Skip to main content
PMC home page
Scientific Reports logoLink to Scientific Reports
. 2018 Nov 23;8:17324. doi: 10.1038/s41598-018-35762-0

Introduction of a leguminous shrub to a rubber plantation changed the soil carbon and nitrogen fractions and ameliorated soil environments

Chang-An Liu 1,✉, Yu Nie 1,2, Yan-Ming Zhang 3, Jian-Wei Tang 1, Kadambot H M Siddique 4
PMCID: PMC6251934  PMID: 30470791

Abstract

The conversion of monoculture rubber (Hevea brasiliensis) plantations into rubber-based agroforestry systems has become a common trend in forestry management in the past few decades. Rubber–Flemingia macrophylla (a leguminous shrub) systems are popular in southwestern China’s Xishuangbanna region. The biogeochemical cycles of soil carbon and nitrogen in forests are mainly affected by their fractions. This study investigated the effect of introducing Flemingia macrophylla to rubber plantations of different ages on soil carbon and nitrogen fractions. The experimental treatments included R1 (young rubber plantation), RF1 (young rubber–Flemingia macrophylla system), R2 (mature rubber plantation) and RF2 (mature rubber–Flemingia macrophylla system). The results showed that the introduction of Flemingia macrophylla to rubber plantations of different ages significantly changed soil carbon and nitrogen fractions, improved soil labile organic carbon and nitrogen contents, and ameliorated soil environments. The average soil microbial biomass organic carbon, nitrogen and nitrate-nitrogen in the 0–10 cm soil layer during the experimental period was 38.9%, 55.5%, and 214.7% higher in RF1 than R1, respectively, and 22.1%, 22.2%, and 652.2% higher in RF2 than R2, respectively. Therefore, Flemingia macrophylla can be used as an alternative interplanted tree species within rubber plantations in similar environments of southeastern Asia.

Introduction

Monoculture rubber (Hevea brasiliensis) plantations have rapidly expanded in the last few decades in southeastern Asia1,2. Approximately 90% of global natural rubber production is derived from plantations in this region (http://www.rubberstudy.com), accounted for an estimated 84% of the total global rubber plantation area in 20121,3. Rapid growth in the Chinese economy has increased demand for natural rubber. In response to this demand, the natural tropical forests of southwestern China’s Xishuangbanna region were deforested and replaced with more than 470,000 ha of rubber plantations, which equates to more than 24% of the total land area of the region4. The expansion of these rubber plantations has led to water loss and soil erosion5,6, environmental degradation3,7, and threatened environmental biodiversity8. At present, rubber-based agroforestry systems are considered the best way to resolve the environmental problems associated with rubber monoculture. In recent years, the local government of Xishuangbanna proposed the development of environmentally friendly rubber plantations to reduce the water and soil losses and increase environmental biodiversity9,10.

Legume plants could greatly enhance ecosystem services. Lucerne (Medicago sativa) and erect milkvetch (Astragalus adsurgens) have the potential to phytoextract rhenium from coal fly ash-amended alkaline soils11. Alfalfa (Medicago sativa L.) can improve its phosphorus acquisition by increasing specific root length and exuding gcarboxylates into the rhizosphere in phosphorus-deficient environments12. A combination of legumes grass species can enhance soil C and N storage, productivity, and diversity in semi-arid grasslands13. Flemingia macrophylla is used in traditional medicine for various therapeutic uses and is widely planted in the Xishuangbanna area of China. Flemingia macrophylla is a perennial leguminous leafy shrub with strong biological nitrogen fixation and high biomass14. As a result, rubber–Flemingia macrophylla intercropped systems have become popular in the Xishuangbanna area to improve soil carbon and nitrogen storage.

Precise and accurate estimations of carbon and nitrogen levels in forest soil are important for understanding biogeochemical cycles15–17. Some studies have indicated that soil organic carbon (SOC) has been depleted in rubber plantations18,19. Rubber plantations have 15% lower annual surface soil CO2 fluxes than natural forests, because they have lower soil respiration during the dry season20. The conversion of tropical rainforests into rubber plantations has increased N2O emissions, which may potentially enhance local climate warming trends21. Rubber plantations have lower mean CH4 uptake rates than secondary and tropical forests22. These transformations and biogeochemical cycles of soil carbon and nitrogen are mainly affected by their fractions23–27 that play essential roles in the turnover of nutrients in soil, including water-soluble organic carbon (WSOC), light fraction of organic C (LFOC), microbial biomass organic C (MBC), ammonium N (AN), nitrate N (NN), light fraction of organic N (LFON), and microbial biomass organic N (MBN). These fractions are often used to study the impacts of land management and ecological succession26,28,29. Rubber-based agroforestry systems have higher SOC and nitrogen levels and lower carbon and nitrogen losses than rubber plantations due to improved soil macroaggregates30. However, little is known about the effect of these systems on soil carbon and nitrogen fractions.

The objectives of this study were to examine: (1) soil carbon and nitrogen fractions, and (2) the relationships between soil carbon and nitrogen fractionsin rubber and rubber–Flemingia macrophylla plantations of different ages.

Results

Soil carbon fractions

The SOC content in the 0–10 or 10–30 cm soil layers did not differ between the plantation treatments (R1, R2, RF1, RF2) for the duration of the study (April 2014 to January 2017) (Table 1). However, SOC content in the 0–10 cm soil layers was significantly higher than the 10–30 cm soil layers in each plantation type. In the 0–10 cm soil layer, RF1 had consistently higher WSOC content than R1 from June 2015 to January 2017, which differed significantly in August 2016 and January 2017. The WSOC content between R2 and RF2 did not differ significantly for the duration of the experiment (Table 2). In the 10–30 cm soil layer, the introduction of Flemingia macrophylla to the rubber plantations had no significant effect on WSOC content.

Table 1.

