J00392

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Short communication

Net nitrification rate and presence of

Nitrosospira

cluster 2 in acid

coniferous forest soils appear to be tree species specific

R.A. Nugroho

a,

*, W.F.M. Ro¨ling

b

, A.M. Laverman

c

, H.A. Verhoef

a

a_{Faculty of Earth and Life Sciences, Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands} b_{Molecular Cell Physiology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands}

c_{Department of Geochemistry, Faculty of Earth Sciences, University Utrecht, Budapestlaan 4, 3584 CD Utrecht, The Netherlands}

Received 22 April 2005; received in revised form 11 July 2005; accepted 27 September 2005 Available online 2 November 2005

Abstract

The impact of four coniferous tree species and their corresponding soil factors on N transformation rates and presence of ammonia-oxidising

bacteria (AOB) was studied in an acid pine forest soil (Appelscha, The Netherlands). Pine soil had a relatively low net nitrification rate, while

spruce, fir and larch soils showed high net nitrification rates. 16S rRNA and

amoA

sequences were only found in soils with high nitrification rates

and belonged solely to

Nitrosospira

cluster 2. We conclude that tree species, possibly through their effects on soil C/N ratios, determines the

presence of

Nitrosospira

cluster 2. Whenever AOB are present, however, the AOB community composition appears to be similar.

q

Keywords:Acid forest soils; Coniferous tree species; C/N ratios; N transformation rates; Ammonia-oxidising bacteria;Nitrosospiracluster 2

Since tree species have a great impact on soil properties

(

Binkley and Giardina, 1998; Myers et al., 2001; Northup et al.,

1998; Knops et al., 2002; Stark and Firestone, 1996

), they can

determine associated soil microbial biomass, activity and

community structure (

Templer et al., 2003; Bauhus et al., 1998;

Priha and Smolander, 1999; Coˆte et al., 2000; Priha et al., 2001;

Smolander and Kitunen, 2002

). This can be explained by the

differences in leaf litter quality across stands of different tree

species (

Pastor and Post, 1986

).

The type of tree species appears also to be related to the

variation in N transformations across forest soils (e.g.

Chen and

Stark, 2000; Finzi et al., 1998; Lovett et al., 2004; Menyailo

et al., 2003; Priha and Smolander, 1999

). As the nitrification

process is critical to the N transformations in forest soils, the

presence, type and activity of ammonia-oxidising bacteria

(AOB) are likely to be influenced by tree species. Our aim was

to elucidate the relationship between soil factors, N

transform-ation rates and the presence of AOB in soils under four

different coniferous tree species (pine (

Pinus sylvestris

L.),

spruce (

Picea abies

(L.) Karst.), fir (

Pseudotsuga menziesii

(Mirb.) Franco) and larch (

Larix decidua

Mill.)) at short

distances in a forest at Appelscha (53

8

05

0

N, 6

8

40

0

E), The

Netherlands (

Fig. 1

). The tree species were of the same age,

growing in the same soil type, at the same elevation and under

identical climatic conditions, and receiving a similar relatively

low N deposition (14–21 kg ha

K1

y

K1

(

RIVM, 2002

)). The

differences between the soils are, therefore, most likely related

to the different tree species.

At each sampling site, one composite sample of the F layer

was collected and analysed for soil characteristics (total C,

total N, Ca content, moisture content, extractable NH

C4

–N and

NO

K3

–N concentrations, and pH

KCl

) as described by

Nugroho et

al. (2005)

. Soil properties differed between soils under the four

tree species (

Table 1

). Comparisons across all soils revealed

that pine soil had the lowest total N and initial NO

K

3

–N

concentration, and the highest C/N ratio. Spruce soil had the

lowest pH and Ca concentration, and the highest total C and

initial NH

C

4

–N concentration. Fir soil had the lowest total C and

C/N ratio, and the highest initial NO

K

3

–N concentration. Larch

soil had the lowest initial NH

C

4

–N concentration, and the

highest pH, Ca and total N. NH

C4

–N was the predominant form

of inorganic-N, accounting for 65–98% of inorganic N. Net

nitrification rates, measured as described by

Nugroho et al.

