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Soil Biology & Biochemistry xxx (2012) 1e9

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Soil Biology & Biochemistry

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1 56
2 57
3
How does N deposition affect belowground heathland recovery following 58
4 wildfire? 59
5 60
6
Q3 E.R. Green a, **, R.J. Ellis b, c, S.R.M. Gadsdon a, d, A. Milcu c, S.A. Power a, e, * 61
7 62
a
8 Division of Biology, Imperial College London, Silwood Park, Ascot, Berkshire SL75 7PY, UK 63
b
Specialist Scientific Services, Animal Health and Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK
9 c
NERC Centre for Population Biology, Imperial College London, Silwood Park, Ascot, Berkshire SL5 7PY, UK
64
10 d
City of London, Epping Forest Office, The Warren, Loughton, Essex IG10 4RW, UK 65
11 e
Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith NSW 2751, Australia 66
12 67
13 68
14 a r t i c l e i n f o a b s t r a c t 69
15 70
16 Article history: Climate change is predicted to increase the frequency and severity of summer fires in the UK. However, 71
17 Received 29 September 2011 the effects of fire on ecosystem processes are likely to depend on pre-burn vegetation characteristics, soil 72
Received in revised form chemistry and microbiology, all of which are known to be affected by changes in nitrogen (N) deposition.
18 22 August 2012
73
19 The occurrence of a severe summer fire after eight years of experimental N additions at Thursley 74
Accepted 23 August 2012
20 Available online xxx
Common National Nature Reserve (UK) thus provided a unique opportunity to evaluate the impacts of 75
a major perturbation on nutrient and microbial dynamics in soils of contrasting chemistry. Soil N and P
21 76
concentrations were substantially reduced by fire, although significant pre-fire treatment effects on soil
22 Keywords: 77
Lowland heathland
N were still apparent immediately after the fire. Soil nutrient availability, turnover rates and leaching
23 losses were also increased by fire. Concentrations of extractable soil N and leaching losses of Ca2þ, Kþ, Cl 78
Wildfire
24 Nitrogen deposition and SO24 immediately after the fire were significantly higher in plots which had previously received N
79
25 Soil nutrient dynamics additions, demonstrating effects of N loading on post-fire nutrient dynamics and economy. Effects of N 80
26 Microbial community composition addition on the activity of soil microbial populations were influenced by soil moisture levels, with 81
27 Ericoid mycorrhizal fungi significantly higher activity in þN plots compared to controls when soil moisture levels were relatively 82
28 high (>15%). Microbial community composition varied considerably between sampling dates, although 83
29 clear N treatment differences were apparent, particularly for bacterial communities, both immediately 84
30 after and one year on from the fire. Persistent effects of wildfire and N deposition on the stocks and 85
availability of N, and microbial community dynamics, are likely to have longer-term consequences for the
31 86
functioning of heathlands and other similar semi-natural ecosystems.
32 87
Ó 2012 Elsevier Ltd. All rights reserved.
33 88
34 89
35 90
36 91
37 1. Introduction latitude areas (Solomon et al., 2007). Such conditions are often 92
38 associated with an increase in the frequency of wildfire events 93
39 Human activities, particularly those associated with agriculture, (Dale et al., 2001) and there is indeed evidence linking a recent 94
40 industry, transport and urban expansion have transformed the land upsurge in the number and intensity of wildfires to human influ- 95
41 surface and resulted in widespread changes to the structure and ence and climate change (Flannigan et al., 2000; Dale et al., 2001; 96
42 functioning of terrestrial ecosystems (Vitousek, 1997). Climate Barcenas-Moreno et al., 2011). Fire has major impacts on the global 97
43 change is a strong driver of ecological change (Sala et al., 2000; carbon (C) cycle, converting organic C stores to CO2 (Cochrane, 98
44 Stocker et al., 2010), with predicted increases in temperature likely 2003; Jones and Cox, 2005; Kasischke et al., 2005; Randerson 99
45 to affect the composition, distribution and functioning of ecosys- et al., 2005). In this way, fire acts as a positive feedback on both 100
46 tems across the globe (Gitay et al., 2002). Changes in precipitation climate change and the likelihood of ecologically damaging wild- 101
47 regimes are also predicted with, in particular, drier summers in mid fires (Borghesio, 2009). Resulting changes in temperature, soil 102
48 moisture and organic matter quality will strongly influence many 103
49 important ecosystem processes including rates of decomposition 104
* Corresponding author. Division of Biology, Imperial College London, Silwood and mineralisation (Swift and Heal, 1979; Schmidt et al., 2004).
50 Park, Ascot, Berkshire SL75 7PY, UK. Tel.: þ44 (0)207 5942318; fax: þ44 (0)207
105
51 5942339.
Anthropogenic disruption of the global nitrogen (N) cycle is also 106
52 Q1 ** Corresponding author. Tel.: þ44 (0)207 5942318; fax: þ44 (0)207 5942339. responsible for widespread change in the diversity and functioning 107
53 E-mail address: s.power@imperial.ac.uk (S.A. Power). of terrestrial ecosystems (Galloway et al., 2008; Gruber and 108
54 109
0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
55 110
http://dx.doi.org/10.1016/j.soilbio.2012.08.025

