Cum

ulat

ive

N2

(µm

ols

N2

h-1)

0.00

0.10

0.20

0.30

Cum

ulat

ive

N2O

(µ

mol

s N

2O h

-1)

0.00

0.03

0.06

NecromassSterile Media

(a) (b)

Live Fungi NecromassSterile Media Live Fungi

Unveiling Fungal Contributions to Agricultural Soil Nitrogen Cycling Following Application of Organic and Inorganic Fertilizers

Rebecca L. Phillips1,2*, Bongkeun Song3, Craig Tobias4, Andrew McMillan1,

Gwen Grelet1, Thilak Palmada1, Mikhail Forkin1, Bevan Weir1 1Landcare Research, NZ, 2Ecological Insights, 3Virginia Institute of Marine Science, 4University of Connecticut

Rebecca Phillips: phillipsr@landcareresearch.co.nz rebecca.phillips@ecologicalinsights.org

Relevance

Removal of excess nitrogen (N) can best be achieved through denitrification processes that transform fixed N to di-nitrogen (N2) gas in various ecosystems. The greenhouse gas nitrous oxide (N2O) is considered an intermediate or end-product in denitrification pathways. Some fungi reportedly produce N2 by combining two N sources through codenitrification. The only other known source of hybrid N2 is anammox , so formation of hybrid N2 is reported as evidence of anammox or codenitrification (Fig. 1). However, fungal codenitrification reports are inconsistent. We used Bipolaris sorokiniana as a model fungal species to examine fungal denitrification and codenitrification using established techniques at nuetral pH. In addition, we rigorously investigated abiotic N2 and N2O production under not only anoxic, but also 20% oxygen conditions.

Objectives 1.  Examine effects of N (both organic and inorganic N) on N2

and N2O production and the expression of N metabolism genes in Bipolaris sorokiniana, a common cereal pathogenic fungus.

2.  Determine if O2 (anoxic vs. 20% O2) affects production of N2 and N2O.

3.  Examine how the absence of live fungi and presence of necromass affects production of N2 and N2O.

4.  Determine if N2O and N2 are produced abiotically in the presence or absence of O2, give N source and sterile media commonly used in fungal codenitrification experiments.

Results

Summary

1.  Di-nitrogen was produced in the presence of live and dead fungi and in sterile media only with no evidence of N2O consumption.

2.  Isotope pairing experiments indicated N2 was produced abiotically and all N2 was formed using a combination of glutamine N and nitrite N.

3.  Di-nitrogen was produced abiotically under anaerobic and fully aerobic conditions.

4.  Differential gene expression of N uptake in B. sorokiniana was observed under different N conditions while the gene expression in N dissimilation is still under investigation.

5.  These results call into question the assumptions that (1) N2O is an intermediate required for N2 formation, (2) production of N2 and N2O requires anaerobiosis, and (3) hybrid N2 is evidence of codenitrification and/or anammox.

Acknowledgements This research is funded by the AFRI program of National Institute of Food and Agriculture and Landcare Research Ltd, New Zealand. Special thanks to Veronica Rollinson, Megan Peterson and Duckchul Park for contributing their technical expertise needed for this project.

Table 1. Average (SD) of 29N2 and 30N2 recovered in the headspace following aerobic and anaerobic incubation of sterile media at three levels of N addition, where added N comprised 50% N from unlabelled C5H10N2O3 and 50% N from labelled 15N-NaNO2 (n=5). Values were adjusted according to helium or heliox blanks.

Figure 3. Abiotic and biotic production rates of (a) N2O and (b) N2 accumulated per hour following N addition under both aerobic and anaerobic conditions for sterile medium, necromass and live fungi. Boxes represent the 90th percentile data; error bars, s.d.; n=4. Median values are the lines horizontally bisecting each box.

Figure 1. Schematic of codenitrification, anammox and known chemical denitrification pathways, a modified adaptation from Butterbach-Bahl et al. (2013). Selected processes potentially leading to N2O and N2 formation, involved N compounds, their reaction pathways as well as their oxidation states are shown. Closed circles are biotic and open circles are abiotic reactions. The last process, abiotic hybrid N2 formation, was observed in this study.

Impact 1.  Findings will encourage development of new approaches to

removal of excess nitrogen in aquatic and terrestrial ecosystems.

2.  Findings challenge researchers to re-assess abiotic contributions to what is known as codenitrification.

3.  Unveiling chemical, rather than fungal, formation of hybrid di-nitrogen may help explain discrepancies in the N budget.

N addition

(mmol N)

29N2 (14N,15N)

(µmol) aerobic

30N2 (15N,15N)

(µmol) aerobic

29N2 (14N,15N)

(µmol) anaerobic

30N2 (15N,15N)

(µmol) anaerobic

0 -0.012

(<0.001) 0.000

(0.000) -0.005 (0.005)

<0.001 (<0.001)

0.5 16.274 (2.045)

0.001 (<0.001)

15.759 (0.995)

0.003 (0.002)

1.0 40.289 (2.318)

0.002 (<0.001)

31.025 (3.144)

0.007 (0.001)

1

Figure 2. Experimental set-up to test how live fungi, necromass and media only incubated in an anoxic and 20% O2 atmosphere affects production of N2O and N2 following addition of both organic and inorganic forms of N to pure cultures under aseptic conditions. Terms are defined as: Glut, glutamine; NaNO2, sodium nitrite; He, helium; HeOx heliox.

Methods log2FPKM

6 4 2 0

Glutamine Nitrite N depleted

Nitrate/nitrite transporter

Nitrate reductase

Nitrite reductase

Glutamine synthase A

Glutamine synthase B

Glutamine dehydrogenase

Glutamine dehydrogenase (NADP+)

Glutamine synthase

Figure 4. Differential gene expression of the nitrogen uptake metabolism in B. sorokiniana under anoxic conditions for three N treatments. RNA-seq was conducted with triplicate samples of each incubation conditions using Illumina HiSeq. Transcriptomes were analyzed with Topha2/Bowtie 2 and Cufflinks pipeline.

NH2-OH

FungalDenitrifica4on

Codenitrifica4onwithNO

ANAMMOX

Chemicaldecomposi4onNH2OH

Chemodenitrifica4on-abio4c

Surfacedecomposi4onNH4NO3

Abio4chybridN2forma4on

-3-1+5+3+2+10Oxida4veState

N-OrgR-NH2

NH4+

NH3

NO3- NO2

- N2ONO N2

?

NH2OH