Science of the Total Environment 497–498 (2014) 91–96
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Effects of different types of N deposition on the fungal decomposition activities of temperate forest soils Shushan Li 1, Yuhan Du 1, Peng Guo ⁎, Lida Guo, Kaiyue Qu, Jianping He Hebei College of Industry and Technology, Hongqi Street 626, Shijiazhuang 050091, China
H I G H L I G H T S • • • •
Fungi were seriously inhibited by inorganic N and significantly promoted by organic N. Greater organic N converted soil microbial communities into fungi-dominated system. Greater organic N addition significantly alleviated soil fungal N-limitation. Excessive organic N deposition caused N saturation and repressed fungal activities.
a r t i c l e
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Article history: Received 25 June 2014 Received in revised form 25 July 2014 Accepted 25 July 2014 Available online xxxx Editor: E. Capri Keywords: Nitrogen deposition Forest soil Fungi Inorganic nitrogen Organic nitrogen Enzymatic activity
a b s t r a c t Nitrogen (N) deposition significantly affects soil microbial activities and litter decomposition processes in forest ecosystems. However, the changes in soil fungi during litter decomposition remain unclear. In this study, ammonium nitrate was selected as inorganic N (IN), whereas urea and glycine were selected as organic N (ON). N fertilizer with different IN-to-ON ratios (1:4, 2:3, 3:2, 4:1, and 5:0) was mixed in equal amounts and then added to temperate forest soils. Half of each treatment was simultaneously added with streptomycin to inhibit soil bacteria. The activities of enzymes involved in litter decomposition (invertase, β-glucosidase, cellulase, polyphenol oxidase, and phosphatase) were assayed after a three-year field experiment. The results showed that enzymatic activities were inhibited by IN addition but accelerated by ON addition in the nonantibiotic addition treatments. An increase in ON in the mixed N fertilizer also shifted enzymatic activities from N inhibition to N stimulation. Similarly, in the antibiotic addition treatments, fungal activities revealed the same trends, but they were seriously inhibited by IN and significantly accelerated by ON. These results indicated that soil fungi were more sensitive to N deposition, particularly to ON. A large amount of ON may convert soil microbial communities into a fungi-dominated system. However, excessive ON deposition (20% IN + 80% ON) caused N saturation and repressed fungal activities. These results suggested that soil fungi were sensitive to N type and that different IN-to-ON ratios may induce diverse ecological effects on soil fungi. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Atmospheric nitrogen (N) deposition is a major threat to ecosystems (Van Den Berg et al., 2011). It affects nearly every aspect of forest ecosystems including microbial enzymatic function, composition, activities and biodiversity, especially for litter decomposition processes (Du et al., 2014; Nilsson et al., 2007). Soil microbial communities are important contributors to organic matter decomposition as various microbial groups take part in the breaking down of organic molecules. However, information on the functions of various microbial groups in breaking down organic molecules and nutrient cycling is limited. Among them, saprophytic fungi have a major function in decomposition because ⁎ Corresponding author. Tel./fax: +86 311 85239006. E-mail address: [email protected] (P. Guo). 1 Each author contributed equally to the work presented here.
http://dx.doi.org/10.1016/j.scitotenv.2014.07.098 0048-9697/© 2014 Elsevier B.V. All rights reserved.
these organisms use dead organic matter as their sources of carbon (C) and energy (Lucas et al., 2007). In soil, fungi produce polyphenol oxidase, cellulase, manganese peroxidase, lignin peroxidase, and other broad-spectrum peroxidases that are known for depolymerizing cellulose and lignin (Sinsabaugh, 2010). The distribution of these enzymes is based on the traditional functional classification of wooddegrading fungi as white rot (predominantly Basidiomycota), brown rot (Ascomycota and Basidiomycota), and soft rot (Ascomycota) (Burns et al., 2013). It has been shown that N deposition has significantly effected on soil fungal communities and activities, however, the findings of previous studies remain controversial. Edwards et al. (2011) showed that simulated N deposition increased the proportion of basidiomycetes recovered from the forest floor and significantly decreased the proportion of ascomycetes in the community. Smolander et al. (1994) found that N deposition increases fungal biomass and changes fungal
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population structure in a Norwegian spruce forest. Nilsson et al. (2007) showed that N input exhibits no effect on the total fungal biomass. However, Carreiro et al. (2000) found that the N inhibition of ligninolytic enzyme activities occurs in litter with high lignin contents and abundant white rot fungi. Allison et al. (2007) suggested that high levels of N availability can affect fungal communities and alter lignocellulolytic enzyme production. Previous research concentrated on inorganic N (IN) deposition and its ecological effects, but interest in organic N (ON) deposition has recently increased because of the increasingly significant contribution (30%) to total atmospheric N deposition (Cornell, 2011). In China, the average contribution of ON to total N is 28% and ranges from 7% to 67% (Zhang et al., 2012). Many reports have revealed rapid turnover rates and tremendous effects of ON on forest ecosystems (Jones et al., 2004; Neff et al., 2002). González et al. (2010) found that ON fertilizers can increase microbial activity in soils between 16% and 20% compared with IN fertilizers. Thirukkumaran and Parkinson (2000) reported larger soil microbial biomass and accelerated litter decomposition after urea fertilization than after NH4NO3 fertilization. Furthermore, N deposition with different IN-to-ON ratios induces different effects on soil microbes. Knorr et al. (2005) reported that single NH4NO3 fertilization inhibits litter decomposition, urea fertilization alone