Soil organic carbon (SOC) (g kg−1) in rubber and rubber–Flemingia macrophylla plantations from April 2014 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Apr 2014 Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 12.50 ± 0.47abA 12.45 ± 0.31aA 12.64 ± 0.79abA 12.50 ± 0.34aA 12.77 ± 0.46abA
RF1 12.77 ± 2.53aA 13.04 ± 1.23aA 13.22 ± 1.42aA 13.59 ± 1.77aA 13.45 ± 1.65aA
R2 13.79 ± 0.31aA 13.53 ± 0.60aA 14.40 ± 1.02aA 14.11 ± 1.35aA 14.20 ± 0.41aA
RF2 13.16 ± 0.90 aA 12.74 ± 0.97 aA 13.04 ± 0.68abA 12.76 ± 1.36 aA 13.24 ± 1.88 aA
10–30 cm R1 9.89 ± 0.71cA 9.65 ± 0.06bA 9.73 ± 0.94cA 9.73 ± 0.43bA 9.98 ± 0.63cA
RF1 10.49 ± 0.31cA 10.61 ± 1.02bA 10.25 ± 2.43cA 9.84 ± 1.85bA 10.08 ± 0.69cA
R2 10.22 ± 0.33cA 10.90 ± 0.18bA 11.00 ± 0.49bcA 10.24 ± 0.42bA 10.67 ± 0.64cA
RF2 10.84 ± 1.03bcA 10.51 ± 0.49bA 10.20 ± 0.37cA 11.16 ± 1.34bA 10.92 ± 1.32bcA
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010.Values within a column followed by the same letter (lower case) or within the same row (upper case) do not differ significantly at P ≤ 0.05.

Table 2.

Soil water-soluble organic carbon (WSOC) (mg kg−1) in rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 70.7 ± 3.7b 78.3 ± 1.0a 67.5 ± 2.2b 67.7 ± 6.7b
RF1 72.3 ± 1.9b 86.0 ± 5.5a 84.9 ± 7.0a 80.5 ± 7.8a
R2 84.5 ± 6.1a 86.6 ± 10.1a 75.1 ± 5.0ab 50.1 ± 4.4d
RF2 82.5 ± 1.7a 80.5 ± 2.7a 76.9 ± 6.2ab 57.7 ± 8.0bcd
10–30 cm R1 71.2 ± 5.5b 80.7 ± 8.4a 72.8 ± 8.0b 66.1 ± 1.5b
RF1 70.7 ± 7.3b 82.5 ± 4.2a 75.6 ± 7.1ab 63.2 ± 8.7bc
R2 88.8 ± 3.4a 82.3 ± 8.2a 75.3 ± 4.6ab 50.4 ± 6.0d
RF2 85.9 ± 2.3a 87.8 ± 14.8a 70.6 ± 7.7b 55.0 ± 1.2 cd
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: The Flemingia macrophylla introduced to R2 in 2010. Values within a column followed by the same letter do not differ significantly at P ≤ 0.05.

The 0–10 cm soil layer had significantly higher LFOC content than the 10–30 cm soil layer in each plantation type. In the 0–10 cm soil layer, RF1 had consistently higher LFOC content than R1 from June 2015 to January 2017, which differed significantly in August 2016 and January 2017. In the same layer, RF2 had consistently lower LFOC content than R2, with significant differences observed in January 2016 and 2017 (Table 3). In the 10–30 cm soil layer, the introduction of Flemingia macrophylla to the rubber plantations had no significant effect on LFOC content but increased MBC content and the ratios of MBC/SOC in the 0–10 and 10–30 cm soil layers (Table 4 and Fig. 1). In the 0–10 cm soil layer, RF1 had consistently higher ratios of LFOC/SOC than R1 from January 2016 to January 2017, but no significant differences were observed between the RF2 and R2 for the duration of the study (Fig. 2). In the 10–30 cm soil layer, the introduction of Flemingia macrophylla to the rubber plantations had no significant effect on the ratios of LFOC/SOC.

Table 3.

Light fraction organic carbon (LFOC) (g kg−1) in rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 1.21 ± 0.15b 1.13 ± 0.15b 1.14 ± 0.25c 1.03 ± 0.06b
RF1 1.19 ± 0.10b 1.36 ± 0.21b 2.38 ± 0.23a 1.34 ± 0.11a
R2 1.56 ± 0.13a 1.75 ± 0.30a 1.61 ± 0.26b 1.45 ± 0.14a
RF2 1.45 ± 0.09a 1.31 ± 0.09b 1.51 ± 0.13b 1.00 ± 0.12b
10–30 cm R1 0.45 ± 0.07c 0.36 ± 0.02c 0.54 ± 0.10d 0.61 ± 0.01c
RF1 0.45 ± 0.07c 0.50 ± 0.07c 0.59 ± 0.06d 0.51 ± 0.16c
R2 0.61 ± 0.09c 0.59 ± 0.12c 0.61 ± 0.11d 0.57 ± 0.18c
RF2 0.54 ± 0.05c 0.47 ± 0.08c 0.60 ± 0.04d 0.48 ± 0.11c
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Values within a column followed by the same letter do not differ significantly at P ≤ 0.05.

Table 4.

Soil microbial biomass carbon (MBC) (mg kg−1) in rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 122.0 ± 7.5e 203.9 ± 23.9de 126.5 ± 16.5de 115.6 ± 17.7c
RF1 141.8 ± 15.0e 256.3 ± 24.3bc 245.0 ± 30.4c 145.9 ± 20.3c
R2 174.3 ± 23.0d 281.3 ± 21.0ab 341.8 ± 25.7b 179.7 ± 17.6b
RF2 236.1 ± 11.1b 314.0 ± 31.6a 420.7 ± 65.2a 222.4 ± 31.5a
10–30 cm R1 47.4 ± 5.9f 182.2 ± 17.3e 72.1 ± 4.2e 68.2 ± 13.4d
RF1 54.9 ± 11.8f 234.7 ± 18.1cd 164.4 ± 27.8d 70.3 ± 7.9d
R2 199.7 ± 18.1c 136.9 ± 14.6f 310.6 ± 39.6bc 138.1 ± 17.9c
RF2 280.9 ± 3.6a 192.8 ± 19.0e 354.5 ± 61.8ab 194.5 ± 7.2ab
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Values within a column followed by the same letter do not differ significantly at P ≤ 0.05.