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildfire?, Soil
Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025
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2 E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

111 Galloway, 2008; Bobbink et al., 2010). N is a limiting factor for plant ii) N addition rapidly increases rates of nutrient availability and 176
112 growth in many semi-natural ecosystems, including forest, grass- turnover, and modifies microbial community composition 177
113 land and heathland, and many plant species associated with these following fire. 178
114 systems are adapted to low levels of N (Bobbink et al., 1998; Aerts 179
115 and Chapin, 2000). Indeed, 65e80% of Central European red list 2. Materials and methods 180
116 species are restricted to ecosystems with the lowest N status, since 181
117 many of these plants can only compete in low nutrient situations 2.1. Site description and experimental design 182
118 (Bobbink et al., 2010). Responses to elevated N deposition include 183
119 faster rates of nutrient cycling, species-specific changes in plant Thursley Common National Nature Reserve is an area of lowland 184
120 productivity and associated loss of diversity and N accumulation heathland in Surrey, UK (Ordnance Survey Grid Reference SU 185
121 (Vitousek, 1997; Carroll et al., 2003; Galloway et al., 2008; Skogen 910404, Longitude 0 41.90 W, Latitude 519.30 N). The experimental 186
122 et al., 2011). area is a virtual monoculture of building phase Calluna (National 187
123 Interactions between N deposition and predicted increases in Vegetation Classification type H2, Rodwell (1991), EUNIS site code 188
124 the frequency of summer drought and associated wildfires are UK930017, EUNIS (2011)) which was eight years old at the time of 189
125 likely for several reasons. Lowland dry heaths are typically domi- the wildfire (Pic. S1). The soil is a podsol derived from Lower 190
126 nated by ericaceous dwarf-shrubs and, due to the large volume of Greensand, comprising a thin (<1 cm) humus layer overlying 191
127 fuel present, they tend to burn readily and produce relatively high a sandy mineral layer, with pure sand below 20 cm depth (Power 192
128 temperature wildfires (Tucker, 2003). N deposition has frequently et al., 1998a; Barker, 2001). Pre- and post-fire soil physical and 193
129 been associated with increased biomass (e.g. Green, 2005; Carroll chemical properties are outlined in Table 1. 194
130 et al., 1999; Gordon et al., 1999; Power et al., 1998a,b) and the In February 1998 a total of 16 plots (4 m  4 m) were set up in 195
131 likelihood, severity, temperature and duration of a fire may, a factorial blocked design with 4 replicate blocks and 4 plots per 196
132 therefore, be increased under elevated N deposition as a result of block. Each plot is further sub divided into four sub-plots (2  2 m); 197
133 a greater fuel load. However, wildfire also has the potential to management treatments were applied randomly to each of these 198
134 remove a large proportion of accumulated N stores, potentially 2 m  2 m sub-plots in February 1998 by Barker (2001) to provide 199
135 slowing or even reversing the process of nutrient build up associ- a gradient of nutrient removal. As very few significant management 200
136 ated with ecosystem N loading. effects were detected, these are not considered here. Post-fire 201
137 Controlled burning is a management tool carried out under sampling and analysis focused on two of these sub-plots which 202
138 strict guidelines governing the season and intensity of burning, as had previously been subjected to either a low intensity mow or 203
139 described by Gimingham (1972). However, uncontrolled wildfires a high temperature burn. From April 1998, half of the plots received 204
140 tend to occur in the spring and summer and may burn intensely for increased N deposition (þN: 30 kg N ha1 yr1) and the remaining 205
141 a long time (Bullock and Webb, 1995). Flame temperatures between plots served as controls (no extra N). Background deposition of N 206
142 225 and 1225  C have been recorded during wildfires, although has been calculated at approximately 8e12 kg N ha1 yr1 207
143 maximum temperatures are believed to reach as high as 2220  C following on site measurements of bulk deposition and gaseous 208
144 (Dennison et al., 2006). The number and extent of heathland concentrations of NH3 and NO2 (Power and Barker, 2003). 209
145 wildfires is very dependent on weather conditions; in 2006 e one Treatments, in the form of (NH4)2SO4, have been applied every 210
146 of the warmest summers on record (Prior and Beswick, 2007) e two weeks, by adding 15 L N solution per 16 m2 plot on each 211
147 there were 923,000 such fires recorded in the UK, an increase of occasion. Ammonium sulphate was used for comparability of 212
148 27% compared to the preceding year (McEvoy and Gamble, 2008). results with an earlier experiment at the same site which started in 213
149 Whilst the devastating effects of wildfire on vegetation and 1989, when NHþ 2
4 co-deposition with SO4 occurred widely across 214
150 wildlife are immediately apparent, its impact on the belowground the UK (NEGTAP, 2001). Control plots receive an artificial rain 215
151 system, especially the microbial community and the ecological solution consisting of Kþ, Mg2þ, Naþ, SO2 
4 , Cl , Ca
2þ
at concen- 216
152 processes they control, are poorly understood. Several studies have trations found in rainfall measured at a nearby site (Uren, 1992), 217
153 shown that soil heating as a result of wildfire decreases numbers of with additions representing 7% of the annual average rainfall at this 218
154 soil microorganisms and, in severe cases, can even temporarily site (Barker et al., 2004). Values for mean annual/growing season 219
155 sterilise the belowground environment (Neary et al., 1999; Mataix- rainfall and temperature for the experimental period are presented 220
156 Solera et al., 2009). The loss of nutrients (especially N) through in Table 2. 221
157 combustion (Gärdenäs et al., 2011) and production of charred N, The wildfire occurred from 14the17th July 2006, after more 222
158 a less accessible form of this element (Knicker, 2007), following fire than 8 years of N addition had taken place. The fire resulted in the 223
159 can result in low soil nutrient availability, slowing recovery of loss of all aboveground vegetation and the litter layer, as well as 224
160 microbial populations. When populations do recover, changes in partial loss of the humus horizon in all experimental plots. The 225
161 the soil chemical environment associated with heat and ash average ash layer depth was 2e3 mm, though this was largely 226
162 deposition e such as increased pH and soluble carbohydrate levels washed away following a heavy rainfall event a month after the fire. 227
163 (Raison, 1979) e may make conditions more favourable for bacteria Treatment additions of N were suspended from JulyeOctober 2006, 228
164 than for fungi. Therefore, not only will the soil environment be but resumed again after this time. The effect of fire was determined 229
165 altered immediately by fire, subsequent feedbacks are also likely as by comparing nutrient stocks, availabilities and microbial activity 230
166 a result of changes in the microbial community (Raison, 1979). with their pre-fire values. 231
167 In this paper we present unique data from a long term heath- 232
168 land N addition experiment in southern England which experi- Table 1 233
169 enced a severe, accidental summer fire in 2006. This study aimed to Pre- and post-fire soil chemical and physical properties at Thursley Common. 234
170 determine the effects of wildfire on soil nutrient status and the Soil properties Pre-fire Post-fire 235
171 impacts of N addition on nutrient and microbial community Total N mg kg1 227.3 66.2 236
172 dynamics following fire. We hypothesise that: Total P mg kg1 127.4 79.4 237
173 Bulk density g cm3 1.35 1.40 238
174 i) Wildfire removes pre-existing treatment related differences pH 4.8 5.0 239
Loss on ignition (%) 7.8 1.8
175 in soil nutrient concentrations. 240

