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Christopher L Schardl - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass-
2016Co-Authors: Epichloe ̈ Symbiota, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Juan Pan, Christopher L SchardlAbstract:The Lolines are a class of bioprotective Alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These Alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group:-NH2 (norLoline),-NHCH3 (Loline),-N(CH3)2 (N-methylLoline),-N(CH3)Ac (N-acetylLoline),-NHAc (N-acetylnorLoline), and-N(CH3)CHO (N-formylLoline). Other than the LolP cytochrome P450, which is required for conversion of N-methylLoline to N-formylLoline, the enzymatic steps for Loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorLoline is the first fully cyclized Loline Alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in Loline Alkaloid diversification. Two genes of the Loline Alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorLoline, and complementation with the two wild-type genes restored production of N-formylLoline and N-acetylLoline. Thes
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enzymes from fungal and plant origin required for chemical diversification of insecticidal Loline Alkaloids in grass epichloe symbiota
PLOS ONE, 2014Co-Authors: Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:The Lolines are a class of bioprotective Alkaloids that are produced by Epichloe species, fungal endophytes of grasses. These Alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norLoline), -NHCH3 (Loline), -N(CH3)2 (N-methylLoline), -N(CH3)Ac (N-acetylLoline), -NHAc (N-acetylnorLoline), and -N(CH3)CHO (N-formylLoline). Other than the LolP cytochrome P450, which is required for conversion of N-methylLoline to N-formylLoline, the enzymatic steps for Loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorLoline is the first fully cyclized Loline Alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in Loline Alkaloid diversification. Two genes of the Loline Alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorLoline, and complementation with the two wild-type genes restored production of N-formylLoline and N-acetylLoline. These results indicated that lolN and lolM are required in the steps from N-acetylnorLoline to other Lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norLoline to Loline, and of Loline to N-methylLoline. One of the more abundant Lolines, N-acetylLoline, was observed in some but not all plants with symbiotic Epichloe siegelii, and when provided with exogenous Loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylLoline, suggesting that a plant acetyltransferase catalyzes N-acetylLoline formation. We conclude that although most Loline Alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.
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Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass-Epichloë Symbiota
2014Co-Authors: Juan Pan, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:The Lolines are a class of bioprotective Alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These Alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norLoline), -NHCH3 (Loline), -N(CH3)2 (N-methylLoline), -N(CH3)Ac (N-acetylLoline), -NHAc (N-acetylnorLoline), and -N(CH3)CHO (N-formylLoline). Other than the LolP cytochrome P450, which is required for conversion of N-methylLoline to N-formylLoline, the enzymatic steps for Loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorLoline is the first fully cyclized Loline Alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in Loline Alkaloid diversification. Two genes of the Loline Alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorLoline, and complementation with the two wild-type genes restored production of N-formylLoline and N-acetylLoline. These results indicated that lolN and lolM are required in the steps from N-acetylnorLoline to other Lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norLoline to Loline, and of Loline to N-methylLoline. One of the more abundant Lolines, N-acetylLoline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous Loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylLoline, suggesting that a plant acetyltransferase catalyzes N-acetylLoline formation. We conclude that although most Loline Alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.
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GC-MS chromatogram of Loline Alkaloids after application of Loline to asymbiotic plants.
2014Co-Authors: Juan Pan, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:Shown are Loline Alkaloids extracted from Loline applications to (A and B) endophyte-free (E-) meadow fescue (MF), and (C and D) E- perennial ryegrass (PRG), (E) mass spectrum of N-acetylLoline (NAL) from application of Loline to E- meadow fescue, and (F) proposed scheme of NAL formation from Loline. Quinoline was added as internal standard (istd). Unlabeled peaks are non-Loline Alkaloid compounds. Numbers after MF or PRG indicate independent trials.
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Loline Alkaloid profiles and LOL-gene screening results for endophyte isolates. a
2014Co-Authors: Juan Pan, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:aAbbreviations are: tr = trace amount; + = Alkaloid detected or full gene present; − = Alkaloid not detected or gene not present; Ψ = pseudogene; nd = gene not detected in PCR screen.bTwo nonsynonymous mutations found in otherwise conserved sites G495D and E551K.Loline Alkaloid profiles and LOL-gene screening results for endophyte isolates. a
Adrian Leuchtmann - One of the best experts on this subject based on the ideXlab platform.