(2005)

, was nearly zero in pine, but much higher in spruce and

fir, and larch soils (

Table 1

). Net ammonification rates

decreased in the following sequence: pine

O

spruce

O

fir

O

www.elsevier.com/locate/soilbio

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larch. Net N mineralisation rates were also different between

different tree species, pine soil had the highest rate and the

other soils were indistinguishable.

The AOB populations associated with these samples were

studied based on 16S rRNA and

amoA

genes analyses. In the

16S rRNA gene-based analysis of AOB communities, DNA

extraction, purification, nested PCR, Temporal Temperature

Gradient Gel Electrophoresis (TTGE), band excision and

cloning were carried out as described by

Nugroho et al.

(2005)

. Clones were screened for inserts of the correct size

by PCR amplification and one correctly sized clone, per

independent electrophoresis profile and per sample, was

sequenced using an ABI PRISM

w

3100 Genetic Analyzer.

Sequences were analysed as described by

Nugroho et al.

(2005)

.

The TTGE banding patterns for AOB 16S rRNA gene

fragments from spruce, fir and larch soils were similar

(

Fig. 2

(a)), indicating low diversity of the AOB community

with only one discernible doublet band detected. Doublet bands

resulted from an ambiguous position in primer CTO 654r, used

to amplify AOB 16SrRNA gene fragments (

Kowalchuk et al.,

1997

). PCR products from these soils co-migrated with

products from cloned standards representing

Nitrosospira

clusters 2 and 3. Phylogenetic analysis of the 16S rRNA

gene fragments of these soils (

Fig. 3

) assigned them (99–100%

similarity) to

Nitrosospira

cluster 2 and were closest related

(99% similarity) to that of

Nitrosospira

sp. strains III7 and

AHB1, isolated from acid soils. In contrast, the band pattern

from pine soil was different from the other soils (

Fig. 2

(a)). The

bands did not clearly co-migrate with any of the cloned

standards.

In addition to the 16S rRNA analysis,

amoA-1F/amoA-2R-TC and amoA-1F-Clamp/amoA-amoA-1F/amoA-2R-TC primer sets

targeting the ammonia monooxygenase subunit A gene

[image:2.595.51.287.70.276.2]

(

amoA

) (

Nicolaisen and Ramsing, 2002

) were used for

Fig. 1. Location of four different tree species in Appelscha, The Netherlands.

Tabl e 1 Soil ch aracteristics and N transformat ion rates of the forest soils studied Tree spec ies PH (KC l)

Calcium (mmol

g K 1 dry soil) Tot al C (%) Tot al N (% ) C/N NH

C–N4

( m gg K 1dry soil) NO

K–N3

( m gg K 1dry soil) Densi ty of AOB (M PN counts g K 1 dry soil) NN R a( m gg K 1 dr y soi l week K 1) NAR b( m gg K 1 dry soi l we ek K 1) NMR c ( m gg K 1dry soil week K 1) P. sylvestris 2.94 (0.02) 23.6 (1.78) 47.8 (0.08) 1.8 (0.01) 27.1 (0.15) 28.5 (0 .25) 0.4 (0.03) 0 0.5 (0.25) 37.3 (0.47) 37.7 (0 .42) P. abies 2.87 (0.02) 22.8 (0.26) 48.6 (0.09) 2.2 (0.01) 22.2 (0.05) 106. 1 (0 .21) 14.8 (0.30) 405 (271) 10.4 (0.87) 22.7 (1.60) 33.1 (2 .45) P. menziesii 2.90 (0.02) 42.2 (0.23) 45.5 (0.26) 2.2 (0.02) 20.4 (0.06) 61.3 (0 .90) 30.9 (0.76) 37 (64) 11.3 (0.31) 22.1 (1.86) 33.4 (2 .14) L. dec idua 3.07 (0.01) 79.1 (1.58) 46.9 (0.20) 2.3 (0.02) 20.8 (0.05) 24.2 (0 .24) 12.8 (0.26) 354 (313) 17.8 (1.96) 15.3 (1.15) 33.1 (1 .39) Values represent means of 3 replic ates G SD. Extractable NH

C–N4

and

NO

K–N3

conce ntratio ns were deter mined by extra ction of the samp les in 1 M KCl (15 g field-moist soil:100 ml 1 M K Cl). A fter fil tration , the extract was analy sed for N H

C–N4

and

NO

K–N3

concent rations and pH KC l . Concent rations of extractable N O

K–N3

in soi l at ti me zero and after 3 weeks we re used to calculate net nitr ification rate. The ne t ammon ifica tion rate and net mine ralisation rate we re calculated in the same man ner; subtrac ting initi al concent ratio ns of NH

C–N4

and ð NH C 4 C NO

KÞ3

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comparison, and profiled by Denaturing Gradient Gel

Electrophoresis (DGGE) as described by

Nicolaisen and

Ramsing (2002)

. Results from

amoA

genes analyses

confirmed the observations made based on analysis of 16S

rRNA genes. Amplified

amoA

gene fragments from spruce,

fir and larch soils migrated to similar end-positions in DGGE

and also co-migrated with products from

amoA

cluster 2

(

Nitrosospira

sp. AHB1) and cluster 3 (

Nitrosospira briensis

)

(

Fig. 2

(b)). Phylogenetic analysis of the

amoA

gene

fragments of these soils (

Fig. 4

) grouped them (99–100%

similarity) within

Nitrosospira

cluster 2, in accordance with

phylogenetic tree analysis of the 16S rRNA gene fragments.

These

amoA

sequences were also closely related (97–100%

similarity) to that of

Nitrosospira

sp. strains III7 and AHB1.

On the other hand, pine soil sample did not generate PCR

product using the

amoA

primers even though a nested PCR

was carried out for this sample.

Therefore, molecular genetic analysis revealed that

Nitrosospira

cluster 2 was the only sequence cluster detected

in spruce, fir and larch forest soils. This result was supported

by the MPN culturing data, enumerated with microtitre plates

(

Rowe et al., 1977

) using 12 fivefold dilution series with

eight replicates at each dilution and ammonium–calcium

carbonate medium (

Alexander and Clark, 1965

). AOB could

be cultured from spruce, fir and larch soils, but were

undetectable in pine soil. Thus, differences in soil properties,

or tree species, did not overtly influence the composition of

the AOB community. Conversely, the presence of

Nitrosos-pira

cluster 2 could not be detected in pine soil, where net

nitrification rate was low. Regarding these results, low C/N

ratio (or high total N) in acidic forest soils is probably

favourable for the presence of

Nitrosospira

cluster 2.

Although AOB are autotrophic and do not depend on

organic matter input directly, differences in the magnitude or

range of organic matter input may result in different or more

variable rates of soil nitrogen mineralisation (

McLaugherty et

al., 1985; Hobbie, 1996

), the main process that provides

ammonium to soil AOB. We do not believe that the absence

of

Nitrosospira

cluster 2 in pine soil is due to spatial

heterogeneity in AOB community composition at Appelscha.

Little temporal and spatial variation in AOB community

composition, with the exclusive detection of a single

Nitrosospira

16S rRNA sequence cluster, was observed in

a nitrogen-saturated pine forest soil (

Laverman et al., 2001,

2005

).

Nitrosospira

cluster 2 (as well as other AOB) was

also absent in other pine forest soils receiving low N

deposition, while it was present in forests in soils with high

N deposition (

Nugroho et al., 2005

).

In conclusion, at our study site, members of

Nitrosospira

cluster 2 dominate in acidic soils with high nitrification rates

regardless of the tree species. Low C/N ratio (or high total

N) of soils are probably favourable for the presence of

Nitrosospira

cluster 2. Thus, tree species can affect N

transformation rates in acid coniferous forest soils with low

N deposition, possibly through the species’ effects on soil

C/N ratios. Moreover, this study shows that large differences

in N transformation rates and presence of

Nitrosospira

cluster 2 can occur within relatively short (0.5–5 km)

distances.

Acknowledgements

The authors thank Paul L.E. Bodelier and Manuela Coci for

providing reference clones and pure cultures of the AOB. Rik

Zoomer and Janine Marie¨n are acknowledged for assistance in

the field and laboratory.

[image:3.595.42.274.66.457.2]

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Cluster 8 Cluster 6a Cluster 6b

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Wekr-c, AJ245989

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Nitrosomonas sp. F5, AJ298691 Nitrosomonas sp. F6, AJ298693 100

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