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Admixture of alder (Alnus formosana) litter can improve the decomposition of eucalyptus (Eucalyptus grandis) litter
Soil Biology & Biochemistry 73 (2014) 115e121
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Admixture of alder (Alnus formosana) litter can improve the decomposition of eucalyptus (Eucalyptus grandis) litter Fuzhong Wu a, c, Changhui Peng b, c, Wanqin Yang a, *, Jian Zhang a, Yu Han a, Tao Mao a a
Key Laboratory of Ecological Forestry Engineering, Institute of Ecology & Forestry, Sichuan Agricultural University, Chengdu 611130, China Laboratory for Ecological Forecasting and Global Change, College of Forestry, Northwest A & F University, Yangling, Shaanxi 712100, China c Department of Biology Sciences, Institute of Environment Sciences, University of Quebec at Montreal, C.P. 8888, Succ. Centre-Ville, Montreal H3C 3P8, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2013 Received in revised form 18 February 2014 Accepted 21 February 2014 Available online 11 March 2014
The slow nutrient turnover of eucalyptus (Eucalyptus grandis) plantations has been well documented. To examine whether the admixture of alder (Alnus formosana) litter could improve the decomposition of eucalyptus litter, a field litterbag experiment was conducted on a new eucalyptus plantation in southwestern China. We investigated the mass loss rate from an alder and eucalyptus foliar litter mixture every half month from May 1st to October 1st, 2009. Five mixture proportions were examined: pure eucalyptus litter (10E), 70% eucalyptus litter mixed with 30% alder litter (7E:3A), 50% eucalyptus litter mixed with 50% alder litter (5E:5A), 30% eucalyptus litter mixed with 70% alder litter (3E:7A) and pure alder litter (10A). Over 169 days of decomposition, approximately 79.22%, 70.23%, 62.82%, 49.95% and 48.59% of mass was lost from the 3E:7A, 10A, 5E:5A, 7E:3A and 10E litter mixtures, respectively. Compared with pure eucalyptus litter, 3E:7A, 10A, 5E:5A and 7E:3A litter mixtures increased 63.04%, 44.54%, 29.29% and 2.80% of accumulated mass loss. The admixture of alder litter can significantly improve eucalyptus litter decomposition, and a small proportion of eucalyptus litter (3E:7A) may also promote alder litter decomposition. As the decomposition proceeded, the litter mixture displayed exactly additive effects in the initial stage and positive non-additive effects in the middle stage. However, negative non-additive effects were detected in the 7E:3A litter mixture in the later stage, although positive non-additive effects were maintained throughout decomposition in the 5E:5A and 3E:7A mixtures. Compared to pure eucalyptus litter, mixtures containing alder litter presented increased microbial biomass carbon and bacterial DGGE (Denaturing Gradient Gel Electrophoresis) bands, but the litter mixture decomposition relied more on microbial biomass than on microbial diversity. The results imply that alder litter can improve material cycling on eucalyptus plantations and that alder could be a potential species for mixed planting with eucalyptus. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Alder litter Decomposition stage Eucalyptus plantation Litter mixture decomposition Microbial biomass Non-additive effects
1. Introduction The development of eucalyptus (normally Eucalyptus grandis) plantations has been increasing in the global commercial timber industry (Forrester et al., 2006, 2013; Zhang et al., 2010; Leslie et al., 2012). Because of the plant’s short rotation (fast growth) and high consumption of water and soil nutrients, nutrient cycling is one of the limitations to establishing sustainable eucalyptus plantation ecosystems (Lemma et al., 2007). Unfortunately, a thick leaf litter layer often accumulates on the floor of eucalyptus plantations, indicating a slow litter decomposition rate because of * Corresponding author. Tel./fax: þ86 28 86290957. E-mail address: [email protected] (W. Yang). http://dx.doi.org/10.1016/j.soilbio.2014.02.018 0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
low litter quality (Guo and Sims, 2001; Forrester et al., 2006). Several previous studies have reported that mixed-species plantations of eucalyptus with a dinitrogen (N2) fixation species have the potential to increase productivity while maintaining soil fertility, enhancing soil organic carbon sequestration and accelerating nutrient cycling (Forrester et al., 2006, 2013; le Maire et al., 2013). It is therefore important to select N2 fixation species with readily decomposable litter and high rates of nutrient cycling. However, both synergistic and antagonistic interactions (review from Gartner and Cardon, 2004) and even non-significant effects (Perez-Harguindeguy et al., 2008) have been observed in litter mixture decomposition. Climate, litter quality and the decomposer community are known to be the main controllers of organic matter decomposition
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(Couteaux et al., 1995). The non-additive effects on the decomposition of litter mixtures compared to that of monoculture litter can be mainly attributed to the changes of the chemical environment and the physical alteration of the total litter surface where decomposition occurs (Hansen and Coleman, 1998; Kaneko and Salamanca, 1999; Hector et al., 2000). In general, higher-quality litter can stimulate decomposition in adjacent, more recalcitrant litters, and conversely, leaf litter decomposition can be slowed by an admixture of lower-quality litter (Fyles and Fyles, 1993; McTiernan et al., 1997; Salamanca et al., 1998). The transfer of nitrogen between the litters has been documented as a key mechanism in the interaction between decomposing litters (Berglund and Ågren, 2012; Berglund et al., 2013). Moreover, an increase in microhabitats may also be correlated with increased mass loss because of the creation of a more diverse and abundant decomposer community (Hansen and Coleman, 1998; Gartner and Cardon, 2004). Thus, chemical and physical changes in the leaf mixture can influence decomposition rates both directly (physically) and indirectly (through the decomposer community and its activities). However, this superior chemical and physical diversity disappears as decomposition proceeds because the liable components are lost and the litter shape is destroyed after early rapid decomposition (Berg and McClaugherty, 2008). The remaining substrate is rich in water-soluble defenses or inhibitory compounds (such as lignin and tannin), leading to co-limits with each other in the litter mixture (Ostrofsky, 2007). Therefore, trends in the rates of decomposition and nutrient loss from mixtures of litter from different species are complex and inconsistent when compared to those of monoculture litter. Moreover, litter in mixtures with N2 fixation species does not necessarily decay faster than monoculture litters of non-N2 fixation species (Rothe and Binkley, 2001; Binkley et al., 2003). As a result, additional research is required on the decomposition of litter mixtures of eucalyptus and N2 fixation species with the goal of providing effective information for mixedspecies plantations of eucalyptus. Alder (Alnus spp.) is an N2 fixation tree with a wide distribution from the boreal zone to the subtropical zone, and its leaf litter has been well documented as possessing desirable decomposition characteristics (Chapman et al., 1988; Gartner and Cardon, 2004). The available information indicates that the admixture of alder litter can improve the decomposition of other litter, such as that of Populus tremuloides (Taylor et al., 1989) and Pseudotsuga menziesii (Fyles and Fyles, 1993). Alder’s ability to improve the nutrient cycling of litter mixture decomposition makes it a candidate tree species for mixed planting with eucalyptus, but little information on this particular combination is available. Therefore, it is hypothesized that the admixture of alder litter can improve the decomposition of eucalyptus litter, which is potentially beneficial for eucalyptus plantations. To test this hypothesis, a field litterbag experiment was conducted on a new eucalyptus plantation in southwestern China, where eucalyptus plantations cover more than 200 000 ha (Zhang et al., 2010, 2012). We measured the accumulated mass loss from alder (Alnus formosana) and eucalyptus (E. grandis) foliar litter mixtures every half month. Five mixed proportions were examined: pure eucalyptus litter (10E), 70% eucalyptus litter mixed with 30% alder litter (7E:3A), 50% eucalyptus litter mixed with 50% alder litter (5E:5A), 30% eucalyptus litter mixed with 70% alder litter (3E:7A) and pure alder litter (10A). The objectives were (1) to determine whether the admixture of alder litter could improve eucalyptus litter decomposition and (2) to identify the aspects of the litter mixture decomposition. The results will be useful in determining the practicality of alder as a candidate species for mixed planting with eucalyptus.