Figure 1.

Figure 1

Open in a new tab

Ratio of soil microbial biomass carbon (MBC) to soil organic carbon (SOC) (MBC/SOC); and ratio of soil microbial biomass nitrogen (MBN) to soil total nitrogen (TN) (MBN/TN) in the rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017. R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Vertical bars are LSD at P ≤ 0.05.

Figure 2.

Figure 2

Open in a new tab

Ratio of soil light fraction carbon (LFOC) to soil organic carbon (SOC) (LFOC/SOC); and ratio of soil light fraction nitrogen (LFON) to soil total nitrogen (TN) (LFON/TN) in the rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017. R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Vertical bars are LSD at P ≤ 0.05.

Soil nitrogen fractions

The TN content in the 0–10 or 10–30 cm soil layers did not differ between the plantation treatments (R1, R2, RF1, RF2) for the duration of the study (April 2014 to January 2017) (Table 5). However, the 0–10 cm soil layer had significantly higher TN contents than the 10–30 cm soil layer in each plantation type. The introduction of Flemingia macrophylla to the rubber plantations generally decreased AN content and increased NN content in the 0–10 and 10–30 cm soil layers (Fig. 3). In the 0–10 cm soil layer, RF1 had consistently higher LFON content than R1 from June 2015 to January 2017, with significant differences observed from January 2016 to January 2017. In the same layer, no significant differences in LFON content were observed between R2 and RF2, except for January 2017 when the LFON content was significantly higher in R2 than RF2 (Table 6). In the 10–30 cm soil layer, the introduction of Flemingia macrophylla to the rubber plantations had no significant effect on LFON content. The 0–10 cm soil layer had significantly higher LFON contents than the 10–30 cm soil layer in each plantation type.

Table 5.

Soil total nitrogen (TN) (g kg−1) in rubber and rubber–Flemingia macrophylla plantations from April 2014 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Apr 2014 Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 1.48 ± 0.03aA 1.50 ± 0.10aA 1.44 ± 0.06abA 1.50 ± 0.06aA 1.45 ± 0.04aA
RF1 1.45 ± 0.20abA 1.45 ± 0.07aA 1.42 ± 0.15abA 1.55 ± 0.35aA 1.46 ± 0.1aA
R2 1.48 ± 0.04aA 1.45 ± 0.07aA 1.52 ± 0.06aA 1.46 ± 0.11aA 1.55 ± 0.05aA
RF2 1.48 ± 0.11aA 1.43 ± 0.11aA 1.50 ± 0.04aA 1.41 ± 0.13abA 1.47 ± 0.14aA
10–30 cm R1 1.15 ± 0.10cA 1.12 ± 0.03bA 1.17 ± 0.07cA 1.16 ± 0.10bcA 1.17 ± 0.06bA
RF1 1.15 ± 0.07cA 1.18 ± 0.09bA 1.19 ± 0.12cA 1.12 ± 0.16cA 1.10 ± 0.06bA
R2 1.28 ± 0.07cA 1.21 ± 0.03bAB 1.29 ± 0.05bcA 1.17 ± 0.04bcB 1.22 ± 0.04bAB
RF2 1.29 ± 0.08bcA 1.24 ± 0.06bA 1.23 ± 0.09cA 1.18 ± 0.12bcA 1.23 ± 0.09bA
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Values within a column followed by the same letter (lower case) or within the same row (upper case) do not differ significantly at P ≤ 0.05.

Figure 3.

Figure 3

Open in a new tab

Soil ammonium N (AN) and nitrate N (NN) content levels in the rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017. R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Vertical bars are LSD at P ≤ 0.05.

Table 6.

Light fraction organic nitrogen (LFON) (g kg−1) in rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 0.057 ± 0.010b 0.051 ± 0.010b 0.049 ± 0.012c 0.044 ± 0.003b
RF1 0.062 ± 0.009b 0.070 ± 0.014a 0.117 ± 0.016a 0.068 ± 0.005a
R2 0.077 ± 0.013a 0.085 ± 0.014a 0.077 ± 0.009b 0.068 ± 0.013a
RF2 0.078 ± 0.005a 0.069 ± 0.003a 0.077 ± 0.005b 0.051 ± 0.004b
10–30 cm R1 0.016 ± 0.003c 0.011 ± 0.002c 0.020 ± 0.004d 0.020 ± 0.003c
RF1 0.021 ± 0.005c 0.019 ± 0.002c 0.026 ± 0.004d 0.019 ± 0.005c
R2 0.024 ± 0.004c 0.023 ± 0.006c 0.024 ± 0.004d 0.022 ± 0.007c
RF2 0.025 ± 0.004c 0.021 ± 0.003c 0.027 ± 0.002d 0.020 ± 0.005c
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Values within a column followed by the same letter do not differ significantly at P ≤ 0.05.

The introduction of Flemingia macrophylla to the rubber plantations increased MBN content and the ratios of MBN/TN in the 0–10 and 10–30 cm soil layers (Table 7 and Fig. 1). In the 0–10 cm soil layer, RF1 had consistently higher ratios of LFON/TN than R1 from January 2016 to January 2017, but no significant differences were observed between the RF2 and R2 for the duration of the study (Fig. 2). In the 10–30 cm soil layer, the introduction of Flemingia macrophylla to the rubber plantations had no significant effect on the ratios of LFON/TN. The introduction of Flemingia macrophylla to the rubber plantations decreased the ratios of LFOC/LFON in the 0–10 and 10–30 cm soil layers (Fig. 4).

Table 7.

Soil microbial biomass nitrogen (MBN) (mg kg−1) in rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017 (mean ± SD, n = 3).