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildfire?, Soil
Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025
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E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9 3

241 Table 2 (18  C) within 12 h of sample collection for subsequent DNA 306
242 Rainfall and temperature data for Thursley Common over the study period. extraction. Total dehydrogenase activity was determined on fresh 307
243 Year Annual Growing season Growing season soil (pre-fire: August 2004, post-fire: August 2006, October 2006, 308
244 rainfall (ApreSept) (ApreSept) February 2007, May 2007, August 2007) as an indicator of overall 309
245 (mm) rainfall (mm) mean temp ( C) microbial activity, using the method described by Ellis et al. (2001). 310
246 2003 690 203 14.4 One ml of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium 311
2004 740 307 14.0
247 chloride (INT, SigmaeAldrich, UK) in 0.85% NaCl was added to 9 ml 312
2005 589 239 13.8
248 2006 850 335 14.7 of the homogenized soil sample. This was shaken at 20  C for 24 h 313
249 2007 925 443 14.1 before formalin (0.5 ml) was added and the sample centrifuged at 314
250 5000 g for 20 min. The optical density (OD490) of the supernatant 315
251 was determined and the pellet extracted with 10 ml methanol 316
252 with 1% (v/v) Triton X-100 and shaken for 1 h at 20  C. The 317
2.2. Soil chemistry and nutrient analyses
253 sample was then centrifuged at 5000 g for 20 min and the OD480 318
254 of the supernatant was determined. The extent of formazan 319
Soil samples (0e10 cm) were collected from eight plots for
255 production was then calculated according to standard conversion 320
Control and þN treatments. Measurements included total N and
256 figures (Merlin et al., 1995). 321
phosphorus (P) concentrations (August 2006, October 2006 and
257 Molecular methods were used to assess total bacterial and 322
February 2007), and extractable concentrations of nitrate, ammo-
258 fungal diversity, using primers targeted to the 16S and 18S ribo- 323
nium and phosphate (August 2006, October 2006, February 2007
259 somal RNA genes, respectively (Muyzer et al., 1993, van Elsas et al., 324
and May 2007). Dried, ground soil samples were prepared for total
260 2000). Post-fire samples were collected in August 2006, October 325
N and P analyses using the Total Kjeldahl Nitrogen (TKN) method,
261 2006, February 2007, May 2007 and August 2007 but no pre-fire 326
based on the wet oxidation of organic material (Allen, 1989). 3 ml of
262 data were available. In addition, post-fire diversity of 16S rRNA 327
sulphuric acid was added to 250 mg of soil, with a selenium cata-
263 genes specific to ammonia-oxidizing bacteria within the b-pro- 328
lyst, in order to convert organic N into the ammonium form. NO 3
264 teobacteria (Kowalchuk et al., 1997), the functional genes required 329
and NHþ 4 ions were extracted from fresh soil samples using 1 M KCl
265 for N-fixation, nifH (Lovell et al., 2000) and those associated with 330
(Allen, 1989). PO4 was also extracted from fresh soil samples, using
266 a key step in denitrification (nitrous oxide reduction), nosZ 331
Truog’s reagent (Allen, 1989). All samples were analysed colori-
267 (Throbäck et al., 2004) was quantified in October 2006, February 332
metrically using a SKALAR San Flow þþ segmented flow analyser
268 2007 and August 2007 for Control and þN plots (Table S1). 333
(Skalar San Flow þþAutoanalyser, SKALAR, UK). Pre-fire data for
269 Furthermore, following recent evidence demonstrating the nega- 334
total (August 2003) and extractable (August 2004) soil nutrient
270 tive impacts of fire on mycorrhizal fungi (Basitas et al., 2006) and 335
concentrations were obtained using identical procedures. August
271 the importance of ericoid mycorrhizae for growth of heather in 336
data are presented to minimise the effect of seasonality when
272 restored lowland heath (Diaz et al., 2006) specific PCR primers were 337
