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Effects of modified nutrient availability on

nitrogen-fixing legumes in a sub-alpine silvo-

pastoral system in south eastern Australia

Internship Report

Héctor Bahamonde

Supervised bySebastian Pfautsch and Tina Bell

Rio Gallegos, Argentina

December 2012

Supported by

Faculty of Agriculture and Environment



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1. Introduction

The sub-alpine regions of mainland south eastern Australia stretch from southernNew South Wales to the central parts of the Great Dividing Range in Victoria,covering an area of approximately 1200 km 2 (Costin et al ., 2000). The landscape isfrequently affected by fire – even prior to European settlement – at intervals of aslittle as 50 years (Gill and Catling, 2002). Common vegetation communities includecgrasslands, heathland and open woodland, each of which is easily identifiedaccording to dominant species, height, growth form and structure (Williams andCostin, 1981; Costin et al ., 2000). Mosaics of these three vegetation types are

characteristic of gently undulating terrain at elevations between 1300 and 1800 masldue to small variations in climatic conditions, with one vegetation type replacing

another over short distances (Costin, 1962).

Snowgum (Eucalyptus pauciflora Sieber ex Spreng.) is the single most dominant tree

species across the sub-alpine region at elevations of 1200 masl. As a resultSnowgum woodlands are unique among the many types of tree-dominatedecosystems within Australia.

The average annual precipitation in sub-alpine areas is ~1600 mm and there is snowcover for approximately 3-4 months of the year from May until September. Thewarmest month is January (~20.0 ºC maximum average) while July the coldestmonth (-4.0 ºC minimum average). Frosts are frequent (greater than 100 potentialfrost days per annum on average) and happen in all months of the year, includingsummer (Costin et al ., 2000). Accordingly, the growing season is short in comparison

with that of forests and woodlands at lower elevations.

Concentrations of plant-available forms of N in soils of most native Australianecosystems, including sub-alpine Australian systems (e.g. Huber et al., 2011), are

low by world standards. On the other hand, native legumes are moderately common.Heathland and woodland communities in sub-alpine areas often contain species ofthe genera Acacia , Daviesia and Bossiaea . As with other legumes, these mostly

woody shrubs can putatively access atmospheric N (N 2) through symbioses withRhizobium bacteria (a process known as biological nitrogen fixation, BNF), and

might be expected to have advantages over non-legumes in N-limited environments(Lafay and Burdon, 1998).



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BNF is well known for its limitation by phosphorus (P). Phosphorus (P) too is found inlow concentrations in most Australian soils, especially the plant-available forms of P(Wild, 1958; Nix, 1981). In part this is due to the processes of cycling of P whichserve to sequester P in plant biomass and soil microbial communities as organic P,

while physio-chemical processes serve to bind inorganic forms of P rendering mostof it unavailable to plants.

Phosphorus is a key requirement for nodule formation and maintenance of effectiveBNF (Brockwellet al. , 2005), and N 2-fixing plants have a greater requirement for P

than non-N2-fixers (Vitousek and Field, 1999). For example, activity of the mainenzyme involved in BNF, nitrogenase, increased with addition of P in Medicago

sativa, a common N 2-fixing legume crop (Crews, 1993). The trace elementmolybdenum (Mo) is also essential for successful N 2-fixation as it is a keycomponent of nitrogenase enzyme (Shah et al ., 1984). As demonstrated by theresults of additions of P to other legumes, increased BNF has been reported for

soybean supplied with higher levels of Mo (Campo and Latmann, 1998).

In many ecosystems in Australia, some, even most of the P is bound in plant matterbecomes available again after fire (Adams et al ., 1994; Rau et al., 2007). This pulsemay persist for some time. For example, Romanya et al. (1994) found higherconcentrations of P in soil from Eucalyptus forest seven months after burning

associated with forest harvesting. Much less is known about the effect of fire on soilmicronutrients such as Mo (Certini, 2005).I investigated the effects of simulated pulses of P and Mo on nodule formation andBNF associated with the native legume, Bossiaea foliosa in sub-alpine Australia. A

key goal is to increase understanding of the role of fire in nutrient cycles in sub-alpine ecosystems with a focus on the potential for increasing productivity of nativelegume species.

I hypothesized that (i) a pulse of P and Mo added to the soil would increaseconcentration of both elements in biomass of B. foliosa ; (ii) increased availability of Pand Mo will improve nodule formation and BNF in B. foliosa , and (iii) improvedcapacity for BNF in B. foliosa (such as following a post-fire pulse of macro- and

micronutrients) will confer a competitiveness for this species. It follows that increased



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productivity of leguminous species may lead to a change in composition ofunderstory species due to increased fire frequency under future climate.

2. Materials and methods

2.1 Site descr ip t io n

The study area was located west of Cooma in the sub-alpine area of southern NewSouth Wales, Australia (36° 05´ 33” S, 148° 31‟ 39” E). The general area is locallyreferred to as the Snowy Plains and much of this ecosystem is protected within theKosciusko National Park (Fig. 1). The study area was located on private propertyimmediately adjacent to the Kosciusko National Park and accordingly used for cattlegrazing during the summer months (November to March). The study site representsa typical silvo-pastoral system that has been in use in the area for at least 100 years.

The vegetation in the study area was dominated by Snowgum woodland with aheathy understorey composed of a mix of leguminous and non-leguminous shrubsand grasses interspersed among the trees (Fig. 2). The site had not been burnt for atleast 10 years and woody shrubs were in a mature state. The long-term averageminimum and maximum air temperatures, rain and potential evapotranspiration forthe area are presented in the Figure 3 (data obtained from Bureau of Meteorology).

The soil in the study area is sub-alpine humus termed Chernic Tenosols (AustralianSoil Classification (Isbell, 2002)), also known as Umbrisol (Food and Agriculture

Organization, 1995). This soil type is rich in organic matter and is slightly acidic (pH

≤5) . There is no sharp boundary between horizons and the soil contains “floaters” of

non-decomposed rock throughout the profile (Costin et al., 1952). The soils aregenerally deeper in the valley bottoms (characterized by grassland vegetation) andshallower and rockier on the upper ridges (characterized by Snowgum woodlandvegetation).



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Figure 1 : Location of the research site used in this study.



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Figure 2 : The Snowy Plains near the research site used in this study. Woodlands of matureSnowgum trees ( Eucalyptus pauciflora ) dominate the slopes and ridges, while heathland andgrassland persist in the flat valley bottoms. The landscape undulated gently and mosaics ofvegetation are due to subtle variations in climatic conditions.

Month

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

AirTemperature

(°C)

-3

0

3

6

9

12

15

18

21

24

MaximumMinimum

(A)

Month

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Prec-Evap

(mm)

0

20

40

60

80

100

120

140

160

180PrecipitationEvapotranspiration

(B)

Figure 3: Monthly average (since 1889 to 2010) data of (A) maximum and minimum air temperature

(C°) and (B) precipitation and potential evapotranspiration (mm) associated with sub-alpine Snowgumwoodland.



