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N2O production, a widespread trait in fungi.

Maeda K, Spor A, Edel-Hermann V, Heraud C, Breuil MC, Bizouard F, Toyoda S, Yoshida N, Steinberg C, Philippot L - Sci Rep (2015)

Bottom Line: The N2O (15)N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰, and we observed a significant taxa effect, with Penicillium strains displaying lower SP values than the other fungal genera.Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect.The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains, and its genetic variability showed a strong congruence with the ITS phylogeny, indicating vertical inheritance of this trait.

View Article: PubMed Central - PubMed

Affiliation: 1] NARO, Hokkaido Agricultural Research Center, Dairy Research Division, 1 Hitsujigaoka, Sapporo 062-8555, Japan [2] INRA, UMR 1347 Agroécologie, 17 rue Sully, 21065 Dijon Cedex, France.

ABSTRACT
N2O is a powerful greenhouse gas contributing both to global warming and ozone depletion. While fungi have been identified as a putative source of N2O, little is known about their production of this greenhouse gas. Here we investigated the N2O-producing ability of a collection of 207 fungal isolates. Seventy strains producing N2O in pure culture were identified. They were mostly species from the order Hypocreales order-particularly Fusarium oxysporum and Trichoderma spp.-and to a lesser extent species from the orders Eurotiales, Sordariales, and Chaetosphaeriales. The N2O (15)N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰, and we observed a significant taxa effect, with Penicillium strains displaying lower SP values than the other fungal genera. Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect. The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains, and its genetic variability showed a strong congruence with the ITS phylogeny, indicating vertical inheritance of this trait. Taken together, this comprehensive set of findings should enhance our knowledge of fungi as a source of N2O in the environment.

No MeSH data available.


Neighbour-joining phylogenetic tree of nirK amino acid sequences constructed by Clustal W with 1000 bootstrap samplings.Strain names in bold indicate the sequences obtained in this study. The numbers in parentheses indicate the number of the strains. Bootstrap values greater than 75% are indicated as black circles.
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f3: Neighbour-joining phylogenetic tree of nirK amino acid sequences constructed by Clustal W with 1000 bootstrap samplings.Strain names in bold indicate the sequences obtained in this study. The numbers in parentheses indicate the number of the strains. Bootstrap values greater than 75% are indicated as black circles.

Mentions: Both copper-containing nitrite reductase (encoded by the nirK gene) and P450nor (nitric oxide reductase) are key enzymes involved in fungal denitrification. However, P450nor belongs to a superfamily of proteins that are widely distributed among fungi and known to be involved in a wide variety of physiological reactions10, which prevents the use of the corresponding genes as molecular markers to target denitrifying fungi. To date, several primers targeting the nirK denitrification gene have been described in the literature, but none of them was designed to amplify denitrifying eukaryotes2549. Despite the low number of fungal nirK sequences available in the databases (less than 30) and the high diversity of the tested fungal strains, the amplification of the fungal nirK denitrification gene was successful in 45 out of 70 strains using our newly designed primer set EunirK-F1 and EunirK-R2. This supports our findings that these fungal strains are capable of denitrification and that N2O was not produced by other processes. Notably, when used to amplify DNA extracted from soil, our primer set also amplified the bacterial nirK in part due to the lower proportion of fungi in soil compared to bacteria (data not shown). A phylogenetic tree was constructed using these fungi nirK sequences and bacterial nirK sequences from available databases (Fig. 3). Our fungal nirK sequences clustered with the other fungal nirK homologues retrieved from the database, and were distinct from the bacterial nirK sequences. The phylogeny also shows that fungal nirK sequences are closer to other eukaryotic sequences (amoeba, protozoa or green alga) than to bacterial ones, indicating that fungal nirK sequences branched from bacterial nirK sequences at a very early stage of their evolutional history, as suggested by Kim et al.50. In addition, we also found a strong congruence between the ITS and the nirK phylogenies (Fig. S2), indicating a vertical inheritance of nirK genes. Interestingly, we found no correlation between the genetic distance of the nirK genes and the N2O production rates. Similarly, weak or no relationships were observed between the bacterial genotypes and denitrification phenotypes in previous studies3151. Phenotypic convergence within similar ecological niches of distantly related organisms can lead to such a discrepancy between genetic and phenotypic distances. In depth investigation of the ecology of denitrifying fungi would undoubtedly help clarify which environmental factors lead to a convergent denitrification phenotype.