comparing pre-fire conditions with those in August 2006, imme-
273 also developed to assess the presence and abundance of this 338
diately after the fire.
274 important group of microorganisms following fire. DNA was 339
Short term investigations into net mineralisation rates were
275 extracted from 250 mg of previously frozen soil, using Powersoil 340
carried out using in situ soil incubations over four month periods
276 DNA isolation kits (MO BIO Laboratories Inc., USA). The PCR mix 341
from MayeSeptember 2004 (pre-fire) and MayeSeptember 2007
277 (50 ml/reaction) comprised 10 ml of each primer, 5 ml 1 Immo- 342
(post-fire). On each occasion, paired soil samples were taken using
278 buffer, 2 mM MgCl2, 0.4 ml of 100 mM deoxynucleoside triphos- 343
PVC tubes (2.0 cm diameter, 10 cm length). One of each pair of tubes
279 phates (dNTP) mix, 1 unit of Immolase DNA polymerase (Bioline, 344
was taken for immediate analysis in the laboratory while the other
280 London, UK) and 1 ml of extracted DNA. The cycle conditions used 345
was sealed and left in the field for the incubation period. On return
281 for each primer are described in the source article listed above for 346
to the laboratory, 40 g of fresh soil was extracted using 250 ml 1 M
282 each gene. Amplified DNA fragments were separated using dena- 347
KCl, and analysed for NHþ 
4 and NO3 by colorimetric analysis. Net N
283 turing gradient gel electrophoresis (DGGE) as described in Ellis 348
mineralisation was taken as the increase in NHþ 
4 eN plus NO3 eN,
284 et al. (2003), using the INGENY phorU2 system (Netherlands). 349
relative to concentrations in their initial paired samples (Van
285 Samples were loaded onto 10% acrylamideebisacrylamide (37.5:1) 350
Vuuren and Van Der Eerden, 1992).
286 gels with denaturing gradients from 30% to 60% (where 100% is 7 M 351
Leachate samples were obtained using porous cup tension
287 urea and 40% [v/v.] deionized formamide) in 1 TAE electropho- 352
lysimeters installed to a depth of 45 cm. Pre-fire data were collected
288 resis buffer. Electrophoresis was performed at 100 V at 60  C for 353
monthly from April 1998 to May 1999 and from June to August
289 16 h. Gels were then stained with SBYR Gold (Cambridge BioSci- 354
2003; post-fire data were collected monthly from September 2006
290 ence, UK) in 1 TAE for 30 min at room temperature and visualised 355
until February 2007. Samples were stored at 5  C overnight and
291 under UV illumination. 356
then NHþ 
4 and NO3 concentrations were determined colorimetri-
292 357
cally using a Skalar San Flow þþAutoanalyser (Skalar, UK). Sub-
293 2.4. Data analyses 358
samples from the collected leachate were also analysed for cation
294 359
content using ion chromatography on a Dionex DX 500 HPLC/Ion
295 Mixed effects models, as implemented in the R Statistical 360
Chromatography System (Thermo Scientific, USA). The pH of fresh
296 package (lme function, R version 2.10.1; Pinheiro and Bates, 2009), 361
soil samples (0e10 cm depth) was determined in August 2004 (pre-
297 were used to analyse the fixed effects of N addition (N), fire and 362
fire), August 2006 and October 2006, using a 1:2 ratio of soil to
298 their interaction, on soil chemistry and dehydrogenase activity. The 363
deionised water and a 3 point calibrated Mettler Toledo MP230 pH
299 random-effects of the initial (maximal) model, fit by maximum 364
meter (Mettler Toledo International, UK).
300 likelihood, had a plot within block structure to account for 365
301 temporal nesting and repeated measurements [R model: lme 366
302 2.3. Soil microbial community analyses (y w Nitrogen*Fire*Time, random ¼ w1jBlock/plot), where time 367
303 represents different sampling dates]. Following this approach (for 368
304 All microbial analyses were carried out on a single (0e10 cm) each response variable) we performed two analyses, one 369
305 soil sample per plot. A sub-sample of soil (250 mg) was frozen comparing differences between the treatments pre-fire and 370

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildfire?, Soil
Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025
 SBB5265_proof ■ 15 September 2012 ■ 4/9

4 E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

371 immediately after the fire, whilst in the second analysis we Controls (post-fire) (F1,10 ¼ 7.01, P < 0.05). Effects of N additions on 436
372 compared the dynamics of the post-fire recovery across several post-fire total P concentrations were, however, not significant 437
373 sampling dates. In order to explore the N addition impact on each (F1,10 ¼ 0.35, P > 0.5). 438
374 sampling occasion a contrast (planned post-hoc test) analysis was No discernable effects of either fire or N treatments were found 439
375 performed to test for differences between Control and þN plots. for extractable PO4 levels, although analyses showed that PO4 440
376 Soil moisture data were used as covariates in the model for concentrations were higher when soil moisture levels were high 441
377 extractable N and dehydrogenase activity measurements. Simpli- (F1,20 ¼ 18.29, P < 0.001). The temporal variation in extractable soil 442
378 fication of the maximal model was achieved by removing non- NHþ 
4 and NO3 concentrations also reflected the dynamic nature of 443
379 significant terms, comparing models using the Akaike informa- moisture availability (Fig. 2). Post-fire extractable NHþ 4 and NO3

444
380 tion criterion (AIC) until reaching the minimum adequate model concentrations were in fact higher than pre-burn (Aug 2004) values 445
381 (Crawley, 2007). Digital images of DGGE gels were analysed using in Control and þN treatments (NHþ 
4 : F1,9 ¼ 13.0, P < 0.005; NO3 : 446
382 GelCompar II V5.10 software (Applied Maths, Belgium). Pairwise F1,9 ¼ 200.4, P < 0.001). Pre-burn extractable N concentrations 447
383 similarities in microbial community characteristics between typically ranged from 0.2 to 0.6 mg kg1 during winter months, 448
384 samples were calculated using the dice similarity coefficient and rising to 0.5e1.9 mg kg1 in the spring/autumn and with the lowest 449
385 allowing for 0.5% variation in band position. Graphical represen- values (0.15e0.19 mg kg1) during the drier summer months 450
386 tations of similarity matrices were produced using UPGMA (Green, 2005); post-fire values were, therefore, high for the season 451
387 (unweighted pair group method using arithmetic averages). (and soil moisture levels). Repeated measures analysis demon- 452
388 Throughout the paper, data are presented as mean  SE and the strated a significant overall effect of N addition on post-fire 453
389 threshold for statistical significance is P  0.05. extractable NHþ 4 concentrations (F1,21 ¼ 12.9, P < 0.01), with 454
390 significant treatment effects seen individually on three of the four 455
391 separate measurement occasions (Fig. 2a). Significantly higher 456
3. Results
392 levels of extractable NO3 were measured in þN plots immediately 457
393 after the fire (Aug 2006) (F1,12 ¼ 5.08, P < 0.05), however, levels of 458
3.1. Soil nitrogen and phosphorus dynamics
394 extractable NO 3 had returned to pre-fire low values by the 459
395 following spring (Fig. 2b). 460
Immediately following the fire (Aug 2006), total N and P
396 461
concentrations for the 0e10 cm soil layer significantly dropped
397 462
below pre-fire (Aug 2003) values [(N: F1,11 ¼ 36.1, P < 0.001);
398 463
(P: F1,12 ¼ 5.51, P < 0.05)] (Fig. 1). However, earlier N treatment-
399 464
related differences in soil N concentrations were still apparent
400 465
immediately after the fire (Aug 2006: F1,9 ¼ 6.06, P < 0.05). Soil
401 466
samples taken shortly after N additions resumed in October 2006
402 467
also had higher total N concentrations in þN plots, relative to
403 468
404 469
405 470
406 a 471
407 472
408 473
409 474
410 475
411 476
412 477
413 478
414 479
415 480
416 481
417 482
418 483
419 484
420 b 485
421 486
422 487
423 488
424 489
425 490
426 491
427 492
428 493
429 494
430 495
431 496
432 497
433 498
Fig. 1. a) Pre- and post-fire total soil content of a) nitrogen and b) phosphorus in Fig. 2. Temporal and treatment-related patterns for KCl extractable a) NHþ 4 and b)
434 relation to N treatment. Error bars represent  1 SEM. Significance codes ‘***’ 0.001, NO3 . Error bars represent  1 SEM. Significance codes ‘***’ 0.001, ‘**’ 0.01, ‘*’ 0.05, ‘ns’
499
435 ‘**’ 0.01, ‘*’ 0.05, ‘ns’ not significant. not significant. 500

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildfire?, Soil
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E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9 5

