Plant and Soil

, Volume 368, Issue 1, pp 407–417

Effects of nitrogen fertilization and root interaction on the agronomic traits of intercropped maize, and the quantity of microorganisms and activity of enzymes in the rhizosphere

Authors

  • Xiangqian Zhang
    • Agricultural CollegeNanjing Agricultural University
    • Research Center on Ecological SciencesJiangxi Agricultural University
  • Xinmin Bian
    • Agricultural CollegeNanjing Agricultural University
  • Qiguo Zhao
    • Nanjing Institute of Soil ScienceChinese Academy of Sciences
Regular Article

DOI: 10.1007/s11104-012-1528-5

Cite this article as:
Zhang, X., Huang, G., Bian, X. et al. Plant Soil (2013) 368: 407. doi:10.1007/s11104-012-1528-5

Abstract

Aims

To elucidate the mechanisms of the beneficial effects of below-ground root interactions in maize plus legume intercropping system,

Methods

A pot experiment was conducted using root separation techniques.

Results

It is shown that root interaction and nitrogen fertilization increased chlorophyll content and improved plant characteristics of maize, and the effect of root interaction was significant (p<0.05). Compared to a full root separation treatment, no root separation increased the leaf and grain nitrogen contents, and economic and biological yields per maize plant by 9.3  %, 6.0  %, 14.0  %, and 6.5  %, respectively. Root interaction and nitrogen fertilization enhanced the numbers of bacteria, fungi, actinomycetes and Azotobacteria and the activities of urease, invertase, acid-phosphatase and protease in soil. Correlation analyses revealed that the quantity of microorganisms and the activity of the aforementioned enzymes were all positively or significantly (p<0.05) positively correlated with chlorophyll content, plant height and economic and biological yields per maize plant.

Conclusions

The findings demonstrate that root interactions are important in improving the soil micro-ecological environment, increasing microbial quantity and enzyme activity in soil, and enhancing crop yield.

Keywords

Root interactionChlorophyll contentYieldMicrobial quantityEnzyme activities

Abbreviations

NS

no root separation

PS

partial root separation with nylon nets

FS

full root separation with plastic film

Introduction

Intercropping is an important cropping system that has been practised by farmers around the world for many years because of its high overall productivity, good pest and disease control, provision of ecological services, and economic profitability (Gómez-Rodrı́guez et al. 2003; Malézieux et al. 2009; Vandermeer 1989; Fang et al. 2010). In China, cereal-legume intercropping is widely used because of its yield benefits and high rates of resource utilization (water, light, nutrients), and it is considered important in the development of sustainable agriculture (Li et al. 2009; Zuo et al. 2000).

The advantages of cereal-legume intercropping are a consequence of above-ground and below-ground interactions of the different crop species. In intercropping systems, two or more crops are planted to grow simultaneously in close proximity with a period of overlapping growth long enough to optimize interactions between the intercropped species (Hauggaard-Nielsen and Jensen 2005). Below-ground interactions are common in intercropping systems, and the mutual benefits when different plants species are grown together are attracting increasing interest, particularly among ecologists and agronomists (Li et al. 2003a; Callaway 1995). Among the beneficial inter-species interactions, rhizosphere effects play an important role in enhancing yield advantage of intercropping. Zhang and Li (2003) observed a 53  %increase of grain yield in wheat intercropped with soybean system and 23  % was attributed to below-ground interactions.

Below-ground root interactions in intercropping can help to improve crop agronomic traits and yield, mainly by improving of soil micro-ecological environment and nutrient availability. Microbial numbers and enzyme activity are usually used as indicators of soil fertility and health (Zhang et al. 2010; Franchini et al. 2007; Parthasarathi and Ranganathan 2000). Soil microorganisms are important agents in nutrient cycling and energy flow, and they also play a key role in the regulation of decomposition of soil organic matter (Marschner et al. 2002; Berg et al. 1998). Soil enzymes derived primarily from microorganisms, plant roots and soil animals are involved in nutrient cycling, and as such, their activities can be used as potential indicators of soil biological activity and fertility. They participate in specific types of chemical reactions (Parthasarathi and Ranganathan 2000; Tan et al. 2008). To help understand the mechanisms behind beneficial inter-species below-ground interactions, we have examined the effects of root interaction on maize agronomic traits and on quantities of microorganisms and activities of enzymes in rhizosphere soils for soybean/maize intercropping systems with the same above-ground environmental conditions but different degrees of root separation below ground. The objectives were (i) to investigate the impact of root interaction on the soil micro-ecological environment and maize agronomic traits, (ii) to identify the correlations between soil microbial quantities, enzyme activities and maize agronomic traits, and (iii) to elucidate the advantageous mechanisms of root interactions in intercropping systems using no root separation (NS), partial root separation with nylon nets (PS), and full root separation with plastic film (FS). We hypothesized that the effects of three root separation methods on improving the soil micro-ecological environment and maize agronomic traits consistently followed the order NS>PS>FS.