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large scale screening of epichloe endophytes infecting schedonorus pratensis and other forage grasses reveals a relation between microsatellite based haplotypes and Loline Alkaloid levels
Frontiers in Plant Science, 2019Co-Authors: Giovanni Cagnano, Niels Roulund, Christian Sig Jensen, Flavia Pilar Forte, Torben Asp, Adrian LeuchtmannAbstract:Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloe. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus, and Lolium perenne. All plants were screened for the presence of Epichloe endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of Loline Alkaloids were measured in 218 infected meadow fescue plants. Average amount of total Loline and the proportions of the single Loline Alkaloids differed significantly among endophyte haplotypes (P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and Alkaloid variation observed in E. uncinata.
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large scale screening of epichloe endophytes infecting schedonorus pratensis and other forage grasses reveals a relation between microsatellite based haplotypes and Loline Alkaloid levels
Frontiers in Plant Science, 2019Co-Authors: Giovanni Cagnano, Niels Roulund, Christian Sig Jensen, Flavia Pilar Forte, Adrian LeuchtmannAbstract:Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloe. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analysed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the U.S. Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus and Lolium perenne. All plants were screened for the presence of Epichloe endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of Loline Alkaloids were measured in 218 infected meadow fescue plants. Average amount of total Loline and the proportions of the single Loline Alkaloids differed significantly among endophyte haplotypes (P<0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and Alkaloid variation observed in E. uncinata.
Giovanni Cagnano - One of the best experts on this subject based on the ideXlab platform.
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large scale screening of epichloe endophytes infecting schedonorus pratensis and other forage grasses reveals a relation between microsatellite based haplotypes and Loline Alkaloid levels
Frontiers in Plant Science, 2019Co-Authors: Giovanni Cagnano, Niels Roulund, Christian Sig Jensen, Flavia Pilar Forte, Torben Asp, Adrian LeuchtmannAbstract:Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloe. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus, and Lolium perenne. All plants were screened for the presence of Epichloe endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of Loline Alkaloids were measured in 218 infected meadow fescue plants. Average amount of total Loline and the proportions of the single Loline Alkaloids differed significantly among endophyte haplotypes (P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and Alkaloid variation observed in E. uncinata.
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large scale screening of epichloe endophytes infecting schedonorus pratensis and other forage grasses reveals a relation between microsatellite based haplotypes and Loline Alkaloid levels
Frontiers in Plant Science, 2019Co-Authors: Giovanni Cagnano, Niels Roulund, Christian Sig Jensen, Flavia Pilar Forte, Adrian LeuchtmannAbstract:Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloe. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analysed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the U.S. Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus and Lolium perenne. All plants were screened for the presence of Epichloe endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of Loline Alkaloids were measured in 218 infected meadow fescue plants. Average amount of total Loline and the proportions of the single Loline Alkaloids differed significantly among endophyte haplotypes (P<0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and Alkaloid variation observed in E. uncinata.
Padmaja Nagabhyru - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass-
2016Co-Authors: Epichloe ̈ Symbiota, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Juan Pan, Christopher L SchardlAbstract:The Lolines are a class of bioprotective Alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These Alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group:-NH2 (norLoline),-NHCH3 (Loline),-N(CH3)2 (N-methylLoline),-N(CH3)Ac (N-acetylLoline),-NHAc (N-acetylnorLoline), and-N(CH3)CHO (N-formylLoline). Other than the LolP cytochrome P450, which is required for conversion of N-methylLoline to N-formylLoline, the enzymatic steps for Loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorLoline is the first fully cyclized Loline Alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in Loline Alkaloid diversification. Two genes of the Loline Alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorLoline, and complementation with the two wild-type genes restored production of N-formylLoline and N-acetylLoline. Thes
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enzymes from fungal and plant origin required for chemical diversification of insecticidal Loline Alkaloids in grass epichloe symbiota
PLOS ONE, 2014Co-Authors: Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:The Lolines are a class of bioprotective Alkaloids that are produced by Epichloe species, fungal endophytes of grasses. These Alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norLoline), -NHCH3 (Loline), -N(CH3)2 (N-methylLoline), -N(CH3)Ac (N-acetylLoline), -NHAc (N-acetylnorLoline), and -N(CH3)CHO (N-formylLoline). Other than the LolP cytochrome P450, which is required for conversion of N-methylLoline to N-formylLoline, the enzymatic steps for Loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorLoline is the first fully cyclized Loline Alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in Loline Alkaloid diversification. Two genes of the Loline Alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorLoline, and complementation with the two wild-type genes restored production of N-formylLoline and N-acetylLoline. These results indicated that lolN and lolM are required in the steps from N-acetylnorLoline to other Lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norLoline to Loline, and of Loline to N-methylLoline. One of the more abundant Lolines, N-acetylLoline, was observed in some but not all plants with symbiotic Epichloe siegelii, and when provided with exogenous Loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylLoline, suggesting that a plant acetyltransferase catalyzes N-acetylLoline formation. We conclude that although most Loline Alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.
