Associations between leguminous plants and symbiotic nitrogen-fixing bacteria (rhizobia) are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Rhizobia improve plant yield furnishing fixed nitrogen; therefore, they are highly used as inoculants, especially in sustainable agriculture. Though this association is species-specific, legume roots are exposed to heterogeneous rhizobial populations where different compatible strains are present and, could be infected by more than one strain. It is known that within the same rhizobial species different strains may have different competition capabilities, but detailed analyses able to predict the rhizobial competitive phenotype based exclusively on their genome are still lacking. In this thesis, we performed a bacterial genome-wide association analysis (GWAS) to define which genetic traits are responsible for an improved competitive phenotype in the model species Sinorhizobium meliloti. A panel of thirteen S. meliloti strains, whose genome is completely sequenced, has been selected and tested against three S. meliloti reference competitor strains (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. The measure of competition phenotypes previously obtained in the competition tests, in combination with strains genomic sequences of strains tested, were used to build-up a k-mer-based statistical models for each set of competition experiments. The obtained models were then applied to evaluate the accuracy in predicting the competition abilities of strains in the three competition patterns (vsRm1021, vsAK83 and vsBL225C). The competitive abilities of S. meliloti strains against two partners, BL225C and Rm1021, were well predict by the predictive models, as shown by the coefficient of determination R2 (equal to 0.96 and 0.84, respectively). Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with the competition phenotype. The most significantly associated k-mers (p <0.05) were mapped on the genomic sequences of the S. meliloti strains used. Most of the k-mers were located on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components. The identification of the best rhizobial inoculants in two new effective breeding lines of pea (Pisum sativum) used in Lithuania (DS 3637-2 and DS 3795-3) was also investigated. Six rhizobial strains, isolated from pea plants, which could be used as potential inoculants, were phylogenetically identified and extensive phenotypically characterized by Phenotype Microarray. All the strains belonged to the Rhizobium leguminosarum group, and were subdivided into three groups related to Rhizobium anhuiense, Rhizobium leguminosarum bv. viciae and R. sophorae/R. laguerreae. Differences observed with Phenotype microarray were linked to different phylogeny of the strains. In terms of symbiotic efficiency, six strains showed different symbiotic performances depending on the breeding line used. In particular, Rhizobium anhuiense strain Z1 (the reference strain) and Rhizobium leguminosarum bv. viciae 14ZE were the best symbiotic inoculants with breeding lines DS 3637-2 and DS 3795-3, respectively.

Into the wild: how rhizobia compete and survive in the early stage of symbiosis / Agnese Bellabarba. - (2022).

Into the wild: how rhizobia compete and survive in the early stage of symbiosis

Agnese Bellabarba
2022

Abstract

Associations between leguminous plants and symbiotic nitrogen-fixing bacteria (rhizobia) are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Rhizobia improve plant yield furnishing fixed nitrogen; therefore, they are highly used as inoculants, especially in sustainable agriculture. Though this association is species-specific, legume roots are exposed to heterogeneous rhizobial populations where different compatible strains are present and, could be infected by more than one strain. It is known that within the same rhizobial species different strains may have different competition capabilities, but detailed analyses able to predict the rhizobial competitive phenotype based exclusively on their genome are still lacking. In this thesis, we performed a bacterial genome-wide association analysis (GWAS) to define which genetic traits are responsible for an improved competitive phenotype in the model species Sinorhizobium meliloti. A panel of thirteen S. meliloti strains, whose genome is completely sequenced, has been selected and tested against three S. meliloti reference competitor strains (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. The measure of competition phenotypes previously obtained in the competition tests, in combination with strains genomic sequences of strains tested, were used to build-up a k-mer-based statistical models for each set of competition experiments. The obtained models were then applied to evaluate the accuracy in predicting the competition abilities of strains in the three competition patterns (vsRm1021, vsAK83 and vsBL225C). The competitive abilities of S. meliloti strains against two partners, BL225C and Rm1021, were well predict by the predictive models, as shown by the coefficient of determination R2 (equal to 0.96 and 0.84, respectively). Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with the competition phenotype. The most significantly associated k-mers (p <0.05) were mapped on the genomic sequences of the S. meliloti strains used. Most of the k-mers were located on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components. The identification of the best rhizobial inoculants in two new effective breeding lines of pea (Pisum sativum) used in Lithuania (DS 3637-2 and DS 3795-3) was also investigated. Six rhizobial strains, isolated from pea plants, which could be used as potential inoculants, were phylogenetically identified and extensive phenotypically characterized by Phenotype Microarray. All the strains belonged to the Rhizobium leguminosarum group, and were subdivided into three groups related to Rhizobium anhuiense, Rhizobium leguminosarum bv. viciae and R. sophorae/R. laguerreae. Differences observed with Phenotype microarray were linked to different phylogeny of the strains. In terms of symbiotic efficiency, six strains showed different symbiotic performances depending on the breeding line used. In particular, Rhizobium anhuiense strain Z1 (the reference strain) and Rhizobium leguminosarum bv. viciae 14ZE were the best symbiotic inoculants with breeding lines DS 3637-2 and DS 3795-3, respectively.
Carlo Viti
ITALIA
Agnese Bellabarba
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2158/1280999
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