Materials and methods

Experimental design and management

A pot experiment was conducted outdoors at the Red Soil Experimental Station of Jiangxi Agricultural University, Nanchang City, Jiangxi province in China (28°46′N, 115°36′E) at an altitude of 22 m above sea level. The annual mean temperature is 16–18 °C and accumulated mean daily temperatures above 10 °C are 5300–5800 °C. The frost-free period is 265–280 days each year. Annual mean total solar radiation is 2450–2550 h. The region is classified as having a subtropical monsoon climate. Annual precipitation is 1450–1650 mm, and 50 % occurs from April to August. The soil for the experiment came from the red soil experimental field of Jiangxi Agricultural University, and its parent material was the Quaternary red clay. Soil pH was 6.1, organic matter 17.9 kg−1, total N 0.69 g kg−1, total P 0.86 g kg−1, available N 69.5 mg kg−1, available P 13.6 mg kg−1, available K 169 mg kg−1 and soil water capacity 25.7 %.

Plastic buckets 50 cm high, with upper and lower diameters of 45 cm and 35 cm, respectively, were used. Nylon net (30 μm) or plastic film dividers were positioned in the middle of some buckets with PVC adhesive to produce two chambers (and waterproof-glue smeared over joins to make them watertight). This produced three root separation methods: no root separation (NS, roots of crop species are allowed to completely intermingle), partial root separation with nylon nets (PS, direct contact of roots is prevented but soil solution allowed to flow to the root surfaces across the nylon nets), and full root separation with plastic film (FS, both direct root contact and the movement of any substances between the two root systems are prevented). All test buckets were divided into 4 rows and each row had 6 buckets. The distance between two buckets was always 1.0 m.

Soil well-mixed with all fertilizers (50 kg) was filled into each bucket and each chamber contained 25 kg soil. Fertilizers were applied at 0 or 0.15 g N/kg dry soil, and 0.1 g P2O5/kg dry soil and 0.1 g K2O/kg dry soil for all treatments. N was supplied as urea. P and K were applied as (NH4)2HPO4 and K2SO4, respectively. The experimental treatments were two levels of nitrogen fertilization (0 and 0.15 g N/kg soil) and three root separation methods (NS, PS and FS). This experimental design yielded 6 treatments (i.e. 2×3), and each treatment was replicated four times.

The selected cultivars of maize and soybean were Kenuo986 (Zea mays L. cv, a local variety) and Zao50 (Glycine max). Maize and soybean were all sown on 10 April 2011, with 4 maize and 4 soybean seeds in each chamber of the bucket, after seedling emergence each chamber was left with 2 maize or 2 soybean plants, and all were harvested on 20 July 2011 (7 maize plants were randomly selected from 8 plants in each treatment for statistical analysis). Water was supplied during crop growth to meet crop needs. The above-ground environmental condition for each treatment was the same since the above-ground portions of each bucket had 2 maize and 2 soybean plants.

Sampling and measurements

Chlorophyll content was measured using a hand-held chlorophyll meter (SPAD-502,Manufactured by Konica Minolta Company in Japan, measuring area: 2 mm×3 mm); the same parts of leaves at the centre of the maize plants were selected and analysed at bell-mouthed stage, silking stage, filling stage and maturity stage. Leaf lengths and widths were measured on ear leaves of the maize.