2. Materials and methods 2.1. Study site The study was conducted in the Leshan region (E103 360 , N29 370, 413 m a.s.l) in western Sichuan Province, southwestern China. The climate is subtropical, with an annual mean temperature of 18.0 C and precipitation of 1137 mm. From a local weather station near the sample site, the monthly average air temperature was higher than 20 C from May to October 2009, with the highest average of 28.2 C in August. The majority of precipitation falls between June and August, whereas only 48.1 mm and 74.3 mm rainfall occurred in May and October 2009, respectively. The soil is classified as ferralsol (Soil Census Office, 1993), is derived from Pleistocene alluvium and has a yellow color, loamy texture and granular structure. To avoid topological heterogeneity and the various effects of tree shadow, three new eucalyptus plantations of at least a 50 m 50 m plot were established in the study site. The plantations were planted with E. grandis at a density of 2.5 m 2.5 m between seedlings in March 2009. The soil had a pH of 4.95 and contained 30.21 g kg1 of soil organic carbon, 0.73 g kg1 of soil nitrogen and 0.67 g kg1 of soil phosphorus. 2.2. Experimental design Litter decomposition was studied using the common litterbag procedure. In March 2009, fresh foliar litter of alder and eucalyptus was collected from the floor of each pure plantation close to the sample plots. To avoid litter structure damage during oven drying, the fresh litter was air-dried for more than two weeks at room temperature. Five samples with approximately 20 g of air-dried litter were oven-dried at 70 C to determine the moisture content, which was 9.65 0.04% and 9.03 0.02% for air-dried alder litter and eucalyptus litter, respectively. A 20 g sample (based on the ovendried mass) of the air-dried alder and eucalyptus litter mixture was then placed in a 20 cm 20 cm nylon bag of a 1 mm mesh, and the edges of the bag were sealed. The following five mixtures were placed in the litterbags: 20 g pure eucalyptus litter (10E), 14 g eucalyptus litter and 6 g alder litter (7E:3A), 10 g eucalyptus litter and 10 g alder litter (5E:5A), 6 g eucalyptus litter and 14 g alder litter (3E:7A), or 20 g pure alder litter (10A). The initial alder litter contained 437.09 12.51 g kg1 organic carbon, 8.73 0.17 g kg1 nitrogen and 1.55 0.01 g kg1 phosphorus; the initial eucalyptus litter contained 513.74 7.57 g kg1 organic carbon, 8.59 0.02 g kg1 nitrogen and 0.78 0.01 g kg1 phosphorus. The experiment began on April 15th, 2009. A total of 825 litterbags (five proportions eleven sampling times five replicates three sampling plots) were placed on the floor of the three sampled plots. To check the litter mixture decomposition processes, we sampled the litterbags every half month from May 1st through the end of the growing season on October 1st, 2009. After four months of decomposition, the remaining litter in several litterbags was insufficient to conduct the mass loss and microbial analyses. We combined five litterbags into three on September 15th and October 1st, 2009. The retrieved litter was separated into two parts after being completely mixed in each litterbag. One part was stored in a refrigerator at 4 C and prepared for the microbial biomass and bacterial DGGE (Denaturing Gradient Gel Electrophoresis) analyses, whereas the remaining part was oven-dried at 70 C for 48 h to determine the dry mass. 2.3. Microbial biomass analysis The microbial biomass carbon (MBC) in the litter was determined according to the differences between unfumigated and
F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121
fumigated samples following extraction with 0.5 mol L1 K2SO4. An efficiency factor (Kc ¼ 0.38) was used to correct for the incomplete extractability of the samples (Vance et al., 1987).
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2.6. Calculations The accumulated mass loss (Ri) during litter decomposition before each stage were calculated using the following equation:
2.4. DNA extraction
Ri ð%Þ ¼ 100 ðM0 Mi Þ=M0
Bacterial DNA was extracted from the litter samples using the method described by Griffiths et al. (2000) in which litter samples were extracted with 0.5 g of glass beads (0.1 mm), 500 ml CTAB extraction buffer, and 500 ml phenol-chloroform isoamyl alcohol (25:24:1) (pH 8.0) by beadbeating at full speed for 30 s with MiniBeadbeater (Biospec Products, Bartlesville, OK, USA). Separation of the phases and precipitation of the nucleic acids was performed, and the separation was then treated using RNAase (Takara) according to the manufacturer’s instructions.
where M0 is the dry mass of the initial litter and Mi is the dry mass of the remaining litter in the bag at each stage after sampling. The theoretical mass loss rates of the litter mixtures were calculated using the following formula (Hoorens et al., 2003):
2.5. PCR-DGGE The variable V3 region of the 16S rRNA gene sequence was amplified by PCR (polymerase chain reaction) using the universal primers 341f and 534r and the touchdown protocol (Muyzer et al., 1993). The extracted DNA was amplified with a PCR mixture (50 ml) containing 37.55 ml of sterilized Milli-Q water, 5 ml of Mg-containing buffer, 4 ml of deoxynucleoside triphosphate mixture, 1 ml of each primer (20 mM), 1 ml of the DNA solution, and 0.45 ml of HotStart Version ExTaq DNA polymerase (Takara). PCR was performed with a Bio-Rad iCycler thermocycler using the following protocol: 1 min at 94 C (denaturation), 1 min at 65 C (annealing), and 3 min at 72 C (elongation), with a 1 C decrease in the annealing temperature every second cycle as a “touchdown” for 20 cycles, followed by 10 cycles at an annealing temperature of 55 C and a final cycle consisting of 10 min at 72 C. After gel electrophoresis (1.5% [wt/vol] agarose gel) of 4 ml subsamples from the PCR product, the amount of amplified DNA was quantified by comparing the band intensities to the standard curves obtained with a Low DNA Mass Ladder (Takara). The band intensities were measured with Quantity One analysis software (Bio-Rad Laboratories, Hercules, CA). Profiles of the amplified 16S rRNA gene sequences were produced by DGGE using the CBS DGGE system (CBS Scientific, USA) (Muyzer et al., 1993). The PCR products were loaded onto a polyacrylamide gel (8% [wt/vol] acrylamide in 1 TAE buffer with a 45e 65% denaturant gradient (100% denaturant was 7 M urea and 40% [vol/vol] deionized formamide)). The wells were loaded with 25 ml of PCR product, and electrophoresis was conducted in the TAE buffer at 100 V for 17 h at 60 C. The DNA fragments were stained by silver staining as described by Radojkovic and Kusic (2000). The gel was destained in distilled water for 5 min. Images of the gels were obtained after destaining using a Bio-Rad GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Hercules, CA). The band patterns were analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA) (Zhang and Jackson, 2008). Each detected band was defined as an operational taxonomic unit (OTU), and the number of bands was defined as the genotypic richness of each sample (Bell et al., 2005). The pixel intensity for each band was detected by Quantity One software and was expressed as the relative abundance (Pi) (Reche et al., 2005). The richness index (ShannoneWiener index, H0 ) (Trevors et al., 2010) was calculated using the relative abundance data based on the following equation:
H ¼
s X
pi ln pi
i¼1
where Pi ¼ ni/S, ni is the abundance of the ith OTU and S is the total abundance of all OTUs in the sample.
