Phylogenetic, taxonomic and functional diversity of fungal denitrifiers and associated N2O production efficacy

Mothapo, Nape V.; Chen, Huaihai; Cubeta, Marc A.; Grossman, Julie; Fuller, Frederick J.; Shi, Wei

doi:10.1016/j.soilbio.2015.02.001

Cited by 116 publications

(110 citation statements)

References 92 publications

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“…2). The p450nor sequences recovered from isolate DNA and soil DNA clustered with p450nor sequences from members of the Leotiomycetes (one sequence), Eurotiomycetes (one sequence), and Sordariomycetes (47 sequences), three diverse fungal lineages within the phylum Ascomycota that are known to harbor denitrifying representatives (33).…”

Section: Fungal Isolationmentioning

confidence: 99%

“…PAL2NAL (31) was used to generate a codon-aware nucleotide alignment, and conserved sites were selected for forward and reverse primer binding (Table 1; see the supplemental material for additional details about primer site selection). The p450nor sequences of A. terreus strain NIH2624 and A. flavus strain NRRL 3357 were among the 38 sequences used for primer design (see Data Set S1 in the supplemental material), and p450nor sequences derived from A. terreus strain NRRL 255 and A. flavus strain NRRL 3357 served as positive controls in all p450nor-targeted PCR assays (32,33). Genomic DNA from the bacterium Dehalogenimonas lykanthroporepellens strain BLDC-9 (ATCC BAA-1523) was used as a negative control in PCR assays.…”

Section: Medium Preparationmentioning

confidence: 99%

“…Morphotyping (e.g., appearance of aerial hyphae with a white, feathery appearance on agar plates) and 18S rRNA gene sequence analysis assigned two additional isolates to the order Mortierellales. Other fungi isolated in this study included members of the genera Verticillium (1 isolate), Humicola (2 isolates), Chaetomium (3 isolates), Podospora (1 isolate), Lecythophora (1 isolate), and Guehomyces (1 isolate) (see Data Set S3 in the supplemental material), all of which belong to fungal lineages known to harbor denitrifying representatives (33).…”

Section: Fungal Isolationmentioning

confidence: 99%

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Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

Higgins

Welsh

Orellana

et al. 2016

Appl Environ Microbiol

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Members of the Fungi convert nitrate (NO 3؊ ) and nitrite (NO 2 ؊ ) to gaseous nitrous oxide (N 2 O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N 2 O from added NO 3 ؊ or NO 2 ؊ in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N 2 O from NO 2 ؊ . Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N 2 O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N 2 O from NO 2 ؊ , whereas nirK (encoding the NO-forming NO 2 ؊ reductase) was amplified in only 13 to 74% of the N 2 O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2 O formation. IMPORTANCEA comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N 2 O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N 2 O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N 2 O emissions. Denitrification is a key process responsible for loss of fixed nitrogen (N) in soils and sediments and is mediated by both abiotic and microbial processes (1, 2). Of the microorganisms involved in denitrification, members of the Bacteria are well studied and considered key contributors; however, some saprotrophic fungi also conserve energy from the reduction of nitrate (NO 3 Ϫ ) or nitrite (NO 2 Ϫ ) to nitrous oxide (N 2 O), the main end product of fungal denitrification (3-5). Fungi have been implicated in N turnover for over 30 years (1,3,6), but the ecological importance of fungal contributions to denitrification remain uncertain. Potential roles of fungi in soil N loss and greenhouse gas (i.e., N 2 O) emission are underexplored, and monitoring tools (e.g., quantitative or endpoint PCR) to specifically address the presence and abundance of denitrif...

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Section: Fungal Isolationmentioning

confidence: 99%

Section: Medium Preparationmentioning

confidence: 99%

Section: Fungal Isolationmentioning

confidence: 99%

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Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

Higgins

Welsh

Orellana

et al. 2016

Appl Environ Microbiol

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“…Most studies have attempted to identify denitrifying fungi from soils and culture collections based on their ability to produce N 2 O (13,14). These studies have demonstrated that some fungal genera produce N 2 O and are routinely isolated from soil, including Fusarium, Aspergillus, Bionectria, Trichoderma, Chaetomium, and Penicillium (4,8,15,16,17).…”

mentioning

confidence: 99%

“…Molecular-based methods have been developed and extensively used to target bacterial denitrifiers; however, analogous tools have only recently been developed for fungal denitrifiers (13,27,28,29). In this context, the P450nor gene appears to be a better molecular target for detecting and characterizing fungal denitrifiers due to the lack of homology existing between the bacterial cnorBqnorB and fungal P450nor genes (14). Therefore, the first aim of this study was to develop a novel P450nor gene detection assay, and the second was to examine the prevalence and diversity of cultivable fungal denitrifiers found in various soil ecosystems using the developed assay.…”