Biological yield per plant was assessed from above-ground dry matter weight per maize plant. Economic yield per plant was evaluated from grain yield per maize plant after harvest. The maize leaves and grain were separated at the end of the experiment, dried at 105 °C for 30 min and then dried at 80°C to constant weight. The nitrogen content of the leaves and grain was analyzed by the Kjeldahl nitrogen determination method (Wang et al. 2006). Nitrogen accumulation (g plant−1) was defined as the nitrogen content of samples (leaves and grain) x the total biomass of samples per plant.

The numbers of soil culturable bacteria, fungi, actinomycetes and Azotobacter were counted at the maturity stage of maize. Soil cores near the maize roots were collected by using an auger. The top 1 cm soil layer was removed and the remaining soil core to a depth of 20 cm was sampled. Soil samples were stored at 4 °C in sealed plastic bags for no more than 2 days. After air-drying, samples were sieved through a 1-mm sieve. Ten grams of each fresh soil sample was added to 95 mL of sterile distilled water. After homogenization for 30 min, each soil suspension was sequentially diluted and 50 μL of the resulting solutions were plated on appropriate isolation culture media. After incubation at 28 °C for 4–5 days for bacteria, 3–4 days for fungi, 6–8 days for actinomycetes or 6–9 days for Azotobacter, the colony forming units (CFU) were counted (Vieira and Nahas 2005). Soil bacteria, fungi, actinomycetes and Azotobacter were respectively cultured on beef extract + peptone + agar medium, Martin medium, improved Gauss No. 1 medium and Waksman No. 77 medium.

Activities of the soil enzymes invertase, urease, acid-phosphotase and protease were determined in air-dried soils according to the method of Guan et al. (1986) using a spectrophotometer (722 s, Shanghai Precision & Scientific Instrument Co. Ltd.). Invertase activity was measured colorimetrically using 3,5-dinitrosalicylic acid and was expressed in mg glucose g−1 soil at 37 °C over 24 h. Five grams of soil sample were mixed with 1 ml of methylbenzene for 15 min, then added to 15 ml of 18% sucrose solution and 5 ml of phosphate buffer (pH 5.5) before incubation at 37 °C for 24 h. Then 5 ml of distilled water and 3 ml of 3,5-dinitrosalicylic acid solution were added into 0.5 ml of filtrate. The tubes were heated in a boiling water bath for 5 min and then cooled to room temperature with tap water. Finally, the solution was diluted with distilled water to 50 ml and absorbance was measured at a wavelength of 508 nm.

Urease activity was determined colorimetrically using indophenol blue and expressed in mg NH3-N g−1 soil at 37 °C over 24 h. Ten grams of soil sample were mixed with 2 ml of methylbenzene for 15 min, then added to 20 ml of citrate buffer (pH 6.7) and 10 ml of 10 %(w/v) urea solution before incubation at 37 °C for 24 h. Next 4 ml of sodium hydroxybenzene solution and 3 ml of sodium hypochlorite solution were added to 1 ml of filtrate and the solution was left for 20 min. Finally, the solutions were diluted with distilled water to 100 ml and the released ammonium was determined at a 578 nm using a spectrophotometer.

Acid−phosphatase activity was measured using the di-sodium phenyl phosphate method and expressed in mg PhOH g−1 soil at 37 °C over 24 h. Five grams of soil sample were mixed with 2 ml of methylbenzene for 15 min, then 20 ml of 0.5 %(w/v) disodium phenyl phosphate solution was added. Suspensions were then incubated in a pH 5.0 acetate buffer solution at 37 °C for 24 h. Next, 0.2 ml of 2,6-dibromoquinone chloride solution and 5 ml of buffer solution were added to 1 ml of filtrate. Finally, the solution was diluted with distilled water to 50 ml, and the released phenol measured colorimetrically at 660 nm.

