. arboris strain might be partially due to a more efficient protein biosynthesis. The genes related to the induction of LCO synthesis and LCO secretion were duplicated in wild-type S. meliloti strains, which is possibly connected to the efficient nodulation capabilities of the strains. The LCO production of a salt-stressed S. arboris HAMBI 2361 was approximately one tenth of that of the non-stressed one, partially explaining the decrease in nodulation under salt stress. The Leucaena isolates were phenotypically diverse, and the S. arboris and wild-type S. meliloti strains diverged from the model rhizobium S. meliloti Rm1021 mainly in the accessory genome implying that the divergence was important in shaping the adaptability of the strains. However, it is impossible to conclude if the diversity and divergence gave the strains any competitive advantage.Kasvit eivät kasva ilman typpeä. Maaperän typen tärkeimmät lähteet ovat teollinen ja biologinen typensidonta. Tässä työssä tutkittiin biologiseen typensidontaan kykeneviä ritsobi-bakteereja. Palkokasvi-ritsobisymbioosissa kasvin juureen muodostuu nystyrä, jonka sisällä ritsobit erilaistuvat typpeä sitoviksi bakteroideiksi. Symbioosissa kasvi ja ritsobi käyvät kemiallista keskustelua tuottamiensa molekyylien välityksellä. Kasvin juuren erittämät fenoliset yhdisteet saavat ritsobin tuottamaan viestimolekyylejä, jotka koostuvat N-asetyyliglukosamiini-ketjusta ja siihen liittyneestä rasvahapporyhmästä. Lisäksi viestimolekyylissä voi olla muita sivuryhmiä. Jotkin kasvit ovat erittäin valikoivia ja kelpuuttavat kumppaneikseen vain harvoja ritsobikantoja. Toiset kasvit valikoivat vain vähän ja tekevät nystyröitä lähes kaikkien ritsobien kanssa, mutta nystyrät ovat usein tehottomia typen sidonnassa. Siksi palkokasvit kasvavat monesti paremmin mikäli ne siirrostetaan sopivalla ritsobikannalla. Epäedulliset kasvuolosuhteet, kuten esimerkiksi maan suolaantuminen, häiritsee nystyröintiä. Tämän työn tarkoituksena oli saada uutta tietoa siirrostamiseen sopivien ritsobikantojen ominaisuuksista. Tarkempina tavoitteina oli tutkia ritsobin viestimolekyylien rakenteiden vaikutusta isäntäspesifisyyteen ja suolastressin vaikutusta viestimolekyylien tuottoon, löytää sopiva ritsobi vähän valikoivalle palkokasvipuulle, ja selvittää siirrostamiseen sopivien ritsobikantojen ominaisuuksien taustalla olevia geneettisiä piirteitä. Työssä määritettiin kahden trooppisia palkokasvipuita (Prosopis chilensis ja Acacia senegal) nystyröivän ritsobikannan viestimolekyylien rakenteet massaspektrometrisesti. Suolastressin vaikutus viestimolekyylien tuottoon selvitettiin analysoimalla typen pysyvillä isotoopeilla leimattuja viestimolekyylejä. Trooppisesta Leucaena leucocephala palkokasvipuusta eristettiin ritsobikantoja, joita tutkittiin fenotyyppisin ja genotyyppisin menetelmin. Siirrostamiseen sopivien S. arboris ja S. meliloti -ritsobikantojen geenejä analysoitiin vertailevalla genomihybridisaatiolla (CGH), jossa vertailukantojen genomi hybridisoitiin malliritsobin S. meliloti Rm1021 DNA-mikrosiruun. Prosopis- ja akaasia-puita nystyröivien ritsobien viestimolekyylit olivat samanlaisia kuin muilla akaasioita nystyröivillä ritsobeilla. L. leucocephala-puun nystyröistä eristetyistä ritsobeista suurin osa kuului Ensifer (Sinorhizobium)-bakteerisukuun. Ensifer-kantojen viestimolekyylien tuottoon liittyvä nodC-geeni oli kaikilla kannoilla samanlainen, joten niiden tuottamat viestimolekyylit saattavat myös olla samanlaisia. Siirrostamiseen sopivien S. meliloti-ritsobikantojen nystyröintiin liittyvät geenit olivat samanlaisia kuin malliritsobilla. Vaikka saman kasvi-isäntää nystyröivien kantojen viestimolekyylit ja nystyröintigeenit olivat samanlaisia, viestimolekyylien vertailu eri kasveja nystyröivien kantojen välillä antoi aiheen olettaa että viestimolekyyleillä on vain pieni vaikutus isäntäspesifisyyteen. L. leucocephala-puun nystyröistä eristetyistä ritsobikannoista yksitoista sitoi tehokkaasti typpeä. Kymmenen kantaa hidasti taimien kasvua. Koska kantojen välillä oli suuria eroja typen sidonnan tehokkuudessa, siirrostamiseen sopivan kannan pitää olla myös kilpailukykyinen; kannasta ei ole hyötyä, mikäli tehottomasti typpeä sitovat kannat syrjäyttävät sen. Ritsobikantojen lajikirjo poikkesi muualla tutkituista. Siirrostamiseen sopivien kantojen valinnassa täytyy painottaa myös kantojen sopivuutta viljelypaikan olosuhteisiin. L. leucocephala-puusta eristettyjen ritsobikantojen feno- ja genotyyppiset ominaisuudet eivät erottaneet tehokkaasti typpeä sitovia kantoja tehottomista. CGH-analyysin tulosten perusteella S. arboris- ja villityypin S. meliloti-kantojen vaste osmoottiseen shokkiin poikkeaa malliritsobin vasteesta. S. arboris -kannan stressinsieto saattaa osin johtua tehokkaasta proteiinisynteesistä. Osa villityypin S. meliloti-kantojen viestimolekyylien tuoton indusointiin ja molekyylien erittämiseen liittyvistä geeneistä oli monistunut, mikä saattaa liittyä kantojen tehokkaaseen nystyröintiin. Suolastressi vähensi S. arboris -kannan viestimolekyylien tuottoa kymmenekseen vertailuolosuhteesta, mikä osittain selittää suolastressin aiheuttaman nystyröinnin vähenemisen. L. leucocephala-puun ritsobikannat olivat fenotyyppisesti monimuotoisia. S. arboris- ja villityypin S. meliloti-kannat poikkesivat malliritsobista eniten ympäristöön sopeutumiseen liittyvien geenien osalta. On kuitenkin mahdotonta päätellä antavatko edellä mainittu monimuotoisuus ja eroavaisuus mainituille kannoille siirrostamiseen liittyviä, toivottuja ominaisuuksia