has no significant effect on decomposition, and NH4NO3 plus urea significantly stimulates decomposition. It has been shown that the type and ratio of N are important factors in controlling soil microbial activities, microbial biomass, and soil C cycling (Du et al., 2014; Guo et al., 2011a,b; Knorr et al., 2005). However, the changes in soil fungi community, biomass, activities and their role in litter decomposition remain unclear. In this work, mixed N with different IN-to-ON ratios was added to temperate forest soils in Northern China, while microbial inhibitor (streptomycin) was added on half of each treatment to inhibit bacterial activity. Soil microbial enzymatic activities were assayed after a three-year field experiment. This study aims to investigate the effects of different types of N fertilization on fungal activities in forest soil. We assume that ON deposition will stimulate soil fungi decomposition activities than that of IN. 2. Materials and methods 2.1. Site description and experiment design The study was conducted in Fenglong Mountain Forest Park (37.9° N, 114.3° E) in Shijiazhuang, China. The study site has an area of 30 km2 and a peak of 812 m. It also has a temperate humid climate with an annual mean temperature of 13.3 °C and an annual precipitation of 520 mm. The dominant species in the forest site are Sophora japonica, Populus tomentosa, Armeniaca vulgaris, Salix babylonica, Larix gmelinii, and Platycladus orientalis. The N deposition level in this area is approximately 2.40 g N m−2 yr−1, and ON comprises ~ 35% of the N species in the field (Zhang et al., 2012). A forest site where P. tomentosa is dominant was chosen as the sampling site. Four 2.0 m × 1.5 m plots with a buffer zone of 20 m between any two plots were randomly established in January 2011. Afterward, each plot was subdivided into six subplots (1.0 m × 0.5 m) and was treated with various N solutions. In this study, NH4NO3 was chosen as the IN source, whereas urea and glycine were chosen and mixed equally as the ON source. A mixture of these sources was added to each subplot at different ratios. The subplots received the following treatments: N1 (IN:ON = 1:4), N2 (IN:ON = 2:3), N3 (IN:ON = 3:2), N4 (IN:ON = 4:1), and N5 (IN:ON = 5:0). The Control involved the addition of deionized water only. During the field experiment, each subplot was sprayed with the mixed N solutions at the total amount of 0.9 g N every 3 months, which is equivalent to 7.2 g N m−2 yr−1. Then, each subplot was divided into two parts (0.5 m × 0.5 m). One part, labeled as IS, was added with 2.00 g m−2 streptomycin to inhibit soil bacteria. Another part, labeled as I0, was added with an equal amount of deionized
water. The inhibitor was also added every 3 months. The study was conducted from January 2011 to January 2014. 2.2. Microbial enzymatic activity assays When harvested, soil cores from each subplot were randomly sampled to a depth of 5 cm using a metal corer with a diameter of 5 cm. These cores were stored in sealed bags and immediately transported to the laboratory. In the laboratory, fresh soil samples were passed through a 2 mm mesh to eliminate leaves, plant root, gravel, and were used for soil enzymatic activity assays. The activities of enzymes involved in decomposition were assayed using a UV–vis spectrophotometer. These enzymes include invertase, β-glucosidase, cellulase, polyphenol oxidase, and phosphatase. Table 1 lists the methods of enzymatic assays and international unit (IU) definition. All data were expressed by dry soil weight. 2.3. Statistical analyses All data were statistically analyzed by ANOVA. Significant differences were accepted at the P b 0.05 level of probability. Factor analysis was conducted to understand the effects of IN (Factor 1, F1) and ON (Factor 2, F2) on soil enzymatic activities. The codified model for factorial design is as follows: ηEA ¼ A0 þ A1 F1 þ A2 F2 ; where A0 represents the global mean and A1 and A2 represent the regression coefficients of F1 and F2, respectively. In this model, both IN and ON were assumed to exhibit positive effects on soil enzymatic activities. All statistical analyses were performed using SPSS 17.0 for Windows. 3. Results In the end of field experiment, cultivable soil bacterial count was determined by the plate count method. Bacteria were isolated in IS treatments, but the count was significantly less than that in I0 treatments (0.88 vs. 10.8 × 106 colony g−1 soil). In this work, the activities of most enzymes in I0 treatments were higher than those in Is treatments and soil enzyme activities were variously affected by N fertilizer with different IN-to-ON ratios. In I0 treatment groups, the invertase activity of Control (4.01 IU) was lower than those of all N treatments (4.21 IU to 4.31 IU) and N3 (4.31 IU) showed the highest invertase activity among all treatment. In IS treatment groups, the activity of Control (3.62 IU) was lower than those of N1 and N2 (3.81 and 3.98 IU, respectively) and higher than those of others (3.15 IU to 3.19 IU). While, the IS/I0 value of Control (0.902) was higher than those of all N treatments except for N2 (0.944), N5 (0.731) had the lowest ratio among all treatments (Fig. 1a). In I0 treatment groups, the β-glucosidase activity of Control (0.689 IU) was lower than that of N1 (0.759 IU) and higher than those of the others (0.503 IU to 0576 IU). In IS treatment groups, the activity of Control (0.518 IU) was lower than those of N1 (0.558 IU) and N2 (0.548 IU), and significantly higher than those of the other treatments (0.287 IU to 0.346 IU, P b 0.05). Similar to invertase, the IS/I0 value of the Control (0.751) was lower than that of N2 (0.953). N4 (0.548) and N5 (0.571) had the lowest IS/I0 values among all treatments (Fig. 1b). In I0 treatment groups, the cellulase activity of Control (10.74 IU) was higher than those of all N treatments except for N1 (11.56 IU). In IS treatment groups, the cellulase activity of Control (7.43 IU) was lower than those of N1 (9.59 IU) and N2 (10.12 IU) but higher than those of the other treatments (4.55 IU to 6.06 IU). The IS/I0 value of the Control (0.691) was lower than those of all N treatments (0.829 to 1.038), except for N4 (0.628) and N5 (0.675) (Fig. 1c).
