Fertility practices and rhizosphere effects alter ammonia oxidizer community structure and potential nitrification activity in pepper production soils

Applied Soil Ecology 99 (2016) 70–77

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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Fertility practices and rhizosphere effects alter ammonia oxidizer community structure and potential nitrification activity in pepper production soils Matt A. Rudisilla , Ron F. Turcob , Lori A. Hoaglanda,* a b

Department of Horticulture and Landscape Architecture, Purdue University, 625 Agriculture Mall Dr., West Lafayette, IN, United States Department of Agronomy, 915 W. State St., West Lafayette, IN, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 July 2015 Received in revised form 11 October 2015 Accepted 13 October 2015 Available online xxx

Increasing nitrogen use efficiency is critical to the productivity and long-term sustainability of vegetable production systems in the US Midwest. Understanding the impact of alternative fertility sources on the ecology of the soil nitrogen cycle has potential to allow for management that will increase crop N uptake and reduce loss. We determined how repeated applications of urea, composted chicken litter, hairy vetch green manure with alfalfa meal, and an unfertilized control affected the structure and potential nitrification activity (PNA) of ammonia oxidizers in bulk and rhizosphere soil when N needs were expected to be high. PNA was greater in animal and green manure treatments relative to urea and the unfertilized control in bulk soil, and greater in the rhizosphere relative to bulk soil regardless of fertility treatment. A strong correlation was observed between PNA and ammonia oxidizing bacteria (AOB) abundance, suggesting that AOB rather than ammonia oxidizing archaea (AOA) controlled nitrification in this system. However, AOB abundance did not differ between bulk and rhizosphere soil, and PNA was lower in the urea treatment despite greater AOB abundance indicating that other factors could have affected PNA. For example, greater availability of labile carbon (C) could have stimulated PNA through various mechanisms, and lower pH and/or specific nitrification potential per AOB cell could have reduced PNA in the urea treatment. AOB community structure was more diverse in all fertility treatments relative to the unfertilized control in bulk soil, and community structure differed between bulk and rhizosphere soil indicating niche differentiation. However, differences in AOB community structure and PNA were only observed in rhizosphere relative to bulk soil indicating that the rhizosphere had a greater effect on nitrification dynamics than fertility practices. These findings indicate that organic fertility amendments stimulate PNA, but they could also increase N loss and should be investigated further. The rhizosphere appears to play a greater role in nitrification dynamics than fertility practices, and more detailed investigations at this key plant-soil interface are warranted. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Rhizosphere Vegetable production Sweet pepper (Capsicum annuum) Ammonia oxidizing bacteria (AOB) Ammonia oxidizing archaea (AOA)

Introduction Demand for fresh, locally sourced vegetables is growing rapidly in the US (Timmons and Wang, 2010), resulting in increased production in non-traditional areas like the Midwest which is currently dominated by cereal crops. Nitrogen (N) is the most limiting nutrient in vegetable crops and growers apply substantial amounts of fertilizer to meet plant needs. Only 50% of fertilizer N is generally utilized by most crops (Smil, 1999), however, and this

* Corresponding author. Fax: +1 765 494 0391. E-mail address: [email protected] (L.A. Hoagland). http://dx.doi.org/10.1016/j.apsoil.2015.10.011 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

could be even lower in vegetables because they have less extensive root systems and are more intensively managed than cereals. For example, Zhu et al. (2005) found that only 10% of fertilizer N was recovered in aboveground pepper biomass, and 52% was lost from the soil–plant system. Fertilizer N not recovered by crops is subject to loss via nitrate (NO3
) leaching and evolution of nitrous oxide (N2O), a potent greenhouse gas. Vegetable growers often utilize organic fertility sources because of positive impacts on soil quality (Hoagland et al., 2008; Rudisill et al., 2015), and potential to reduce NO3
leaching and N2O emissions relative to inorganic fertilizers (Drinkwater et al., 1998; Kramer et al., 2006). However, unlike inorganic N fertilizers, organic fertility amendments must be