Faba Bean (Vicia faba L.) Nodulating Rhizobia in Panxi, China, Are Diverse at Species, Plant Growth Promoting Ability, and Symbiosis Related Gene Levels

We isolated 65 rhizobial strains from faba bean (Vicia faba L.) from Panxi, China, studied their plant growth promoting ability with nitrogen free hydroponics, genetic diversity with clustered analysis of combined ARDRA and IGS-RFLP, and phylogeny by sequence analyses of 16S rRNA gene, three housekeeping genes and symbiosis related genes. Eleven strains improved the plant shoot dry mass significantly comparing to that of not inoculated plants. According to the clustered analysis of combined ARDRA and IGS-RFLP the isolates were genetically diverse. Forty-one of 65 isolates represented Rhizobium anhuiense, and the others belonged to R. fabae, Rhizobium vallis, Rhizobium sophorae, Agrobacterium radiobacter, and four species related to Rhizobium and Agrobacterium. The isolates carried four and five genotypes of nifH and nodC, respectively, in six different nifH-nodC combinations. When looking at the species-nifH-nodC combinations it is noteworthy that all but two of the six R. anhuiense isolates were different. Our results suggested that faba bean rhizobia in Panxi are diverse at species, plant growth promoting ability and symbiosis related gene levels.Peer reviewe

Bacterial community changes in response to oil contamination and perennial crop cultivation