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Table 1 Soil enzymatic activity assay methods, their enzyme commission number (EC), corresponding substrates, active international unit (IU) definition, and references. Enzyme
EC
Substrate
IU definition
References
Invertase Cellulase Polyphenol oxidase β-Glucosidase Phosphatase
EC 3.2.1.26 EC 3.2.1.4 EC 1.10.3.1 EC 3.2.1.21 EC 3.1.3.1
Sucrose Sodium tylose Catechol p-Nitrophenyl glucoside p-Nitrophenyl phosphate
1 mg glucose released 24 h−1 g−1 soil 1 mg glucose released min−1 g−1 soil absorbance change of 0.01 U min−1 g−1 soil 1 mg pNP released h−1 g−1 soil 1 mg pNP released h−1 g−1 soil
Frankenberger and Johanson (1983) Ghose (1987) Perucci et al. (2000) Eivazi and Tabatabai (1988) Turner et al. (2002)
In I0 treatment groups, the polyphenol oxidase activity of the Control (3.17 IU) was significantly lower than that of N1 (3.57 IU, P b 0.05) but higher than those of others (2.99 IU to 3.16 IU). In IS treatment groups, the activity of the Control (2.71 IU) was lower than those of all N treatments except for N1 (2.65 IU). N2 (3.53 IU, P b 0.05) showed the highest polyphenol oxidase activity among all treatments. The activities of N2 (3.53 IU) and N3 (3.09 IU) in IS treatment groups were higher than those in I0 treatment groups (3.17 and 2.99 IU, respectively). The Control (0.823) had the lowest IS/I0 value among all treatments (0.876 to 1.114) (Fig. 1d).
In I0 treatment groups, the phosphatase activities of all N treatments (0.351 IU to 0.394 IU) were higher than that of Control (0.321 IU). N2 (0.394 IU) showed the highest phosphatase activities among all treatments. Similarly, the Control (0.269 IU) in IS treatment groups exhibited the lowest activities among all N treatments. However, no significant difference in phosphatase activities was detected among the N treatments (0.279 IU to 0.314 IU). The IS/I0 value of the Control (0.838) was higher than those of all N treatments (0.783 to 0.803). N1 (0.783) had the lowest IS/I0 value among all treatments (Fig. 1e).
Fig. 1. Responses of soil enzymatic activities to various mixed N fertilization treatments: (a) invertase, (b) β-glucosidase, (c) cellulase, (d) polyphenol oxidase and (e) phosphatase. The treatments were Control (deionized water), N1 (IN:ON = 1:4), N2 (IN:ON = 2:3), N3 (IN:ON = 3:2), N4 (IN:ON = 4:1) and N5 (IN:ON = 5:0). The international unit (IU) of each enzyme is defined in Table 1. Error bars indicate standard deviation (SD, n = 4).
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Factor analysis revealed that F1 (IN) in I0 treatments negatively affected (A1 = − 0.973 to − 2.258) all enzymes, except for invertase (A1 = 0.662) and phosphatase (A1 = 0.924). By contrast, F2 (ON) positively affected (A2 = 0.504 to 1.579) all enzymes. In IS treatments, the F1 values were negative (A1 = −2.175 to −1.441) for all enzymes, except for polyphenol oxidase (A1 = 0.074) and phosphatase (A1 = 0.340). By contrast, the F2 values were positive (A2 = 1.026 to 2.350). Meanwhile, the A2 values were higher in IS than in I0 for all enzymes (Table 2, Supplemental material 1).
4. Discussion Soil enzymatic activities are important indicators of soil microbial activities (Kaiser et al., 2010). The changes in enzymatic activities reflect the response of soil microorganisms to N deposition. Most studies have focused on the effect of N deposition on soil microbial activities. It has been shown that soil microbial biomass, litter decomposition process, enzymatic activities, and their relationship are all affected by N type and IN-to-ON ratio (Du et al., 2014; Guo et al., 2011a,b; Knorr et al., 2005; Thirukkumaran and Parkinson, 2000). However, the changes in different microorganisms remain unknown. In the current study, streptomycin was the antibiotic used to selectively kill bacteria (leaving only fungi). Our results showed that fungal activities showed different responses to the different types and ratios of N deposition. Invertase is a ubiquitous enzyme in soil. It is widely distributed in bacteria, fungi, and other microorganisms. This enzyme is closely related to soil microbial biomass (Guo et al., 2011b) and is partially responsible for the breakdown of plant litter in soils (Gianfreda et al., 1995). In the current study, N deposition accelerated soil invertase activities in non-antibiotic addition treatments. The present results are similar to the reports of Manning et al. (2008) and Wang et al. (2008) who used NH4NO3 and urea separately as fertilizer. The activities also increased with increasing of ON. Fungal invertase activities were significantly inhibited by single IN fertilization but significantly promoted by ON. However, a large amount of ON addition (20% IN + 80% ON) inhibited fungal invertase activities. Similar results were reported by Guo et al. (2011b) who found reduced soil enzymatic activities after fertilization with extremely high ON percentage in mixed N (10% IN + 90% ON). β-Glucosidase, cellulase, and polyphenol oxidase are important enzymes involved in the decomposition of complex organic matter (cellulose and lignin), which are the main components of soil organic C in soil (Plessis et al., 2010). These enzymes are mainly secreted by fungi, particularly white rot, brown rot, and soft rot fungi (Allison et al., 2009; Rineau et al., 2013; Wurzburger et al., 2012). Previous studies revealed that β-glucosidase showed positive (Weand et al., 2010), negative (Freeman et al., 2001) or non-significant (Thomas et al., 2012) responses to N fertilizations. Meanwhile, N addition accelerated cellulase activity (Geisseler and Horwath, 2009) but repressed polyphenol oxidase activity (Hofmockel et al., 2007; Weand et al., 2010). In the current study, β-glucosidase, cellulase, and polyphenol oxidase activities were inhibited by sole IN addition. These responses are consistent with those reported by Weand et al. (2010) and Table 2 Factor analysis of IN and ON on soil enzymatic activities of IS and I0 treatment groups. Enzyme
Invertase β-Glucosidase Cellulase Polyphenol oxidase Phosphatase
I0
IS
A0
A1
A2
A0
A1
A2
−0.516 0.823 0.935 0.050 −0.988
0.662 −1.982 −2.258 −0.973 0.924
0.555 0.504 0.583 1.309 1.579
0.365 0.746 0.183 −0.820 −0.707
−1.575 −2.175 −1.542 0.074 0.340
1.267 1.026 1.764 2.350 1.611
Legend: A0 represents the global mean, A1 and A2 represent the regression coefficients of F1 (inorganic N, IN) and F2 (organic N, ON), respectively.