Protease activity was measured colorimetrically using ninhydrin and was expressed as μg NH2 produced g−1 soil after incubation for 2 h at 50 °C. Five grams of soil sample were mixed with 5 ml of Tris-HCl buffer ( 0.05 mol l−1, containing 0.1 mol l−1 of CaCl2, pH 7.6) and 1 ml of methylbenzene. After 15 min, 5 ml of 2 %(w/w) sodium caseinate was added, then the mixture was shaken at 200 rpm for 2 h at 50 °C. A control without adding sodium caseinate during shaking but added after shaking was prepared, and incubated at 0~4 °C for 30 min. 1.5 ml of 0.6 mol l−1 lead acetate solution was added, and the mixture was centrifuged at 17,000 g for 10 min. Next, 1.5 ml of the supernatant was added to 457 μl of a mixed solution of sodium oxalate and acetic acid, centrifuged at 17,000 g for 10 min, and 1 ml of the supernatant was added to 2 ml of acetate buffer (0.01 mol l−1 , pH 5.8) and 1.5 ml of ninhydrin reagent (0.02 g of ascorbic acid and 2 g of ninhydrin dissolved in 100 ml of ethanol). After 16 min of heating in boiling water, the solution was cooled with tap water for 15 min, 1 ml of 0.2 %(w/w) KIO3 was added, and the mixture adjusted to 10 ml with distilled water, absorbance was then measured at 570 nm.

Statistical analysis

The experiment was conducted as a randomized block design. Analysis of variance (ANOVA) was performed using the general linear model-univariate (mixed model) procedure from SPSS 17.0 software. ANOVAs were done with N fertilization level, root separation method and growth stage as the main effects and including two- or three-way interactions. All the treatment means were compared for any significant differences using the LSD’s multiple range tests at significant level of P=0.05.

Results

Effects of nitrogen fertilization and root separation on maize plant characteristics

As shown in Table 1, fertilization level had a significant effect (p<0.05) on maize plant height, leaf length and width. When compared to the no fertilization treatment, fertilization increased maize plant height, leaf length and width by 11.5  %, 14.0  %, 15.0  % and 11.5  %, 11.1  %, 9.0  % respectively at silking and maturity stages. The separation method also had significant effects (p<0.05) on plant height, leaf length and width. Compared to the FS treatment (full root separation with plastic film), PS (partial root separation with nylon nets) increased maize plant height, leaf length and width by 3.3  %, 5.1  %, 4.5  % and 4.1  %, 4.1  %, 2.6  % at silking and maturity stages, respectively. For the NS treatment (no root separation) the corresponding increases compared to the FS treatment were 5.3  %, 8.3  %, 6.6  % and 6.6  %, 6.7  %, 3.9  % at silking and maturity stages, respectively. Therefore, nitrogen fertilization and root interaction can help to increase maize plant height, leaf length and width, but the effect of their interaction was insignificant.
Table 1

Effects of fertilization and root separation on plant characteristics of maize

Treatment

Plant height /m

Leaf length /cm

Leaf width /cm

Silking stage

No fertilization

1.48±0.12

50.28±5.03

5.41±0.34

Fertilization

1.65±0.11

57.30±3.41

6.22±0.48

FS

1.52±0.14

51.49±6.00

5.61±0.59

PS

1.57±0.16

54.11±5.32

5.86±0.55

NS

1.60±0.13

55.77±4.73

5.98±0.59

Maturity stage

No fertilization

1.92±0.09

63.19±3.88

7.23±0.34

Fertilization

2.14±0.12

70.17±2.99

7.88±0.48

FS

1.96±0.15

64.36±5.07

7.40±0.49

PS

2.04±0.15

67.01±4.34

7.59±0.55

NS

2.09±0.14

68.67±4.64

7.69±0.53

Significance test

Growth stage

**

**

**

Fertilization level

**

**

**

Separation method

**

**

*

Growth stage × Fertilization level

NS

NS

NS

Growth stage × Separation method

NS

NS

NS

Fertilization level × Separation method

NS

NS

NS

Growth stage × Fertilization level × Separation method

NS

NS

NS

values are means ± SD (n=7). Analysis of variance (ANOVA) P values and symbols were defined as: *P<0.05; **P<0.01; NS: P>0.05. FS, PS, and NS represent full root separation with plastic film, partial root separation with nylon nets, and no root separation, respectively

Effects of nitrogen fertilization and root separation on chlorophyll content of maize