N2O production, a widespread trait in fungi.

Maeda K, Spor A, Edel-Hermann V, Heraud C, Breuil MC, Bizouard F, Toyoda S, Yoshida N, Steinberg C, Philippot L - Sci Rep (2015)

Neighbour-joining phylogenetic tree of nirK amino acid sequences constructed by Clustal W with 1000 bootstrap samplings.Strain names in bold indicate the sequences obtained in this study. The numbers in parentheses indicate the number of the strains. Bootstrap values greater than 75% are indicated as black circles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403702&req=5

f3: Neighbour-joining phylogenetic tree of nirK amino acid sequences constructed by Clustal W with 1000 bootstrap samplings.Strain names in bold indicate the sequences obtained in this study. The numbers in parentheses indicate the number of the strains. Bootstrap values greater than 75% are indicated as black circles.
Mentions: Both copper-containing nitrite reductase (encoded by the nirK gene) and P450nor (nitric oxide reductase) are key enzymes involved in fungal denitrification. However, P450nor belongs to a superfamily of proteins that are widely distributed among fungi and known to be involved in a wide variety of physiological reactions10, which prevents the use of the corresponding genes as molecular markers to target denitrifying fungi. To date, several primers targeting the nirK denitrification gene have been described in the literature, but none of them was designed to amplify denitrifying eukaryotes2549. Despite the low number of fungal nirK sequences available in the databases (less than 30) and the high diversity of the tested fungal strains, the amplification of the fungal nirK denitrification gene was successful in 45 out of 70 strains using our newly designed primer set EunirK-F1 and EunirK-R2. This supports our findings that these fungal strains are capable of denitrification and that N2O was not produced by other processes. Notably, when used to amplify DNA extracted from soil, our primer set also amplified the bacterial nirK in part due to the lower proportion of fungi in soil compared to bacteria (data not shown). A phylogenetic tree was constructed using these fungi nirK sequences and bacterial nirK sequences from available databases (Fig. 3). Our fungal nirK sequences clustered with the other fungal nirK homologues retrieved from the database, and were distinct from the bacterial nirK sequences. The phylogeny also shows that fungal nirK sequences are closer to other eukaryotic sequences (amoeba, protozoa or green alga) than to bacterial ones, indicating that fungal nirK sequences branched from bacterial nirK sequences at a very early stage of their evolutional history, as suggested by Kim et al.50. In addition, we also found a strong congruence between the ITS and the nirK phylogenies (Fig. S2), indicating a vertical inheritance of nirK genes. Interestingly, we found no correlation between the genetic distance of the nirK genes and the N2O production rates. Similarly, weak or no relationships were observed between the bacterial genotypes and denitrification phenotypes in previous studies3151. Phenotypic convergence within similar ecological niches of distantly related organisms can lead to such a discrepancy between genetic and phenotypic distances. In depth investigation of the ecology of denitrifying fungi would undoubtedly help clarify which environmental factors lead to a convergent denitrification phenotype.

Bottom Line: The N2O (15)N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰, and we observed a significant taxa effect, with Penicillium strains displaying lower SP values than the other fungal genera.Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect.The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains, and its genetic variability showed a strong congruence with the ITS phylogeny, indicating vertical inheritance of this trait.

View Article: PubMed Central - PubMed

Affiliation: 1] NARO, Hokkaido Agricultural Research Center, Dairy Research Division, 1 Hitsujigaoka, Sapporo 062-8555, Japan [2] INRA, UMR 1347 Agroécologie, 17 rue Sully, 21065 Dijon Cedex, France.

ABSTRACT
N2O is a powerful greenhouse gas contributing both to global warming and ozone depletion. While fungi have been identified as a putative source of N2O, little is known about their production of this greenhouse gas. Here we investigated the N2O-producing ability of a collection of 207 fungal isolates. Seventy strains producing N2O in pure culture were identified. They were mostly species from the order Hypocreales order-particularly Fusarium oxysporum and Trichoderma spp.-and to a lesser extent species from the orders Eurotiales, Sordariales, and Chaetosphaeriales. The N2O (15)N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰, and we observed a significant taxa effect, with Penicillium strains displaying lower SP values than the other fungal genera. Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect. The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains, and its genetic variability showed a strong congruence with the ITS phylogeny, indicating vertical inheritance of this trait. Taken together, this comprehensive set of findings should enhance our knowledge of fungi as a source of N2O in the environment.

No MeSH data available.