Geisseler and Horwath (2009). These findings can be attributed to the nitrogenous compounds that can condense with carbohydrates and promote melanoidin production, which may increase polyphenol polymerization (Treseder, 2008). However, the repression on these enzymatic activities weakened with the increase of ON and even converted to stimulation, particularly with the addition of a large amount of ON. This finding may be due to the presence of directly utilizable ON, such as glycine, which was turned over, assimilated very rapidly and promotes microbial activities (Jones et al., 2004). Alternatively, lower soil pH decline was shown after urea addition than after NH4NO3 addition, as decreases in soil pH may have deleterious effects on soil microbial communities (Zhang et al., 2008). For soil fungi, these enzymatic activities were also inhibited by IN. However, these activities were more seriously inhibited than those of non-antibiotic addition treatments. Frey et al. (2004) found the declined fungal biomass after NH4NO3 addition and significant reduction in phenol oxidase activity. However, these fungal enzymatic activities increased and accelerated more rapidly than those of non-antibiotic addition treatments as fertilizer of ON increased. Exceptionally, cellulase and polyphenol oxidase activities were inhibited by excessive ON addition (20% IN + 80% ON). Keyser et al. (1978) found that the lignindecomposing enzymatic activity of white rot fungi (e.g., Phanerochaete chrysosporium) declined when amino acids accumulate. This result can be attributed to the fact that soil microbes prefer to directly use amino acid as C and N resources, indicating a decline in nutrient uptake by the process of decomposition. Therefore, extreme ON deposition can repress litter decomposition by fungi and promote humus accumulation in the surface layer of the forest soil. Litter decomposition is an enzymatically complex process, which is mainly mediated by a species-rich community of saprotrophic fungi. It has been shown that litter decomposition rates are sensitive to N availability in the environment (Edwards et al., 2011; Osono, 2007). It also has been demonstrated that increasing N inputs may alter microbial community structure activities, functions, and fungi-to-bacterial ratios (Compton et al., 2004; Waldrop et al., 2004). In the current study, IS-to-I0 ratios were used to compare the responses of fungi and total microbes to different N types. The decline of the ratio after single IN addition also indicated that soil fungi were seriously inhibited by IN. This finding is consistent with the reports of Tietema (1998) and Holland and Coleman (1987) − who suggested that N (NH+ 4 and NO3 ) addition can shift a soil microbial community from a fungi-dominated system into a bacteria-dominated system. Meanwhile, the ratio increased when ON was added. Factor analysis also showed the same conclusion as the A1 values of IS treatments were higher than those of I0 treatments for all enzymes. This finding may be attributed to the stoichiometric differences between fungi and other microbes because fungi have higher C/N ratios than other microbes and therefore could have lower N demands (Griepentrog et al., 2014). Consequently, a shift toward bacterial biomass is expected when N limitation is reduced due to atmospheric N deposition while C sources remain equally accessible (Strickland and Rousk, 2010). These results indicated that the N limitation of soil fungi was easily alleviated and ON significantly accelerated this alleviation. We hypothesized that microbial community soil may even be converted into a fungi-dominated system with increasing ON percentage in atmosphere N. However, the ratio declined in the treatment with excessive ON percentage (20% IN + 80% ON). This may be attributed to the higher soil pH after urea addition than after NH4NO3 addition under the same amount (data not shown), as Caldwell (2005) has demonstrated that fungal extracellular enzymes have acidic optima while bacterial sources generally have neutral-alkaline optima. Phosphatase activity is a good indicator of the organic P mineralization potential and biological activity of soils (Dick et al., 2000). In contrast to the responses of other enzymes, phosphatase activities increased after IN addition. This finding is consistent with the results of previous studies (Hogan et al., 2010; Ochoa-Hueso et al., 2013; Turner
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et al., 2003). Similar to other enzymes, phosphatase activities also increased with increasing ON addition. This finding indicated that both IN and ON can promote P mineralization. However, for soil fungi, the influence of different N types on phosphatase activity was not significant. Excessive ON even inhibited its activity, although phosphatase activities increased in all N treatments compared to the Control. These findings indicated that the promoting effect of N addition on phosphatase activities in fungi was not more significant than in other microbes such as bacteria and actinobacteria. Meanwhile, the declined IS-to-I0 value of phosphatase indicated that other soil microbes (such as bacteria) may be the dominated decomposer of complex P compounds after N addition, as we found significant increase in phosphobacteria after N addition, particularly after excessive ON addition (data not shown). As the result, fungi may be seriously limited by P after N deposition, specifically after ON deposition. This finding may explain the decline in some enzymatic activities in the treatments with excessive ON. In summary, soil fungi showed different responses from other microorganisms in response to N deposition. Fungi were seriously inhibited by IN and significantly accelerated by ON. Greater ON accelerated the conversion of soil microbial communities into a fungidominated system, but excessive ON deposition (20% IN + 80% ON) caused N saturation and badly repressed fungal activities. These results suggested that soil fungi were sensitive to N type and that different IN-to-ON ratios may induce diverse ecological effects on soil fungi. However, the three-year field experiment only reflected the shortterm responses of different N depositions to forest soils of fungi. A ten-year observation has been conducted. Furthermore, whether fungal diversity shows different responses to various mixed N additions should also be investigated. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.07.098.
Acknowledgments This work was supported by Scientific Foundation for the Introduction of Researcher of Hebei College of Industry and Technology, China (No. BZ1101).