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2.2 Exper imental des ign

Five plots were established in October 2010 along a gentle south-west facing slope.Each plot consisted of three sub-plots representing two treatments and a control:

(i) +P+Mo : addition of fertilizer containing 8.8% w/w P and 0.025% w/w Moas sodium molybdate. The quantity applied was equivalent to 100 kg ha -1 of P and 0.1 kg ha -1 of Mo

(ii) +P –Mo : addition of fertilizer containing the same quantity of P and noadditional Mo

(iii) –P –Mo : no application of fertilizer(„control treatment‟)

The vegetation in each plot included a mix of N 2-fixing (legume) and non-N2-fixing

(reference) plants. Each sub-plot covered an area of 2 x 2 m and included the N 2-fixing target species ( Bossiaea foliosa A. Coon.) and two grass species (non-N 2-fixing) that served as reference plants ( Poa costiniana Vickery and Poa hiemata

Vickery).

2.3 Collect ion and analys is of p lant and soi l sam ples

Soil and plant samples were collected at three times:(i) before application of fertilizer (October 2010)

(ii) 7 weeks after application of fertilizer (December 2010)(iii) 12 weeks after application of fertilizer (January 2011)

Soil samples were taken from four depths (0-2, 2-5, 5-10, 10-20 cm) using a straight-sided shovel and trowel. Soil was excavated from three areas in each sub-plot and

bulked for each depth. Samples were thoroughly homogenized, sieved to 2 mm androots and rock fragments were removed manually. All soil samples were stored in re-sealable plastic bags and kept dark and cool until further analysis.

Soil bulk density (sbd) was determined during the first sampling date prior toapplication of fertilizer by extracting one soil core of known volume (90.5 cm 3) fromeach sub-plot and drying to constant weight at 105 °C once they had been returned

to the laboratory. To calculate sbd the next formula was used:



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vw

sbd

Where w is the dry weight (in grams) of each soil sample and v is the volume of thesample (in cm 3).Gravimetric water content of all other soil samples was determined by weighing 20 gof fresh soil into aluminum cups, oven-drying at 105 °C for 3 days and recording dryweights. Soil pH was measured for all soil samples collected during December 2010.

A sub-sample (7.5 g) was mixed with deionized water (15 ml), shaken for 1 hour and

rested for a period of 30 minutes before pH was measured using a standard meter(PH240, cdm 230, Radiometer Analytical SAS, France) fitted with the appropriate

sensor (labCHEM-CP, TPS Pty. Ltd., Australia). All remaining analyses used fresh

and air-dried soil samples collected during December 2010 and January 2011.

Plant material from N2-fixing and reference species were collected before (October2010) and after application of fertilizer (December 2010 and January 2011). A mix ofgreen leaves was collected from at least 10 individuals of each target species ineach sub-plot at each sampling date. The leaves were stored in re-sealable plasticbags and kept dark and cool until analyzed. In the laboratory, leaf material was oven

dried at 60 ºC for 72 h. Leaf material from N 2-fixing species was ground to a finepowder using a Matrix Mill (Vibration mixer Mill, Type MM 301, Retsch, Haan,Germany). Grass material was fibrous and needed to be ground by hand using aceramic mortar and pestle and liquid nitrogen.

2.4 Extractabl e NO 3 - , NH 4

+ and plan t-available P

An amount of fresh soil equivalent to 10 g dry soil (calculated according to

gravimetric water content) was weighed into 50 ml plastic tubes. Nitrate (NO 3-) was

extracted with deionized water and subsequently analyzed using a HPLC anionseparation column (Hamilton PRP-X100) with a flow rate of 1 ml min-1, a column of 5cm length and 4 mm diameter and a mobile phase of 2 μM potassium hydrogenphthalate (pH 5.5). Using this set of conditions, the detection limit was 20 µmol NO 3

-.

Ammonia (NH4+) was extracted from fresh soil samples (equivalent to 10 g dry

weight) by adding 2M KCl to a volume of 50 ml and mixing during 2 hours in agyratory shaker to 150 rpm. After that, the sample were transferred to tubes and



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centrifuged during 5 minutes and filtered with paper filter N° 40. Standards wereprepared using ammonium sulphate at concentrations from 0 to 100 mg L -1 NH4

+.The absorbance of soil extracts and standards were measured with aspectrophotometer (UV mini 1240, Shimadzu, Japan) at 650 nm wavelength. This

extraction followed the protocol of Baethgen and Alley (1989).

Plant-available P was extracted from 1 g of fresh soil samples mixed with 7 ml of anextracting solution, which was obtained as follow: 37 g of NH4F were dissolved indistilled water to 1 liter (NH4F 1.0N ); 20.2 ml of HCl were diluted to a volume of 500ml with distilled water (HCl 0.5N ). Then 15 ml of 1.0N NH4F and 25 ml of 0.5 N HCl

were added to 460 ml of distilled water.The mixture of soil and extracting solution was shaken 1 min and then filtered

through Whatman no. 42 paper. Standard phosphate solutions were produceddissolving 0.4393 g of oven-dry (100 ° C) KH2PO4 in distilled water and diluting to 1liter. One ml of this solution contained 100 ug of P. Then this solution was used toprepare standards in the range of 0 and 100 µg of P ml -1 by diluting suitable aliquotsof the solution with distilled water. Absorbance of soil extracts and standard solutionswere measured with a spectrophotometer (UV mini 1240, Shimadzu, Japan) at 660

nm wavelength. This method is detailed in Olsen and Sommers (1982).

2.5 Total phos phorus and molybdenum

Total P and Mo concentration were measured in soil from each sub-plot and twodepths (0-5 and 5-10 cm) collected in December 2010 and January 2011 (n = 60)and plant material collected at all sampling times (n = 70). Following a nitric andperchloric acid block digestion, concentration of both nutrients was determined using

an inductively coupled plasma atomic emission spectrometer (ICP-AES, Varian VistaPro-Axial, Varian Instruments, Palo Alto, USA). Dried soil and leaves were finelyground to <0.5 mm. Aliquots of 0.3 g soil or leaves were weighed into 75 ml digestiontubes and 1 ml concentrated nitric acid and 3 ml concentrated perchloric acid added.Digestion tubes were placed in an aluminium digestion block and heated slowly to

210 °C at approximately 6 °C minute -1. After complete digestion, samples werecooled, 10 ml of Type-I water added and mixed thoroughly using a vortex. After

standing overnight, samples were centrifuged for 5 minutes at 3000 rpm then



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measured on the ICP-AES. More details about the analytical method can be found inSommers and Nelson (1972) and Bowman (1988).