Soil depth (cm) Treatments Jun 2015 Jan 2016 Aug 2016 Jan 2017
0–10 cm R1 18.2 ± 1.3d 35.2 ± 14.1b 21.2 ± 3.2c 20.5 ± 4.6cd
RF1 28.8 ± 4.6bc 42.5 ± 11.9ab 44.1 ± 10.5b 32.5 ± 5.4ab
R2 24.0 ± 4.8c 54.7 ± 10.2a 38.6 ± 3.9b 34.0 ± 1.4ab
RF2 34.0 ± 3.2b 55.2 ± 3.3a 59.3 ± 7.1a 36.1 ± 7.1a
10–30 cm R1 5.3 ± 0.2e 32.7 ± 3.4b 17.1 ± 4.1c 17.0 ± 6.2de
RF1 10.8 ± 3.3e 36.6 ± 3.9b 25.2 ± 1.7c 12.3 ± 3.9e
R2 30.4 ± 2.1b 32.8 ± 5.9b 49.4 ± 15.5ab 27.1 ± 0.6bc
RF2 37.1 ± 3.3a 35.8 ± 6.4b 45.8 ± 4.7b 32.2 ± 0.6ab
Open in a new tab

R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Values within a column followed by the same letter do not differ significantly at P ≤ 0.05.

Figure 4.

Figure 4

Open in a new tab

Ratio of organic C to total N in the soil light fractions (LFOC/LFON) in the rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017. R1: rubber plantations established in 2006; R2: rubber plantations established in 1994; RF1: Flemingia macrophylla introduced to R1 in 2010; RF2: Flemingia macrophylla introduced to R2 in 2010. Vertical bars are LSD at P ≤ 0.05.

Relationships between soil carbon and nitrogen fractions

MBC and MBN had significant positive correlations with LFOC, LFON, and the ratios of MBC/SOC, LFOC/SOC, MBN/TN and LFON/TN (Table 8). MBC and MBN also had negative correlations with the ratio of LFOC/LFON. AN and NN had significant positive correlations with LFOC and LFON, and the ratios of LFOC/SOC and LFON/TN. Furthermore, AN and NN had significant negative correlations with LFOC/LFON.

Table 8.

Correlation coefficients (R) among different soil carbon and nitrogen fractions in rubber and rubber–Flemingia macrophylla plantations from June 2015 to January 2017.

Measurement LFON/TN LFOC/SOC LFOC/LFON MBN/TN MBC/SOC NN AN LFON MBN LFOC MBC
MBC 0.45* 0.40* −0.47** 0.91*** 0.95*** 0.26 0.39* 0.43* 0.92*** 0.39*
LFOC 0.97*** 0.99*** 0.69*** 0.20 0.13 0.39* 0.41* 0.99*** 0.44*
MBN 0.48** 0.44* −0.46** 0.96*** 0.85*** 0.28 0.20 0.48**
LFON 0.99*** 0.98*** −0.75*** 0.23 0.17 0.45** 0.41*
AN 0.43* 0.42* −0.45** 0.13 0.30 0.25
NN 0.47*** 0.42* −0.55** 0.17 0.16
MBC/SOC 0.19 0.15 0.28 0.92***
MBN/TN 0.25 0.21 0.28
LFOC/LFON 0.76*** 0.67***
LFOC/SOC 0.98***
LFON/TN
Open in a new tab

MBC: soil microbial biomass carbon; LFOC: light fraction organic carbon; MBN: soil microbial biomass nitrogen; LFON: light fraction organic nitrogen; AN: soil ammonium nitrogen; NN: soil nitrate-nitrogen; MBC/SOC: ratio of MBC to SOC; MNB/TN: ratio of MBN to TN; LFOC/LFON: ratio of LFOC to LFON; LFOC/SOC: ratio of LFOC to SOC; LFON/TN: ratio of LFON to TN. Statistical significance: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Discussion

Soil carbon and nitrogen fractions in the rubber and rubber–Flemingia macrophylla plantation systems

Plantations with suitable native, broad-leaved species (for example, Alnus subcordata C. A. Mey.) along with planned forestation management could potentially rehabilitate the degraded natural forests of northern Iran31. Mo and Sha32 reported that adding carbon-sink plants into rubber plantations increased the soil organic carbon storage. The presence of legumes in semi-arid grasslands increased soil C and N storage by increasing the above- and below-ground biomass, litter biomass, plant species richness, and diversity13. In 2014 in present study, biomass of Flemingia macrophylla accumulated in the young and mature rubber plantations at 23.02 and 0.55 t ha−1, respectively14 but had no significant effect on SOC or TN contents. There are four possible explanations for this response: (1) the rubber–Flemingia macrophylla system had only been established for seven years, and it is unlikely that soil C and N storage would have changed significantly in such a short time; (2) the high-temperature and high-humidity environments in the rainy season would promote the decomposition of plant residues and nutrient leaching, which is not conducive to C and N storage; (3) the introduction of Flemingia macrophylla to the rubber plantations reduced plant species richness and diversity (Fig. 5) and the input of litter biomass of other species; (4) most rubber plantations in this area have been planted on sloping land20. In our study, the rubber and rubber–Flemingia macrophylla plantations were planted on sloping land between 47 and 58%, and the nutrients of the litter decomposition and some un-decomposed litter of Flemingia macrophylla would have been lost due to runoff.

Figure 5.

Figure 5

Open in a new tab

Photographs of the rubber and rubber–Flemingia macrophylla plantations. (A) rubber plantation; (B) rubber–Flemingia macrophylla plantation; (C) Flemingia macrophylla after mowing in the rubber–Flemingia macrophylla plantation.

While the introduction of Flemingia macrophylla to the rubber plantations had no significant effect on the C and N contents in the soil, the fractions of C and N changed significantly. The introduction of Flemingia macrophylla in the young rubber plantations was propitious for improving the labile organic C (WSOC and LFOC) and LFON contentin the 0–10 cm soil layersdue to the input of large amounts of litter from Flemingia macrophyllaeach year (Fig. 5C). The light fraction of the soil mainly consisted of plant residues, small animals, and microorganisms in various stages of decomposition. It served as a readily decomposable substrate for soil microorganisms and a short-term reservoir for plant nutrients. It potentially served as an early indicator of the effects of the management practices33,34. The WSOC also played a dominant role in several soil processes and was more sensitive for land use than the total SOC pool28,35. The WSOC indicated that the introduction of Flemingia macrophylla to the young rubber plantations could change the soil C fractions in the 0–10 cm soil layer within a short time. However, the introduction of Flemingia macrophylla into the mature rubber plantations had no significant effect on WSOC, LFOC, or LFON content in the short term, due to the lower input levels of Flemingia macrophylla litter each year.