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Effects of different types of N deposition on the fungal decomposition activities of temperate forest soils Shushan Li 1, Yuhan Du 1, Peng Guo ⁎, Lida Guo, Kaiyue Qu, Jianping He Hebei College of Industry and Technology, Hongqi Street 626, Shijiazhuang 050091, China
H I G H L I G H T S • • • •
Fungi were seriously inhibited by inorganic N and significantly promoted by organic N. Greater organic N converted soil microbial communities into fungi-dominated system. Greater organic N addition significantly alleviated soil fungal N-limitation. Excessive organic N deposition caused N saturation and repressed fungal activities.
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 25 July 2014 Accepted 25 July 2014 Available online xxxx Editor: E. Capri Keywords: Nitrogen deposition Forest soil Fungi Inorganic nitrogen Organic nitrogen Enzymatic activity
a b s t r a c t Nitrogen (N) deposition significantly affects soil microbial activities and litter decomposition processes in forest ecosystems. However, the changes in soil fungi during litter decomposition remain unclear. In this study, ammonium nitrate was selected as inorganic N (IN), whereas urea and glycine were selected as organic N (ON). N fertilizer with different IN-to-ON ratios (1:4, 2:3, 3:2, 4:1, and 5:0) was mixed in equal amounts and then added to temperate forest soils. Half of each treatment was simultaneously added with streptomycin to inhibit soil bacteria. The activities of enzymes involved in litter decomposition (invertase, β-glucosidase, cellulase, polyphenol oxidase, and phosphatase) were assayed after a three-year field experiment. The results showed that enzymatic activities were inhibited by IN addition but accelerated by ON addition in the nonantibiotic addition treatments. An increase in ON in the mixed N fertilizer also shifted enzymatic activities from N inhibition to N stimulation. Similarly, in the antibiotic addition treatments, fungal activities revealed the same trends, but they were seriously inhibited by IN and significantly accelerated by ON. These results indicated that soil fungi were more sensitive to N deposition, particularly to ON. A large amount of ON may convert soil microbial communities into a fungi-dominated system. However, excessive ON deposition (20% IN + 80% ON) caused N saturation and repressed fungal activities. These results suggested that soil fungi were sensitive to N type and that different IN-to-ON ratios may induce diverse ecological effects on soil fungi. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Atmospheric nitrogen (N) deposition is a major threat to ecosystems (Van Den Berg et al., 2011). It affects nearly every aspect of forest ecosystems including microbial enzymatic function, composition, activities and biodiversity, especially for litter decomposition processes (Du et al., 2014; Nilsson et al., 2007). Soil microbial communities are important contributors to organic matter decomposition as various microbial groups take part in the breaking down of organic molecules. However, information on the functions of various microbial groups in breaking down organic molecules and nutrient cycling is limited. Among them, saprophytic fungi have a major function in decomposition because ⁎ Corresponding author. Tel./fax: +86 311 85239006. E-mail address: [email protected] (P. Guo). 1 Each author contributed equally to the work presented here.
http://dx.doi.org/10.1016/j.scitotenv.2014.07.098 0048-9697/© 2014 Elsevier B.V. All rights reserved.
these organisms use dead organic matter as their sources of carbon (C) and energy (Lucas et al., 2007). In soil, fungi produce polyphenol oxidase, cellulase, manganese peroxidase, lignin peroxidase, and other broad-spectrum peroxidases that are known for depolymerizing cellulose and lignin (Sinsabaugh, 2010). The distribution of these enzymes is based on the traditional functional classification of wooddegrading fungi as white rot (predominantly Basidiomycota), brown rot (Ascomycota and Basidiomycota), and soft rot (Ascomycota) (Burns et al., 2013). It has been shown that N deposition has significantly effected on soil fungal communities and activities, however, the findings of previous studies remain controversial. Edwards et al. (2011) showed that simulated N deposition increased the proportion of basidiomycetes recovered from the forest floor and significantly decreased the proportion of ascomycetes in the community. Smolander et al. (1994) found that N deposition increases fungal biomass and changes fungal
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population structure in a Norwegian spruce forest. Nilsson et al. (2007) showed that N input exhibits no effect on the total fungal biomass. However, Carreiro et al. (2000) found that the N inhibition of ligninolytic enzyme activities occurs in litter with high lignin contents and abundant white rot fungi. Allison et al. (2007) suggested that high levels of N availability can affect fungal communities and alter lignocellulolytic enzyme production. Previous research concentrated on inorganic N (IN) deposition and its ecological effects, but interest in organic N (ON) deposition has recently increased because of the increasingly significant contribution (30%) to total atmospheric N deposition (Cornell, 2011). In China, the average contribution of ON to total N is 28% and ranges from 7% to 67% (Zhang et al., 2012). Many reports have revealed rapid turnover rates and tremendous effects of ON on forest ecosystems (Jones et al., 2004; Neff et al., 2002). González et al. (2010) found that ON fertilizers can increase microbial activity in soils between 16% and 20% compared with IN fertilizers. Thirukkumaran and Parkinson (2000) reported larger soil microbial biomass and accelerated litter decomposition after urea fertilization than after NH4NO3 fertilization. Furthermore, N deposition with different IN-to-ON ratios induces different effects on soil microbes. Knorr et al. (2005) reported that single NH4NO3 fertilization inhibits litter decomposition, urea fertilization alone has no significant effect on decomposition, and NH4NO3 plus urea