2.6 Measurem ent of 13 C and 15 N and to ta l C and N

For the analysis of 13C and 15N and total C and N, approximately 3.0 mg of each soil(air-dried and finely ground) and 0.9 mg of plant material were carefully weighed intotin foil capsules following standard protocols and weights recorded accurately to fourdecimal places. Samples were analyzed using a Delta V Advantage Isotope RatioMass Spectrometer (IRMS) coupled to a Flash HT Elemental Analyzer (ThermoFisher Scientific, Bremen, Germany) operated with separate combustion andreduction columns. Samples were dropped into the combustion oven at 1060 °Cusing an autosampler (IVA Analysentechnik, Meerbusch, Germany). Complete

combustion was achieved by injection of oxygen into the combustion oven. Gasproducts from the combustion were transported by carrier gas (helium) at a flow rateof 100 ml min-1 into the reduction column at 680 °C to remove excess oxygen andreduce nitrogen oxides to elemental nitrogen. The gas stream was dried using amagnesium perchlorate trap before being introduced into a chromatographic column(Porapak) that separated N 2 and CO 2. The gases passed through a thermal

conductivity detector (TCD) before being introduced into the IRMS for the analysis ofδ 15N and δ 13C. Carbon dioxide was diluted to varying degrees depending on thecarbon content of the sample to match the detection range of the IRMS. Fivelaboratory standards of known isotopic and elemental composition were used (pureamino acids and carbohydrates). Measurement precision of the IRMS was 0.06-

0.14‰ for 13C, 0.04-0.19‰ for 15N, 2.74-11.88% for C and 0.09-0.48% for N.Percentages of samples were calculated using the known weights of standard

materials. Isotopic composition is expressed relative to the Vienna Pee DeeBelemnite (VPDB) scale (international standard):

δ 13C (‰) = ((Rsample – RVPDB)/ RVPDB) * 1000

Where R sample and R VPDB represent the abundance of 13C in the sample and

international standard, respectively. Results are expressed as δ13

C in part perthousand (‰ ) because the absolute abundance of the heavier isotope is very small.



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2.7 Biolo gical n i t rog en f ixat ion

Biological nitrogen fixation of B. foliosa was estimated using the 15N natural

abundance method. The natural abundance of 15N of atmospheric N2 is constant(0.3663 at% 15N; Junk and Svec, 1958) and does not vary spatially (Mariotti, 1983).

Small differences in δ 15N abundance between soil or plant (sample) N and theatmosphere are usually expressed as δ 15N or parts per thousand (‰) relative to the15N composition of atmospheric N 2:

3663.0)3663.015%(1000

)000(15

Nsampleat N

An effectively nodulated legume growing in a medium free of combined N (i.e.mineral N and/or organic N) is assumed to be completely reliant upon symbiotic N 2-fixation for growth. The isotopic composition of the legume would therefore beexpected to be similar to atmospheric N ( δ 15N = 0) (Unkovich et al ., 2008). If a non-N2-fixing plant (reference plant) is grown in a soil containing mineral N, its δ15N valueshould be similar to that of the soil. In this way, the %Ndfa (%N 2 fixed from theatmosphere) of the legume can then be calculated from its δ 15N value using thefollowing equation (Shearer and Kohl, 1986):

)15()1515(

100% B Nref

nt Nfixingpla Nref Ndfa

where „ δ 15 Nref ‟ represents the δ 15N (‰) detected in a reference plant growing in the

same soil at the same time as the legume, „ δ 15 Nfixingplant ‟ is the δ 15N of the legume

and B is the δ 15N of the legume (or its shoots) grown obtaining all of its N from N2-fixation (Boddey et al ., 2000, Unkovich et al., 2008). The use of a B value correction

factor is based on the observation that δ 15N can vary across plant organs as result ofvarying rates of isotopic discrimination during metabolic processes. Numerous fieldstudies have reported slightly negative δ 15N values when only the aerial parts of theplants were analyzed (Unkovich et al., 2008) which leaves the false impression that

these legumes also took up soil N. This can be corrected using the B-value and

applies to the current study where only aerial parts of the legumes were sampled. In



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cases where negative values of N 2-fixation were derived as result of δ 15N inreference plants being less negative than legume plants, it is not possible to assumeN2 fixation and values were converted to zero as indicated in Pons et al. (2007).

After a thorough literature search (see also Unkovich et al., 2008), no B-value couldbe identified for B. foliosa , or more generally, for legumes in sub-alpine Australia. A

B-value of zero was then chosen. The potential effect of variation in B-values was

tested by substituting for the selected zero, values ranging from -0.5 to 0.2. Therange of selected B values was based on %Ndfa values, which neither exceed 100%

nor were negative. To estimate the %Ndfa over time the δ 15N of legume andreference plant (grass) was measured in October 2010 in plants before application offertilizer. In the next sampling dates (December 2010 and January 2011) the δ15N ofthe plants was measured for each treatment (-P-Mo, +P-Mo and +P+Mo) to compare

the %Ndfa among treatments.

2.8 Nitrogenase act iv i ty

It was anticipated that the number and weight of nodules from B. foliosa and their

nitrogenase activity (using acetylene reduction assays) would be determined foreach treatment during the sampling campaign in January 2011. However, we could

not find nodules on roots of Bossiaea in any of the treatments. In addition, roots ofother legumes such as Trifolium growing in the research site were assessed but no

nodules were found.

2.9 Statist ical analyses

Three-way analysis of variance (ANOVA) was used with a significance level of P

<0.05 to test differences among dates (before and after fertilizer applications),

treatments (+P+Mo, +P-Mo, -P-Mo), soil depth for nutrient status and naturalabundance of 15N and 13C. A similar statistical analysis was used to test differencesamong collection dates, treatments and types of plants (N 2-fixer and reference).Differences in rates of nitrogen fixation (estimated by δ15N natural abundance) weretested using a two-way ANOVA (P <0.05 significance level) among treatment and

date. Post-hoc Tukey tests were used to determine significant differences amonggroup means as indicated by ANOVA. All statistic analyses were done using SPSS

Statistics 17.0 software.



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3. Results

3.1 Soi l physical and chemical pro per t ies

3.1.1 Bulk density and pH

Bulk density of soil ranged from 0.75 to 0.84 mg m -3 but did not differ significantlyamong treatments (ANOVA, P = 0.58, Table 1). Soil collected in October 2010, prior

to any addition of fertilizer, was slightly less acidic in the +P+Mo treatment (4.6)compared to other treatments (4.4, Table 1).

3.1.2 Moisture content, extractable NH 4+, carbon and nitrogen

Despite sampling during the summer period, soil moisture content (pooled for

December 2010 and January 2011) was somewhat high. Soil moisture contentdecreased significantly with depth (ANOVA, P = 0.04) from close to 50% at the

surface to around 40% at 5 cm depth where it remained constant until 20 cm (Fig.4A). Ammonium concentration in soil varied from 1 to 5 mg NH4

+ kg-1 soil but was not

significantly different among treatments or with depth (Fig. 4B). No plant availableNO3

- was detected in soil extracts, regardless of sample date, soil depth or treatment.

Soil Moisture (%)

20 30 40 50 60 70

Depth

(cm)

0

5

10

15

20

-P -Mo+P -Mo+P +Mo

2 a

b

b

b

(A)

Extractable NH 4

(mg kg -1)

0 1 2 3 4 5 6 7 8

Depth

(cm)

0

5

10

15

20

2 a

a

a

a

(B)

Figure 4: Changes in soil (A) moisture and (B) NH 4

+ concentration with depth associated withsub-alpine Snowgum woodland. Samples from each depth were pooled (n = 5) for each of thethree treatments and two sample dates (December 2010 and January 2011). Letters indicatesignificant differences among sampling depths ( P >0.05); error bars represent SD.

Total C and N amount (Mg C ha -1) and C content (%) in the top 20 cm of soil weresignificantly different among collection dates (ANOVA, P = 0.01, Table 1). Total C

and N content varied significantly with depth (ANOVA, P = 0.014, Fig. 5), with thegreatest range measured in samples collected in December 2010 (data not shown).