Table 2 shows that fertilization level had a significant effect (p<0.05) on maize chlorophyll content. Compared to the no fertilization treatment, fertilization increased maize chlorophyll content by 9.9  %, 8.0  %, 6.3  % and 8.8  %, respectively at bell-mouthed stage, silking stage, filling stage and maturity stage. Separation method also had significant effects (p<0.05) on maize chlorophyll content, and the effect of three root separation methods on maize chlorophyll content at different growth stages was consistently NS>PS>FS. Compared to the FS treatment, NS increased maize chlorophyll content by 3.9  %, 4.8  %, 5.1  % and 7.6  % respectively at bell-mouthed stage, silking stage, filling stage and maturity stage. Therefore, nitrogen fertilization and root interaction can help to increase maize chlorophyll content, but the effect of their interaction was insignificant.
Table 2

Effects of fertilization and root separation on maize chlorophyll content (SPAD values)

Treatment

Growth stage

Bell-mouthed stage

Silking stage

Filling stage

Maturity stage

Fertilization level

No fertilization

45.41±2.82

53.89±2.90

57.83±4.14

45.07±3.52

Fertilization

49.92±2.57

58.21±4.26

61.47±2.68

49.05±2.46

Separation method

FS

46.78±3.22

54.66±4.14

58.00±3.44

45.21±3.36

PS

47.60±3.37

56.21±4.00

60.00±3.69

47.32±3.46

NS

48.62±3.91

57.29±4.37

60.95±4.22

48.64±3.39

Significance test

    

Growth stage

**

Fertilization level

**

Separation method

*

Growth stage × Fertilization level

NS

Growth stage × Separation method

NS

Fertilization level × Separation method

NS

Growth stage × Fertilization level × Separation method

NS

values are means ± SD (n=7). Analysis of variance (ANOVA) P values and symbols were defined as: *P<0.05; **P<0.01; NS: P>0.05. FS, PS, and NS represent full root separation with plastic film, partial root separation with nylon nets, and no root separation, respectively

Effects of nitrogen fertilization and root separation on nitrogen accumulation and yields at maturity

As shown in Table 3, compared to the no fertilization treatment, fertilization increased leaf nitrogen content, grain nitrogen content, economic and biological yields per plant by 35.9  % , 18.9 %, 28.6  % and 10.1  % respectively, and the effect of nitrogen fertilization was significant (p<0.05). The effects of the root separation methods on the leaf nitrogen content, grain nitrogen content, economic and biological yields per plant of maize at maturity stage were consistently NS>PS>FS. Compared to the FS treatment, NS increased leaf nitrogen content, grain nitrogen content, economic and biological yields per plant by 9.3  %, 6.0  %, 14.0  % and 6.5  %, respectively. Therefore, nitrogen fertilization and root interaction not only can help to increase nitrogen accumulation in maize leaf and grain, but also help to enhance the economic and biological yields per plant at maturity. However the effect of their interaction was insignificant.
Table 3

Effects of fertilization and root separation on nitrogen accumulation and yields

Treatment

Nitrogen accumulation

Yields

Leaf nitrogen content (g plant-1)

Gain nitrogen content (g plant-1)

Economic yield per plant (g)

Biological yield per plant (g)

Fertilization level

No fertilization

0.39±0.06

1.43±0.10

105.50±11.94

555.45±32.50

Fertilization

0.53±0.07

1.70±0.10

135.68±14.48

611.31±26.59

Separation method

FS

0.43±0.10

1.51±0.17

112.18±20.34

564.49±39.72

PS

0.46±0.10

1.58±0.17

121.69±18.96

584.71±40.16

NS

0.47±0.10

1.60±0.17

127.90±19.21

600.93±36.40

Significance test

    

Fertilization level

**

**

**

**

Separation method

NS

*

**

**

Fertilization level × Separation method

NS

NS

NS

NS

values are means ± SD (n=7). Analysis of variance (ANOVA) P values and symbols were defined as: *P<0.05; **P<0.01; NS: P>0.05. FS, PS, and NS represent full root separation with plastic film, partial root separation with nylon nets, and no root separation, respectively

Effects of nitrogen fertilization and root separation on the quantity of microorganisms