In this study, the introduction of Flemingia macrophylla to the rubber plantations decreased AN content and increased NN content, which may have been due to the increased rate of nitrification from improved microbial activity in the rubber–Flemingia macrophylla systems. The NN content in soil is important for the growth of rubber trees, as well as rubber production36. In this region, soil NN content in rubber plantations is generally <1 mg kg−1 37. In our study, soil NN content in the rubber plantations ranged from 0.02–0.93 mg kg−1. These findings suggest that Flemingia macrophylla is important for improving NN content in the soil of the rubber plantations37.

Relationships between soil carbon and nitrogen fractions

The microbial biomass of soil controls organic matter sequestration and decomposition and is generally used as an ecological sensitivity indicator for changes in soil nutrients due to land use practices38,39. For example, high MBC/SOC and MBN/TN ratios indicate that the organic matter decomposed quickly40. In the current study, the introduction of Flemingia macrophylla to the rubber plantations increased MBC and MBN, along with the MBC/SOC and MBN/TN ratios in the 0–10 and 10–30 cm soil layers. These results agree with those of Wen et al.41, who suggested that the conversions of monocultures into mixed forests had a strong positive effect on soil microbial biomass, and increased the efficiency of microbes in soil carbon decomposition along the profile by improving the ratios of the MBC/SOC and MBN/TN. Interestingly, the introduction of Flemingia macrophylla to the rubber plantations in our study decreased the ratio of LFOC/LFON. It was observed that large amounts of leaves from the Flemingia macrophylla were incorporated into the soil each year, which tended to decrease the LFOC/LFON ratio in the soil due to the low C/N ratio in the leaves of Flemingia macrophylla. Generally speaking, the C/N ratio of Flemingia macrophylla is approximately 15:114. Chen42 and Huang43 reported that low C/N ratios in soil could increase microbial biomass, and accelerate the decomposition of soil organic matter. We also observed a significant negative correlation between the ratio of LFOC/LFON, and MBC and MBN (P ≤ 0.01). These results suggest that the decreased ratios of LFOC/LFON in the rubber–Flemingia macrophylla plantations enhanced microbial activity.

Conclusions

While the introduction of Flemingia macrophylla to the rubber plantations did not significantly increase total soil carbon or nitrogen levels over a short period, it changed the carbon and nitrogen fractions, improved labile organic carbon and nitrogen contents, and ameliorated the soil environment. We recommend that local governments and farmers in southeastern Asia use Flemingia macrophylla as alternative interplanted tree species within rubber plantations.

Materials and Methods

Description of the study site

This study was conducted in the Xishuangbanna region (21°33′N, 101°28′E; 880 to 900 m asl) of Yunnan Province in southwestern China. This region has a typical tropical monsoon climate, with an annual mean temperature of 21.8 °C. The area receives mean annual precipitation of ~1,500 mm, 80% of which occurs in the rainy season (May to October)44. Furthermore, Xishuangbanna contains the largest area of tropical rainforests in China. Its biodiversity is rich as it is part of the Indo-Burma world biodiversity hotspot45. The soil has been classified as laterite (Oxisol), which developed from arenaceous shale sediment44,46.

In 1991 and 2003, the tropical forests with slopes ranging from 47–58% were deforested. Sugarcane (Saccharum officinarum L.) was then planted annually. Rubber trees were plantedon these sites in May 1994 and 2006 at a density of 450 rubber trees ha−1, with 8 m spacing between adjacent rows. In accordance with the local practices for rubber trees less than three years of age, fertilizers were applied between the rubber trees at depths of 20 cm using spades at rates of 27.0 kg ha−1 N, 5.9 kg ha−1 P, and 11.2 kg ha−1 K, which were split into two applications per year (May and October). Once the rubber trees were more than three years of age, the fertilizer application rates changed to 54.0 kg ha−1 N, 11.8 kg ha−1 P, and 22.4 kg ha−1 K. The rubber plantation farmers generally sprayed sulfur powder at 30–60 kg ha−1 yr−1 to control powdery mildew on the rubber trees. Weeds in the plantations were cut using a sickle twice per year (April/May and November/December) and left on the ground. In July 2010, Flemingia macrophylla was introduced into the differently aged rubber plantations (4 and 16 years of age) at a density of 10,830 plants ha−1. From 2011 onwards, the Flemingia macrophylla in the different rubber plantations was cut using a sickle in December each year and left as ground cover. From 2012 onwards, given the strong biological nitrogen fixation of Flemingia macrophylla, no additional N was applied in the rubber–Flemingia macrophylla plantations. The inputs of P, K, and S in the rubber–Flemingia macrophylla plantations remained the same as those in the adjacent rubber plantations.

Experimental design, sampling, and measurements

In this study, three replicate sites were selected within each rubber and rubber–Flemingia macrophylla plantation (Fig. 5). Each replication site consisted of 20 × 25 m2 survey plots (four rows of rubber trees, and three 8-m wide hedgerows) containing nine sampling subplots (8 × 6 m2), with three located at each slope position (upper, middle, and lower slope).

