Chapter 20

Influence of Intercropping andIntercropping plus RhizobialInoculation on Microbial Activityand Community Composition inRhizosphere of Alfalfa (Medicagosativa L.) and Siberian Wildrye(Elymus sibiricus L.)

YanMei Sun and NanNan ZhangState Key Lab for Agrobiotechnology, College of Biological Sciences and Center forBiomass Engineering, China Agricultural University, China

En Tao WangDepartamento de Microbiologıa, Escuela Nacional de Ciencias Biologicas, InstitutoPolitecnico Nacional, Mexico

HongLi Yuan, JinShui Yang, and WenXin ChenState Key Lab for Agrobiotechnology, College of Biological Sciences and Center forBiomass Engineering, China Agricultural University, China

20.1 INTRODUCTION

Intercropping, which means to grow two or more cropssimultaneously in a given area, has been widely appliedbecause of its economic, ecological, and environmentalbenefits (Whitmore and Schroder, 2007) compared tomonoculture. For example, intercropping has been foundto improve the mobilization and uptake of phosphorus(Cu et al., 2005; Li et al., 2003, 2007; Wang et al., 2007),potassium, and micronutrients through interspecificrhizosphere interactions (Inal et al., 2007; Zhang et al.,

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

2004). Most recent studies suggest that intercroppingcould reduce nitrate leaching and nitrate pollution togroundwater (Li et al., 2005; Whitmore and Schroder,2007). Besides, intercropping is also used to control pestsand plant diseases (Fenandez-Aparicioa et al., 2007; Renet al., 2008). Legume and cereal plants are the mostpopular combination in intercropping systems becausethe nitrogen fixed by legumes may become available tothe cereals (Doereiner, 1997), and this combination hasbeen widely used in forage production. Alfalfa is themost common forage grown in China because of its high

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212 Chapter 20 Influence of Intercropping and Intercropping plus Rhizobial Inoculation

adaptability and high protein content (Yu et al., 2006).Some studies have suggested that alfalfa–cereal grassintercropping increased the yield of forage and improvednutritional value (Lauriault and Kirksey, 2004).

Recently, the yield increase in intercropping and inter-cropping with rhizobial inoculation have been related tothe effective use of space, time, and light (Zhang et al.,2008), planting density (Muoneke and Mbah, 2007), effi-ciency of water utilization (Jahansooz et al., 2007; Xuet al., 2008), and the relationship between interactions ofdifferent crop roots and nutrient mobilization. Meanwhile,a few studies on soil microorganisms in intercropping sys-tems or in intercropping with rhizobial inoculation havebeen reported (Song et al., 2007; Wang et al., 2007). Itis well known that the crop growth depends largely onsoil fertility while soil fertility is closely related to soilmicroorganisms and soil enzymatic activities because theyplay a crucial role in biogeochemical cycling of essen-tial plant nutrients and the turnover of organic carbon(Obbard, 2001). Vice versa, soil management by intercrop-ping and intercropping plus rhizobial inoculation may alsohave effects on soil enzyme activity, the size of microbialbiomass, and microbial composition.

Soil microbes could be measured using C contentdetermination in the microbial biomass (Benintende et al.,2008), which is not only an important source and sink ofnutrients but also a significant indicator of microbiologi-cal properties and soil fertility (Vasquez-Murrieta et al.,2007; Qu and Wang, 2008).

Soil enzymes are predominantly from microorgan-isms and are closely related to microbial abundance and/oractivity (Insam, 2001). They are responsible for organicmatter decomposition, and therefore affect soil nutrientcycles and the growth of plants. Soil urease, phosphatase,and invertase play essential roles in the cycles of mainnutrients (N, P, C), and therefore the enzyme activity canbe used as an index of soil function (Saha et al., 2008;Zeng et al., 2007).

Microbial community composition is also relatedto soil functions and ecosystem sustainability becauseof their involvement in organic matter dynamics andnutrient cycling (Acosta-Martınez et al., 2008). Ammoniaoxidation to nitrite by ammonia-oxidizing bacteria (AOB)is the rate-limiting step of nitrification, which plays animportant role in plant growth and N-cycling in soil.Therefore, enhancement of the performance of terres-trial ecosystems requires a better understanding of themicrobiology and ecology of AOB. Changes in microbialcommunity composition influence the potential of soilenzyme and may reflect the functional integrity of the soil(Iyyemperumal and Shi, 2008). Thus, characterizationof microbial activity and community composition mayprovide a better way to understand and manipulateecosystem functions.