As shown in Fig. 1, the differences in the numbers of bacteria, fungi, actinomycetes and Azotobacteria in soil between no fertilization and fertilization treatments were significant (p<0.05). When compared to the no fertilization treatment, fertilization increased the number of bacteria, fungi, actinomycetes and Azotobacteria in soil by 38.3  %, 58.4  %, 48.7  % and 16.3  %, respectively. The effects of three root separation methods on the microbial quantities mentioned above were always NS>PS>FS. Compared to the FS treatment, NS increased the numbers of bacteria, fungi, actinomycetes and Azotobacteria in soil by 70.7  %, 50.1  %, 66.4  % and 75.1  %, respectively; the difference between FS and NS treatments was significant. Therefore, nitrogen fertilization and root interaction can help to increase microbial quantity in maize rhizosphere.
https://static-content.springer.com/image/art%3A10.1007%2Fs11104-012-1528-5/MediaObjects/11104_2012_1528_Fig1_HTML.gif
Fig. 1

Effects of fertilization and root separation on the number of bacteria (a), fungi (b), actinomycetes (c) and Azotobacteria (d) in soils. Data points are means ± SD (n=7). Different letters indicate significant differences between two fertilization levels or among three root separation methods. Root separation methods: FS, full root separation with plastic film; PS, partial root separation with nylon nets; NS, no root separation

Effects of nitrogen fertilization and root separation on enzyme activities

As shown in Table 4, fertilization level had a significant effect (p<0.05) on the activities of invertase, protease and urease, but insignificant effect on the activity of acid-phosphatase (p>0.05). Compared to the no fertilization treatment, fertilization increased the activities of invertase, protease, urease and acid-phosphatase in maize rhizosphere soil by 30.3  %, 58.6  %, 56.9  % and 9.0  %, respectively. The effects of three root separation methods on the activities of the four enzymes consistently followed the order NS>PS>FS. Compared to the FS treatment, NS increased the activities of invertase, protease, urease and acid-phosphatase in soil by 47.2  %, 22.2  %, 20.0  % and 53.8  % respectively and the effect of separation method was significant (p<0.05). Therefore, nitrogen fertilization and root interaction increase the activity of enzymes in the maize rhizosphere, but the effect of their interaction was insignificant.
Table 4

Effects of fertilization and root separation on the activity of soil enzymes

Treatment

Invertase (glucose mg g-1 soil )

Protease (NH2–N μg g-1 soil )

Urease (NH3–N mg g-1 soil )

Acid-phosphatase (phenol mg g-1 soil )

Fertilization level

No fertilization

5.32±1.29

52.45±12.51

0.65±0.12

1.00±0.26

Fertilization

6.93±1.05

83.16±11.27

1.02±0.10

1.09±0.22

Separation method

FS

4.83±1.22

60.25±18.78

0.75±0.21

0.80±0.18

PS

6.42±1.15

69.52±18.76

0.86±0.20

1.10±0.14

NS

7.11±0.78

73.65±19.80

0.90±0.21

1.23±0.16

Significance test

    

Fertilization level

**

**

**

NS

Separation method

**

*

**

**

Fertilization level × Separation method

NS

NS

NS

NS

values are means ± SD (n=7). Analysis of variance (ANOVA) P values and symbols were defined as: *P<0.05; **P<0.01; NS: P>0.05. FS, PS, and NS represent full root separation with plastic film, partial root separation with nylon nets, and no root separation, respectively

Correlation analysis

As shown in Table 5, the number microorganism (bacteria, fungi, actinomycetes, Azotobacteria) and the activities of enzymes (urease, invertase, acid-phosphatase, protease) in soil, as well as the chlorophyll content, plant height, biological and economic yields per maize plant were all positively correlated with each other, indicating that there was a positive interaction of mutual promotion between each factor. Chlorophyll content,plant height,biological and economic yields were all significantly (p<0.05) or markedly significantly (p<0.01) correlated with the number of microorganisms and the activity of enzymes in the soil except with the number of Azotobacteria and the activity of acid-phosphatase. In addition, the numbers of the four kinds of microorganisms and the activities of four enzymes in the soil were all significantly or markedly significantly related to each other, except for fungi to acid-phosphatase, Azotobacteria to urease, Azotobacteria to protease, urease to acid-phosphatase and acid-phosphatase to protease. Therefore the relationship between soil microbial quantity and enzyme activities may be exploited by appropriate intercropping patterns to increase soil microbial quantity and enzyme activities and thus improve crop agronomic traits.
Table 5

Correlation analyses between quantity of microorganisms, enzyme activities, chlorophyll content, plant height, and economic and biological yields of maize per plant