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The C:N ratio also varied significantly among treatments (ANOVA, P <0.001, Table1), with no differences among depth ( P = 0.65, Table 1, Fig. 5).

Total C (Mg ha -1)

0 15 30 45 60 75 90 105 120

Depth

(cm)

0

5

10

15

20

-P -Mo+P -Mo+P +Mo

2 a

ab

b

C

(A)

Total C

(Mg ha -1)

0 15 30 45 60 75 90 105 120

Depth

(cm)

0

5

10

15

20

2 a

ab

b

C

(B)

Total N(Mg ha -1)

0.0 1.5 3.0 4.5 6.0 7.5 9.0

Depth

(cm)

0

5

10

15

20

2 a

ab

b

c

(C)

Total N

(Mg ha -1)

0.0 1.5 3.0 4.5 6.0 7.5 9.0

Depth

(cm)

0

5

10

15

20

2 a

ab

b

c

(D)

C:N ratio

13 14 15 16 17 18

Depth

(cm)

0

5

10

15

20

2 a

a

a

a

(E)

Figure 5: Changes in soil carbon amount, nitrogen amount with samples taken on December2010 (A and C) and January 2011 (B and D); and C:N ratio (E) with depth associated with sub-alpine Snowgum woodland. In C:N ratio samples from each depth were pooled (n = 5) for each ofthe three treatments and two sample dates (December 2010 and January 2011). Letters indicatesignificant differences among sampling depths ( P >0.05); error bars represent SD.



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3.1.3 Phosphorus and molybdenum

Greater concentration of available P was measured in soil from treatment with addedP (+P-Mo) in samples taken in January 2011 with a concentration of 13.2 mg kg -1,whilst the other treatments and dates did not exceed 9.8 mg kg -1 (ANOVA,P <0.001,

Table 1). As expected, greater amounts of total P were measured in soil fromtreatments with added P (+P+Mo, +P-Mo) averaging values of 519 kg ha -1, whilst thecontrol treatment (with not P added) averaged 409 kg ha -1 (ANOVA,P <0.001, Table

1). Similarly, greater concentration of Mo were measured in soil from the +P+Motreatment in December 2010 and January 2011 averaging values of 2.8 mg kg -1 (ANOVA,P = 0.007, Table 1).

The concentration of Mo differed with soil depth and was greater in the upper 5 cmcompared to soil below 5 cm depth (Fig. 6A). This was not the case for available P

(data not shown). Concentration of total P in soil differed among depth of collectionwith the greatest concentration in the upper 5 cm (Fig. 6B).

Total Mo (mg kg -1)

1.0 1.5 2.0 2.5 3.0 3.5 4.0

Depth

(cm

)

0

5

10

-P-Mo+P-Mo+P +Mo

a

b

(A)

Total P

(mg kg -1)

750 1000 1250 1500 1750 2000

Depth

(cm)

0

5

10

a

b

(B)

Figure 6: Changes in (A) molybdenum and (B) total phosphorus with depth associated with sub-alpine Snowgum woodland. Samples from each depth were pooled (n = 5) for each of the threetreatments and two sample dates (December 2010 and January 2011). Letters indicatesignificant differences among sampling depths ( P >0.05); error bars represent SD.

3.1.4 13 C and 15 N signatures Both, δ 13C and δ 15N signatures of soil did not differ significantly (ANOVA,P >0.05)among treatments with ranges of -25.5 to -25.7 and 3.2 to 3.8 for δ 13C and δ 15N,

respectively (Table 1). In contrast, significant differences among soil depths weredetected in both δ 13C and δ 15N (ANOVA,P <0.001, Fig. 7).



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13C (‰)

-26.8 -26.4 -26.0 -25.6 -25.2

Depth

(cm)

0

5

10

15

20

-P-Mo+P-Mo+P+Mo

2 a

b

c

d

(A)

15N

(‰)

0 1 2 3 4 5 6

Depth

(cm)

0

5

10

15

20

2 a

b

c

d

(B)

Figure 7: Changes of (A) δ13C and (B) δ 15N signatures with depth associated with sub-alpineSnowgum woodland. Samples from each depth were pooled (n = 5) for each of the threetreatments and two sample dates (December 2010 and January 2011). Letters indicate

significant differences among sampling depths ( P >0.05); error bars represent SD.



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Table 1 . Soil properties for three treatments (mean values for 0-20 cm depth, n = 5) collected at two sampling dates. For each sampling date, the mean followed by thesame letter indicates no significant difference ( P >0.05); * = indicates if the factor is significantly different; ns = not significant. Standard deviations are given inparenthesis.

Date andtreatment

Bulkdensity

pH Soilmoisture

ExtractableNH4+

Plant-availableP

TotalP

TotalMo

TotalC

TotalN

C:N δ 13 C δ 15 N

(mg m -3) (%) (ppm) (kg ha -1) (ppm) (kg ha -1) (ppm) (kg ha -1) (ppm) (kg ha -1) (%) (Mg ha -1) (%) (Mg ha -1) ( ‰ ) ( ‰ )

December

-P-Mo 0.87

(0.24)

4.4

(0.1) a

42.4

(11.1)

2.9

(2.8)

1.9

(2.0)

8.3

(4.8) a

5.0

(3.7)

965

(141) a

414

(75) a

2.4

(0.4) b

1.0

(0.4)

9.4

(1.5)

56

(25)

0.6

(0.1)

3.8

(1.7)

15.0

(0.9) a

-25.7

(0.4)

3.2

(1.6)

+P-Mo 0.84(0.22)

4.4(0.3) a

41.5(9.3)

3.0(2.6)

1.8(1.6)

9.8(7.8) a

4.9(2.9)

1336(415) b

573(267) b

2.5(0.5) b

1.1(0.4)

9.6(2.5)

54(28)

0.6(0.2)

3.6(1.9)

15.1(1.2) a

-25.7(0.6)

3.5(1.4)

+P+Mo 0.75

(0.11)

4.6

(0.2) b

43.0

(5.3)

2.8

(2.4)

1.4

(1.1)

8.2

(6.1) a

4.2

(2.3)

1336

(261) b

498

(88) ab

2.7

(0.4) a

1.0

(0.2)

10

(1.9)

49

(22)

0.7

(0.1)

3.4

(1.5)

14.6

(0.7) b

-25.6

(0.4)

3.4

(1.0)

January

-P-Mo 38.2

(10.9)

3.9

(1.6)

2.5

(1.6)

8.2

(3.8) a

4.8

(2.6)

921

(135) a

405

(88) a

2.5

(0.4) b

1.1

(0.4)

7.5

(2.2)

41

(17)

0.5

(0.1)

2.9

(1.3)

14.5

(1.3) b

-25.5

(0.5)

3.7

(1.7)

+P-Mo 38.1

(10.9)

1.9

(1.5)

1.3

(1.4)

13.2

(13.1) b

5.8

(4.4)

1137

(247) ab

488

(174) ab

2.4

(0.6) b

1.0

(0.3)

8.2

(2.5)

44

(21)

0.5

(0.2)

2.9

(1.5)