For each of the nine subplots, soil samples were collected using a soil auger, avoiding the fertilization holes, at two depths (0–10 cm and 10–30 cm) after carefully removing the litter-fall and/or grass layer. For each replicate site in the different rubber and rubber–Flemingia macrophylla plantations, soil core samples were collected in April 2014, June 2015, January and August 2016, and January 2017. The nine soil cores were combined into a composite sample, which were air-dried, ground, and sieved (at <2 mm) for analysis of ammonium nitrogen (AN) and nitrate-nitrogen (NN), and the light fraction organic carbon (LFOC) and nitrogen (LFON). The sieved samples were sieved again (at <0.25 mm) for determination of soil organic carbon (SOC) and total nitrogen (TN). For soil water-soluble organic carbon (WSOC), microbial biomass carbon (MBC) and nitrogen (MBN) measurements, the samples were taken to the laboratory and stored at 4 °C for subsequent analyses.

The SOC and TN of the bulk soil were determined using a Vario MAX CN-Analyzer (Elementar Analysensysteme GmbH, Germany). A density fractionation scheme for light was used following the method described by Gregorich and Ellert47. During fractionation, 25 g of air-dried soil was shaken with 50 mL of NaI solution (sp.Gr. = 1.70) for 60 min. After centrifugation, the supernatant was passed through a Millipore filter (0.45 μm) and the light fraction collected. The soil residue in the centrifuge was extracted again with NaI, and the additional light fraction collected. The light fraction was oven-dried at 60 °C of 72 h. The concentration of organic carbon and nitrogen was determined by dry combustion using a Vario MAX CN-Analyzer (Elementar Analysensysteme GmbH, Germany). Dried samples, each weighing 5 g, were added to 50 mL of 2 M KCl, shaken for one hour, and analyzed with an Auto Analyzer 3 (SEAL Analytical GmbH, Germany) to determine AN and NN contents34,48. Microbial biomass carbon (MBC) and nitrogen (MBN) in the soil were estimated using a fumigation-extractionmethod49 that included a purified CHCl3 treatment, followed by a 0.5 M K2SO4 extraction of fumigated and unfumigated soil50. After which, soil samples (equivalent to 25 g of dry soil weight) were fumigated for 24 h at 25 °C with CHCl3 (ethanol-free). Following fumigant removal, the soil was extracted with 100 mL of 0.5 M K2SO4 by shaking for 1 h at 200 rpm, followed by filtering. The non-fumigated portions were extractedat similar time intervals. Following the extraction, MBC and MBN contents were measured by determining the C and N masses in the filtrate using a Vario TOC cube-Analyzer (Elementar Analysensysteme GmbH, Germany). Microbial biomass carbon was calculated as follows: MBC = (Corg(fum) − Corg(non))/0.3851. Microbial biomass nitrogen wascalculated as follows: MBN = (TN(fum) − TN(non))/0.4551. The ratios of MBC to total carbon (MBC/SOC) and MBN to total nitrogen (MBN/TN) were then calculated. Water-soluble organic carbon (WSOC) was extracted from field-moist samples within 24 h of sampling by shaking 15 g soil with 30 mL distilled water for 2 h at 25 °C, followed by centrifugation at 5000 r min−1 at 4 °C for 15 min. The supernatant was filtered through a 0.45 μm carbon-free membrane. The filtrates were stored at 4 °C and analyzed within 24 h using a Vario TOC cube-Analyzer (Elementar Analysensysteme GmbH, Germany)35.

Statistical analysis

The data were subjected to analysis of variance (ANOVA) using SAS statistical analysis software version 8.0. One-factor ANOVA was deployed to compare treatment effects. The least significant difference (LSD at 0.05 level of probability) test was applied to assess the differences between means. Pearson’s coefficient analysis was used for correlation.

Acknowledgements

We would like to acknowledge the Public Technology Service Center of XTBG, CAS, for their support with field measurements and soil analyses. This study was funded by the National Natural Science Foundation of China (31470639), the Natural Science Foundation of Yunnan Province (2017FB059 and 2016FA047), the Key Program of CAS (KFZD-SW-312) and the Project of Xishuangbanna Science and Technology Bureau (200915, 201116).

Author Contributions

C.A.L. conducted the experiments and wrote the manuscript; Y.N. analyzed the data assisted. J.W.T., K.H.M.S. and Y.M.Z. assisted with writing the manuscript.

Competing Interests

The authors declare no competing interests.