Theoretical mass loss rateð%Þ ¼ Ra Pa þ Re Pe ; where Ra and Re are the accumulated mass loss of pure alder and eucalyptus litter at each stage, respectively, and Pa and Pe are the initial proportions of alder and eucalyptus litter in the mixture, respectively. 2.7. Statistical analysis All of the variables, including the measurements and calculations among the different litter mixtures, were tested by one-way analysis of variance (ANOVA). When significant differences were detected, the LSD multiple range test was used to determine where the differences existed. Differences were considered significant at P < 0.05. Linear regressions were used to examine the relationships between the accumulated mass loss and the microbial biomass and bacterial DGGE bands. All of the statistical analyses were performed using the SPSS (Statistical Package for the Social Sciences) software package (Standard release version 16.0 for Windows, SPSS Inc., IL, USA). 3. Results 3.1. Mass loss Over 169 days of decomposition, the accumulated mass loss was significantly different in the pattern 3E:7A > 10A > 5E:5A > 7E:3A > 10E. Approximately 79.22%, 70.23%, 62.82%, 49.95% and 48.59% of mass was lost from the 3E:7A, 10A, 5E:5A, 7E:3A and 10E litter mixtures, respectively (Fig. 1). Compared with pure eucalyptus litter, 3E:7A, 10A, 5E:5A and 7E:3A litter mixtures increased 63.04%, 44.54%, 29.29% and 2.80% of accumulated mass loss. Few differences in the accumulated mass loss were observed between the different litter mixtures in the half month of decomposition before May 1st, and the 3E:7A litter mixture had a higher accumulated mass loss only in the last two months of the study (after August 1st). The accumulated mass loss of pure eucalyptus litter (10E) was significantly lower than those of the other litter mixtures after May 15th, but few differences were detected between the accumulated mass loss of the 10E and 7E:3A litter mixtures after September 1st. Additionally, the observed accumulated mass loss was close to the theoretical values for each litter mixture on May 1st, but a significant non-additive effect was detected over the course of the litter mixture decomposition after May 1st. Although the observed accumulated mass loss in the 7E:3A litter mixture after September 1st was slightly lower than their theoretical values, the observed accumulated mass loss in the other litter mixtures was higher than their theoretical values (Fig. 2). 3.2. Microbial biomass Microbial biomass carbon (MBC) first increased and then decreased as the litter mixture decomposition proceeded (Fig. 3),
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90
10E
7E:3A
5E:5A
3E:7A
10A c
Accumulated mass loss (%)
80
d
70 bb b
60 c
50 40
c
30 20 10
b
aaaaa
bb b aab
a
bb b b
aa
bb b
a
bb
b b bb c
a
c bc c
bb b
a
a
1-Aug
15-Aug
aa
c a b bc a
aa
0 1-May 15-May
1-Jun
15-Jun
1-Jul
15-Jul Date
1-Sep
15-Sep
1-Oct
Fig. 1. The accumulated mass loss in the litterbags during the decomposition of the E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3) and the different letters denote significant differences (P < 0.05) among different litter mixtures.
and its peak value did not appear at the same time for each litter mixture. The highest MBC values during the decomposition of the 10A and 7E:3A litter mixtures were observed on August 15th, whereas the highest values for the other mixtures were observed on September 15th. Few differences were observed among the MBC values of the different litter mixtures during the first two months (until June 1st) of decomposition. After this point, the MBC values of the pure alder litter (10A) decomposition were higher than those of the other litter mixtures from June 1st to August 15th, and the values of the 3E:7A litter mixture were higher after September 1st. Conversely, the MBC values for the pure eucalyptus litter (10E) decomposition were the lowest among the other litter mixtures after July 1st.
3.3. Bacterial community from PCR-DGGE The PCR-DGGE bacterial analysis revealed that there were significant variations between the DGGE bands of the different litter samples during decomposition (Fig. 4). The DGGE bands showed an increase followed by a decreasing tendency as the litter mixture decomposition proceeded, with the exception of the 5A:5E litter mixture, which showed the opposite tendency (Table 1). In comparison with the DGGE bands for the pure eucalyptus litter decomposition, the addition of alder litter increased the number of DGGE bands, and the most DGGE bands were detected for the
Accumulated mass loss (%)
90
Observed
3E:7A litter mixture. The ShannoneWiener indices showed similar patterns as the changes in DGGE bands.
4. Discussion The hypothesis that the admixture of alder litter can improve eucalyptus litter decomposition was confirmed by this study. Both the present study and previous results have demonstrated that mixing eucalyptus litter with more readily decomposable litter can enhance the decomposition of eucalyptus litter (Briones and Ineson, 1996). From a quantitative perspective, this study hypothesized that the litter mixture would decompose more slowly as the amount of slowly decomposing eucalyptus litter increases (Ostrofsky, 2007; Pérez-Suárez et al., 2012). Interestingly, our results indicated that the presence of a certain amount of eucalyptus litter actually enhanced the mass loss rate of the litter mixture (3E:7A) over that of the pure alder litter. Furthermore, a significant non-additive effect was observed in this study, as the mass loss rates of the litter mixtures were higher than their theoretical values at most of the stages of decomposition. This result may be explained as a synergistic effect (Gartner and Cardon, 2004) between alder litter and eucalyptus litter, confirming that these two species facilitate each other’s litter decomposition. Many previous studies have reported that the admixture of higher-quality litter can promote the decomposition of more
Theoretical
80 70 60 50 40 30 20 10
7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A
0
1-May 15-May 1-Jun
15-Jun
1-Jul
15-Jul
1-Aug 15-Aug 1-Sep 15-Sep
1-Oct
Fig. 2. The observed and theoretical values of the accumulated mass loss during the decomposition of E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3).
F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121
1400
10E
7E:3A
5E:5A
3E:7A
119
10A
MBC (mg kg dry mass-1)
1200 1000 800 600 400 200 0 1-May
15-May
1-Jun
15-Jun
1-Jul
15-Jul
1-Aug
15-Aug
1-Sep
15-Sep
1-Oct
Date Fig. 3. The microbial biomass carbon (MBC) during the decomposition of E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3).
Fig. 4. The silver-stained bands obtained by DGGE of bacteria during the decomposition of E. grandis and A. formosana litter mixtures.
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Table 1 ShannoneWiener index (H) and the abundance of DGGE bands (S) during the decomposition of E. grandis and A. formosana litter mixtures. Treatments
10E 7E:3A 5E:5A 3E:7A 10A
Diversity index
Sampling time 1-May
15-May
1-Jun
15-Jun
1-Jul
15-Jul
1-Aug
15-Aug
1-Sep
15-Sep
1-Oct
H S H S H S H S H S
2.5875 14 2.4814 12 3.4453 32 3.4504 33 3.4170 31
2.7474 16 3.3088 28 3.5300 35 3.4821 34 3.3547 29
2.9745 20 3.1609 24 2.7516 27 3.4074 31 3.4237 31
3.0219 21 2.8392 18 2.7578 16 3.5550 36 3.4552 32
2.9705 20 3.6048 38 2.9809 20 3.4954 34 3.5232 34
3.0139 21 3.1887 25 3.1128 23 3.8154 47 3.4300 31
2.9245 19 3.2987 28 3.3390 29 3.8637 50 3.5377 35
2.9751 20 3.6621 40 3.3831 30 3.6211 39 3.6465 40
2.9759 20 3.4755 33 3.1169 23 3.5638 37 3.5726 37
2.9047 19 3.4436 32 3.2798 27 3.6503 40 3.5455 35
3.1636 24 3.4215 33 3.2387 26 3.3709 31 3.5716 33
(Hansen and Coleman, 1998; Hector et al., 2000; Berglund et al., 2013). We also observed that although the number of bacterial DGGE bands was higher in the litter mixtures (5E:5A and 3E:7A before May 15th) than in the pure alder and eucalyptus litters (Table 1), microbial biomass carbon did not significantly change (Fig. 3). As decomposition proceeded, increasing decomposer communication and physical changes in the litter mixture structure contributed to the mass loss of the litter mixture (Hansen and Coleman, 1998; Hector et al., 2000), leading to the positive nonadditive effects (obvious synergistic effects) observed in all of the litter mixtures (Fig. 2) at this stage. However, after the rapid loss of labile components, litter mixture decomposition can be slowed by the decomposition of the remaining inhibitory compounds, such as phenolics and tannins (Fyles and Fyles, 1993; Salamanca et al., 1998). Less decomposable litter and more inhibitory compounds remain in the litter mixture because of the reduction in labile components. This observation is consistent with the 7E:3A litter mixture, which displayed negative non-additive effects (antagonistic interactions) after September 1st. The continuous synergistic effects in the 5E:5A and 3E:7A litter mixtures may be explained by the presence of relatively more labile components, which can maintain the positive interactions for a longer time. These types of litter mixtures may also exhibit antagonistic interactions in the later decomposition stages. Therefore, our present results suggest that the inconsistencies in previous observations (both negative and positive non-additive effects that were reported in the review of Gartner and Cardon, 2004) of litter mixture decompositions might be attributed to the differences in the decomposition stages and mixture proportions. Additional studies and longer decomposition experiments should be conducted to resolve these issues.