significantly stimulates decomposition. It has been shown that the type and ratio of N are important factors in controlling soil microbial activities, microbial biomass, and soil C cycling (Du et al., 2014; Guo et al., 2011a,b; Knorr et al., 2005). However, the changes in soil fungi community, biomass, activities and their role in litter decomposition remain unclear. In this work, mixed N with different IN-to-ON ratios was added to temperate forest soils in Northern China, while microbial inhibitor (streptomycin) was added on half of each treatment to inhibit bacterial activity. Soil microbial enzymatic activities were assayed after a three-year field experiment. This study aims to investigate the effects of different types of N fertilization on fungal activities in forest soil. We assume that ON deposition will stimulate soil fungi decomposition activities than that of IN. 2. Materials and methods 2.1. Site description and experiment design The study was conducted in Fenglong Mountain Forest Park (37.9° N, 114.3° E) in Shijiazhuang, China. The study site has an area of 30 km2 and a peak of 812 m. It also has a temperate humid climate with an annual mean temperature of 13.3 °C and an annual precipitation of 520 mm. The dominant species in the forest site are Sophora japonica, Populus tomentosa, Armeniaca vulgaris, Salix babylonica, Larix gmelinii, and Platycladus orientalis. The N deposition level in this area is approximately 2.40 g N m−2 yr−1, and ON comprises ~ 35% of the N species in the field (Zhang et al., 2012). A forest site where P. tomentosa is dominant was chosen as the sampling site. Four 2.0 m × 1.5 m plots with a buffer zone of 20 m between any two plots were randomly established in January 2011. Afterward, each plot was subdivided into six subplots (1.0 m × 0.5 m) and was treated with various N solutions. In this study, NH4NO3 was chosen as the IN source, whereas urea and glycine were chosen and mixed equally as the ON source. A mixture of these sources was added to each subplot at different ratios. The subplots received the following treatments: N1 (IN:ON = 1:4), N2 (IN:ON = 2:3), N3 (IN:ON = 3:2), N4 (IN:ON = 4:1), and N5 (IN:ON = 5:0). The Control involved the addition of deionized water only. During the field experiment, each subplot was sprayed with the mixed N solutions at the total amount of 0.9 g N every 3 months, which is equivalent to 7.2 g N m−2 yr−1. Then, each subplot was divided into two parts (0.5 m × 0.5 m). One part, labeled as IS, was added with 2.00 g m−2 streptomycin to inhibit soil bacteria. Another part, labeled as I0, was added with an equal amount of deionized
water. The inhibitor was also added every 3 months. The study was conducted from January 2011 to January 2014. 2.2. Microbial enzymatic activity assays When harvested, soil cores from each subplot were randomly sampled to a depth of 5 cm using a metal corer with a diameter of 5 cm. These cores were stored in sealed bags and immediately transported to the laboratory. In the laboratory, fresh soil samples were passed through a 2 mm mesh to eliminate leaves, plant root, gravel, and were used for soil enzymatic activity assays. The activities of enzymes involved in decomposition were assayed using a UV–vis spectrophotometer. These enzymes include invertase, β-glucosidase, cellulase, polyphenol oxidase, and phosphatase. Table 1 lists the methods of enzymatic assays and international unit (IU) definition. All data were expressed by dry soil weight. 2.3. Statistical analyses All data were statistically analyzed by ANOVA. Significant differences were accepted at the P b 0.05 level of probability. Factor analysis was conducted to understand the effects of IN (Factor 1, F1) and ON (Factor 2, F2) on soil enzymatic activities. The codified model for factorial design is as follows: ηEA ¼ A0 þ A1 F1 þ A2 F2 ; where A0 represents the global mean and A1 and A2 represent the regression coefficients of F1 and F2, respectively. In this model, both IN and ON were assumed to exhibit positive effects on soil enzymatic activities. All statistical analyses were performed using SPSS 17.0 for Windows. 3. Results In the end of field experiment, cultivable soil bacterial count was determined by the plate count method. Bacteria were isolated in IS treatments, but the count was significantly less than that in I0 treatments (0.88 vs. 10.8 × 106 colony g−1 soil). In this work, the activities of most enzymes in I0 treatments were higher than those in Is treatments and soil enzyme activities were variously affected by N fertilizer with different IN-to-ON ratios. In I0 treatment groups, the invertase activity of Control (4.01 IU) was lower than those of all N treatments (4.21 IU to 4.31 IU) and N3 (4.31 IU) showed the highest invertase activity among all treatment. In IS treatment groups, the activity of Control (3.62 IU) was lower than those of N1 and N2 (3.81 and 3.98 IU, respectively) and higher than those of others (3.15 IU to 3.19 IU). While, the IS/I0 value of Control (0.902) was higher than those of all N treatments except for N2 (0.944), N5 (0.731) had the lowest ratio among all treatments (Fig. 1a). In I0 treatment groups, the β-glucosidase activity of Control (0.689 IU) was lower than that of N1 (0.759 IU) and higher than those of the others (0.503 IU to 0576 IU). In IS treatment groups, the activity of Control (0.518 IU) was lower than those of N1 (0.558 IU) and N2 (0.548 IU), and significantly higher than those of the other treatments (0.287 IU to 0.346 IU, P b 0.05). Similar to invertase, the IS/I0 value of the Control (0.751) was lower than that of N2 (0.953). N4 (0.548) and N5 (0.571) had the lowest IS/I0 values among all treatments (Fig. 1b). In I0 treatment groups, the cellulase activity of Control (10.74 IU) was higher than those of all N treatments except for N1 (11.56 IU). In IS treatment groups, the cellulase activity of Control (7.43 IU) was lower than those of N1 (9.59 IU) and N2 (10.12 IU) but higher than those of the other treatments (4.55 IU to 6.06 IU). The IS/I0 value of the Control (0.691) was lower than those of all N treatments (0.829 to 1.038), except for N4 (0.628) and N5 (0.675) (Fig. 1c).
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Table 1 Soil enzymatic activity assay methods, their enzyme commission number (EC), corresponding substrates, active international unit (IU) definition, and references. Enzyme
EC
Substrate
IU definition
References
Invertase Cellulase Polyphenol oxidase β-Glucosidase Phosphatase
EC 3.2.1.26 EC 3.2.1.4 EC 1.10.3.1 EC 3.2.1.21 EC 3.1.3.1
Sucrose Sodium tylose Catechol p-Nitrophenyl glucoside p-Nitrophenyl phosphate
1 mg glucose released 24 h−1 g−1 soil 1 mg glucose released min−1 g−1 soil absorbance change of 0.01 U min−1 g−1 soil 1 mg pNP released h−1 g−1 soil 1 mg pNP released h−1 g−1 soil
Frankenberger and Johanson (1983) Ghose (1987) Perucci et al. (2000) Eivazi and Tabatabai (1988) Turner et al. (2002)
In I0 treatment groups, the polyphenol oxidase activity of the Control (3.17 IU) was significantly lower than that of N1 (3.57 IU, P b 0.05) but higher than those of others (2.99 IU to 3.16 IU). In IS treatment groups, the activity of the Control (2.71 IU) was lower than those of all N treatments except for N1 (2.65 IU). N2 (3.53 IU, P b 0.05) showed the highest polyphenol oxidase activity among all treatments. The activities of N2 (3.53 IU) and N3 (3.09 IU) in IS treatment groups were higher than those in I0 treatment groups (3.17 and 2.99 IU, respectively). The Control (0.823) had the lowest IS/I0 value among all treatments (0.876 to 1.114) (Fig. 1d).
In I0 treatment groups, the phosphatase activities of all N treatments (0.351 IU to 0.394 IU) were higher than that of Control (0.321 IU). N2 (0.394 IU) showed the highest phosphatase activities among all treatments. Similarly, the Control (0.269 IU) in IS treatment groups exhibited the lowest activities among all N treatments. However, no significant difference in phosphatase activities was detected among the N treatments (0.279 IU to 0.314 IU). The IS/I0 value of the Control (0.838) was higher than those of all N treatments (0.783 to 0.803). N1 (0.783) had the lowest IS/I0 value among all treatments (Fig. 1e).
Fig. 1. Responses of soil enzymatic activities to various mixed N fertilization treatments: (a) invertase, (b) β-glucosidase, (c) cellulase, (d) polyphenol oxidase and (e) phosphatase. The treatments were Control (deionized water), N1 (IN:ON = 1:4), N2 (IN:ON = 2:3), N3 (IN:ON = 3:2), N4 (IN:ON = 4:1) and N5 (IN:ON = 5:0). The international unit (IU) of each enzyme is defined in Table 1. Error bars indicate standard deviation (SD, n = 4).
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Factor analysis revealed that F1 (IN) in I0 treatments negatively affected (A1 = − 0.973 to − 2.258) all enzymes, except for invertase (A1 = 0.662) and phosphatase (A1 = 0.924). By contrast, F2 (ON) positively affected (A2 = 0.504 to 1.579) all enzymes. In IS treatments, the F1 values were negative (A1 = −2.175 to −1.441) for all enzymes, except for polyphenol oxidase (A1 = 0.074) and phosphatase (A1 = 0.340). By contrast, the F2 values were positive (A2 = 1.026 to 2.350). Meanwhile, the A2 values were higher in IS than in I0 for all enzymes (Table 2, Supplemental material 1).
4. Discussion Soil enzymatic activities are important indicators of soil microbial activities (Kaiser et al., 2010). The changes in enzymatic activities reflect the response of soil microorganisms to N deposition. Most studies have focused on the effect of N deposition on soil microbial activities. It has been shown that soil microbial biomass, litter decomposition process, enzymatic activities, and their relationship are all affected by N type and IN-to-ON ratio (Du et al., 2014; Guo et al., 2011a,b; Knorr et al., 2005; Thirukkumaran and Parkinson, 2000). However, the changes in different microorganisms remain unknown. In the current study, streptomycin was the antibiotic used to selectively kill bacteria (leaving only fungi). Our results showed that fungal activities showed different responses to the different types and ratios of N deposition. Invertase is a ubiquitous enzyme in soil. It is widely distributed in bacteria, fungi, and other microorganisms. This enzyme is closely related to soil microbial biomass (Guo et al., 2011b) and is partially responsible for the breakdown of plant litter in soils (Gianfreda et al., 1995). In the current study, N deposition accelerated soil invertase activities in non-antibiotic addition treatments. The present results are similar to the reports of Manning et al. (2008) and Wang et al. (2008) who used NH4NO3 and urea separately as fertilizer. The activities also increased with increasing of ON. Fungal invertase activities were significantly inhibited by single IN fertilization but significantly promoted by ON. However, a large amount of ON addition (20% IN + 80% ON) inhibited fungal invertase activities. Similar results were reported by Guo et al. (2011b) who found reduced soil enzymatic activities after fertilization with extremely high ON percentage in mixed N (10% IN + 90% ON). β-Glucosidase, cellulase, and polyphenol oxidase are important enzymes involved in the decomposition of complex organic matter (cellulose and lignin), which are the main components of soil organic C in soil (Plessis et al., 2010). These enzymes are mainly secreted by fungi, particularly white rot, brown rot, and soft rot fungi (Allison et al., 2009; Rineau et al., 2013; Wurzburger et al., 2012). Previous studies revealed that β-glucosidase showed positive (Weand et al., 2010), negative (Freeman et al., 2001) or non-significant (Thomas et al., 2012) responses to N fertilizations. Meanwhile, N addition accelerated cellulase activity (Geisseler and Horwath, 2009) but repressed polyphenol oxidase activity (Hofmockel et al., 2007; Weand et al., 2010). In the current study, β-glucosidase, cellulase, and polyphenol oxidase activities were inhibited by sole IN addition. These responses are consistent with those reported by Weand et al. (2010) and Table 2 Factor analysis of IN and ON on soil enzymatic activities of IS and I0 treatment groups. Enzyme
Invertase β-Glucosidase Cellulase Polyphenol oxidase Phosphatase
I0
IS
A0
A1
A2
A0
A1
A2
−0.516 0.823 0.935 0.050 −0.988
0.662 −1.982 −2.258 −0.973 0.924
0.555 0.504 0.583 1.309 1.579
0.365 0.746 0.183 −0.820 −0.707
−1.575 −2.175 −1.542 0.074 0.340
1.267 1.026 1.764 2.350 1.611
Legend: A0 represents the global mean, A1 and A2 represent the regression coefficients of F1 (inorganic N, IN) and F2 (organic N, ON), respectively.