M.A. Rudisill et al. / Applied Soil Ecology 99 (2016) 70–77

mineralized before N is available for crop uptake. Consequently, synchronizing N availability with critical periods of crop uptake is challenging. Greater understanding of ecological factors that regulate the soil N cycle is needed to increase N uptake and reduce loss in emerging vegetable systems. Nitrification is one of the first steps in the soil N cycle ultimately directing the pathway of N in agricultural systems. During nitrification ammonium (NH4+) is oxidized to nitrite (NO2
) and then NO3
in a two-step process. Nitrate is a highly water soluble compound that can be taken up by plant roots, immobilized in microbial biomass, leached to aquatic systems, or emitted as N2O via nitrification or subsequent denitrification processes (Cameron et al., 2013). It is now clear that both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) contribute to the first and rate limiting step in nitrification, but there is still much debate about the relative contribution of each group as well as factors that influence their activity in agricultural soils. For example, potential nitrification rates have been correlated with AOA population size in some studies (Hallin et al., 2009; He et al., 2007), while in others rates are correlated with AOB population size (Ai et al., 2013; Glaser et al., 2010; Jia and Conrad, 2009). AOB and AOA belong to separate phylogenetic domains with different cell metabolic and biochemical processes which could lead to niche differentiation and specialization in response to various soil biotic and abiotic factors (Prosser and Nicol, 2012; Wessen et al., 2010). Alternatively, wide ecophysiological diversity within each group could contribute to functional redundancy (Schauss et al., 2009). Studies comparing inorganic fertilizers and raw animal manures have observed changes in ammonia oxidizer populations and potential nitrification activity (PNA) (Ai et al., 2013; Chu et al., 2008; Enwall et al., 2007; Hallin et al., 2009; Wessen et al., 2010). Results varied widely given soils and the type of fertilizer applied, however, and the mechanisms regulating these differences are still unclear. In some studies, AOB populations and nitrification activity were more stimulated by inorganic fertilizers than animal manure (Chu et al., 2008; Strauss et al., 2014), whereas in another incorporation of animal manure in conjunction with an inorganic fertilizer resulted in greater nitrification activity and abundance of AOA and AOB compared to inorganic fertilizer alone (He et al., 2007). Vegetable growers rarely use raw animal manure because of food safety concerns and instead rely on composted materials and leguminous cover crops to supply N. These amendments are generally more stable and have less immediately available NH4+ than raw animal manure which is likely to influence the soil N cycle. For example, while raw animal manure often increases N2O emissions relative to inorganic N fertilizer, solid organic fertilizers have been found to produce an average of 28% fewer emissions than inorganic fertilizer (Aguilera et al., 2013). Few studies have compared composted animal manure or cover crops with inorganic fertilizers on nitrification dynamics, however, particularly in intensively managed vegetable systems. The plant rhizosphere is a zone of intense microbial activity that can have profound effects on plant nutrient acquisition and health (Berendsen et al., 2012). Nitrifying populations (Chen et al., 2008; Hussain et al., 2011; Kleineidam et al., 2011) and PNA (Ai et al., 2013; Enwall et al., 2007) are often enhanced in the rhizosphere compared to bulk soil. These differences are thought to result from rhizodeposition, a process by which plant roots release organic C stimulating ammonification from soil organic matter (Herman et al., 2006). Such relationships are likely to be critical to enhancing crop N uptake, particularly in the presence of organic fertility amendments. However, limited knowledge exists of how alternative fertilizer sources influence nitrification dynamics in the rhizosphere (Ai et al., 2013), and additional studies are needed to optimize N dynamics at this key plant-soil interface.

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The objective of this study was to determine how three years of repeated applications of urea, composted chicken litter, green manure based on cover crop of hairy vetch (Vicia villosa) plus alfalfa meal (Medicago sativa), and an unfertilized control affected the structure and potential nitrification activity of ammonia oxidizing organisms in bulk soil and the rhizosphere of sweet pepper (Capsicum annuum). We hypothesized that (1) PNA activity would be stimulated by organic relative to inorganic fertility amendments, (2) PNA activity would be greater in the rhizosphere vs. bulk soil, and (3) differences in PNA activity would be correlated with increased abundance and diversity of ammonia oxidizing communities. 2. Materials and methods 2.1. Site description, soil treatments, and sampling The field trial was conducted at Meigs Horticulture Research Farm (40 170 21.05100 –86 530 3.1200 ) in Tippecanoe County, Indiana. Soils were from the Drummer series (fine-silty, mixed, superactive, mesic Typic Endoaquolls). The experiment was conducted for three years with the amendments applied in each growing season (2011–2013) at rates estimated to supply sufficient N for the pepper crop. Four treatments were: urea, partially composted and dehydrated chicken litter, green manure (fall-seeded hairy vetch supplemented with dehydrated alfalfa meal), and an unamended control. The carbon:nitrogen (C:N) ratio of the chicken litter, hairy vetch, and alfalfa meal amendments were 7:1, 11:1, and 15:1, respectively, resulting in 5233 and 10,669 kg ha
1 C added over three years to the AM and GM plots respectively. Details of the initial soil characteristics, climate, cropping history, and amendment application rates are explained in Rudisill et al. (2015). Plots were arranged in a randomized complete block design with four replicates. All plots were tilled each spring to incorporate amendments and prepare beds for transplanting. Ten replicate soil cores were collected and pooled within each plot to a depth of 15.0 cm on August 10, 2013, when plant were actively growing and producing fruit and N needs were expected to be high. Rhizosphere samples were obtained by removing two randomly selected plants from each plot, shaking roots, and collecting soil adhering to plant roots. Replicate samples were pooled in polyethylene bags, placed on ice during transport, and stored at -80  C, 4  C, or air-dried upon arrival for downstream DNA isolation, potential enzymatic assays, and chemical analyses, respectively. 2.2. Soil geochemistry Mineral N (ammonium (NH4+) and total NOx (NO3
and NO2
) was quantified spectrophotometrically using a SEAL AQ2 discrete analyzer (SEAL Analytical, Mequon, WI) following 1.0 M KCl extraction (1:2.5 soil to solution ratio w/v) of air-dried, 2.0 mm sieved soil samples. Soil solution pH was measured in a slurry containing a 1:2 ratio of air-dried, 2.0 mm sieved soil to deionized water (Kalra, 1995). Total C and N were quantified by combustion on a FlashEA 1112 (Thermo Scientific, Waltham, MA, USA). Gravimetric soil moisture content was determined by drying 25 g of field moist soil at 80  C. 2.3. Potential nitrification activity Assays for potential nitrification activity (PNA) were carried out using a microscale method based on ISO 15685 (Hoffmann et al., 2007). Briefly, 2.5 g of fresh soil was placed in a 50 mL flask, and test medium (300 mM KH2PO4, 700 mM K2HPO4, 10 mM sodium chlorate, and 1.5 mM (NH4)2SO4; pH 7.2) was added to create slurry with exact volume of 10 mL. The reaction was incubated at