501 N mineralisation rates following the fire were not significantly 566
502 higher in þN (4.4 g N m2) relative to Control (1.5 g N m2) plots. 567
503 Values were, however, significantly higher than pre-fire minerali- 568
504 sation rates measured over a similar period in 2004 (0.16 g N m2 569
505 Control, 0.37 g N m2 þN; F1,13 ¼ 14.1, P < 0.01) (Fig. 3). Leaching 570
506 losses of NO þ
3 and NH4 were generally small, and did not differ 571
507 significantly between Control and þN plots (NHþ 4 F1,21 ¼ 1.85, 572
508 P > 0.05; NO 3 F1,21 0.81, P > 0.05). However, significantly higher Ca
þ
573
þ 
509 (F1,21 ¼ 6.00, P < 0.05), K (F1,21 ¼ 23.4, P < 0.001) Cl (F1,21 ¼ 8.77, 574
510 P < 0.01) and SO2 4 (F1,21 ¼ 11.5, P < 0.01) ion concentrations were 575
511 present in leachate collected from þN plots following the fire (Sept 576
512 2006eFeb 2007), compared to Control plots. The biggest treatment 577
513 effects were seen shortly after the fire (Sept 2006), with Control/þN 578
514 plot values of 81/869 mg l1, 52/222 mg l1, 363/869 mg l1 and 54/ 579
515 194 mg l1 for Caþ, Kþ, Cl and SO2 4 , respectively, at this time. 580
516 Soil pH was significantly higher after the fire than pre-fire values 581
517 recorded in August 2004 (control: 4.8, þN: 4.3, Green, 2005); 582
518 values in October 2006 increased by 0.2 unitse5.0 and 4.5 in 583
519 Control and þN plots, respectively (F1,10 ¼ 29.6, P < 0.001). 584
520 However, pre-fire N treatment differences in pH remained highly 585
521 significant after the fire (F1,10 ¼ 17.3, P < 0.001). 586
Fig. 4. Pre- and post-fire soil dehydrogenase activity. Error bars represent  1 SEM.
522 587
Significance codes ‘***’ 0.001, ‘**’ 0.01, ‘*’ 0.05, ‘ns’ not significant.
523 588
3.2. Soil microbial communities
524 589
525 overall variation, but that there was overlap between different 590
Overall levels of microbial activity, as indicated by hydrogenase
526 time/treatment groups (Fig. S1). Cluster analysis, however, revealed 591
activity, appeared to be strongly influenced by weather conditions
527 strong treatment differences between Control and þN plots one 592
preceding sampling; when post-fire soil moisture levels were
528 year after the fire, highlighting the persistence of treatment effects 593
<15%, activity was generally low (Aug 06, Oct 06, May 07;
529 after a major disturbance event (Fig. 5). There was greater vari- 594
<8 mM INT g1 h1) and there were no significant effects of N at these
530 ability between sample dates than between N treatments for fungal 595
times. Following relatively wetter weather (soil moisture > 15%),
531 communities (Fig. S2) although N treatment effects were apparent 596
microbial activity increased, particularly in the þN plots (Feb 07, Aug
532 in the cluster analysis (Fig. 6) 12 months after the fire occurred. 597
07; >13 mM INT g1 h1) and N treatment effects were significant
533 Although populations of ammonia-oxidizing bacteria belonging 598
(F1,9 ¼ 8.53, P < 0.01) (Fig. 4). Interestingly, whilst measurements of
534 to the b-proteobacteria were not particularly diverse they were 599
soil microbial activity in August 2004 (pre-fire) and August 2006
535 highly variable, even within treatments, so definitive effects N 600
(immediately post-fire) e both times when soil moisture levels and
536 addition could not be determined (results not shown). In contrast, 601
microbial activity were low e were not significantly different, effects
537 there was little variation in the functional genes for N fixation (nifH) 602
of N addition were significant before (F1,9 ¼ 32.5, P < 0.001), but not
538 and denitrification (nosZ) which were relatively stable over time 603
after the fire.
539 and unaffected by N treatment (results not shown). The use of PCR 604
Assessment of post-fire microbial molecular diversity showed
540 primers specific for Rhizoscyphus ericae indicated that this 605
effects of N treatment on various components of the soil microbial
541 606
community. For total bacterial and fungal communities, cluster
542 607
analysis and multidimensional scaling differentiated between
1 00

543 608
90
40

50

60

70

80

samples from Control and þN plots. Multidimensional scaling of

544 609
bacterial communities from Aug 06, May 07 and Aug 07 showed
545 610
that the variation between replicate samples from the same
546 611
treatment taken at the same time was small compared to the
547 612
548 613
549 614
550 615
551 616
552 617
553 618
554 619
555 620
556 621
557 622
558 623
559 624
560 625
561 626
562 627
563 628
Fig. 3. Pre- (MayeSept 2004) and post-fire (MayeSept 2007) net mineralisation rates.
564 Error bars represent  1 SEM. Significance codes ‘***’ 0.001, ‘**’ 0.01, ‘*’ 0.05, ‘ns’ not Fig. 5. UPGMA cluster analysis of bacterial communities one year (Aug 2007) after the 629
565 significant. fire, as assessed by PCR/DGGE of 16S ribosomal RNA genes. 630

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Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025
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6 E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9

631 of accumulated organic material (Raison, 1979) e they were also 696

1 00
50

60

90
70

80
632 significantly higher in þN plots, demonstrating a negative effect of 697
633 earlier N additions on the economy of these ions following a major 698
634 perturbation. 699
635 The high concentrations of extractable NHþ 4 present in þN plots 700
636 immediately after the fire at Thursley may have contributed to 701
637 a replacement of cations (Mgþ, Caþ and Kþ) at soil exchange sites 702
638 (Brady and Weil, 1996) and thus account for the increased 703
639 concentrations of Ca2þ and Kþ in leachate, relative to both control 704
640 plot values (this study) and pre-fire levels (C. Barker, unpublished 705
641 data). Rainfall levels after the fire were consistent with the long 706
642 term seasonal average at the site (1998e2009, Meteorological 707
643 Office, 2011) and are, therefore, unlikely to have resulted in 708
644 Fig. 6. UPGMA cluster analysis of fungal communities one year (Aug 2007) after the 709
unrepresentative dilution/concentration of lysimeter samples
fire, as assessed by PCR/DGGE of 16S ribosomal RNA genes.
645 during the post-fire analysis period. Niemeyer et al. (2005) also 710
646 found increased leaching of Caþ and Kþ following a prescribed burn 711
647 mycorrhizal fungus was abundant in the soil of burnt areas one year treatment, although these soil leaching losses were low compared 712
648 after the fire, although, again, no effects of N treatment were to combustion losses from aboveground vegetation, and are likely 713
649 apparent. to be replenished from precipitation within a few years (Allen, 714
650 1964; Robertson and Davies, 1965; Chapman et al., 1989). 715
651 Typically, levels of available plant nutrients in a heathland 716
652 4. Discussion system decline with time as they become immobilised in woody 717
653 plant material (Tucker, 2003) and microbial biomass (Green, 2005), 718
654 4.1. Changes in biogeochemistry and are turned over only slowly. The immediate post-fire increase 719
655 in extractable NHþ 
4 and NO3 concentrations found in this study in 720
656 The literature suggests that up to 95% of the N and 20e30% of both Control and þN plots is likely to be a consequence of the rapid 721
657 the other principal plant nutrients in the standing vegetation and oxidation of organic material and absence of a vegetation sink, as 722
658 accumulated litter can be lost during heathland fires (Neary et al., has been reported following wildfires elsewhere (Allen, 1964; 723
659 1999; de Luis et al., 2003). In the current study, fire resulted in Kenworthy, 1964; Lloyd, 1971; Tucker, 2003). Significant treatment- 724
660 a decrease in soil total N and P concentrations by an average of 72% related differences in NHþ 4 concentrations were, however, in line 725
661 and 42%, respectively, although pre-treatment differences in soil N with those found on multiple measurement occasions prior to the 726
662 concentrations were still apparent. The smaller proportional fire, highlighting the consistency of response in extractable 727
663 reduction in soil P stocks, compared to N, may reflect the rapid nutrient concentrations across seasons and years. 728
664 incorporation of P deposited in ash (Christensen and Muller, 1975; Increased microbial activity after the fire, compared to pre-fire 729
665 Grove et al., 1986; Smithwick et al., 2005). In areas of moderate to levels measured during the same season, imply high levels of 730
666 high N deposition where N losses are quickly replaced, wildfire may oxidative activity in the soil (Taylor et al., 2002) and the retention of 731
667 act to exacerbate P limitation in lowland heathland as replacement at least small amounts of soil organic matter following the wildfire 732
668 of P losses is a relatively slow process (Chapman et al., 1989). For (Table 1). Although post-fire mineralisation rates were not affected 733
669 example, Chapman (1967) suggest that following loss of soil by N treatment, they were significantly higher than pre-burn values 734
670 organic matter at sites (such as Thursley Common) where the (MayeSept 2004). Similar increases in mineralisation rates 735
671 adsorption capacity is low, it may take c. 20 years for the lost P to be following wildfires have been reported elsewhere for upland 736
672 replaced. Phosphorus availability may, therefore, exercise control moorland (Pilkington et al., 2007), chaparral (Singh et al., 1991), 737
673 on the N economy of the system following wildfire, particularly in tropical forest (Matson et al., 1987) and grassland (Hobbs and 738
674 areas of high N deposition (Aerts and Bobbink, 1999; Gordon et al., Schimel, 1984), indicating that more frequent fires are likely to 739
675 2001; Turner et al., 2002). Under high rates of N deposition and affect nutrient turnover and economy across a wide range of 740
676 concurrent P depletion (through fire or controlled management terrestrial systems. 741
677 removal of nutrients), soil conditions may also favour invasion and A review by Smithwick et al. (2005) evaluated 24 studies 742
678 eventual dominance by species that are well adapted to P limitation investigating the effects of various fire types on forest ecosystems. 743
679 (e.g. Molinia caerulea, Tomassen et al., 2003), with implications for They found that soil NHþ 
4 and NO3 concentrations generally 744
680 the diversity, structure and conservation value of heathland increased between 2 and 26 times following a fire, with the pulse 745
681 ecosystems. lasting less than two years. Conflicting results were found with 746
682 Leaching losses of soil nutrients have been shown elsewhere to regard to mineralisation rates but, where increases were observed, 747
683 be high following a fire if the soil has a low adsorption capacity they were attributed to a much reduced uptake of nutrients by 748
684 (Robertson and Davies, 1965). Post-fire losses of NHþ 4 and NO3