Geisseler and Horwath (2009). These findings can be attributed to the nitrogenous compounds that can condense with carbohydrates and promote melanoidin production, which may increase polyphenol polymerization (Treseder, 2008). However, the repression on these enzymatic activities weakened with the increase of ON and even converted to stimulation, particularly with the addition of a large amount of ON. This finding may be due to the presence of directly utilizable ON, such as glycine, which was turned over, assimilated very rapidly and promotes microbial activities (Jones et al., 2004). Alternatively, lower soil pH decline was shown after urea addition than after NH4NO3 addition, as decreases in soil pH may have deleterious effects on soil microbial communities (Zhang et al., 2008). For soil fungi, these enzymatic activities were also inhibited by IN. However, these activities were more seriously inhibited than those of non-antibiotic addition treatments. Frey et al. (2004) found the declined fungal biomass after NH4NO3 addition and significant reduction in phenol oxidase activity. However, these fungal enzymatic activities increased and accelerated more rapidly than those of non-antibiotic addition treatments as fertilizer of ON increased. Exceptionally, cellulase and polyphenol oxidase activities were inhibited by excessive ON addition (20% IN + 80% ON). Keyser et al. (1978) found that the lignindecomposing enzymatic activity of white rot fungi (e.g., Phanerochaete chrysosporium) declined when amino acids accumulate. This result can be attributed to the fact that soil microbes prefer to directly use amino acid as C and N resources, indicating a decline in nutrient uptake by the process of decomposition. Therefore, extreme ON deposition can repress litter decomposition by fungi and promote humus accumulation in the surface layer of the forest soil. Litter decomposition is an enzymatically complex process, which is mainly mediated by a species-rich community of saprotrophic fungi. It has been shown that litter decomposition rates are sensitive to N availability in the environment (Edwards et al., 2011; Osono, 2007). It also has been demonstrated that increasing N inputs may alter microbial community structure activities, functions, and fungi-to-bacterial ratios (Compton et al., 2004; Waldrop et al., 2004). In the current study, IS-to-I0 ratios were used to compare the responses of fungi and total microbes to different N types. The decline of the ratio after single IN addition also indicated that soil fungi were seriously inhibited by IN. This finding is consistent with the reports of Tietema (1998) and Holland and Coleman (1987) − who suggested that N (NH+ 4 and NO3 ) addition can shift a soil microbial community from a fungi-dominated system into a bacteria-dominated system. Meanwhile, the ratio increased when ON was added. Factor analysis also showed the same conclusion as the A1 values of IS treatments were higher than those of I0 treatments for all enzymes. This finding may be attributed to the stoichiometric differences between fungi and other microbes because fungi have higher C/N ratios than other microbes and therefore could have lower N demands (Griepentrog et al., 2014). Consequently, a shift toward bacterial biomass is expected when N limitation is reduced due to atmospheric N deposition while C sources remain equally accessible (Strickland and Rousk, 2010). These results indicated that the N limitation of soil fungi was easily alleviated and ON significantly accelerated this alleviation. We hypothesized that microbial community soil may even be converted into a fungi-dominated system with increasing ON percentage in atmosphere N. However, the ratio declined in the treatment with excessive ON percentage (20% IN + 80% ON). This may be attributed to the higher soil pH after urea addition than after NH4NO3 addition under the same amount (data not shown), as Caldwell (2005) has demonstrated that fungal extracellular enzymes have acidic optima while bacterial sources generally have neutral-alkaline optima. Phosphatase activity is a good indicator of the organic P mineralization potential and biological activity of soils (Dick et al., 2000). In contrast to the responses of other enzymes, phosphatase activities increased after IN addition. This finding is consistent with the results of previous studies (Hogan et al., 2010; Ochoa-Hueso et al., 2013; Turner
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et al., 2003). Similar to other enzymes, phosphatase activities also increased with increasing ON addition. This finding indicated that both IN and ON can promote P mineralization. However, for soil fungi, the influence of different N types on phosphatase activity was not significant. Excessive ON even inhibited its activity, although phosphatase activities increased in all N treatments compared to the Control. These findings indicated that the promoting effect of N addition on phosphatase activities in fungi was not more significant than in other microbes such as bacteria and actinobacteria. Meanwhile, the declined IS-to-I0 value of phosphatase indicated that other soil microbes (such as bacteria) may be the dominated decomposer of complex P compounds after N addition, as we found significant increase in phosphobacteria after N addition, particularly after excessive ON addition (data not shown). As the result, fungi may be seriously limited by P after N deposition, specifically after ON deposition. This finding may explain the decline in some enzymatic activities in the treatments with excessive ON. In summary, soil fungi showed different responses from other microorganisms in response to N deposition. Fungi were seriously inhibited by IN and significantly accelerated by ON. Greater ON accelerated the conversion of soil microbial communities into a fungidominated system, but excessive ON deposition (20% IN + 80% ON) caused N saturation and badly repressed fungal activities. These results suggested that soil fungi were sensitive to N type and that different IN-to-ON ratios may induce diverse ecological effects on soil fungi. However, the three-year field experiment only reflected the shortterm responses of different N depositions to forest soils of fungi. A ten-year observation has been conducted. Furthermore, whether fungal diversity shows different responses to various mixed N additions should also be investigated. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.07.098.
Acknowledgments This work was supported by Scientific Foundation for the Introduction of Researcher of Hebei College of Industry and Technology, China (No. BZ1101).
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