15.5

(1.2) a

-25.5

(0.5)

3.8

(1.5)

+P+Mo 40.3

(5.5)

1.6

(1.7)

0.9

(0.8)

8.7

(7.8) a

4.0

(2.3)

1367

(241) b

516

(117) b

2.9

(0.6) a

1.1

(0.3)

8.5

(2.4)

41

(17)

0.6

(0.2)

2.8

(1.1)

14.5

(0.7) b

-25.6

(0.6)

3.7

(1.7)

Treatment ns * ns ns ns * ns * * * ns ns ns ns ns * ns ns

Date - - ns ns ns ns ns ns ns ns ns * * ns * ns ns ns

Treatment× date

- - ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns



20

Month

October December January

Ccontent

(%)

0

10

20

30

40

50

60

GrassLegume

a

a

b

a

b

a(A)

Month


N content

(%)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

a

a

b

a

b

a

(B)

Figure 9: Mean (A) carbon and (B) nitrogen content in leaves of Bossiaea foliosa (legume, n = 35)and Poa spp. (grass, n = 35) sampled from sub-alpine Snowgum woodland. Samples were collectedprior to treatment application (October 2010) and twice after treatments had been applied (December2010, January 2011). Letters above bars indicate significant differences ( P < 0.05) among the twoplant types collected during the same sampling campaign; error bars represent SD.

Addition of fertilizer showed no effect of the C:N ratio of either the legume or grass atany sampling date (Fig. 10A). In contrast, N:P ratios differed significantly amongtreatments and date of collection and type of plant (Fig. 10B). For example, grass

from the control treatment (-P-Mo) had the highest N:P ratio (18 versus 7.5 in theothers treatments) in January , but not in December, whilst for legumes there wereno differences between treatments at any date.

Type of plant (month)

grass (D) grass (J) legume (D) legume (J)

C:N ratio

0

10

20

30

40

-P-Mo+P-Mo+P+Mo

(A)



N:Pratio

0

10

20

30

40

aa

a

aa

a

a

b b

a

a a

(B)

Figure 10: Ratios of (A) C:N and (B) N:P in leaves of Bossiaea foliosa (legume, n = 30) and Poa spp.(grass, n = 30) collected from sub-alpine Snowgum woodland. Both plant types were subjected tothree treatments (indicated by different colored bars) and collected during sampling campaigns inDecember 2010 (D) and January 2011 (J). No significant differences ( P >0.05) in C:N ratio due to



21

treatments were detected for either plant type collected during individual campaigns. Significantdifferences among treatments within both plant type and individual sampling dates in N: ratio areindicated by letters (error rate 0.05); error bars represent SD.

Grasses showed greater C:N ratios than N2-fixing plants in December and January

but not in October (Fig. 11A). The C:N ratio of grass did not vary over time, whereasthe same ratio declined significantly from October 2010 (prior to treatmentapplication) to December 2010 ( P = 0.004) and remained at this reduced value in

January 2011.

Leaf material of legumes had by far greater N:P ratios when compared to that ofgrass at every date (Fig. 11B), however grass did not show a change in N:P ratiobetween any of the dates ( P = 0.5). For legumes, the N:P ratio in October was higherthan in January ( P = 0.01), but not than in December that had intermediate N:P

ratios (P = 0.5) compared to the others sampling dates.

Month


C:N ratio

0

5

10

15

20

25

30

35

GrassLegumea

a

b

a

b

a(A)

Month


N:Pratio

0

10

20

30

40

50

b

a

b

a

b

a

(B)

Figure 11: Mean (A) C:N and (B) N:P ratios in leaves of Bossiaea foliosa (legume, n = 35) and Poa spp. (grass, n = 35) sampled from sub-alpine Snowgum woodland. Samples were collected prior totreatment application (October 2010) and twice after treatments had been applied (December 2010,January 2011). Letters above bars indicate significant differences ( P < 0.05) between the two planttypes collected during the same sampling campaign; error bars represent SD.

3.2.2 Phosphorus and molybdenum Both P and Mo concentration in plant tissues varied significantly among treatmentand plant type ( P <0.05) and treatment and collection date for grass ( P <0.05).

However, concentrations of P and Mo did not differ among treatments or sampling

date for legumes, averaging concentrations of 910 and 0.19 mg kg -1 for P and Mo,



22

respectively (Fig. 12). The concentration of P in leaves was highest in grass in thetreatments with added P (+P-Mo and +P+Mo) in both December and January,averaging 1856 mg kg -1 versus 1060 mg kg -1 in the control treatment (-P-Mo) (Fig.12A). Similarly, the highest concentration of Mo was found in grass in December in

the +P+Mo treatment (Fig. 12B).



P concentration

(mgkg

-1)

0

500

1000

1500

2000

2500

-P-Mo+P+P+Mo

b

a

a

b

b

bb

a a

b

b

b

(A)



Mo concentration

(mgkg

-1)

0.0

0.2

0.4

0.6

0.8

1.0

b

b

a

b

ab

b

bb

a

b

b

b

(B)

Figure 12: Mean (A) phosphorus and (B) molybdenum concentration in leaves of Bossiaea foliosa (legume, n = 30) and Poa spp. (grass, n = 30) sampled from sub-alpine Snowgum woodland. Bothplant types were subjected to three treatments (indicated by different colored bars) and collectedduring sampling campaigns in December 2010 (D) and January 2011 (J). Significant differencesamong treatments within both plant type and individual sampling dates are indicated by letters (errorrate 0.05); error bars represent SD.

Leaf material of grasses contained significantly more P ( P = 0.006) than leaf material

of the legume species, regardless of the sampling date (Fig 13A). Phosphorus

concentrations were significantly lower (P = 0.006) in October for grasses, whilst forthe legumes, the concentration of P was significantly higher ( P = 0.007) in January.

Grasses generally had greater concentrations of Mo compared to legumes. However,these differences were significant only in material collected during December 2010(Fig. 13B).



23

Month


P concentration

(mgkg

-1)

0

500

1000

1500

2000 GrassLegume

a

b

b

a

b

a(A)

Month


Mo concentration

(mgkg

-1)

0.0

0.2

0.4

0.6

0.8

a

a

b

a

a

a

(B)

Figure 13: Mean (A) phosphorus and (B) molybdenum concentration in leaves of Bossiaea foliosa (legume, n = 35) and Poa spp. (grass, n = 35) sampled from sub-alpine Snowgum woodland.Samples were collected prior to treatment application (October 2010) and twice after treatments hadbeen applied (December 2010, January 2011). Letters above bars indicate significant differences ( P <0.05) among the two plant types collected during the same sampling campaign; error bars representSD.

3.2.3 13C and 15 N signatures

In general, values of δ 13C and δ 15N did not differ among treatments. The δ 13C in

grasses averaged - 27.8 ‰. The one exception was for legumes sampled inDecember 2010 when the δ 13C value was the lowest in plant tissues from the controltreatment (Fig. 14A). δ15N values averaged -2.1 and - 1.2 ‰ for grasses and legume,

respectively (Fig. 14B).