We investigated bacterial community dynamics in response to used motor oil contamination and perennial crop cultivation by 16S rRNA gene amplicon sequencing in a 4-year field study. Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were the major bacterial phyla, and Rhodococcus was the most abundant genus. Initially, oil contamination decreased the overall bacterial diversity. Actinobacteria, Betaproteobacteria, and Gammaproteobacteria were sensitive to oil contamination, exhibiting clear succession with time. However, bacterial communities changed over time, regardless of oil contamination and crop cultivation. The abundance difference of most OTUs between oil-contaminated and non-contaminated plots remained the same in later sampling years after the initial abundance difference induced by oil spike. The abundances of three oil-favored actinobacteria (Lysinimonas, Microbacteriaceae, and Marmoricola) and one betaproteobacterium (Aquabacterium) changed in different manner over time in oil-contaminated and non-contaminated soil. We propose that these taxa are potential bio-indicators for monitoring recovery from motor oil contamination in boreal soil. The effect of crop cultivation on bacterial communities became significant only after the crops achieved stable growth, likely associated with plant material decomposition by Bacteroidetes, Armatimonadetes and Fibrobacteres.Peer reviewe

Advantages of grass-legume mixture for improvement of crop growth and reducing potential nitrogen loss in a boreal climate

Peer reviewe

Future images of data in circular economy for textiles

Rapid expansion of digitalization and in the volume of data available constitutes a major driver toward circular economy. In the textile industry, with its vast quantities of waste and huge environmental impact, transformation toward such circularity is necessary but challenging. To explore how the use of data could support building sustainability-aligned pathways to circular economy of textiles, a study employing a two-round disaggregative Delphi approach (engaging 33 experts in the first round, in May 2021, and 26 in the second, in June 2021) articulated alternative images of the future. The three images, dubbed Transparency, Conflicting Interests, and Sustainable Textiles, imply that the role for data is intertwined with sustainability aspirations. The results highlight that exploiting data in pursuit of circular economy is a collaborative effort involving business value networks that include consumers and regulators. Availability and sharing of accountability-affording, meaningful data on textiles' life cycle and value network function as a key enabler. By working with the images developed, actors can better assess their circular-economy commitments, planned actions, and the consequences of these. Furthermore, the images provide a tool for mutual discussion of the development desired and of related re-sponsibilities and uncertainties.Peer reviewe

Response of Soil Bacterial Community Diversity and Composition to Time, Fertilization, and Plant Species in a Sub-Boreal Climate

Pastures are an important part of crop and food systems in cold climates. Understanding how fertilization and plant species affect soil bacterial community diversity and composition is the key for understanding the role of soil bacteria in sustainable agriculture. To study the response of soil bacteria to different fertilization and cropping managements, a 3-year (2013-2015) field study was established. In the split-plot design, fertilizer treatment (unfertilized control, organic fertilizer, and synthetic fertilizer) was the main plot factor, and plant treatment [clear fallow, red clover (Trifolium pratense), timothy (Phleum pratense), and a mixture of red clover and timothy] was the sub-plot factor. Soil bacterial community diversity and composition, soil properties, and crop growth were investigated through two growing seasons in 2014 and 2015, with different nitrogen input levels. The community diversity measures (richness, Shannon diversity, and Shannon evenness) and composition changed over time (P<0.05) and at different time scales. The community diversity was lower in 2014 than in 2015. The temporal differences were greater than the differences between treatments. The overall correlations of Shannon diversity to soil pH, NO3-, NH4+, and surplus nitrogen were positive and that of bacterial richness to crop dry matter yield was negative (P<0.05). The major differences in diversity and community composition were found between fallow and planted treatments and between organic and synthetic fertilizer treatments. The differences between the planted plots were restricted to individual operational taxonomic units (OTUs). Soil moisture, total carbon content, and total nitrogen content correlated consistently with the community composition (P<0.05). Compared to the unfertilized control, the nitrogen fertilizer loading enhanced the temporal change of community composition in pure timothy and in the mixture more than that in red clover, which further emphasizes the complexity of interactions between fertilization and cropping treatments on soil bacteria.Peer reviewe