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Table 1 Thermocycling conditions, qPCR standard range, and qPCR efficiencies of the ammonia monooxygenase amoAfor archaea (AOA) and bacteria (AOB). Group

Cycling conditions

Standard range

qPCR efficiency

AOA AOB

30 s at 95  C followed by 40 cycles of 30 s at 95  C, 30s at 53  C, 60 s at 72  C (data acquisition) 30 s at 95  C followed by 40 cycles of 30 s at 95  C, 30 s at 57  C, 60 s at 72  C; 30 s at 81  C (data acquisition)a

1E3–1E7 1E2–1E6

72 79

a

Denotes step not used in PCR amplification of T-RFLP samples.

25  C and shaken at 175 rpm. At 2 and 4 h, 1.0 mL samples were collected and added to 1.0 mL 4.0 M KCl, briefly vortexed and centrifuged to separate soil particles. The resulting supernatant was removed and stored at
20  C until analysis. Nitrite concentrations were determined spectrophotometrically at 540 nm using a BioTek Epoch plate reader (BioTek, Winooski, VT, USA).

standard deviation of 1.0, and peaks were aligned with a clustering threshold of 0.5 bp in T-REX (Culman et al., 2009). Total peak area was relativized within each sample, and relative abundance across samples was averaged to create a matrix for use in subsequent analysis. 2.7. Statistical analysis

2.4. DNA extraction and normalization Community genomic DNA (cgDNA) was isolated from two 0.5 g soil samples per plot using PowerSoil DNA Extraction Kits (MoBio Laboratories, CA, USA) with a modified bead-beating protocol. The two replicates from each plot were pooled and analyzed for purity using a NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), and concentrations of total extracted ds-DNA determined using a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA). Samples were adjusted to 10 ng/mL using filter-sterilized ultra-pure H2O. 2.5. Quantitative PCR and standard preparation All qPCR reactions were carried out on a StepOne Real-Time PCR system (Applied Biosystems, CA, USA) in triplicate 15 mL reactions containing the following ingredients: 7.5 mL of Fast-SYBR Green Master Mix (Life Technologies, CA, USA), 4.5 mL ultra-pure H2O, 1.0 mL each of 0.4 nM forward and reverse primers for AOA (Francis et al., 2005) and AOB (Rotthauwe et al., 1997), and 1.0 mL of diluted cgDNA. Cycling protocols given in Table 1. The specificities of each amplification were confirmed using a melting curve across the temperature range 60–95  C. Quantitative-PCR standards were created from purified pCR2.1-TOPO (Life Technologies) plasmid DNA including the target gene isolated from OneShot Top10 chemically competent E. coli(Life Technologies). Gene identity and size were confirmed through sequencing followed by nBLAST. Standard ranges, and qPCR efficiencies are given in Table 1. All R2 values were 0.99 for both AOA and AOB amoAstandard curves. Soil nitrification potential per AOB amoAgene copy number was calculated to estimate differences in metabolic activity in response to fertility treatments in bulk and rhizosphere soil following Chu et al. (2008). 2.6. Terminal restriction fragment length polymorphism of bacterial amoA The bacterial ammonia monooxygenase, amoA, was PCR amplified in triplicate 50 mL reactions with 50 -6-FAM forward primers used for qPCR, and the thermocycling conditions presented in Table 1 without the data acquisition step (using 30 amplification cycles instead of 40). Replicate reactions were pooled and checked for amplification specificity on a 1.0% agarose gel. The amoAproducts were purified using ethanol precipitation, and resuspended in sterile 18.0 MV H2O. One microgram of amoAproduct was digested using HaeIII(New England Biolabs, Beverly, MA, USA) according to the manufacturer's instructions. Digested samples were submitted to the University of Illinois Core DNA Sequencing Facility for fragment analysis. Restriction fragments greater than 50 bp and less than 500 bp were used in downstream analysis. Noisy peaks were filtered from true peaks using a