Footnotes

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Warren-Thomas E, Dolman PM, Edwards DP. Increasing demand for natural rubber necessitates a robust sustainability initiative to mitigate impacts on tropical biodiversity. Conservation Letters. 2015;8:230–241. doi: 10.1111/conl.12170. [DOI] [Google Scholar]
  • 2.Tangena JAA, Thammavong P, Wilson AL, Brey PT, Lindsay SW. Risk and control of mosquito-borne diseases in Southeast Asian rubber plantations. Trends in Parasitology. 2016;32(5):402–415. doi: 10.1016/j.pt.2016.01.009. [DOI] [PubMed] [Google Scholar]
  • 3.Ahrends A, et al. Current trends of rubber plantation expansion may threaten biodiversity and livelihoods. Global Environmental Change. 2015;34:48–58. doi: 10.1016/j.gloenvcha.2015.06.002. [DOI] [Google Scholar]
  • 4.Mei, C. C. The spatial-temporal evolution of rubber plantation and the distribution patterns of aboveground biomass carbon storage in Xishuangbanna. M.Sc degree thesis, University of Chinese Academic of Sciences (2015).
  • 5.Tan ZH, et al. Rubber plantations act as water pumps in tropical China. Geophysical Research Letters. 2011;38:L24406. doi: 10.1029/2011GL047702. [DOI] [Google Scholar]
  • 6.Guillaume T, Damris M, Kuzyakov Y. Losses of soil carbon by converting tropical forest to plantations: erosion and decomposition estimated byδ13C. Global Change Biology. 2015;21(9):3548–3560. doi: 10.1111/gcb.12907. [DOI] [PubMed] [Google Scholar]
  • 7.Zheng G, Li S, Yang X. Spider diversity in canopies of Xishuangbanna rainforest (China) indicates an alarming juggernaut effect of rubber plantations. Forest Ecology and Management. 2015;338:200–207. doi: 10.1016/j.foreco.2014.11.031. [DOI] [Google Scholar]
  • 8.Yi ZF, Cannon CH, Chen J, Ye CX, Swetnam RD. Developing indicators of economic value and biodiversity loss for rubber plantations in Xishuangbanna, southwest China: A case study from Menglun township. Ecological Indicators. 2014;36:788–797. doi: 10.1016/j.ecolind.2013.03.016. [DOI] [Google Scholar]
  • 9.Bai JK. Developing environmental-friendly rubber plantations in Yunnan province for promoting the sustainability of ecological rubber. China State Farms. 2015;5:20–22. [Google Scholar]
  • 10.Liu W, Zhu C, Wu J, Chen C. Are rubber-based agroforestry systems effective in controlling rain splash erosion? Catena. 2016;147:16–24. doi: 10.1016/j.catena.2016.06.034. [DOI] [Google Scholar]
  • 11.He H, et al. Phytoextraction of rhenium by lucerne (Medicago sativa) and erect milkvetch (Astragalus adsurgens) from alkaline soils amended with coal fly ash. Science of the Total Environment. 2018;630:570–577. doi: 10.1016/j.scitotenv.2018.02.252. [DOI] [PubMed] [Google Scholar]
  • 12.He H, et al. Growth, morphological and physiological responses of alfalfa (Medicago sativa) to phosphorus supply in two alkaline soils. Plant and Soil. 2017;416:565–584. doi: 10.1007/s11104-017-3242-9. [DOI] [Google Scholar]
  • 13.Wu GL, Liu Y, Tian FP, Shi ZH. Legumes functional group promotes soil organic carbon and nitrogen storage by increasing plant diversity. Land Degradation & Development. 2017;28:1336–1344. doi: 10.1002/ldr.2570. [DOI] [Google Scholar]
  • 14.Wang, F. J. The content of nitrogen fixed by Flemingia macrophylla and its effects on the growth of rubber and concentration of soil total nitrogen in rubber–Flemingia macrophylla intercropped systems in different stand ages. M.Sc degree thesis, University of Chinese Academic of Sciences (2015).
  • 15.Janssens IA, et al. Reduction of forest soil respiration in response to nitrogen deposition. Nature Geoscience. 2010;3:315–322. doi: 10.1038/ngeo844. [DOI] [Google Scholar]
  • 16.Li D, Niu S, Luo Y. Global patterns of the dynamics of soil carbon and nitrogen stocks following afforestation: a meta-analysis. New Phytologist. 2012;195(1):172–181. doi: 10.1111/j.1469-8137.2012.04150.x. [DOI] [PubMed] [Google Scholar]
  • 17.Tashi S, Singh B, Keitel C, Adams M. Soil carbon and nitrogen stocks in forests along an altitudinal gradient in the eastern Himalayas and a meta-analysis of global data. Global Change Biology. 2016;22(6):2255–2268. doi: 10.1111/gcb.13234. [DOI] [PubMed] [Google Scholar]
  • 18.Zhang M, Fu XH, Feng WT, Zou XM. Soil organic carbon in pure rubber and tea-rubber plantations in southwestern China. Tropical Ecology. 2007;48:201–207. [Google Scholar]
  • 19.de Blécourt M, Hänsel VM, Brumme R, Corre MD, Veldkamp E. Soil redistribution by terracing alleviates soil organic carbon losses caused by forest conversion to rubber plantation. Forest Ecology and Management. 2014;313:26–33. doi: 10.1016/j.foreco.2013.10.043. [DOI] [Google Scholar]
  • 20.Goldberg SD, et al. Soil respiration in sloping rubber plantations and tropical natural forests in Xishuangbanna, China. Agriculture, Ecosystem & Environment. 2017;249:237–246. doi: 10.1016/j.agee.2017.08.001. [DOI] [Google Scholar]
  • 21.Zhou WJ, et al. The effects of nitrogen fertilization on N2O emissions from a rubber plantation. Scientific Reports. 2016;6:28230. doi: 10.1038/srep28230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Werner C, et al. N2O, CH4 and CO2 emissions from seasonal tropical rainforests and a rubber plantation in Southwest China. Plant and Soil. 2006;289(1–2):335–353. doi: 10.1007/s11104-006-9143-y. [DOI] [Google Scholar]
  • 23.Shi Y, Chen X, Shen HM. Light fraction carbon and water-stable aggregates in black soils. Pedosphere. 2007;17(1):97–100. doi: 10.1016/S1002-0160(07)60013-7. [DOI] [Google Scholar]
  • 24.Demisie W, Liu Z, Zhang M. Effect of biochar on carbon fractions and enzyme activity of red soil. Catena. 2014;121:214–221. doi: 10.1016/j.catena.2014.05.020. [DOI] [Google Scholar]
  • 25.Shahid M, et al. Carbon and nitrogen fractions and stocks under 41 years of chemical and organic fertilization in a sub-humid tropical rice soil. Soil & Tillage Research. 2017;170:136–146. doi: 10.1016/j.still.2017.03.008. [DOI] [Google Scholar]