1

3C

(‰)

-32

-31

-30

-29

-28

-27

-26

-P- Mo+P- Mo+P+ Mo

a

aa

b

a a

aa a

a

a a

(A)



1

5N

(‰)

-4

-3

-2

-1

0

(B)



24

Figure 14: Abundance of (A) δ 13C and (B) δ 15N in leaves of Bossiaea foliosa (legume, n = 30) andPoa spp. (grass, n = 30) sampled from sub-alpine Snowgum woodland. Both plant types weresubjected to three treatments (indicated by different colored bars) and collected during samplingcampaigns in December 2010 (D) and January 2011 (J). Significant differences among treatmentswithin both plant type and individual sampling dates for δ 13C are indicated by letters ( P < 0.05); Nosignificant differences (P >0.05) in δ 15N due to treatments were detected for either plant type collected

during individual campaigns; error bars represent SD.

The δ 13C values varied between type of plant at all sampling dates with the highestvalues (less negative) for grass, while δ 15N values were highest in legumes sampledin December 2010 and January 2011, but not in October (Fig. 15).

Month


1

3C

(‰)

-31

-30

-29

-28

-27

GrassLegume

a

bb

a

b

a

(A)

Month


1

5N

(‰)

-4

-3

-2

-1

0

a

a b

a

b

a

(B)

Figure 15: Abundance of (A) δ 13C and (B) δ 15N in leaves of Bossiaea foliosa (legume, n = 35) andPoa spp. (grass, n = 35) sampled from sub-alpine Snowgum woodland. Samples were collected priorto treatment application (October 2010) and twice after treatments had been applied (December 2010,January 2011). Letters above bars indicate significant differences ( P < 0.05) between the two planttypes collected during the same sampling campaign; error bars indicate SD.

3.3 Nitrogen f ixat ion (%Ndfa)

Nitrogen fixation did not vary among treatments in December 2010 and January2011 when a B value of 0 (zero) was used (Fig. 16). There were also no significantdifferences between sampling dates, including mean proportions of N 2-fixation of

23% when measured in October in the control (data not shown). When we comparedN2-fixation using different B values there were some differences among treatments.Using a B value of -0.5 for plants from the full treatment (+P+Mo), N2-fixation

capacity reached of 62.2% compared to 40% for other treatments in December(Table 2). Similarly, in January when we used negative B values, the differencebetween the control and the other treatments was greater (Table 2). Nevertheless,



25

none of the B values tested showed significant differences among treatments inDecember or January ( P >0.05).

Date

December January

N fixation

(%)

0

20

40

60

80

100

-P -Mo+P -Mo+P +Mo

Figure 16: Mean proportions of N2-fixation of Bossiaea foliosa (n = 30), using a “ B” value of zero, sampled from sub-alpine Snowgum woodland. The plants were subjected to three treatments(indicated by different colored bars) and collected during sampling campaigns in December 2010 andJanuary 2011. No significant differences ( P >0.05) in N2-fixation due to treatments were detectedduring individual campaigns; error bars represent SD.

Table 2. Nitrogen fixation (%) for three treatments (+P+Mo, +P-Mo, -P-Mo) and two sampling dates(December 2010 and January 2011) using different B values (range from -0.5 to 0.2). Standarddeviations are given in parenthesis.

Date andtreatment

N2 fixation (%)B value

-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2

December

-P-Mo 38.4

(37.4)

37.0

(36.1)

35.8

(34.9)

34.6

(33.8)

33.4

(32.7)

32.4

(31.7)

31.4

(30.7)

30.5

(29.9)

+P-Mo 44.1

(25.2)

41.4

(23.8)

39.0

(22.7)

36.9

(21.7)

35.1

(20.8)

33.4

(20.1)

32.0

(19.4)

30.6

(18.7)

+P +Mo 62.2

(29.9)

40.4

(30.6)

38.8

(29.5)

37.4

(28.5)

36.0

(27.6)

34.7

(26.7)

33.5

(25.9)

32.4

(25.1)

January

-P-Mo 81.6

(19.0)

79.0

(19.0)

76.6

(19.0)

74.4

(19.4)

72.3

(19.7)

50.5

(30.5)

48.7

(29.4)

47.1

(28.4)

+P-Mo 54.6

(28.1)

50.6

(27.9)

47.3

(27.6)

44.5

(27.2)

42.1

(26.6)

40.0

(26.1)

38.1

(25.5)

36.3

(24.9)

+P +Mo 51.8

(29.2)

49.0

(27.6)

46.4

(26.3)

44.1

(25.1)

42.1

(24.1)

40.2

(23.0)

38.5

(22.2)

37.0

(21.4)

Treatment ns ns ns ns ns ns ns ns

Date ns ns ns ns ns ns ns ns

Treatment ×date

ns ns ns ns ns ns ns ns



26

4. Discussion

4.1 Soi l and plant prop er t ies

Total and available P in soil from the control treatment (-P-Mo+) was in the range ofpreviously reported values for the study area (Jenkins, 2009). The addition of Palone and in combination with Mo increased total P (and Mo) in soil, but not in leafbiomass of B. foliosa . The first hypothesis therefore has to be rejected. Some of the

P and Mo that was added may have been adsorbed onto soil surfaces (immobilized)or leached from the soil (McLaughlin, 1996). However, the increased concentrationof P and Mo in leaf biomass of the grass ( Poa spp., the non-legume control)

suggests that at least some of the added nutrients in the simulated nutrient pulsewas taken up and incorporated into plant material.

Neither P nor Mo additions as fertilizer affected total soil C or N at any depth (to 20cm). Values measured here were in the range of those previously published for thestudy area (Jenkins, 2009) and showed a matching pattern of decreasing C and Nwith increasing soil depth. The greater concentrations of soil C and N in December

may simply be the result of litter decomposition and subsequent incorporation intoupper soil layers. It is documented that the largest percentage of C and N is releasedduring the very early stages of litter decomposition where, dependent ofenvironmental factors and litter type, easily soluble compounds leach out of theorganic material after which they rapidly degrade (Cornelissen, 1996; Berg et al., 1996; Arunachalam et al., 1998; Brand et al., 2007). In the Snowy Plains, this

process is likely to begin at the end of September when snowmelt is complete andair and soil temperatures are increasing (Jenkins, 2009).

Leaf N and P content in grass and legumes found in this study are similar to valuesreported for other species of Poa (Peri and Lasagno, 2010) and woody legumes(Grove, 1990 Yasmeen, 2010). The ratio of N:P in plant tissues can be used as

indicator of either N or P limitation. According to Aerts and Chapin (2000) empiricaldata shown that N:P ratio in leaves of close to 10 may be optimal for plant growth,which suggest that deviations from this value would imply N or P limitation for

plants .However this N:P ratio value varies among species. For example, Tessier



27

and Raynal (2003) found that upland vegetation is limited solely by P at lower N:Pratios than wetland vegetation. We do not know the threshold values indicatinglimitation of P or N in the plants studied here, but the decrease of the N:P ratio ingrass over time could indicate that they had P limitation in natural conditions (before

the application of phosphorus). The concentration of P and the N:P ratio in leaves oflegumes did not change with addition of P indicating that grasses may have agreater competitive ability for acquisition of P or that the legume was not limited by Pdespite its greater N:P ratio.

Despite a greater N:P ratio in foliage of B. foliosa , the concentration of P (but not N,see below) in leaves was greater compared to values reported for Bossiaea

laidlawiana growing in south-western Australia (Grove, 1990). Townsend et al.

(2007) provided evidence that foliage of legumes in general had a N:P ratio around30 (matching those reported in the current study) resulting from the generally greaterconcentration of N in legumes compared to non-legumes. In general, despite verylow availability of P in soil, N2-fixing plants have developed specialized strategies tomeet their P requirements (Adams et al ., 2002; Houlton et al., 2008). Suchadaptations include anatomical or morphological plasticity (formation of root clusters

or nodules), as well as physiological adaptations (capacity to uptake P in organicform).

Responses of legumes to the addition of Mo include increased activity of the enzymenitrogenase (Barron et al., 2008). Although the current study did not involve enzymeactivity assays, we found that Mo concentration in grasses in the +P+Mo treatmentplots increased while the legume in the same treatment plots did not. This may have

several causes and these will be discussed in the following paragraphs.

Results of studies that have examined the capacity of legumes and non-legumes totake up additional Mo are scarce. According to Reith et al . (1984), application of Moto soil as sodium molybdate resulted in increased concentration of Mo in clover

(legume) compared to ryegrass (non-legume). Also, the choice of plant tissueanalysed for Mo is of particular importance. Fleming and Murphy (1968) reported

greater concentration of Mo in leaf biomass compared to other plant parts forgrasses (as flowering heads and stems), while other studies reported greater



28

concentrations of Mo in stem compared to leaf material in clover (Fleming, 1963;Gupta and Lipsett, 1981) or lower concentrations in stems than in roots for alfalfa(John et al, 1972, cited in Whitehead, 2000).

Generally, there is limited information available about levels at which plants sufferfrom Mo deficiency and, when Mo is readily available; there is little information aboutthe range of responses to increased Mo availability. The concentration of Mo in soilsfrom our control sub-plots was lower (2.5 times less) than other soils that havedeveloped from granitic parent material (Whitehead, 2000). There was a small butsignificant change in Mo concentration of soil after application of the +P+Motreatment. The lowest values of Mo in foliage of B. foliosa (0.2 mg Mo kg-1 leafbiomass) represent a range where Mo of soils should not be limiting (Taiz and Zeiger,

2006). Here we sampled only leaves from the studied plants, and according to theabove information this choice may have contributed to the differences found in Moconcentration among legume and grasses, because Mo partitioning (proportionallocated to roots, stem or leaves) could be different among them. We recommendthat future studies targeting Mo concentrations in plants extend collection of planttissues and include stems and if feasible, root material in the analyses, and at

different times throughout the year.

Similar to the results regarding soil C and N, the addition of P and Mo (+P+Mo and+P-Mo treatments) did not alter C and N concentration in leaf material of either thelegume or the grass in this study. However, both C and N concentrations weregreater in N2-fixing compared to the reference plants, a result that has been reportedpreviously (Cramer et al ., 2007; Garten et al ., 2008). The concentration of C in leaf

tissue of the reference species was also in the range reported for other species ofthe genus Poa and other grasses (Peri and Lasagno, 2010). Also our values of Nconcentration in leaves of B. foliosa were similar to those reported for B. laidlawiana

(Grove, 1990). As shown in a previous study by Grove and Malajczuk (1992),addition of P may not necessarily lead to increased concentration of N in leaves of a

legume. However, the authors of this study found that addition of P increased BNFfor this species and their reported increase in N content was solely a result of greater

aboveground biomass.



29

The overall values of δ 13C in soil in this study were in the range reported for soilsfrom Eucalyptus forests (Ladd et al., 2009). Similar to our results, δ 13C regularly

becomes less depleted with increasing soil depth (Volkoff and Cerri, 1987). Theseauthors have been interpreted depth-related changes as a consequence of carbon

isotopic discrimination during the humification process and humus biodegradation insoil. δ 13C values for grasses were in the range of those found by Craine and Lee(2003) for both native and exotic grasses along a broad environmental gradient.Similarly, the δ 13C signature in leaves of B. foliosa was comparable values of Acacia

dealbata and A. melanoxylon (N2-fixers) in south-eastern Australia (Pfautsch, 2007).

It is well known that the δ 15N signature in soil is the product of several processes thatare involved in N cycling and the values depend on which process is dominating N

mineralization (Hobbie and Ouimette, 2009). At our study sites, the dominatingprocesses should be (a) N 2-fixation, assimilation and denitrification at 0-2 cm depth;and (b) denitrification at 2-20 cm depth (Högberg, 1997; Kendall, 1998; Pörtl et al.,

2007). In addition, the increase of δ 15N with depth in soil forest has beendocumented (Nadelhoffer and Fry, 1988). Boddey et al. (2000) indicate that natural15N abundance in soil of a wide array of terrestrial ecosystems is enriched compared

to leaves from plants growing in the same environment. Our results confirm thisobservation.

The δ 15N signatures in soil from our study site remained unchanged with progressionof summer (October to January). This observation supports the concept that δ 15N ofthe soil is dominated by the isotopic signature of stable N, and therefore is unlikely tochange with time (Högberg, 1997). The literature noted that the changes of 15N

abundance of soil N in periods less than 1 year are very difficult to detect in naturalsystems, due to the recalcitrant nature of the soil organic N ( Boddey et al. , 2000).

Nevertheless, the abundance of 15N in soils has been reported to increase (Handleyand Scrimgeour, 1997) and also to decrease (Marriot et al. , 1997). We conclude thatpulse fertilization of shallow soils in Austr alia‟s sub-alpine region, either naturally

following fire or simulated as per the current study, does not trigger a change in 15Nabundance in soils.



30

Different values of δ 15N have been reported for both grasses and legumes. Craineand Lee (2003) measured values of δ 15N in a range of 5 to -9 ‰ for an altitudegradient (45 to 1205 m above sea level) in New Zealand. They found a significant

correlation between δ 15N values in grasses and altitude, with the lower values of

δ 15N at higher elevations and related this pattern to lower rates of N supply at highaltitude. More generally, there is a trend evident for enrichment of foliage in 15N forvegetation occurring in environments that are not nutrient limited (Vitousek et al.,

1989; HÖgberg, 1990). This is consistent with the negative values of δ15N that wefound in our study. Also is consistent with the negative correlation reported by Austinand Sala (1999) (based on data from Schulze et al., 1998) between foliar 15N and

rainfall in Australia. Austin and Sala (1999) suggest that annual rainfall may be animportant factor influencing the ecosystem nitrogen cycling. This would occur

because an increase in rainfall increase the input of N (through more rapid turnover),although in a long term also the output would be increase, but in a lower proportionthan drier sites, implying lower potential for loss from sites with higher rainfall. In our

study site the annual rainfall is in the range of the indicated as “high” precipitation in

the mentioned correlation.

4.2 N 2 f ixat ion

In this study it was shown that addition of P and Mo did not increase abundance ofthese elements in leaves of the legume species leading to the rejection of the first

hypothesis. The second hypothesis was related to the addition of P and Moincreasing BNF of B. foliosa and leading to an alteration in the abundance of 15N in

leaves. The results of this study do not support the first part of this hypothesis.

However, a decreasing abundance of 15N in leaf material was measured fromDecember 2010 to January 2011. Unfortunately, this reduction was observed in bothtreatments and the control, which indicates that the reduction was not specificallydue to the application of P and Mo. Consequently the second part of our secondhypothesis must also be rejected.

It was shown that B. foliosa derived around 40% of its N from the atmosphere. This

was a smaller proportion of that reported for B. laidlawiana growing in south-western Australia with rates of 52 and 84% Ndfa (Grove and Malajczuk, 1992). Other rates



31

of %Ndfa reported for native legumes include 50 to 80% for Acacia dealbata inVictoria, Australia (May and Attiwill, 2003), and 46 to 83% for two species of Acacia from African savannas (Cramer et al. , 2007). It should be noted that the latter twostudies used the 15N natural abundance method while rates of %Ndfa of B.

laidlawiana were evaluated using acetylene reduction. Both methods have beenshown to yield comparable results (Boddey, 2000). Reasoning of our somewhatlower values of N2-fixation for B. foliosa could be the result of generally low

nodulation and for a short period in spring only, evidenced by the fact that we did notfind any nodules during the sampling campaign in January 2010. However, thegenerally lower δ15N signature in leaf material of B. foliosa compared to the non-legume Poa spp. verifies that N 2 fixation is a feature of native legumes of Australia.

Evidence for nodulation of B. foliosa in summer from sub-alpine environments hasbeen observed (Lafay and Burdon, 1998), making it difficult to provide a reasonableexplanation for the absence of nodules in the present study. If we consider the δ 15Nsignatures in leaves as indicator for N 2 fixation, sic nodulation, one explanation for

observations in this study could be an initially low formation (possibly due acombination of low soil pH and low temperatures), a short life span and rapid

decomposition of nodules from the onset of spring until our sampling in mid-summer.Fast rates of nodule decomposition (75 % in 28 days) were reported from tropicalenvironments (Nygren et al. , 2000). However, other factors may have contributed to

our observation.

The most common cause for low or no nodule formation in N 2-fixing plants is highconcentration of plant available N (NO 3

- + NH4+) in the soil. Although high levels of

NO3- can inhibit N2 fixation (Dakora, 1998), a recent study has shown that elevatedconcentrations of soil N form a negative exponential relationship with N 2-fixation inlegume crops (Salvagiotti et al., 2008). However, due to the absence of NO 3

- and

very low NH4+ availability in the soil of the studied ecosystem, we can comfortably

reject the possibility of high soil N as the cause for low levels of nodulation. Other

environmental factors can also lead to inhibition of nodule formation. These includehigh soil temperatures (between 27 and 40 °C depending of the rizhobial strain;

Hungria and Vargas, 2000); low temperatures (Bordeleau and Prévost 1994 indicatethat in controlled environment nodule development is totally inhibited at 8°C), but



32

also the literature note that there are species adapted to nodulation in extreme lowtemperature environments (Schulman et al., 1988); soil water deficit (Lawrie, 1981;

Purcell and King, 1996) and soil acidity (Parker and Harris, 1977, Hungria andVargas 2000). By reviewing all available information of this research project,

including soil nutrient status and environmental data, we conclude that a combinationof low temperature and soil acidity is the most likely constraint for the absence ofnodules on roots of B. foliosa .

Despite the range of B-value used to calculate %Ndfa, it is surprising that addition ofP and Mo did not increase N 2 fixation, given the importance of these elements in theprocess of N 2 fixation and the low availability of N, P and Mo in the soils investigatedhere. The addition of P has been shown to increase N 2 fixation in B. laidlawiana

(Grove and Malajezuk, 1992). These authors showed that addition of 30 and 200 kgP ha -1 increased rates of N 2-fixation by up to 99%. They further reported that soil-extractable P rose from 7 and 23 ppm, resulting in greater number of nodules formedrather than increased nitrogenase activity in nodules. Additionally, they reported thatN concentration in leaves did not change with added P, but biomass increasemarkedly. Similarly, Crews (1993) measured increase of nitrogenase activity with P

addition to alfalfa, but the effect of P addition was not reflected in increased ethyleneproduction per unit root mass. The effect of phosphorus on N 2-fixation resultedprimarily from an increase in host plant biomass rather than a direct effect of P onthe N2-fixation capacity of Rhizobium . In this study we did not measure biomass

production of legume N2-fixer or grasses, so we do not know if the quantity of N 2 fixed per unit area could be modified by the treatments. Nevertheless, if this hadoccurred it should be consistent with the %Ndfa calculated (Sanginga et al., 1995).

Studies that have measured the effect of the addition of Mo on N 2-fixation arerelatively scarce. Barron et al. (2008) measured nitrogenase activity in free-living

heterotrophic bacteria in tropical soils with addition of different levels of P and Mo.They found that Mo addition resulted in an increase in nitrogenase activity and

therefore N2-fixation even when Mo was applied without additional P. Barron andcolleagues (2008) concluded that soil acidity plays and important role where addition

of Mo does not increase N 2-fixation.



33

Considering all of the results from this study it can be hypothesized that there aretwo main reasons that could explain why addition of P and Mo had no direct effect onN2-fixation. Firstly, the finding that concentrations of both added nutrients onlyincreased in the non-fixing reference plant could indicate greater competitiveness of

the non-legume over the legume species to scavenge these elements that arenaturally scarce in soils of sub-alpine ecosystems in Australia. It has previously beenshown that non-legume species can indeed acquire P more efficiently compared tolegumes (Jackman and Mouat, 1972; Föhse et al. , 1988). Secondly, the fact thatroots of B. foliosa were not nodulated in January could indicate that environmental

conditions constrained the process of N 2-fixation, making our treatments largelyineffective. Here, soil acidity appears to be the most likely limiting condition for BNFas shown in other studies (Wood et al ., 1984; Ledgard and Steele, 1992; Hungria

and Vargas, 2000) but low soil temperatures may have had a role (Bordeleau andPrévost, 1994).

5 . Conc lus ions and ou t look

Effect of fire, followed by nutrient pulse and subsequent beneficial growth conditions

for legumes compared to non-legumes, has not been reflected in the P and Moconcentration of legumes in this study with the addition of fertilizer, as we hadhypothesized. Contrary, the grasses increased its concentrations of P and Mo withthe fertilization. In the same way, we found no evidence that post fire conditions(through pulses of P and Mo) represent an environment where nodules formationand therefore BNF increased competitiveness of B. foliosa compared to associated

grasses, as we have hypothesized.

In a context of prognosticated climate change for the alpine and sub-alpine regionand an under increased fire frequency (as a result of increased frequency ofdroughts and heat waves) is unlikely that at least B. foliosa that will out compete

associated grasses will lead to a change in composition of understory species.Despite in this study we did not evaluate the competition among species (legume

and grasses) from a productivity point of view (dry matter production), the resultssuggest a probable advantages of grasses over B. foliosa , which also could have

implications in terms of use of these environments. For example, under a



34

silvopastoral use, the changes in composition and productivity of different species inthese ecosystems would influence in the livestock performance.However, it is necessary to improve our knowledge about these interactions and itsecological and productive implications, in different environmental conditions and

longer periods.

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