All simple statistics, tests for normality and homogeneity of variance, and analysis of variance (ANOVA) were carried out in SAS 9.2 (SAS Institute, Cary, NC, USA). All data were initially checked for normality according to the Shapiro–Wilk test using PROC UNIVARIATE, and non-normal data were Box-Cox transformed using PROC TRANSREG. Levene’s test was used in order to test for homogeneity of variance when a two-way ANOVA model was used. One-way ANOVA was used to test impacts of fertility treatment within either bulk or rhizosphere soil, and a two-way ANOVA was used to compare experiment-wide differences between fertility treatments and sample location (bulk versus rhizosphere soil) and their interaction using PROC GLM. Relationships between soil chemical properties, AOA and AOB populations, and PNA were determined using spearman rank correlation analyses using PROC CORR. Differences were determined as significant at the p  0.05 probability level, unless otherwise stated. All multiple comparisons were made using Tukey-adjusted least squared means when the ANOVA F-test was statistically significant. Multivariate analysis of bacterial amoAT-RFs was carried out in Canoco v.5 for Windows (Microcomputer Power, Ithaca, NY). Redundancy analysis (RDA) with forward selection of environmental variables was initially conducted to analyze the influence of environmental variables on amoAcommunity structure. RDA revealed that no measured environmental variable significantly explained variation in the amoAcommunity structure, so principal component analysis (PCA) was performed using environmental variables as supplementary data to determine how soil chemical and biological parameters correlated withamoA.

Table 2 Mean soil geochemical properties within bulk soil (0–15 cm) or rhizosphere soil from four fertility treatments. Treatment

pH

Total C (g kg
1)

Total N (g kg
1)

NH4+–N (mg kg
1)

NOx–N (mg kg
1)

Bulk soil Control Urea Animal manure Green manure

7.36 aa 6.81 b 7.29 a 7.10 ab

18.6 a 18.8 a 18.3 a 21.8 a

1.43 1.46 1.54 1.73

b b ab a

2.10 ab 2.24 a 1.97 ab 1.48 b

3.73 a 4.15 a 6.06 a 7.43 a

Rhizosphere Control Urea Animal manure Green manure

7.19 aa 6.87 b 7.04 ab 7.21 a

24.1 a 25.8 a 24.7 a 24.9 a

1.69 1.76 1.95 1.96

b ab a a

1.41 a 1.29 a 1.53 a 1.75 a

4.98 b 17.8 a 12.6 ab 15.0 a

a Different letters within a column represent significant difference between treatments within soil fractions as determined by Tukey adjusted least square means.

M.A. Rudisill et al. / Applied Soil Ecology 99 (2016) 70–77

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3. Results

3.3. Quantification of ammonia-oxidizing archaea and bacteria

3.1. Soil and amendment chemical properties

Impacts of fertility treatments on AOA and AOB amoAgene abundances were more pronounced within bulk than rhizosphere soil. The abundance of AOA amoAwas significantly greater in the bulk soil receiving urea than in the green manure treated soil (Table 4), but gene copy number was not different between fertility treatments within rhizosphere soil. Gene copy number of AOB amoAwas greater in the urea and animal manure treatments compared to the control in bulk soil (Table 4), but was not impacted by fertility treatments within the rhizosphere. When compared across sample locations, no statistically significant differences in gene copy number of either AOA or AOB amoAwere observed between fertility treatments or rhizosphere and bulk soil (Table 5). Soil nitrification potential per AOB amoAgene copy number was lower in the urea treatment relative to the unfertilized control in bulk soil (Table 4), but did not differ between bulk and rhizosphere soil (Table 5).

Both bulk and rhizosphere soil pH was significantly (p > 0.05) impacted by fertility treatment. In bulk soil, pH was lowered by the urea treatment relative to the animal manure and unfertilized control (Table 2). In the rhizosphere soil, the urea treatment lowered pH compared to the green manure and control treatments. When compared across bulk and rhizopshere soils, pH in the urea treatment was lower than all other treatments, and soil pH was not different between bulk and rhizosphere soil (Table 3). Total C was unaffected by fertility treatments within bulk or rhizosphere soil (Table 2). In contrast, greater total C was observed in rhizosphere versus bulk soil when compared across fertility treatments (Table 3). Total N in the green manure treatment was greater than the urea and control treatments in bulk soil, whereas both green manure and animal manure treatments had greater total N than the control in the rhizosphere (Table 2). When compared across sample locations, total N was greater in green manure and animal manure than urea and control treatments, and there was more total N in the rhizosphere than bulk soil (Table 3). Ammonium–N was greater in bulk soil receiving urea than green manure, but no differences were observed between fertility treatments within rhizosphere soil (Table 2), or when treatments were compared across sample locations (Table 3). However, rhizosphere soil contained less NH4+–N than bulk soil. NOx–N was not affected by fertility treatments in bulk soil, but urea and green manure treatments were greater than the control in rhizosphere soil (Table 2). When compared across sample locations, NOx–N was greater in all treatments receiving fertility inputs than the control, and NOx–N was greater in rhizosphere than bulk soil (Table 3). 3.2. Potential nitrification activity Animal and green manure treatments promoted a higher rate of PNA than urea and control treatments in bulk soil, but there were no differences among fertility treatments in rhizosphere soil (Table 4). When compared across sample locations, PNA was greater in soil receiving animal manure than the unamended control, and greater PNA was also observed in rhizosphere compared to bulk soil. (Table 5).

Table 3 Mean soil geochemical properties between bulk soil (0–15 cm) and rhizosphere soil and four fertility treatments. Treatment

pH

Total C (g kg
1)

Total N (g kg
1)

NH4+–N (mg kg
1)

NOx–N (mg kg
1)

Fraction Bulk soil Rhizosphere

7.14 aa 7.08 a

19.4 b 24.9 a

1.54 b 1.84 a

1.95 a 1.50 b

5.34 b 12.6 a

Amendment Control Urea Animal manure Green manure

7.27 aa 6.84 b 7.16 a 7.16 a

21.4 a 22.3 a 21.5 a 23.4 a

1.56 a 1.61 a 1.74 b 1.85 b

1.76 1.77 1.75 1.61

4.36 b 11.0 a 9.33 a 11.2 a

P>F 0.3864 0.0012 0.2252

<0.0001 0.1996 0.2530

<0.0001 <0.0001 0.2830

0.0003 0.6999 0.0034

Factor Fraction (F) Amendment (A) FxA

df 1 3 3

a a a a

<0.0001 0.0028 0.0194

a Different letters within a column represent significant difference as determined by Tukey adjusted least square means.

3.4. Correlations of soil chemical and biological properties When compared across fertility treatments and sample locations, soil PNA was positively correlated with AOB amoAgene abundance, total N, and NOx-N, while a negative correlation was found between PNA and NH4+–N (Table 6). No significant correlations were observed between the abundance of AOB amoAwith soil chemical properties, though the abundance of AOA amoAwas positively correlated with NH4+–N. 3.5. Bacterial amoA community structure and diversity A total of 20 amoAT-RFs were found across all samples (Fig. 1). Bulk soil receiving urea amendments had the greatest number of TRFs (13), while the control treatment in bulk soil was dominated by only two T-RFs. Using PCA, the first principal component (Axis 1) explained 73.03% of the variation in amoAT-RFs, while the second principal component (Axis 2) explained 12.77% (Fig. 2). The PCA ordination biplot revealed clear differences in amoAcommunity structure of the fertility treatments between bulk and rhizosphere soil. Soils receiving animal manure and green manure inputs were separated across Axis 1, while urea amended soils were separated by Axis 2. The control treatment in bulk versus rhizosphere soil was separated by both Axis 1 and Axis 2. However, no clear pattern was observed between fertility treatments within either the bulk or rhizosphere soils. Soil chemical and biological properties are represented on the biplot graph as arrows, and estimated values within a treatment can be made by drawing a perpendicular line from the projected arrows to the treatment. The direction of the arrow indicates predicted increase for that variable. Shannon–Wiener diversity indices based on the relative abundance of amoAT-RFs indicated that addition of all fertility inputs resulted in greater amoAdiversity and eveness than the control in bulk soil samples (data not shown). When compared across fertility treatments and locations, diversity was greater in the animal manure than control treatment, but there was no difference between rhizosphere and bulk soil (data not shown). 4. Discussion Increasing N use efficiency is critical to the productivity and long-term sustainability of emerging vegetable production systems in the US Midwest. In this study, we show that amending soil with partially composted animal and green manure increased PNA relative to soils receiving urea and an unfertilized control in an intensively managed pepper crop. This is consistent with other studies that observed enhanced nitrification activity in soils

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Table 4 Mean potential nitrification activity (PNA), ammonia-oxidizing archaea amoAabundance (AOA), ammonia-oxidizing bacteria amoAabundance (AOB), and specific nitrification potential per AOB cell (SNP) within bulk soil (0–15 cm) or rhizosphere soil from four fertility treatments. Treatment

PNA (nmol NO2
–N g
1 h
1)

AOA (copies g
1 soil)

AOB (copies g
1 soil)

SNP (nmol NO2
–N g
1 h
1 AOB copies
1 g
1 soil)

Bulk soil Control Urea Animal manure Green manure

522.0 ba 487.4 b 1198.9 a 1014.0 a

8.63e6 ab 1.29e7 a 1.10e7 ab 6.26e6 b

1.84e6 b 1.07e7 a 7.73e6 a 6.32e6 ab

3.14e
4 a 5.83e
5 b 1.62e
4 ab 2.02e
4 ab

Rhizosphere Control Urea Animal manure Green manure

877.7 aa 1424.8 a 1221.7 a 1100.9 a

5.90e6 a 7.95e7 a 9.92e7 a 7.41e6 a

5.55e6 a 5.54e6 a 7.57e6 a 6.07e6 a

1.63e
4 a 4.23e
4 a 1.80e
4 a 2.71e
4 a

a

Different letters represent significant difference between treatments within soil fractions as determined by Tukey adjusted least square.

amended with raw animal manure (Chu et al., 2008; Fan et al., 2011; He et al., 2007) or straw mulch (Wessen et al., 2010), compared to those receiving inorganic fertilizer amendments alone. Stimulation of PNA could increase N acquisition and result in greater crop productivity. For example, NO3
has greater mobility in soil than NH4+ resulting in more rapid diffusion to plant roots, and it synergistically promotes uptake of other key nutrients such as K+ and Ca2+, whereas NH4+ competes with these cations for uptake (Boudsocq et al., 2012). Many plants, including sweet pepper (Marti and Mills, 1991), also prefer NO3
over NH4+ as an N source, and NH4+ can be toxic at high concentrations. However, NO3
is also highly subject to leaching, and greater PNA during periods when crops are not actively taking up nutrients could increase N loss. Plots receiving composted animal manure and a legume cover crop have been found to have greater PNA than those receiving inorganic fertilizer in autumn following crop harvest (Kong et al., 2010). Future studies comparing organic and inorganic fertility amendments should account for temporal variation over the growing season to better understand N dynamics and identify effective strategies to reduce N loss. A better mechanistic understanding of factors that regulate nitrification dynamics in response to alternative fertility amendments is needed to optimize trade-offs between N availability and potential for loss. Several factors could explain greater PNA in organic treatments relative to the inorganic and unfertilized control observed in this study. In contrast to inorganic fertilizers like urea, organic amendments must mineralize before they are available for plant uptake or oxidation by nitrifying soil microorganisms—resulting in a more gradual release of NH4+ over the growing season (Burger and Jackson, 2008). Prolonged release of

NH4+ as a result of ammonification could have promoted more continuous expression of genes controlling ammonia oxidation, and possibly explain greater PNA in plots receiving animal or green manure inputs. Total N was greater in the green manure than urea and control treatments, while NH4+ was lower in the green manure than urea treatment. Total N and NH4+ were both correlated with PNA, indicating that greater ammonification in plots receiving organic fertility treatments could be tied to PNA activity in this soil. However, future studies that quantify gene expression are needed to confirm this hypothesis. Another factor that could have contributed to greater PNA in plots receiving organic amendments was stimulation of heterotrophic activity in response to concurrent C additions. Amending soil with C supplements has previously been shown to stimulate PNA, suggesting that mineralization and nitrification could be complementary (Wheatley et al., 2001). While we did not observe differences in total C among fertility treatments in bulk soil despite substantial C applications over the three year study period, we previously reported greater active soil carbon and microbial activity in the animal and green manure treatments relative to the urea and unfertilized control (Rudisill et al., 2015). Greater total C and PNA activity were observed in the rhizosphere relative to bulk soil in this study, which further supports our hypothesis that C availability could have stimulated PNA in this soil. Nitrification has long been thought to be primarily an autotrophic process in agricultural systems with differences in population dynamics driven primarily by changes in NH3 availability, soil pH, moisture, and temperature (Hallin et al., 2009). Several studies have now provided evidence for mixotrophic or heterotrophic growth in AOA (Prosser and Nicol, 2012), however, and in some cases even AOB

Table 5 Mean potential nitrification activity (PNA), ammonia-oxidizing archaea amoAabundance (AOA), and ammonia-oxidizing bacteria amoAabundance (AOB) between bulk soil (0–15 cm) and rhizosphere soil, four fertility amendments, and their interaction. Treatment Fraction Bulk soil Rhizosphere Amendment Control Urea Animal manure Green manure Factor Fraction (F) Amendment (A) FxA a

df 1 3 3

PNA (nmol NO2
–N g
1 h
1)

AOA (copies g
1 soil)

AOB (copies g
1 soil)

SNP (nmol NO2
–N g
1 h
1 AOB copy
1 g
1 soil)

805.6 ba 1156.3 a

9.71e6 a 7.79e6 a

6.37e6 a 6.19e6 a

1.84e
4 a 2.59e
4 a

700.0 ba 956.1 ab 1210.3 a 1057.5 ab

7.27e6 a 1.04e7 a 1.05e7 a 6.84e6 a

3.70e6 a 8.06e6 a 7.65e6 a 5.70e6 a

2.38e
4 a 2.40e
4 a 1.71e
4 a 2.37e
4 a

P>F 0.0205 0.0445 0.1119

0.1084 0.0534 0.3052

0.9686 0.0572 0.0828

0.2471 0.8343 0.0596

Different letters represent significant difference between treatments within soil fractions as determined by Tukey adjusted least square means.

M.A. Rudisill et al. / Applied Soil Ecology 99 (2016) 70–77 Table 6 Spearman correlations of soil chemical properties, potential nitrification activity (PNA), ammonia-oxidizing archaea amoAabundance (AOA), and ammonia-oxidizing bacteria amoAabundance (AOB).

AOA AOB pH Ctot Ntot NH4–N NOx–N * **

PNA

AOA

AOB


0.047 0.583** -0.018 0.312 0.405*
0.353* 0.543**

– 0.210
0.170
0.078
0.115 0.385* 0.005

0.210 –
0.099
0.049 0.038 0.056 0.216

Correlation is significant at the P < 0.05 level. Correlation is significant at the P < 0.01 level.

(Kouki et al., 2011). All AOA and AOB contain genes encoding enzymes required for organotrophic growth which could be an important factor in niche differentiation (Prosser and Nicol, 2012). Alternatively, greater PNA associated with higher amounts of C could be attributed to indirect effects mediated by stimulation of other heterotrophs (Prosser and Nicol, 2012; Wheatley et al., 2001). Finally, it is well known that amending soil with C can alter soil physical properties, and changes in soil moisture and temperature in response to greater C additions could have enhanced PNA in the animal and green manure treatments. Changes in ammonia oxidizing community structure could also be related to differences in PNA observed in this study. AOA and AOB coexist in soil, but their relative abundance and activity is likely to vary in response to changes in environmental conditions induced by alternative fertility amendments. Several studies have observed correlations between increased abundance of AOA and PNA in response to raw animal manure (Ai et al., 2013; He et al., 2007; Schauss et al., 2009) and straw (Wessen et al., 2010) compared to inorganic fertility amendments alone. This has been attributed to greater availability of labile C, and the potential for AOA to utilize mixotrophic or heterotrophic growth strategies (Walker et al., 2010). In this study, AOA relative abundance was greater than AOB which is consistent with others (Jia and Conrad, 2009; Strauss et al., 2014). However, AOA abundance was only elevated in urea relative to the green manure treatment, and AOA were not correlated with PNA. In addition, the ratio of AOB to AOA increased in all fertility treatments relative to the unfertilized

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control, and AOB were strongly correlated with PNA across fertility treatments suggesting that AOB rather than AOA more likely controlled nitrification processes in this soil. Others have also demonstrated that AOB is more likely to contribute to nitrification dynamics in agricultural systems than AOA (Ai et al., 2013; Di et al., 2010; Fan et al., 2011; Jia and Conrad, 2009), which has been attributed to contrasting physiologies between AOA and AOB, as well as soil pH (Prosser and Nicol, 2012). In culture-based studies, AOB have been found to have greater NO2
production rates than AOA (de la Torra et al., 2008; Konneke et al., 2005; Okano et al., 2004; Prosser, 1989; Ward, 1987), suggesting that AOB have potential to respond more rapidly to N fertility inputs. In contrast, AOA appear to exert greater control on nitrification in soils with low pH (Erguder et al., 2009; Yao et al., 2011). There were no correlations between soil pH or AOA and PNA in this study, however, and soil pH was lower in urea-amended soils relative to the green manure which could explain greater abundance of AOA in the urea relative to the green manure treatment. Several studies have observed increased abundance and activity of AOB in response to N fertilization (Mendum et al., 1999; Phillips et al., 2000). In this trial, AOB abundance was greater in the urea and animal manure plots relative to the unfertilized control, though PNA was lower in the urea than animal and green manure treatments, and not different from the unfertilized control. Thus, AOB abundance alone does not appear to be directly related to PNA in this study. It is possible that lower PNA in urea plots could have been related to lower pH, as AOB amoAtranscripts have been shown to decrease in response to reduced soil pH while gene copy numbers remain unaltered (Nicol et al., 2008). However, AOB abundance was not elevated in the rhizosphere relative to the bulk soil despite greater PNA which is consistent with the findings of Towe et al., (2010). Alternatively, differences in PNA could be attributed to changes in the metabolic activity of AOB. For example, Chu et al. (2008) observed lower specific nitrification potential per AOB cell (SNP) in an inorganic fertilizer relative to an organic manure treatment indicating that AOB were less metabolically active in the inorganic fertility treatment. Intense competition for NH4+ with heterotrophs in the presence of greater C availability could select for species of ammonia oxidizers that are more metabolically efficient. In our trial, SNP was lower in the urea relative to the unfertilized control, which could help explain why

Fig. 1. Relative abundance of T-RF’s representing AOB amoA community structure from four fertility treatments: unamended control (UC), urea (UR), animal manure (AM), and green manure (GM), in rhizosphere (RZ) and bulk soil (BS).

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1.0

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additional studies using isotopic approaches and sequencing technologies that can assign taxonomic identity of microbial diversity are needed to confirm these claims.

AM

UC

Axis 2 (12.77%)

UR

PNA

TN

5. Conclusions

TC

UR GM

NOx-N

AM NH4-N pH

-1.0

GM UC

-1.0

Axis 1 (73.03%)

1.0

Fig. 2. Principal component biplot of bacterial amoAT-RFs in bulk soil (0–15 cm; *; filled circles) and rhizosphere soil (&; open squares) from four fertility treatments: unamended control (UC), urea (UR), animal manure (AM), and green manure (GM). Arrows represent soil pH (pH), total carbon (TC), total nitrogen (TN), ammonium (NH4–N), nitrate plus nitrite (NOx–N), and potential nitrification activity (PNA).

PNA activity was not enhanced in this treatment despite higher AOB abundance. However, while quantification of functional genes themselves can shed light on the transcriptional potential for ammonia oxidation, it should not serve as a stand-alone predictor of nitrification dynamics in soil and future studies should quantify gene expression rather than presence alone. Changes in AOB community structure is another factor that could be related to differences in PNA observed in this study. There are at least seven distinct clusters of AOB within the b-subclass of proteobacteria (Kowalchuk et al., 2000; Stephen et al., 1996), with each group likely possessing distinct physiological properties that could be influenced by soil properties or agricultural practices (Enwall et al., 2007). For example, Nitrosospiracluster 4 are prevalent in unfertilized soil (Kowalchuk et al., 2000), while group 3 are common in agricultural soils amended with NH4+ rich fertilizers (Bruns et al., 1999; Phillips et al., 2000). Changes in PNA has been observed in soils where AOB community structure differed in response to different types and rates of fertility amendments (Ai et al., 2013; Enwall et al., 2007; Wang et al., 2009; Wu et al., 2011). In our study, all fertility treatments appeared to have greater AOB diversity than the unfertilized control in bulk soil, though PNA was only stimulated in the animal and green manure treatment and thus do not appear to be directly related in this soil compartment. In contrast, AOB community structure appeared to be significantly different in the rhizosphere relative to bulk soil, and PNA was greater in the rhizosphere indicating that community structure could be correlated with process rates at this location. Root exudates of barley have previously been shown to enhance the growth of some nitrifying strains, while not affecting or suppressing the growth of others (Satoh et al., 2003). Plants compete fiercely for NH4+ in the rhizosphere, and some plant species have even been shown to release nitrification inhibitors from their roots (Subbarao et al., 2007). Intense competition for resources with heterotrophs stimulated by rhizodeposits could also influence community structure at this location. Wessen et al. (2010) proposed that expected competition with heterotrophs in the presence of high C:N ratio conditions likely selects for AOB with high affinities for ammonia. Changes in nitrifier community structure could regulate N uptake and reduce N loss, though

Results of this study demonstrate that nitrification dynamics are influenced by contrasting fertility practices in intensively managed vegetable systems, and organic fertility practices stimulate PNA more than an inorganic fertilizer. This could lead to greater N availability and enhanced crop productivity, but it could also lead to greater losses from the soil–plant system. Additional studies that take temporal dynamics into account and utilize gene expression to better characterized nitrification dynamics in these systems are warranted. Ammonia-oxidizing bacteria appear to play a larger role in nitrification than their functionally equivalent archaeal counterpart, with changes in AOB community structure potentially contributing to differences in PNA activity. Rhizosphere effects appeared to have a greater effect on AOB community structure than the type of fertilizer applied, indicating that nitrification dynamics at this key plant–soil interface should be further studied to increase nutrient-use efficiency in agricultural systems. Acknowledgements This work was supported by Purdue Agriculture Research Programs. We thank Longjie Cheng and the Purdue Statistical Consulting Service for assistance with statistical analyses, and Nate Linder, Jay Young, and Tristand Tucker for assistance with plot management at the Meigs Horticulture Research Farm. References Aguilera, E., Lassaletta, L., Sanz-Cobena, A., Garnier, J., Vallejo, A., 2013. The potential of organic fertilizers and water management to reduce N2O emissions in Mediterranean climate cropping systems. A review. Agric. Ecosyst. Environ. 111164, 32–52. Ai, C., Liang, G., Sun, J., Wang, X., He, P., Zhou, W., 2013. Different roles of rhizosphere effect and long-term fertilization in the activity and community structure of ammonia oxidizers in a calcerous fluvo-aquic soil. Soil Biol. Biochem. 57, 30–42. Berendsen, R., Pieterse, C., Bakker, P., 2012. The rhizosphere microbiome and plant health. Trends Plant Sci. 17, 478–486. Boudsocq, S., Niboyet, A., Lata, C., Raynaud, X., Loeuille, N., Mattieu, J., Blouin, M., Abdadie, L., Barot, S., 2012. Plant preference for ammonium over nitrate: a neglected determinant of ecosystem functioning. Am. Nat. 180, 60–69. Bruns, M.A., Stephen, J.R., Kowalchuk, G.A., Prosser, J.I., Paul, E.A., 1999. Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in nativetilled, and successional soils. Appl. Environ. Microbiol. 65, 2994–3000. Burger, M., Jackson, E.E., 2008. Microbial immobilization of ammonium and nitrate in relation to ammonification and nitrification rates in organic and conventional cropping systems. Soil Biol. Biochem. 35, 29–36. Cameron, K.C., Di, H.J., Moir, J.L., 2013. Nitrogen losses from the soil/plant system: a review. Ann. Appl. Biol. 162, 145–173. Chen, X.P., Zhu, Y.G., Xia, Y., Shen, J.P., He, J.Z., 2008. Ammonia-oxidizing archaea: reduced carbon and nitrogen losses. Nature 396, 262–265. Chu, H., Fujii, T., Morimoto, S., Lin, X., Yaki, K., 2008. Population size and specific nitrification potential of sol ammonia-oxidizing bacteria under long-term fertilization management. Soil Biol. Biochem. 40, 1960–1963. Culman, S.W., Bukowski, R., Gauch, H.G., Cadillo-Quiroz, H., Buckley, D.H., 2009. TREX: software for the processing and analysis of T-RFLP data. BMC Bioinf. 10, 171. de la Torra, J.R., Walker, C.B., Ingalls, A.E., Konneke, M., Stahl, D.A., 2008. Cultivation of thermophillic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ. Microbiol. 10, 810–818. Di, H., Cameron, K.C., Shen, J., Winefield, C.S., O’Callaghan, M., Bowatte, S., He, J., 2010. Ammmonia-oxidizing bacteria and archaea grow under contrasting soil nitrogen conditions. FEMS Microbiol. Ecol 72, 386–394. Drinkwater, L.E., Wagoner, P., Sarrantonio, M., 1998. Legume-based cropping systems have reduced carbon and nitrogen losses. Nature 396, 262–265. Enwall, K., Nyberg, K., Bertilsson, S., Cederlund, H., Stenstrom, J., Hallin, S., 2007. Long-term impact of fertilization on activity and composition of bacterial communities and metabolic guilds in agricultural soil. Soil Biol. Biochem. 39, 106–115.

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