recalcitrant litters, or vice versa (Fyles and Fyles, 1993; Briones and Ineson, 1996; McTiernan et al., 1997), which is partly consistent with the present study’s finding that an admixture of alder litter accelerated the decomposition of eucalyptus litter in the present study, but the addition of eucalyptus litter also stimulated alder litter decomposition when the mixture proportion is considered. This result coincided with the mechanisms proposed by Ostrofsky (Ostrofsky, 2007), who declared that four principles control the decomposition of mixed litter: (1) the presence of rapidly decomposing leaves that favor the rapid colonization of decomposer communities, which accelerate the mass loss of slowly processed recalcitrant leaves; (2) the co-occurring reductions of the mass loss rates of rapidly decomposing leaves and slowly decomposing leaves caused by high concentrations of water-soluble defensive or inhibitory compounds (e.g., tannins and other phenolics); (3) a more persistent habitat for decomposing organisms provided by the structural stability of the litter mixture beds to accelerate the decomposition of slowly decomposing leaves by protecting the abrasion and leaching processes; (4) both facilitative and inhibitory mechanisms that occur simultaneously, as none of these effects are mutually exclusive, so that the observed accumulated mass loss changes in litter mixture decomposition represent the net effect of multiple processes (Ostrofsky, 2007; Pérez-Suárez et al., 2012). However, the different litter mixtures did not follow the same pattern over the course of decomposition. In the initial decomposition stage, the litter mixture mass loss rate equaled the sum of the alder and eucalyptus litter mass loss rates alone, displaying a completely additive effect, shown in Fig. 2. The initially intact shapes of both types of litter may limit their complete mixture, leading to few changes in nutrient transfer and decomposer habitat
(a)
1200
(b)
50
800 600 2
R = 0.8013 P =0.0000
400
DGGE band number
MBC (mg kg dry mass -1)
1000
60
200
40 30 2
R = 0.2710 P =0.007
20 10
0
0 0
20
40
60
80
Accumulated mass loss (%)
100
0
20 40 60 80 Accumulated mass loss (%)
100
Fig. 5. The linear relationships between the accumulated mass loss and microbial biomass carbon (MBC) (a), and between the accumulated mass loss and DGGE band number of bacteria (b).
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Additionally, one of the essential promoters of litter mixture decomposition is the increase of decomposer diversity because of the alteration of substrate diversity and microhabitat complexity (Kaneko and Salamanca, 1999; Wardle et al., 2006). In the current study, we observed that the alder and eucalyptus mixture litter showed a higher number of bacterial DGGE bands as decomposition proceeded compared to pure eucalyptus litter. However, a higher number of bacterial DGGE bands were also detected in the 3E:7A litter mixture than in the pure alder litter at most of the decomposition stages, indicating obvious synergistic effects. This result is in accordance with the conclusion of Gartner and Cardon (2004), who claimed that the interactions of litter from different species in an ecosystem influenced the structure and activity of the decomposer community, which is largely responsible for decomposition. In comparison with bacterial biodiversity, the microbial biomass was more statistically correlated to the litter mixture mass loss (Fig. 5). Microbial biomass refers to the biomass composed of the microorganisms on the suitable substrate, which represents biological activity. In an incubation study, Thiessen et al. (2013) also documented that organic matter decomposition mainly depended on microbial biomass. In conclusion, the admixture of alder litter can stimulate eucalyptus litter decomposition, and a small amount of eucalyptus litter may also promote alder litter decomposition. Over the total investigation period, the decomposition of each litter mixture displayed positive non-additive effects. However, the effects differed over the decomposition process: exactly additive effects were observed in the initial stage, positive non-additive effects were observed in the middle stage, and negative non-additive effects were observed in the later stage and would remain in progress as the decomposition proceeded. Furthermore, litter mixture decomposition relied more on microbial biomass than microbial diversity. The results indicate that alder could be a potential candidate for mixed-species eucalyptus plantations because of its synergistic effects on material cycling. Acknowledgments The authors are very grateful to their colleagues from Ecological Modelling and Carbon Science (ECO-MCS), Institute of Environment Sciences, University of Quebec at Montreal (UQAM) for providing helpful suggestions in manuscript preparation. This research was financially supported by the National Natural Science Foundation of China (31170423 & 31270498), the National Key Technologies R&D of China (2011BAC09B05), and the Sichuan Youth Sci-tech Foundation (2012JQ0008 & 2012JQ0059). References Bell, T., Ager, D., Song, J.I., Newman, J.A., Thompson, I.P., Lilley, A.K., van der Gast, C.J., 2005. Larger islands house more bacterial taxa. Science 308, 1884. Berg, B., McClaugherty, C., 2008. Plant Litter: Decomposition, Humus Formation, Carbon Sequestration, second ed. Springer, New York. Berglund, S.L., Ågren, G.I., 2012. When will litter mixtures decompose faster or slower than individual litters? A model for two litters. Oikos 121, 1112e1120. Berglund, S.L., Ågren, G.I., Ekblad, A., 2013. Carbon and nitrogen transfer in leaf litter mixtures. Soil Biology & Biochemistry 57, 341e348. Binkley, D., Senock, R., Bird, S., Cole, T.G., 2003. Twenty years of stand development in pure and mixed stands of Eucalyptus saligna and N-fixing Facaltaria moluccana. Forest Ecology and Management 182, 93e102. Briones, M.J.I., Ineson, P., 1996. Decomposition of eucalyptus leaves in litter mixtures. Soil Biology & Biochemistry 28, 1381e1388. Chapman, K., Whittaker, J.B., Heal, O.W., 1988. Metabolic and faunal activity in litters of tree mixtures compared with pure stands. Agriculture, Ecosystems & Environment 24, 33e40. Couteaux, M.M., Bottner, P., Berg, B., 1995. Litter decomposition, climate and litter quality. Trends in Ecology & Evolution 10, 63e66. Forrester, D.I., Bauhus, J., Cowie, A.L., Vanclay, J.K., 2006. Mixed-species plantations of eucalyptus with nitrogen fixing trees: a review. Forest Ecology and Management 233, 211e230.
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Forrester, D.I., Pares, A., O’Hara, C., Khanna, P.K., Bauhus, J., 2013. Soil organic carbon is increased in mixed-species plantations of eucalyptus and nitrogen-fixing acacia. Ecosystems 16, 123e132. Fyles, J.W., Fyles, I.H., 1993. Interaction of Douglas-fir with red alder and salal foliage litter during decomposition. Canadian Journal of Forest Research 23, 358e361. Gartner, T.B., Cardon, Z.G., 2004. Decomposition dynamics in mixed-species leaf litter. Oikos 104, 230e246. Griffiths, R.I., Whiteley, A.S., O’Donnell, A.G., Bailey, M.J., 2000. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-Based microbial community composition. Applied and Environmental Microbiology 66, 5488e5491. Guo, L.B., Sims, R.E.H., 2001. Effects of light, temperature, water and meatworks effluent irrigation on eucalypt leaf litter decomposition under controlled environmental conditions. Applied Soil Ecology 17, 229e237. Hansen, R.A., Coleman, D.C., 1998. Litter complexity and composition are determinants of the diversity and species composition of orabatid mites (Acari: Oribatida) in litterbags. Applied Soil Ecology 9, 17e23. Hector, A., Beale, A.J., Minns, A., Otway, S.J., Lawton, J.H., 2000. Consequences of the reduction of plant diversity for litter decomposition: effects through litter quality and microenvironment. Oikos 90, 357e371. Hoorens, B., Aerts, R., Stroetenga, M., 2003. Does initial litter chemistry explain litter mixture effects on decomposition? Oecologia 137, 578e586. Kaneko, N., Salamanca, E.F., 1999. Mixed leaf litter effects on decomposition rates and soil microarthropod communities in an oak-pine stand in Japan. Ecological Research 14, 131e138. le Maire, G., Nouvellon, Y., Christina, M., Ponzoni, F.J., Gonçalves, J.L.M., Bouillet, J.P., Laclau, J.P., 2013. Tree and stand light use efficiencies over a full rotation of single- and