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Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass-Epichloë Symbiota
2014Co-Authors: Juan Pan, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:The Lolines are a class of bioprotective Alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These Alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norLoline), -NHCH3 (Loline), -N(CH3)2 (N-methylLoline), -N(CH3)Ac (N-acetylLoline), -NHAc (N-acetylnorLoline), and -N(CH3)CHO (N-formylLoline). Other than the LolP cytochrome P450, which is required for conversion of N-methylLoline to N-formylLoline, the enzymatic steps for Loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorLoline is the first fully cyclized Loline Alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in Loline Alkaloid diversification. Two genes of the Loline Alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorLoline, and complementation with the two wild-type genes restored production of N-formylLoline and N-acetylLoline. These results indicated that lolN and lolM are required in the steps from N-acetylnorLoline to other Lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norLoline to Loline, and of Loline to N-methylLoline. One of the more abundant Lolines, N-acetylLoline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous Loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylLoline, suggesting that a plant acetyltransferase catalyzes N-acetylLoline formation. We conclude that although most Loline Alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.
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GC-MS chromatogram of Loline Alkaloids after application of Loline to asymbiotic plants.
2014Co-Authors: Juan Pan, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:Shown are Loline Alkaloids extracted from Loline applications to (A and B) endophyte-free (E-) meadow fescue (MF), and (C and D) E- perennial ryegrass (PRG), (E) mass spectrum of N-acetylLoline (NAL) from application of Loline to E- meadow fescue, and (F) proposed scheme of NAL formation from Loline. Quinoline was added as internal standard (istd). Unlabeled peaks are non-Loline Alkaloid compounds. Numbers after MF or PRG indicate independent trials.
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Loline Alkaloid profiles and LOL-gene screening results for endophyte isolates. a
2014Co-Authors: Juan Pan, Minakshi Bhardwaj, Padmaja Nagabhyru, Robert B Grossman, Christopher L SchardlAbstract:aAbbreviations are: tr = trace amount; + = Alkaloid detected or full gene present; − = Alkaloid not detected or gene not present; Ψ = pseudogene; nd = gene not detected in PCR screen.bTwo nonsynonymous mutations found in otherwise conserved sites G495D and E551K.Loline Alkaloid profiles and LOL-gene screening results for endophyte isolates. a
Christian Sig Jensen - One of the best experts on this subject based on the ideXlab platform.
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large scale screening of epichloe endophytes infecting schedonorus pratensis and other forage grasses reveals a relation between microsatellite based haplotypes and Loline Alkaloid levels
Frontiers in Plant Science, 2019Co-Authors: Giovanni Cagnano, Niels Roulund, Christian Sig Jensen, Flavia Pilar Forte, Torben Asp, Adrian LeuchtmannAbstract:Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloe. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus, and Lolium perenne. All plants were screened for the presence of Epichloe endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of Loline Alkaloids were measured in 218 infected meadow fescue plants. Average amount of total Loline and the proportions of the single Loline Alkaloids differed significantly among endophyte haplotypes (P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and Alkaloid variation observed in E. uncinata.
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large scale screening of epichloe endophytes infecting schedonorus pratensis and other forage grasses reveals a relation between microsatellite based haplotypes and Loline Alkaloid levels
Frontiers in Plant Science, 2019Co-Authors: Giovanni Cagnano, Niels Roulund, Christian Sig Jensen, Flavia Pilar Forte, Adrian LeuchtmannAbstract:Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloe. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analysed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the U.S. Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus and Lolium perenne. All plants were screened for the presence of Epichloe endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of Loline Alkaloids were measured in 218 infected meadow fescue plants. Average amount of total Loline and the proportions of the single Loline Alkaloids differed significantly among endophyte haplotypes (P<0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and Alkaloid variation observed in E. uncinata.