aboveground vegetation, reduced microbial immobilisation (Klo- 749
685 were, however, low in the current study as, in fact, they had been on patek et al., 1990), increased soil temperatures (Hobbs and Schimel, Q2 750
686 all measurement occasions since the start of the experiment in 1984), increased anion exchange at depth (Matson et al., 1987) and 751
687 1998. An earlier 15N tracer study at the site demonstrated a very higher pH levels in the soil (Allen, 1964; Christensen and Muller, 752
688 strong N immobilisation capacity in this system, with >75% of 15N 1975; Stevenson et al., 1996). A significant increase in soil pH was 753
689 inputs rapidly immobilised in the soil microbial biomass (Green, observed in both the Control and þN plots compared to pre-fire 754
690 2005). Whilst fire undoubtedly affected the size and activity of levels and this may have contributed to the increase in post-fire 755
691 microbial populations, their rapid recovery and high N sink levels of microbial activity in the experimental plots. An increase 756
692 strength may explain the very low leaching losses of N observed in pH has been reported in many systems following wildfire due to 757
693 after the fire. In contrast, however, not only were Ca2þ, Kþ, Cl and the addition of alkaline salts in ash deposits and the destruction of 758
694 SO2
4 concentrations elevated in the first few months following the organic matter (DeBano et al., 1976; Giovannini et al., 1990; 759
695 fire e a likely consequence of ash deposition following combustion Forgeaard and Frenot, 1996). Overall the results of this study show 760

Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildfire?, Soil
Biology & Biochemistry (2012), http://dx.doi.org/10.1016/j.soilbio.2012.08.025
 SBB5265_proof ■ 15 September 2012 ■ 7/9

E.R. Green et al. / Soil Biology & Biochemistry xxx (2012) 1e9 7

761 that, even though nutrient stocks are undoubtedly reduced by fire, supporting our second hypothesis. Whilst effects on specific 826
762 nutrient availability may be increased in the short term. microbial functional groups were not apparent, clear N-treatment 827
763 and temporal differences in the activity and composition of the soil 828
764 4.2. Soil microbial communities microbial community were found, also proving support for our 829
765 second hypothesis. These findings, combined with previous 830
766 Pre-fire data collected at this site have shown clearly that N evidence of persistent, long term impacts of historical N loading at 831
767 addition affects the structure and activity of soil microbial this site (Power et al., 2006), suggest that belowground effects of N 832
768 communities (Green, 2005). Post-fire assessments revealed strong, and fire may be highly persistent. This study clearly shows that, 833
769 persistent effects of N addition on the structure of bacterial and while severe summer fires have the potential to remove a propor- 834
770 fungal communities. Soil microbial communities were seen to be tion of accumulated soil N stores, deposition-driven differences in 835
771 highly dynamic, changing substantially between successive nutrient availability and microbial community composition are 836
772 measurements, although with clear differences between Control quickly re-established and the long term consequences of elevated 837
773 and þN plots evident on each measurement occasion. Wildfire may N inputs are, therefore, unlikely to be beneficial at an ecosystem 838
774 actually make soil more favourable for bacteria (Raison, 1979; level. 839
775 Alexander, 1977) due to an increase in pH and inputs of soluble 840
776 carbohydrates. Renbuss et al. (1973) studied soil microbiology and Acknowledgements 841
777 nutrient changes following wildfire which sterilised the soil in 842
778 a forest ecosystem over a 15 month period. Bacterial recovery was This study was funded through an Urgency Grant from the UK 843
779 reported to be swift, with populations increasing significantly, to Natural Environment Research Council (NE/E011063/1). We are 844
780 ten times pre-fire levels within a month, although it took a year for grateful to the Site Wardens at Thursley Common, Simon Nobes and 845
781 bacterial community structure to return to pre-fire compositions. In James Giles, for their support for this long term experiment, and to 846
782 the current study, the compositions of both bacterial (Fig. S1) and Kim Prior for assistance in both the field and laboratory. We would 847
783 fungal (Fig. S2) communities were highly dynamic in the year also like to thank Dr Nathan Callaghan for comments on earlier 848
784 following fire, with persistent differences in composition apparent versions on the manuscript and Dr David Ames for analysis of 849
785 between control and þN plots. Measurements of microbial activity leachate chemistry. 850
786 suggest that some of the temporal differences in community 851
787 composition may have been linked to variations in soil moisture. 852
Appendix A. Supplementary data
788 Although the presence of functional genes for ammonia oxida- 853
789 tion, N fixation and nitrous oxide reduction, all of which are key 854
Supplementary data related to this article can be found at http://
790 steps in the N cycle, were not affected by N-treatment, it is probable 855
dx.doi.org/10.1016/j.soilbio.2012.08.025.
791 that the relative abundance and activity of associated organisms are 856
792 related to changes in soil chemistry. Whilst clear treatment-related 857
793 differences in soil chemistry were apparent in August 2006, no N References 858
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Please cite this article in press as: Green, E.R., et al., How does N deposition affect belowground heathland recovery following wildfire?, Soil
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