mixed-species Eucalyptus grandis and Acacia mangium plantations. Forest Ecology and Management 288, 31e42. Lemma, B., Nilsson, I., Kleja, D.B., Olsson, M., Knicker, H., 2007. Decomposition and substrate quality of leaf litters and fine roots from three exotic plantations and a native forest in the southwestern highlands of Ethiopia. Soil Biology & Biochemistry 39, 2317e2328. Leslie, A.D., Mencuccini, M., Perks, M., 2012. The potential for Eucalyptus as a wood fuel in the UK. Applied Energy 89, 176e182. McTiernan, K.B., Ineson, P., Coward, P.A., 1997. Respiration and nutrient release from tree leaf litter mixtures. Oikos 78, 527e538. Muyzer, G., De Wall, E.C., Uitterinden, A.G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rDNA. Applied and Environmental Microbiology 59, 695e700. Ostrofsky, M.L., 2007. A comment on the use of exponential decay models to test non additive processing hypotheses in multispecies mixtures of litter. Journal of the North American Benthological Society 26, 23e27. Pérez-Suárez, M., Arredondo-Moreno, J.T., Huber-Sannwald, E., 2012. Early stage of single and mixed leaf-litter decomposition in semiarid forest pine-oak: the role of rainfall and microsite. Biogeochemistry 108, 245e258. Perez-Harguindeguy, N., Blundo, C.M., Gurvich, D.E., Dlaz, S., Cuevas, E., 2008. More than the sum of its parts? Assessing litter heterogeneity effects on the decomposition of litter mixtures through leaf chemistry. Plant and Soil 303, 151e159. Radojkovic, D., Kusic, J., 2000. Silver staining of denaturing gradient gel electrophoresis gels. Clinical Chemistry 46, 883e884. Reche, I., Pulido-Villena, E., Morales-Baquero, R., Casamayor, E.O., 2005. Does ecosystem size determine aquatic bacterial richness? Ecology 86, 1715e1722. Rothe, A., Binkley, D., 2001. Nutritional interactions in mixed species forests: a synthesis. Canadian Journal of Forest Research 31, 1855e1870. Salamanca, E.F., Kaneko, N., Katagiri, S., 1998. Effects of leaf litter mixtures on the decomposition of Quercus serrata and Pinus densiflora using field and laboratory microcosm methods. Ecological Engineering 10, 53e73. Soil Census Office, 1993. Soil Classification System of China. Agricultural Press, Beijing (in Chinese). Taylor, B.R., Parsons, W.F., Parkinson, D., 1989. Decomposition of Populus tremuloides leaf litter accelerated by addition of Alnus crispa litter. Canadian Journal of Forest Research 19, 674e679. Thiessen, S., Gleixner, G., Wutzler, T., Reichstein, M., 2013. Both priming and temperature sensitivity of soil organic matter decomposition depend on microbial biomass e an incubation study. Soil Biology & Biochemistry 57, 739e748. Trevors, J.T., Kevan, P.G., Tam, L., 2010. Microbial diversity across a Canadian subArctic, isostatically rebounding, soil transect. Polar Science 4, 81e91. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology & Biochemistry 19, 703e707. Wardle, D.A., Yeates, G.W., Barker, G.M., Bonner, K.I., 2006. The influence of plant litter diversity on decomposer abundance and diversity. Soil Biology & Biochemistry 38, 1052e1062. Zhang, D.J., Zhang, J., Yang, W.Q., Wu, F.Z., 2010. Potential allelopathic effect of Eucalyptus grandis across a range of plantation ages. Ecological Research 25, 13e23. Zhang, D.J., Zhang, J., Yang, W.Q., Wu, F.Z., 2012. Effects of afforestation with Eucalyptus grandis on soil physicochemical and microbiological properties. Soil Research 50, 167e176. Zhang, H., Jackson, T.A., 2008. Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae). 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Admixture of alder (Alnus formosana) litter can improve the decomposition of eucalyptus (Eucalyptus grandis) litter Fuzhong Wu a, c, Changhui Peng b, c, Wanqin Yang a, *, Jian Zhang a, Yu Han a, Tao Mao a a
Key Laboratory of Ecological Forestry Engineering, Institute of Ecology & Forestry, Sichuan Agricultural University, Chengdu 611130, China Laboratory for Ecological Forecasting and Global Change, College of Forestry, Northwest A & F University, Yangling, Shaanxi 712100, China c Department of Biology Sciences, Institute of Environment Sciences, University of Quebec at Montreal, C.P. 8888, Succ. Centre-Ville, Montreal H3C 3P8, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2013 Received in revised form 18 February 2014 Accepted 21 February 2014 Available online 11 March 2014
The slow nutrient turnover of eucalyptus (Eucalyptus grandis) plantations has been well documented. To examine whether the admixture of alder (Alnus formosana) litter could improve the decomposition of eucalyptus litter, a field litterbag experiment was conducted on a new eucalyptus plantation in southwestern China. We investigated the mass loss rate from an alder and eucalyptus foliar litter mixture every half month from May 1st to October 1st, 2009. Five mixture proportions were examined: pure eucalyptus litter (10E), 70% eucalyptus litter mixed with 30% alder litter (7E:3A), 50% eucalyptus litter mixed with 50% alder litter (5E:5A), 30% eucalyptus litter mixed with 70% alder litter (3E:7A) and pure alder litter (10A). Over 169 days of decomposition, approximately 79.22%, 70.23%, 62.82%, 49.95% and 48.59% of mass was lost from the 3E:7A, 10A, 5E:5A, 7E:3A and 10E litter mixtures, respectively. Compared with pure eucalyptus litter, 3E:7A, 10A, 5E:5A and 7E:3A litter mixtures increased 63.04%, 44.54%, 29.29% and 2.80% of accumulated mass loss. The admixture of alder litter can significantly improve eucalyptus litter decomposition, and a small proportion of eucalyptus litter (3E:7A) may also promote alder litter decomposition. As the decomposition proceeded, the litter mixture displayed exactly additive effects in the initial stage and positive non-additive effects in the middle stage. However, negative non-additive effects were detected in the 7E:3A litter mixture in the later stage, although positive non-additive effects were maintained throughout decomposition in the 5E:5A and 3E:7A mixtures. Compared to pure eucalyptus litter, mixtures containing alder litter presented increased microbial biomass carbon and bacterial DGGE (Denaturing Gradient Gel Electrophoresis) bands, but the litter mixture decomposition relied more on microbial biomass than on microbial diversity. The results imply that alder litter can improve material cycling on eucalyptus plantations and that alder could be a potential species for mixed planting with eucalyptus. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Alder litter Decomposition stage Eucalyptus plantation Litter mixture decomposition Microbial biomass Non-additive effects
1. Introduction The development of eucalyptus (normally Eucalyptus grandis) plantations has been increasing in the global commercial timber industry (Forrester et al., 2006, 2013; Zhang et al., 2010; Leslie et al., 2012). Because of the plant’s short rotation (fast growth) and high consumption of water and soil nutrients, nutrient cycling is one of the limitations to establishing sustainable eucalyptus plantation ecosystems (Lemma et al., 2007). Unfortunately, a thick leaf litter layer often accumulates on the floor of eucalyptus plantations, indicating a slow litter decomposition rate because of * Corresponding author. Tel./fax: þ86 28 86290957. E-mail address: [email protected] (W. Yang). http://dx.doi.org/10.1016/j.soilbio.2014.02.018 0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
low litter quality (Guo and Sims, 2001; Forrester et al., 2006). Several previous studies have reported that mixed-species plantations of eucalyptus with a dinitrogen (N2) fixation species have the potential to increase productivity while maintaining soil fertility, enhancing soil organic carbon sequestration and accelerating nutrient cycling (Forrester et al., 2006, 2013; le Maire et al., 2013). It is therefore important to select N2 fixation species with readily decomposable litter and high rates of nutrient cycling. However, both synergistic and antagonistic interactions (review from Gartner and Cardon, 2004) and even non-significant effects (Perez-Harguindeguy et al., 2008) have been observed in litter mixture decomposition. Climate, litter quality and the decomposer community are known to be the main controllers of organic matter decomposition
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(Couteaux et al., 1995). The non-additive effects on the decomposition of litter mixtures compared to that of monoculture litter can be mainly attributed to the changes of the chemical environment and the physical alteration of the total litter surface where decomposition occurs (Hansen and Coleman, 1998; Kaneko and Salamanca, 1999; Hector et al., 2000). In general, higher-quality litter can stimulate decomposition in adjacent, more recalcitrant litters, and conversely, leaf litter decomposition can be slowed by an admixture of lower-quality litter (Fyles and Fyles, 1993; McTiernan et al., 1997; Salamanca et al., 1998). The transfer of nitrogen between the litters has been documented as a key mechanism in the interaction between decomposing litters (Berglund and Ågren, 2012; Berglund et al., 2013). Moreover, an increase in microhabitats may also be correlated with increased mass loss because of the creation of a more diverse and abundant decomposer community (Hansen and Coleman, 1998; Gartner and Cardon, 2004). Thus, chemical and physical changes in the leaf mixture can influence decomposition rates both directly (physically) and indirectly (through the decomposer community and its activities). However, this superior chemical and physical diversity disappears as decomposition proceeds because the liable components are lost and the litter shape is destroyed after early rapid decomposition (Berg and McClaugherty, 2008). The remaining substrate is rich in water-soluble defenses or inhibitory compounds (such as lignin and tannin), leading to co-limits with each other in the litter mixture (Ostrofsky, 2007). Therefore, trends in the rates of decomposition and nutrient loss from mixtures of litter from different species are complex and inconsistent when compared to those of monoculture litter. Moreover, litter in mixtures with N2 fixation species does not necessarily decay faster than monoculture litters of non-N2 fixation species (Rothe and Binkley, 2001; Binkley et al., 2003). As a result, additional research is required on the decomposition of litter mixtures of eucalyptus and N2 fixation species with the goal of providing effective information for mixedspecies plantations of eucalyptus. Alder (Alnus spp.) is an N2 fixation tree with a wide distribution from the boreal zone to the subtropical zone, and its leaf litter has been well documented as possessing desirable decomposition characteristics (Chapman et al., 1988; Gartner and Cardon, 2004). The available information indicates that the admixture of alder litter can improve the decomposition of other litter, such as that of Populus tremuloides (Taylor et al., 1989) and Pseudotsuga menziesii (Fyles and Fyles, 1993). Alder’s ability to improve the nutrient cycling of litter mixture decomposition makes it a candidate tree species for mixed planting with eucalyptus, but little information on this particular combination is available. Therefore, it is hypothesized that the admixture of alder litter can improve the decomposition of eucalyptus litter, which is potentially beneficial for eucalyptus plantations. To test this hypothesis, a field litterbag experiment was conducted on a new eucalyptus plantation in southwestern China, where eucalyptus plantations cover more than 200 000 ha (Zhang et al., 2010, 2012). We measured the accumulated mass loss from alder (Alnus formosana) and eucalyptus (E. grandis) foliar litter mixtures every half month. Five mixed proportions were examined: pure eucalyptus litter (10E), 70% eucalyptus litter mixed with 30% alder litter (7E:3A), 50% eucalyptus litter mixed with 50% alder litter (5E:5A), 30% eucalyptus litter mixed with 70% alder litter (3E:7A) and pure alder litter (10A). The objectives were (1) to determine whether the admixture of alder litter could improve eucalyptus litter decomposition and (2) to identify the aspects of the litter mixture decomposition. The results will be useful in determining the practicality of alder as a candidate species for mixed planting with eucalyptus.
2. Materials and methods 2.1. Study site The study was conducted in the Leshan region (E103 360 , N29 370, 413 m a.s.l) in western Sichuan Province, southwestern China. The climate is subtropical, with an annual mean temperature of 18.0 C and precipitation of 1137 mm. From a local weather station near the sample site, the monthly average air temperature was higher than 20 C from May to October 2009, with the highest average of 28.2 C in August. The majority of precipitation falls between June and August, whereas only 48.1 mm and 74.3 mm rainfall occurred in May and October 2009, respectively. The soil is classified as ferralsol (Soil Census Office, 1993), is derived from Pleistocene alluvium and has a yellow color, loamy texture and granular structure. To avoid topological heterogeneity and the various effects of tree shadow, three new eucalyptus plantations of at least a 50 m 50 m plot were established in the study site. The plantations were planted with E. grandis at a density of 2.5 m 2.5 m between seedlings in March 2009. The soil had a pH of 4.95 and contained 30.21 g kg1 of soil organic carbon, 0.73 g kg1 of soil nitrogen and 0.67 g kg1 of soil phosphorus. 2.2. Experimental design Litter decomposition was studied using the common litterbag procedure. In March 2009, fresh foliar litter of alder and eucalyptus was collected from the floor of each pure plantation close to the sample plots. To avoid litter structure damage during oven drying, the fresh litter was air-dried for more than two weeks at room temperature. Five samples with approximately 20 g of air-dried litter were oven-dried at 70 C to determine the moisture content, which was 9.65 0.04% and 9.03 0.02% for air-dried alder litter and eucalyptus litter, respectively. A 20 g sample (based on the ovendried mass) of the air-dried alder and eucalyptus litter mixture was then placed in a 20 cm 20 cm nylon bag of a 1 mm mesh, and the edges of the bag were sealed. The following five mixtures were placed in the litterbags: 20 g pure eucalyptus litter (10E), 14 g eucalyptus litter and 6 g alder litter (7E:3A), 10 g eucalyptus litter and 10 g alder litter (5E:5A), 6 g eucalyptus litter and 14 g alder litter (3E:7A), or 20 g pure alder litter (10A). The initial alder litter contained 437.09 12.51 g kg1 organic carbon, 8.73 0.17 g kg1 nitrogen and 1.55 0.01 g kg1 phosphorus; the initial eucalyptus litter contained 513.74 7.57 g kg1 organic carbon, 8.59 0.02 g kg1 nitrogen and 0.78 0.01 g kg1 phosphorus. The experiment began on April 15th, 2009. A total of 825 litterbags (five proportions eleven sampling times five replicates three sampling plots) were placed on the floor of the three sampled plots. To check the litter mixture decomposition processes, we sampled the litterbags every half month from May 1st through the end of the growing season on October 1st, 2009. After four months of decomposition, the remaining litter in several litterbags was insufficient to conduct the mass loss and microbial analyses. We combined five litterbags into three on September 15th and October 1st, 2009. The retrieved litter was separated into two parts after being completely mixed in each litterbag. One part was stored in a refrigerator at 4 C and prepared for the microbial biomass and bacterial DGGE (Denaturing Gradient Gel Electrophoresis) analyses, whereas the remaining part was oven-dried at 70 C for 48 h to determine the dry mass. 2.3. Microbial biomass analysis The microbial biomass carbon (MBC) in the litter was determined according to the differences between unfumigated and
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fumigated samples following extraction with 0.5 mol L1 K2SO4. An efficiency factor (Kc ¼ 0.38) was used to correct for the incomplete extractability of the samples (Vance et al., 1987).
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2.6. Calculations The accumulated mass loss (Ri) during litter decomposition before each stage were calculated using the following equation:
2.4. DNA extraction
Ri ð%Þ ¼ 100 ðM0 Mi Þ=M0
Bacterial DNA was extracted from the litter samples using the method described by Griffiths et al. (2000) in which litter samples were extracted with 0.5 g of glass beads (0.1 mm), 500 ml CTAB extraction buffer, and 500 ml phenol-chloroform isoamyl alcohol (25:24:1) (pH 8.0) by beadbeating at full speed for 30 s with MiniBeadbeater (Biospec Products, Bartlesville, OK, USA). Separation of the phases and precipitation of the nucleic acids was performed, and the separation was then treated using RNAase (Takara) according to the manufacturer’s instructions.