mentioning

confidence: 99%

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Novinscak

Zebarth

Burton

et al. 2016

Appl Environ Microbiol

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Denitrifying fungi produce nitrous oxide (N 2 O), a potent greenhouse gas, as they generally lack the ability to convert N 2 O to dinitrogen. Contrary to the case for bacterial denitrifiers, the prevalence and diversity of denitrifying fungi found in the environment are not well characterized. In this study, denitrifying fungi were isolated from various soil ecosystems, and novel PCR primers targeting the P450nor gene, encoding the enzyme responsible for the conversion of nitric oxide to N 2 O, were developed, validated, and used to study the diversity of cultivable fungal denitrifiers. This PCR assay was also used to detect P450nor genes directly from environmental soil samples. Fungal denitrification capabilities were further validated using an N 2 O gas detection assay and a PCR assay targeting the nirK gene. A collection of 492 facultative anaerobic fungi was isolated from 15 soil ecosystems and taxonomically identified by sequencing the internal transcribed spacer sequence. Twenty-seven fungal denitrifiers belonging to 10 genera had the P450nor and the nirK genes and produced N 2 O from nitrite. N 2 O production is reported in strains not commonly known as denitrifiers, such as Byssochlamys nivea, Volutella ciliata, Chloridium spp., and Trichocladium spp. The prevalence of fungal denitrifiers did not follow a soil ecosystem distribution; however, a higher diversity was observed in compost and agricultural soils. The phylogenetic trees constructed using partial P450nor and nirK gene sequences revealed that both genes clustered taxonomically closely related strains together. IMPORTANCE A PCR assay targeting the P450nor gene involved in fungal denitrification was developed and validated. The newly developedP450nor primers were used on fungal DNA extracted from a collection of fungi isolated from various soil environments and on DNA directly extracted from soil. The results indicated that approximatively 25% of all isolated fungi possessed this gene and were able to convert nitrite to N 2 O. All soil samples from which denitrifying fungi were isolated also tested positive for the presence of P450nor. The P450nor gene detection assay was reliable in detecting a large diversity of fungal denitrifiers. Due to the lack of homology existing between P450nor and bacterial denitrification genes, it is expected that this assay will become a tool of choice for studying fungal denitrifiers.

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Responses of fungal community composition to long‐term chemical and organic fertilization strategies in Chinese Mollisols

Jiang

Wang

et al. 2018

MicrobiologyOpen

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How fungi respond to long‐term fertilization in Chinese Mollisols as sensitive indicators of soil fertility has received limited attention. To broaden our knowledge, we used high‐throughput pyrosequencing and quantitative PCR to explore the response of soil fungal community to long‐term chemical and organic fertilization strategies. Soils were collected in a 35‐year field experiment with four treatments: no fertilizer, chemical phosphorus, and potassium fertilizer (PK), chemical phosphorus, potassium, and nitrogen fertilizer (NPK), and chemical phosphorus and potassium fertilizer plus manure (MPK). All fertilization differently changed soil properties and fungal community. The MPK application benefited soil acidification alleviation and organic matter accumulation, as well as soybean yield. Moreover, the community richness indices (Chao1 and ACE) were higher under the MPK regimes, indicating the resilience of microbial diversity and stability. With regards to fungal community composition, the phylum Ascomycota was dominant in all samples, followed by Zygomycota, Basidiomycota, Chytridiomycota, and Glomeromycota. At each taxonomic level, the community composition dramatically differed under different fertilization strategies, leading to different soil quality. The NPK application caused a loss of Leotiomycetes but an increase in Eurotiomycetes, which might reduce the plant–fungal symbioses and increase nitrogen losses and greenhouse gas emissions. According to the linear discriminant analysis (LDA) coupled with effect size (LDA score > 3.0), the NPK application significantly increased the abundances of fungal taxa with known pathogenic traits, such as order Chaetothyriales, family Chaetothyriaceae and Pleosporaceae, and genera Corynespora, Bipolaris, and Cyphellophora. In contrast, these fungi were detected at low levels under the MPK regime. Soil organic matter and pH were the two most important contributors to fungal community composition.

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Phylogenetic, taxonomic and functional diversity of fungal denitrifiers and associated N2O production efficacy

Cited by 116 publications

References 92 publications

Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

Novel P450nor Gene Detection Assay Used To Characterize the Prevalence and Diversity of Soil Fungal Denitrifiers

Responses of fungal community composition to long‐term chemical and organic fertilization strategies in Chinese Mollisols

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