Stable nitrogen-cycling capacity in relation to fertilization and intercropping in a sub-boreal grassland

Grasslands are important in sub-boreal climate agricultural systems and are managed with various combinations of N fertilization and plant species. Ammonia-oxidizing and denitrifying microorganisms are key players in determining the fate of nitrogen (N) and thereby also the yield in grassland systems and their impact on gaseous N losses and leaching. We established a three-year field study in southern Finland with fertilizer treatment as a main-plot factor, including organic and synthetic fertilizers and plant species and mixtures thereof as the sub-plot factor. We quantified six genes encoding key N-cycling enzymes by quantitative PCR to determine the abundance of the communities involved in N-transformation processes and also included previously published data on crop yield, soil properties and the overall bacterial community composition. With the exception of ammonia oxidizing bacteria (AOB), which were primarily affected by fertilization, the abundances of all other N-cycling communities changed over time with either an increase or decrease from summer to autumn. Differences in gene abundances between plant species treatments and in fertilizer by plant species interactions were detected mainly in the beginning of the cropping season during the first year. The nirS-type denitrifiers and nosZII nitrous oxide reducers responded more to changes in soil properties than their functional counterpart nirK and nosZI communities. Using structural equation modeling, we show that the overall microbial community composition and diversity played an important role in mediating the management effects on crop yield, genetic potential for N retention and N2O sink capacity. However, a trade-off between the genetic potential for N retention and N2O sink capacity was detected, indicating the challenges in managing grasslands in a sustainable way.Peer reviewe

Stable nitrogen-cycling capacity in relation to fertilization and intercropping in a sub-boreal grassland

Grasslands are important in sub-boreal climate agricultural systems and are managed with various combinations of N fertilization and plant species. Ammonia-oxidizing and denitrifying microorganisms are key players in determining the fate of nitrogen (N) and thereby also the yield in grassland systems and their impact on gaseous N losses and leaching. We established a three-year field study in southern Finland with fertilizer treatment as a main-plot factor, including organic and synthetic fertilizers and plant species and mixtures thereof as the sub-plot factor. We quantified six genes encoding key N-cycling enzymes by quantitative PCR to determine the abundance of the communities involved in N-transformation processes and also included previously published data on crop yield, soil properties and the overall bacterial community composition. With the exception of ammonia oxidizing bacteria (AOB), which were primarily affected by fertilization, the abundances of all other N-cycling communities changed over time with either an increase or decrease from summer to autumn. Differences in gene abundances between plant species treatments and in fertilizer by plant species interactions were detected mainly in the beginning of the cropping season during the first year. The nirS-type denitrifiers and nosZII nitrous oxide reducers responded more to changes in soil properties than their functional counterpart nirK and nosZI communities. Using structural equation modeling, we show that the overall microbial community composition and diversity played an important role in mediating the management effects on crop yield, genetic potential for N retention and N2O sink capacity. However, a trade-off between the genetic potential for N retention and N2O sink capacity was detected, indicating the challenges in managing grasslands in a sustainable way.Peer reviewe

Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.Peer reviewe

Future images of data in circular economy for textiles

Rapid expansion of digitalization and in the volume of data available constitutes a major driver toward circular economy. In the textile industry, with its vast quantities of waste and huge environmental impact, transformation toward such circularity is necessary but challenging. To explore how the use of data could support building sustainability-aligned pathways to circular economy of textiles, a study employing a two-round disaggregative Delphi approach (engaging 33 experts in the first round, in May 2021, and 26 in the second, in June 2021) articulated alternative images of the future. The three images, dubbed Transparency, Conflicting Interests, and Sustainable Textiles, imply that the role for data is intertwined with sustainability aspirations. The results highlight that exploiting data in pursuit of circular economy is a collaborative effort involving business value networks that include consumers and regulators. Availability and sharing of accountability-affording, meaningful data on textiles' life cycle and value network function as a key enabler. By working with the images developed, actors can better assess their circular-economy commitments, planned actions, and the consequences of these. Furthermore, the images provide a tool for mutual discussion of the development desired and of related responsibilities and uncertainties.</p