where M0 is the dry mass of the initial litter and Mi is the dry mass of the remaining litter in the bag at each stage after sampling. The theoretical mass loss rates of the litter mixtures were calculated using the following formula (Hoorens et al., 2003):
2.5. PCR-DGGE The variable V3 region of the 16S rRNA gene sequence was amplified by PCR (polymerase chain reaction) using the universal primers 341f and 534r and the touchdown protocol (Muyzer et al., 1993). The extracted DNA was amplified with a PCR mixture (50 ml) containing 37.55 ml of sterilized Milli-Q water, 5 ml of Mg-containing buffer, 4 ml of deoxynucleoside triphosphate mixture, 1 ml of each primer (20 mM), 1 ml of the DNA solution, and 0.45 ml of HotStart Version ExTaq DNA polymerase (Takara). PCR was performed with a Bio-Rad iCycler thermocycler using the following protocol: 1 min at 94 C (denaturation), 1 min at 65 C (annealing), and 3 min at 72 C (elongation), with a 1 C decrease in the annealing temperature every second cycle as a “touchdown” for 20 cycles, followed by 10 cycles at an annealing temperature of 55 C and a final cycle consisting of 10 min at 72 C. After gel electrophoresis (1.5% [wt/vol] agarose gel) of 4 ml subsamples from the PCR product, the amount of amplified DNA was quantified by comparing the band intensities to the standard curves obtained with a Low DNA Mass Ladder (Takara). The band intensities were measured with Quantity One analysis software (Bio-Rad Laboratories, Hercules, CA). Profiles of the amplified 16S rRNA gene sequences were produced by DGGE using the CBS DGGE system (CBS Scientific, USA) (Muyzer et al., 1993). The PCR products were loaded onto a polyacrylamide gel (8% [wt/vol] acrylamide in 1 TAE buffer with a 45e 65% denaturant gradient (100% denaturant was 7 M urea and 40% [vol/vol] deionized formamide)). The wells were loaded with 25 ml of PCR product, and electrophoresis was conducted in the TAE buffer at 100 V for 17 h at 60 C. The DNA fragments were stained by silver staining as described by Radojkovic and Kusic (2000). The gel was destained in distilled water for 5 min. Images of the gels were obtained after destaining using a Bio-Rad GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Hercules, CA). The band patterns were analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA) (Zhang and Jackson, 2008). Each detected band was defined as an operational taxonomic unit (OTU), and the number of bands was defined as the genotypic richness of each sample (Bell et al., 2005). The pixel intensity for each band was detected by Quantity One software and was expressed as the relative abundance (Pi) (Reche et al., 2005). The richness index (ShannoneWiener index, H0 ) (Trevors et al., 2010) was calculated using the relative abundance data based on the following equation:
H ¼
s X
pi ln pi
i¼1
where Pi ¼ ni/S, ni is the abundance of the ith OTU and S is the total abundance of all OTUs in the sample.
Theoretical mass loss rateð%Þ ¼ Ra Pa þ Re Pe ; where Ra and Re are the accumulated mass loss of pure alder and eucalyptus litter at each stage, respectively, and Pa and Pe are the initial proportions of alder and eucalyptus litter in the mixture, respectively. 2.7. Statistical analysis All of the variables, including the measurements and calculations among the different litter mixtures, were tested by one-way analysis of variance (ANOVA). When significant differences were detected, the LSD multiple range test was used to determine where the differences existed. Differences were considered significant at P < 0.05. Linear regressions were used to examine the relationships between the accumulated mass loss and the microbial biomass and bacterial DGGE bands. All of the statistical analyses were performed using the SPSS (Statistical Package for the Social Sciences) software package (Standard release version 16.0 for Windows, SPSS Inc., IL, USA). 3. Results 3.1. Mass loss Over 169 days of decomposition, the accumulated mass loss was significantly different in the pattern 3E:7A > 10A > 5E:5A > 7E:3A > 10E. Approximately 79.22%, 70.23%, 62.82%, 49.95% and 48.59% of mass was lost from the 3E:7A, 10A, 5E:5A, 7E:3A and 10E litter mixtures, respectively (Fig. 1). Compared with pure eucalyptus litter, 3E:7A, 10A, 5E:5A and 7E:3A litter mixtures increased 63.04%, 44.54%, 29.29% and 2.80% of accumulated mass loss. Few differences in the accumulated mass loss were observed between the different litter mixtures in the half month of decomposition before May 1st, and the 3E:7A litter mixture had a higher accumulated mass loss only in the last two months of the study (after August 1st). The accumulated mass loss of pure eucalyptus litter (10E) was significantly lower than those of the other litter mixtures after May 15th, but few differences were detected between the accumulated mass loss of the 10E and 7E:3A litter mixtures after September 1st. Additionally, the observed accumulated mass loss was close to the theoretical values for each litter mixture on May 1st, but a significant non-additive effect was detected over the course of the litter mixture decomposition after May 1st. Although the observed accumulated mass loss in the 7E:3A litter mixture after September 1st was slightly lower than their theoretical values, the observed accumulated mass loss in the other litter mixtures was higher than their theoretical values (Fig. 2). 3.2. Microbial biomass Microbial biomass carbon (MBC) first increased and then decreased as the litter mixture decomposition proceeded (Fig. 3),
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90
10E
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3E:7A
10A c
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80
d
70 bb b
60 c
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a
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Fig. 1. The accumulated mass loss in the litterbags during the decomposition of the E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3) and the different letters denote significant differences (P < 0.05) among different litter mixtures.
and its peak value did not appear at the same time for each litter mixture. The highest MBC values during the decomposition of the 10A and 7E:3A litter mixtures were observed on August 15th, whereas the highest values for the other mixtures were observed on September 15th. Few differences were observed among the MBC values of the different litter mixtures during the first two months (until June 1st) of decomposition. After this point, the MBC values of the pure alder litter (10A) decomposition were higher than those of the other litter mixtures from June 1st to August 15th, and the values of the 3E:7A litter mixture were higher after September 1st. Conversely, the MBC values for the pure eucalyptus litter (10E) decomposition were the lowest among the other litter mixtures after July 1st.
3.3. Bacterial community from PCR-DGGE The PCR-DGGE bacterial analysis revealed that there were significant variations between the DGGE bands of the different litter samples during decomposition (Fig. 4). The DGGE bands showed an increase followed by a decreasing tendency as the litter mixture decomposition proceeded, with the exception of the 5A:5E litter mixture, which showed the opposite tendency (Table 1). In comparison with the DGGE bands for the pure eucalyptus litter decomposition, the addition of alder litter increased the number of DGGE bands, and the most DGGE bands were detected for the
Accumulated mass loss (%)
90
Observed
3E:7A litter mixture. The ShannoneWiener indices showed similar patterns as the changes in DGGE bands.
4. Discussion The hypothesis that the admixture of alder litter can improve eucalyptus litter decomposition was confirmed by this study. Both the present study and previous results have demonstrated that mixing eucalyptus litter with more readily decomposable litter can enhance the decomposition of eucalyptus litter (Briones and Ineson, 1996). From a quantitative perspective, this study hypothesized that the litter mixture would decompose more slowly as the amount of slowly decomposing eucalyptus litter increases (Ostrofsky, 2007; Pérez-Suárez et al., 2012). Interestingly, our results indicated that the presence of a certain amount of eucalyptus litter actually enhanced the mass loss rate of the litter mixture (3E:7A) over that of the pure alder litter. Furthermore, a significant non-additive effect was observed in this study, as the mass loss rates of the litter mixtures were higher than their theoretical values at most of the stages of decomposition. This result may be explained as a synergistic effect (Gartner and Cardon, 2004) between alder litter and eucalyptus litter, confirming that these two species facilitate each other’s litter decomposition. Many previous studies have reported that the admixture of higher-quality litter can promote the decomposition of more
Theoretical
80 70 60 50 40 30 20 10
7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A 7E:3A 5E:5A 3E:7A
0
1-May 15-May 1-Jun
15-Jun
1-Jul
15-Jul
1-Aug 15-Aug 1-Sep 15-Sep
1-Oct
Fig. 2. The observed and theoretical values of the accumulated mass loss during the decomposition of E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3).
F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121
1400
10E
7E:3A
5E:5A
3E:7A
119
10A
MBC (mg kg dry mass-1)
1200 1000 800 600 400 200 0 1-May
15-May
1-Jun
15-Jun
1-Jul
15-Jul
1-Aug
15-Aug
1-Sep
15-Sep
1-Oct
Date Fig. 3. The microbial biomass carbon (MBC) during the decomposition of E. grandis and A. formosana litter mixtures. Bars indicate SE (n ¼ 3).
Fig. 4. The silver-stained bands obtained by DGGE of bacteria during the decomposition of E. grandis and A. formosana litter mixtures.
120
F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121
Table 1 ShannoneWiener index (H) and the abundance of DGGE bands (S) during the decomposition of E. grandis and A. formosana litter mixtures. Treatments
10E 7E:3A 5E:5A 3E:7A 10A
Diversity index
Sampling time 1-May
15-May
1-Jun
15-Jun
1-Jul
15-Jul
1-Aug
15-Aug
1-Sep
15-Sep
1-Oct
H S H S H S H S H S
2.5875 14 2.4814 12 3.4453 32 3.4504 33 3.4170 31
2.7474 16 3.3088 28 3.5300 35 3.4821 34 3.3547 29
2.9745 20 3.1609 24 2.7516 27 3.4074 31 3.4237 31
3.0219 21 2.8392 18 2.7578 16 3.5550 36 3.4552 32
2.9705 20 3.6048 38 2.9809 20 3.4954 34 3.5232 34
3.0139 21 3.1887 25 3.1128 23 3.8154 47 3.4300 31
2.9245 19 3.2987 28 3.3390 29 3.8637 50 3.5377 35
2.9751 20 3.6621 40 3.3831 30 3.6211 39 3.6465 40
2.9759 20 3.4755 33 3.1169 23 3.5638 37 3.5726 37
2.9047 19 3.4436 32 3.2798 27 3.6503 40 3.5455 35
3.1636 24 3.4215 33 3.2387 26 3.3709 31 3.5716 33
(Hansen and Coleman, 1998; Hector et al., 2000; Berglund et al., 2013). We also observed that although the number of bacterial DGGE bands was higher in the litter mixtures (5E:5A and 3E:7A before May 15th) than in the pure alder and eucalyptus litters (Table 1), microbial biomass carbon did not significantly change (Fig. 3). As decomposition proceeded, increasing decomposer communication and physical changes in the litter mixture structure contributed to the mass loss of the litter mixture (Hansen and Coleman, 1998; Hector et al., 2000), leading to the positive nonadditive effects (obvious synergistic effects) observed in all of the litter mixtures (Fig. 2) at this stage. However, after the rapid loss of labile components, litter mixture decomposition can be slowed by the decomposition of the remaining inhibitory compounds, such as phenolics and tannins (Fyles and Fyles, 1993; Salamanca et al., 1998). Less decomposable litter and more inhibitory compounds remain in the litter mixture because of the reduction in labile components. This observation is consistent with the 7E:3A litter mixture, which displayed negative non-additive effects (antagonistic interactions) after September 1st. The continuous synergistic effects in the 5E:5A and 3E:7A litter mixtures may be explained by the presence of relatively more labile components, which can maintain the positive interactions for a longer time. These types of litter mixtures may also exhibit antagonistic interactions in the later decomposition stages. Therefore, our present results suggest that the inconsistencies in previous observations (both negative and positive non-additive effects that were reported in the review of Gartner and Cardon, 2004) of litter mixture decompositions might be attributed to the differences in the decomposition stages and mixture proportions. Additional studies and longer decomposition experiments should be conducted to resolve these issues.
recalcitrant litters, or vice versa (Fyles and Fyles, 1993; Briones and Ineson, 1996; McTiernan et al., 1997), which is partly consistent with the present study’s finding that an admixture of alder litter accelerated the decomposition of eucalyptus litter in the present study, but the addition of eucalyptus litter also stimulated alder litter decomposition when the mixture proportion is considered. This result coincided with the mechanisms proposed by Ostrofsky (Ostrofsky, 2007), who declared that four principles control the decomposition of mixed litter: (1) the presence of rapidly decomposing leaves that favor the rapid colonization of decomposer communities, which accelerate the mass loss of slowly processed recalcitrant leaves; (2) the co-occurring reductions of the mass loss rates of rapidly decomposing leaves and slowly decomposing leaves caused by high concentrations of water-soluble defensive or inhibitory compounds (e.g., tannins and other phenolics); (3) a more persistent habitat for decomposing organisms provided by the structural stability of the litter mixture beds to accelerate the decomposition of slowly decomposing leaves by protecting the abrasion and leaching processes; (4) both facilitative and inhibitory mechanisms that occur simultaneously, as none of these effects are mutually exclusive, so that the observed accumulated mass loss changes in litter mixture decomposition represent the net effect of multiple processes (Ostrofsky, 2007; Pérez-Suárez et al., 2012). However, the different litter mixtures did not follow the same pattern over the course of decomposition. In the initial decomposition stage, the litter mixture mass loss rate equaled the sum of the alder and eucalyptus litter mass loss rates alone, displaying a completely additive effect, shown in Fig. 2. The initially intact shapes of both types of litter may limit their complete mixture, leading to few changes in nutrient transfer and decomposer habitat
(a)
1200
(b)
50
800 600 2
R = 0.8013 P =0.0000
400
DGGE band number
MBC (mg kg dry mass -1)
1000
60
200
40 30 2
R = 0.2710 P =0.007
20 10
0
0 0
20
40
60
80
Accumulated mass loss (%)
100
0
20 40 60 80 Accumulated mass loss (%)
100
Fig. 5. The linear relationships between the accumulated mass loss and microbial biomass carbon (MBC) (a), and between the accumulated mass loss and DGGE band number of bacteria (b).
F. Wu et al. / Soil Biology & Biochemistry 73 (2014) 115e121
Additionally, one of the essential promoters of litter mixture decomposition is the increase of decomposer diversity because of the alteration of substrate diversity and microhabitat complexity (Kaneko and Salamanca, 1999; Wardle et al., 2006). In the current study, we observed that the alder and eucalyptus mixture litter showed a higher number of bacterial DGGE bands as decomposition proceeded compared to pure eucalyptus litter. However, a higher number of bacterial DGGE bands were also detected in the 3E:7A litter mixture than in the pure alder litter at most of the decomposition stages, indicating obvious synergistic effects. This result is in accordance with the conclusion of Gartner and Cardon (2004), who claimed that the interactions of litter from different species in an ecosystem influenced the structure and activity of the decomposer community, which is largely responsible for decomposition. In comparison with bacterial biodiversity, the microbial biomass was more statistically correlated to the litter mixture mass loss (Fig. 5). Microbial biomass refers to the biomass composed of the microorganisms on the suitable substrate, which represents biological activity. In an incubation study, Thiessen et al. (2013) also documented that organic matter decomposition mainly depended on microbial biomass. In conclusion, the admixture of alder litter can stimulate eucalyptus litter decomposition, and a small amount of eucalyptus litter may also promote alder litter decomposition. Over the total investigation period, the decomposition of each litter mixture displayed positive non-additive effects. However, the effects differed over the decomposition process: exactly additive effects were observed in the initial stage, positive non-additive effects were observed in the middle stage, and negative non-additive effects were observed in the later stage and would remain in progress as the decomposition proceeded. Furthermore, litter mixture decomposition relied more on microbial biomass than microbial diversity. The results indicate that alder could be a potential candidate for mixed-species eucalyptus plantations because of its synergistic effects on material cycling. Acknowledgments The authors are very grateful to their colleagues from Ecological Modelling and Carbon Science (ECO-MCS), Institute of Environment Sciences, University of Quebec at Montreal (UQAM) for providing helpful suggestions in manuscript preparation. This research was financially supported by the National Natural Science Foundation of China (31170423 & 31270498), the National Key Technologies R&D of China (2011BAC09B05), and the Sichuan Youth Sci-tech Foundation (2012JQ0008 & 2012JQ0059). References Bell, T., Ager, D., Song, J.I., Newman, J.A., Thompson, I.P., Lilley, A.K., van der Gast, C.J., 2005. Larger islands house more bacterial taxa. Science 308, 1884. Berg, B., McClaugherty, C., 2008. Plant Litter: Decomposition, Humus Formation, Carbon Sequestration, second ed. Springer, New York. Berglund, S.L., Ågren, G.I., 2012. When will litter mixtures decompose faster or slower than individual litters? A model for two litters. Oikos 121, 1112e1120. Berglund, S.L., Ågren, G.I., Ekblad, A., 2013. Carbon and nitrogen transfer in leaf litter mixtures. Soil Biology & Biochemistry 57, 341e348. Binkley, D., Senock, R., Bird, S., Cole, T.G., 2003. Twenty years of stand development in pure and mixed stands of Eucalyptus saligna and N-fixing Facaltaria moluccana. Forest Ecology and Management 182, 93e102. Briones, M.J.I., Ineson, P., 1996. Decomposition of eucalyptus leaves in litter mixtures. Soil Biology & Biochemistry 28, 1381e1388. Chapman, K., Whittaker, J.B., Heal, O.W., 1988. Metabolic and faunal activity in litters of tree mixtures compared with pure stands. Agriculture, Ecosystems & Environment 24, 33e40. Couteaux, M.M., Bottner, P., Berg, B., 1995. Litter decomposition, climate and litter quality. Trends in Ecology & Evolution 10, 63e66. Forrester, D.I., Bauhus, J., Cowie, A.L., Vanclay, J.K., 2006. Mixed-species plantations of eucalyptus with nitrogen fixing trees: a review. Forest Ecology and Management 233, 211e230.
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