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

Abstract

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.

An evaluation of primers for detecting denitrifiers via their functional genes

“…Several primers have been designed for fungal nirK recently (Long et al, 2015;Maeda et al, 2015;Wei et al, 2015a;Chen et al, 2016;Novinscak et al, 2016). Two fungal denitrification genes have been targeted for development of molecular methods: nirK and P450nor.…”

“…Due to the high similarity of bacterial and fungal nirK sequences, the fungal nirK primers were also tested for specificity against the 361 bacterial and archaeal nirK sequences used for testing bacterial nirK primers. All three primer pairs covered more than 75% of the sequences, with p450nor394F/p450nor1008R (Higgins et al, 2016) and p450nor1F/p450nor1R (Novinscak et al, 2016) having the highest coverage (both 85.3%, Table 1). However, experimental results indicate that specificity remains a problem.…”

“…However, experimental results indicate that specificity remains a problem. Despite the high coverage, the specificity of the primers was not promising, because multiple products appeared when these primers were used in PCR to amplify the target sequence from fungal isolates or soil samples (Higgins et al, 2016;Novinscak et al, 2016). The primer set of Maeda and colleagues (2015) had the poorest specificity, with all the clones sequenced containing sequences from bacterial nirK.…”

“…Two fungal denitrification genes have been targeted for development of molecular methods: nirK and P450nor. Several primers have been designed for fungal nirK recently (Long et al, 2015;Maeda et al, 2015;Wei et al, 2015a;Chen et al, 2016;Novinscak et al, 2016). These primers were tested here for coverage against 159 available fungal nirK sequences from a variety of taxonomy groups (Table 1 and Supporting Information Table S7).…”

“…For P450nor, the fungal gene for cytochrome P450 nitric oxide reductase, the available primers were tested against 68 P450nor nucleotide sequences. All three primer pairs covered more than 75% of the sequences, with p450nor394F/p450nor1008R (Higgins et al, 2016) and p450nor1F/p450nor1R (Novinscak et al, 2016) having the highest coverage (both 85.3%, Table 1). The forward primers of the two studies target the same position, while the reverse primers differ in their target sequences (Supporting Information Table S8).…”