 

Fungi

Actinomycetes

Azotobacteria

Urease

Invertase

Acid-phosphatase

protease

Chlorophyll

Plant height

Biological yield

Economic yield

Bacteria

0.932**

0.981**

0.929**

0.832*

0.961**

0.898*

0.835*

0.933**

0.880*

0.894*

0.858*

Fungi

 

0.975**

0.814*

0.949**

0.956**

0.737

0.943**

0.990**

0.973**

0.989**

0.967**

Actinomycetes

 

0.892*

0.879*

0.958**

0.824*

0.878*

0.959**

0.924**

0.940**

0.904*

Azotobacteria

  

0.623

0.920**

0.976**

0.617

0.811

0.685

0.731

0.669

Urease

   

0.863*

0.563

0.999**

0.961**

0.991**

0.986**

0.997**

Invertase

    

0.888*

0.855*

0.965**

0.896*

0.923**

0.889*

Acid-phosphatase

     

0.559

0.755

0.622

0.660

0.603

Protease

      

0.956**

0.991**

0.982**

0.995**

Chlorophyll

       

0.973**

0.989**

0.976**

Plant height

        

0.994**

0.993**

Biological yield

         

0.994**

*p<0.05; **p<0.01

Discussion

Interspecific facilitation (or positive interaction) is a phenomenon when one plant species enhances the survival, growth, or fitness of another, and has been demonstrated in many natural plant communities (Li et al. 2003a; Hauggaard-Nielsen and Jensen 2005). In our study, maize plant height as well as leaf length and width at silking and maturity stages all had the same trend with respect to root separation: NS>PS>FS, indicating that below-ground root interaction in intercropping systems facilitate increased maize plant height, leaf length and width. Since the above-ground parts of every bucket all had two maize and two soybean plants, the above-ground environmental condition of each treatment were the same, and the changes in each observation index can be interpreted to originate from below-ground root interaction. We also found that nitrogen fertilization increased maize plant height, and leaf length and width, and that this effect was statistically significant (p<0.05). It is well-known that chlorophyll is the material basis for plant photosynthesis and that its levels determine (to some extent) the photosynthetic rate, a parameter that is closely relate to crop yield. Chu et al. (2004) observed that the chlorophyll content of rice leaves under intercropping condition on 8 August and 15 September were 44.3 and 42.8 (SPAD) compared to 38.5 and 30.9 under monocropping conditions, respectively. In this study, the effect of three root separation methods on maize chlorophyll content at different growth stages had the following order of influence: NS>PS>FS, indicating that improvement in the chlorophyll content of intercropped maize was mainly caused by rhizosphere interactions between maize and soybean. Table 2 also shows that nitrogen fertilization increased maize chlorophyll content.

Interspecific interactions in the rhizosphere can facilitate N and P uptake in an intercropping system, resulting in a substantial yield advantage when compared to monocropping (Li et al. 2003b; Richardson et al. 2009; Lupwayi and Haque 1999). In this study, NS increased leaf nitrogen content, grain nitrogen content, economic and biological yields per plant by 9.3 %, 6.0 %, 14.0 %, and 6.5 %, respectively, when compared to FS treatment, suggesting that root interaction in the maize and soybean intercropping system contributes to both increased N uptake by maize but also enhanced yield. A potential mechanism for root interaction to enhance nitrogen accumulation in maize leaf and grain is the fact that in intercropping systems between cereals and legumes, the cereals can obtain some part of the N from the associated legumes. Cereal, on the other hand, can compete for N in the rhizosphere of legumes/cereal mixtures, ultimately leading to N depletion in the rhizosphere of the legumes and stimulation of increased N2 fixation by the legumes (Yu et al. 2010; Lehmann et al. 2002). Root interaction can increase the economic and biological yields of maize mainly through positive interactions where a species can modify the biotic/abiotic environment of its roots (rhizosphere), ultimately benefiting the intercropped species by increasing nutrient availability (Callaway 2007). Most cereal/legume intercropping studies implicitly assume that the cereal will benefit from the legume species because legumes are known to excrete larger amounts of protons, carboxylates, and phosphatases in their rhizosphere (Hinsinger et al. 2011; Pearse et al. 2006; Raynaud et al. 2008). Li et al. (2007) found that intercropped maize overyielding resulted from its uptake of phosphorus mobilized by the acidification of the rhizosphere via release of organic acids and protons from faba bean root. As previously discussed, the transfer of fixed N by legumes to intercropped cereals during joint growing periods may be another reason for the high yield of maize observed. We also found that nitrogen fertilization can increase nitrogen accumulation in maize leaf and grain as well as enhance the economic and biological yields per plant.