  • 26.de Moraes Sá JC, et al. Soil carbon fractions and biological activity based indices can be used to study the impact of land management and ecological successions. Ecological Indicators. 2018;84:96–105. doi: 10.1016/j.ecolind.2017.08.029. [DOI] [Google Scholar]
  • 27.Ren C, et al. Differential responses of soil microbial biomass, diversity, and compositions to altitudinal gradients depend on plant and soil characteristics. Science of the Total Environment. 2018;610–611:750–758. doi: 10.1016/j.scitotenv.2017.08.110. [DOI] [PubMed] [Google Scholar]
  • 28.Tang X, Zhang X, Wang Z, Ci Z. Water-soluble organic carbon (WSOC) and its temperature-resolved carbon fractions in atmospheric aerosols in Beijing. Atmospheric Research. 2016;181:200–210. doi: 10.1016/j.atmosres.2016.06.019. [DOI] [Google Scholar]
  • 29.Liu CA, Siddique KHM, Hua S, Rao X. The trade-off in the establishment of artificial plantations by evaluating soil properties at the margins of oases. Catena. 2017;157:363–371. doi: 10.1016/j.catena.2017.05.031. [DOI] [Google Scholar]
  • 30.Chen C, Liu W, Jiang X, Wu J. Effects of rubber-based agroforestry systems on soil aggregation and associated soil organic carbon: Implications for land use. Geoderma. 2017;299:13–24. doi: 10.1016/j.geoderma.2017.03.021. [DOI] [Google Scholar]
  • 31.Moghimian N, Hossein SM, Kooch Y, Darki BZ. Impacts of changes in land use/cover on soil microbial and enzyme activities. Catena. 2017;157:407–414. doi: 10.1016/j.catena.2017.06.003. [DOI] [Google Scholar]
  • 32.Mo HZ, Sha LQ. The carbon stock and carbon sequestration potential of rubber plantations under different agro-forestry systems in Xishuangbanna, SW China. Mountain Research. 2016;34(8):707–715. [Google Scholar]
  • 33.Liu CA, et al. Effects of water management with plastic film in a semi-arid agricultural system on available soil carbon fractions. European of Journal Soil Biology. 2013;57:9–12. doi: 10.1016/j.ejsobi.2013.03.007. [DOI] [Google Scholar]
  • 34.Zhong Y, Yan W, Shangguan Z. Soil carbon and nitrogen fractions in the soil profile and their response to long-term nitrogen fertilization in a wheat field. Catena. 2015;135:38–46. doi: 10.1016/j.catena.2015.06.018. [DOI] [Google Scholar]
  • 35.Wei ZQ, Wu SH, Zhou SL, Li JT, Zhao QG. Soil organic carbon transformation and related properties in urban soil under impervious surfaces. Pedosphere. 2014;24(1):56–64. doi: 10.1016/S1002-0160(13)60080-6. [DOI] [Google Scholar]
  • 36.Li Y. Accumulated impacts of sulfur spraying on soil nutrient availability and microbial biomass in Rubber Plantations. CLEAN-Soil, Air, Water. 2016;44(8):1001–1010. doi: 10.1002/clen.201400397. [DOI] [Google Scholar]
  • 37.Zhang M, Zou XM. Comparison of soil C and N in rubber plantation and seasonal rainforest. Chinese Journal of Applied Ecology. 2009;20(5):1013–1019. [PubMed] [Google Scholar]
  • 38.Spohn M, Klaus K, Wanek W, Richter A. Microbial carbon use efficiency and biomass turnover times depending on soil depth-Implications for carbon cycling. Soil Biology and Biochemistry. 2016;96:74–78. doi: 10.1016/j.soilbio.2016.01.016. [DOI] [Google Scholar]
  • 39.Buyer JS, Baligar VC, He Z, Arévalo-Gardini E. Soil microbial communities under cacao agroforestry and cover crop systems in Peru. Applied Soil Ecology. 2017;120:273–280. doi: 10.1016/j.apsoil.2017.09.009. [DOI] [Google Scholar]
  • 40.Jiang HM, Li FM, Jiang JP. Soil carbon pool and effects of soil fertility in seeded alfalfa fields on the semi-arid Loess Plateau in China. Soil Biology and Biochemistry. 2006;38(8):2350–2358. doi: 10.1016/j.soilbio.2006.02.008. [DOI] [Google Scholar]
  • 41.Wen L, Lei P, Xiang W, Yan W, Liu S. Soil microbial biomass carbon and nitrogen in pure and mixed stands of Pinus massoniana and Cinnamomum camphora differing in stand age. Forest Ecology and Management. 2014;328:150–158. doi: 10.1016/j.foreco.2014.05.037. [DOI] [Google Scholar]
  • 42.Chen, W. X. Soil Science and Environmental Microbiology. BeijingAgriculture University Press, Beijing (1990).
  • 43.Huang, C. Y. Soil Science. China Agriculture Press, Beijing (2000).
  • 44.Li H, Ma Y, Liu W, Liu W. Soil changes induced by rubber and tea plantation establishment: comparison with tropical rain forestsoil in Xishuangbanna, SW China. Environmental Management. 2012;50(5):837–848. doi: 10.1007/s00267-012-9942-2. [DOI] [PubMed] [Google Scholar]
  • 45.Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J. Biodiversity hotspots for conservation priorities. Nature. 2000;403:853–858. doi: 10.1038/35002501. [DOI] [PubMed] [Google Scholar]
  • 46.Wang, W. F., Qiu, D. Y., Wu, J. C. & Ye, H. M. The Soils of Yunnan. Yunnan Science and Technology Press, Kunming, China (1996).
  • 47.Gregorich, E. G. & Ellert, B. H. Light fraction and macroorganic matter in mineral soils, in: M.R. Carer (Ed.), Soil Sampling and Methods of Analysis, Can Soc Soil Sci, Lewis Publishers, Division of CRC Press, Boca Raton, FL, pp. 397–405 (1993).
  • 48.Zhang Y, Zhao W. Vegetation and soil property response of short-time fencing in temperate desert of the Hexi Corridor, northwestern China. Catena. 2015;133:43–51. doi: 10.1016/j.catena.2015.04.019. [DOI] [Google Scholar]
  • 49.Brookes PC, Landman A, Pruden G, Jenkinson DS. Chloroform fumigation and release of soil nitrogen: rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology and Biochemistry. 1985;17(6):837–842. doi: 10.1016/0038-0717(85)90144-0. [DOI] [Google Scholar]
  • 50.Singh H, Singh KP. Effect of residue placement and chemical fertilizer on soil microbial biomass under tropical dryland cultivation. Biology and Fertility of Soils. 1993;16(4):275–281. doi: 10.1007/BF00369304. [DOI] [Google Scholar]
  • 51.Ocio JA, Brooks PC. An evaluation of methods for measuring the microbial biomass in soils following recent addition of wheat straw, and the characterization of the biomass that develops. Soil Biology and Biochemistry. 1990;22(5):262–685. doi: 10.1016/0038-0717(90)90016-S. [DOI] [Google Scholar]

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES