Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment

Wei, Wei; Isobe, Kazuo; Shiratori, Yutaka; Nishizawa, Tomoyasu; Ohte, Nobuhito; Ise, Yuta; Otsuka, Shigeto; Senoo, Keishi

doi:10.1016/j.soilbio.2014.11.026

Cited by 49 publications

(67 citation statements)

References 16 publications

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“…To date, only a limited number of studies have relied on molecular methods to detect, identify, and/or characterize fungal denitrifiers (13,27,28,29,35,36). The study by Long et al (36) (4,17,37), most strains isolated in the present study were not able to reduce NO 3 Ϫ to N 2 O, with the exception of Fusarium strains (data not shown).…”

Section: Discussionmentioning

confidence: 52%

“…These results are contrary to those for bacterial nirK, where no sequence relatedness based on a bacterium's taxonomic identification was observed (5,43). Congruency of nirK, P450nor, and the 18S gene was reported previously by Wei et al (27), and the ITS region was reported by Maeda et al (13). Therefore, unlike that of bacteria, the P450nor and nirK phylogeny may be able to estimate the taxonomic affiliations of uncultured fungal denitrifiers.…”

Section: Discussionmentioning

confidence: 60%

“…The mismatch at this site was not observed in the 27 P450nor sequences determined in this study and may offer an explanation as to why P450nor sequences from C. rosea were not amplified. The nirK primers developed by Maeda et al (13), Long et al (28), and Wei et al (27) were also used to attempt to amplify the nirK gene from the eight strains of C. rosea using their respective PCR protocols; however, bands of the correct size were not observed for any of the primer pairs used in the eight C. rosea strains (results not shown). The P450nor PCR primers and nirK PCR primers developed in this study detected most denitrifying species tested in the current study, but they show some limitations, like most PCR primer sets targeting functional microbial genes, being incapable of generating amplicons from at least one genus of known fungal denitrifiers, Clonostachys.…”

Section: Discussionmentioning

confidence: 99%

“…With little attention having been focused on denitrifying fungi in soil, their abundance and diversity are not well documented. 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).…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 52%

Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…For example, since our collection of nirK and nirS sequences from databases, sequences have been obtained from more diverse microorganisms, such as nirK from Firmicutes (Murawska et al, 2013) and nirS from Crenarchaeota (Mardanov et al, 2012). Some groups of fungi also have nirK (Wei et al, 2015). PCR techniques still have a greater capacity for the deep analysis of target genes, although efforts are needed to minimize the PCR biases.…”

Section: Discussionmentioning

confidence: 99%

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

et al. 2015

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Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2-6 times more abundant than those revealed with the conventional primers. RNA-and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.

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Increasing livestock wastewater application in alternate-furrow irrigation reduces nitrification gene abundance but not nitrification rate in rhizosphere

et al. 2019

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In water-scarce regions, alternate-furrow irrigation (AFI)-alternately wetting half of the plant roots-has proven to be an effective water-saving approach without compromising yield. However, the extent to which AFI with wastewater affects N cycling genes remains poorly studied. We aimed to investigate changes in main N transformation processes, bacterial and fungal community composition, as well as relative abundance of N cycle-associated genes in soil receiving AFI with swine wastewater. The experimental plan included three irrigation rates, irrigating pepper plants with 50%, 65%, and 80% of the amount of water required under conventional furrow irrigation to prevent the crop suffering water stress. Each treatment had a groundwater irrigation control. We measured edaphic factors, microbial community composition, and relative abundance of genes in rhizosphere and bulk soils. Altering water use in AFI did not exert a significant effect on bacterial and fungal communities. By increasing the irrigation rate of wastewater, relative abundances of nifH, bacterial and archaeal amoA and nosZ genes decreased, whereas those of nirK and nirS genes increased in the rhizosphere soil; nitrification rate did not decrease and the denitrification rate remained unchanged in both rhizosphere and bulk soil, implying that appropriate increase of wastewater use by AFI can improve N use efficiency.

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Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment

Cited by 49 publications

References 16 publications

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

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

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

Increasing livestock wastewater application in alternate-furrow irrigation reduces nitrification gene abundance but not nitrification rate in rhizosphere

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