Different soil management and intercropping systems can increase soil microbial biomass and quantity and activity of soil enzymes (Balota et al. 2003; Wang et al. 2005; kremer and Kussman 2011). A comparison of microbial quantity and diversity could be useful in understanding how rhizosphere communities mediate impacts of different agricultural practices on crop yields. In this study, we found that nitrogen fertilization and root interaction helped increase the quantity of bacteria, fungi, actinomycetes and Azotobacteria in maize rhizosphere soil. The effects of three root separation methods on soil microbial quantity were in the following, decreasing order: NS>PS>FS, indicating that below-ground root interaction in the intercropping system played an important role in increasing the quantity of soil microorganisms, similar to previous studies (Song et al. 2007; Zhang et al. 2010). The reason why interspecific root interaction can increase soil microbial quantity may be because plants in intercropping systems exert species-specific effects on the rhizosphere microbial community and quantity as a result of differences in amount and composition of root exudates (Baudoin et al. 2003; Richardson et al. 2009). Studies of enzyme activities in soil are important as they indicate the soil’s potential to support biochemical processes that are essential for the maintenance of soil quality (Moscatelli et al. 2001). In this study, the activities of invertase, protease, urease and acid-phosphatase in maize rhizosphere soil all decreased in the order NS>PS>FS, indicating that interspecific root interaction can help to increase the activities of soil enzymes. Wang et al. (2005) also demonstrated that tree-crop combinations can enhance soil enzymes activities when compared to sole plantation of Chinese fir. In addition, we also found that nitrogen fertilization can enhance the activities of these soil enzymes.

Correlation analysis revealed that the number of bacteria, fungi, actinomycetes, and Azotobacteria and the activities of urease, invertase, acid-phosphatase, and protease, were all positively or significantly positively (p<0.05) related to maize chlorophyll content, plant height, and economic and biological yields per plant. Mainly this is because soil microbial quantity and enzyme activities are strongly linked to physicochemical properties of soil, so they have an impact on crop physiological characteristics and yield. Tan et al. (2008) indicated that soil enzymes are specific for the types of chemical reactions in which they participate. Van der Heijden et al. (2008) indicated that soil microbes had a strong influence on plant growth and productivity by increasing the availability and supply of limiting nutrients. A study by Alvey et al. (2003) showed that ammonia-oxidizing bacteria, particularly those in the rhizosphere, play a key role in controlling N availability to plants, suggesting they could be important in the interactions between plant species in intercropping. In this study, all data indicated that the economic and biological yields of crops can be increased through improved soil microbial quantity and enzyme activities.

Conclusions

The results of the present study improve our understanding of the mechanisms for intercropping-induced crop enhancements by using a systematic approach. They also provide some theoretical guidance for agricultural production practices. In order to make better use of plant diversity, and especially of niche complementarity and facilitation occurring in the rhizospheres of intercropped species, more consideration should be given to how to optimise cropping patterns in agricultural production practices. This should include increasing crop yield and/or quality by improving the rhizosphere soil micro-ecological environment and enhancing N and P use efficiency of crops. Such an approach is preferable to using a high fertilizer input rate with low use efficiency. The advantages of cereal-legume intercropping partially come from how cereals can obtain some part of their N from the associated legumes, while simultaneously stimulating increased N2 fixation by the legumes. Therefore, the advantageous effects of root interaction may be partially inhibited or enhanced when changing the nitrogen fertilization levels. Other than this mechanism, there are still many complicated and internal relationships between different nitrogen fertilization levels and the effects of root interaction that require further study to fully utilize the advantages of root interaction in intercropping systems under optimal fertilization levels.

Acknowledgments

This study was supported by research grants from the Chinese National Natural Science Fund (U1033004).

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© Springer Science+Business Media Dordrecht 2012