In the present study, alfalfa and Siberian wildrye,a combination of excellent perennial leguminous andcereal forages, were chosen as test plants to investigatethe effects of intercropping and intercropping with rhi-zobial inoculation on rhizosphere soil enzyme activities,microbial biomass, bacterial composition, and importantgroups of N-cycling bacteria (AOB).

20.2 MATERIALS AND METHODS

20.2.1 Experimental DesignField experiments were established in 2006 and thestudy site was at the Experimental Agricultural Ecosys-tem Station of Inner Mongolia Agricultural University(40 ◦49′N, 111 ◦39′E) located in the Inner MongoliaAutonomous Region, Northern China. This site hasloam soil with 22.4 g/kg organic matter, 34 mg/kgalkali-hydrolysable N, 16.1 mg/kg available phosphorus,144 mg/kg available potassium, and pH 7.9. Fourtreatments were included: Siberian wildrye–alfalfa stripcropping, Siberian wildrye–alfalfa intercropping plusrhizobial inoculation, Siberian wildrye monoculture, andalfalfa monoculture. Each treatment had three replicateplots (4 m × 5 m) that were arranged randomly withinthe field. Following the local agricultural tradition, alltreatments received 220 kg urea/ha and the plots were0.5 m apart and separated by bare ground. Alfalfa wasinoculated with Sinorhizobium meliloti CCBAU01199and the inoculum level was approximately 109 CFU foreach plant. In intercropping treatments, one row of alfalfawas intercropped with one row of Siberian wildrye with0.20 m interrow distance. The monoculture plots wereplanted in similar strips but with only one plant species,either Siberian wildrye or alfalfa.

20.2.2 Soil SamplesSoil samples were collected on June 20 in the second yearat the flowering stage of alfalfa. The soil samples were col-lected and gathered according to the method described inSong et al. (2007). In each field replicate of the treatments,five plants were excavated randomly and, after looselyadhering soil was shaken off, the tightly adhering soilwas taken as rhizosphere soil from the five plants to formone sample. Each of the soil samples sieved through a1-mm screen was divided into two parts: one was storedat 4 ◦C for later analyses of soil microbiological propertiesincluding soil enzymes and microbial biomass; the otherwas stored at −20 ◦C for total DNA extraction. The mois-ture content was measured by drying at 105 ◦C for 24 h.Soil pH was measured in air-dried samples with a pHmeter 103 (Horiba, Ltd., Japan) in soil–H2O suspension(1:2.5, w/v).

20.2 Materials and Methods 213

20.2.3 Microbial Biomass CDeterminationMicrobial biomass C (MBC) was determined by thechloroform–fumigation–extraction method (Lin and Liu,1999). A moist sample equivalent to 20 g of oven-drysoil was fumigated for 24 h at 25 ◦C with ethanol-freechloroform. After the fumigant was removed, the soilwas extracted by horizontal shaking at 200 rpm with80 ml of K2SO4 (0.5 M) for 30 min and then wasfiltered to obtain the extract solution. The non-fumigatedcontrol was extracted under the same conditions whenfumigation began. The difference of extracted organicC was calculated as the non-fumigated control valuesubtracted from the fumigated (24 h) value. Accordingto the soil characters, a conversion factor of 2.64 wasused to convert extracted organic C to biomass C (Vanceet al., 1987). Each sample had triplicate analyses and theresults were expressed on a moisture-free basis.

20.2.4 Enzyme AssaysThe relative quantities of enzymes in soils were measuredby colorimetric determination. Urease and invertase activ-ities were estimated according to the method described byGuan (1986). Urease activity was determined by estimat-ing the ammonium equivalent after the incubation of thesoil sample in the presence of urea at 37 ◦C for 24 h and itwas expressed as mg NH4

+ –N/g soil/h. Invertase activitywas analyzed by the released glucose equivalent. Phos-phomonoesterase (acid and alkaline phosphatase) activitywas determined using a modified method adopted fromSardans et al. (2006) with para-nitrophenyl phosphate(pNPP) as substrate and phenol as product. Briefly, 1 gof each soil sample (wet equivalent) in a 50-ml Erlen-meyer flask was mixed with 4 ml of universal buffer (pH6.5 for acid phosphatase assay and pH 11 for alkalinephosphatase) and 1 ml of pNPP solution. After incuba-tion for 1 h at 37 ◦C, the enzyme reaction was stopped byadding 1 ml of 0.5 M CaCl2 and 4 ml of 0.5 M NaOH.Then the soil suspension was filtered and its absorbanceat 410 nm was measured; enzyme activity was expressedas µg p-nitrophenol released/g soil/h.

20.2.5 DNA Extraction and 16SrDNA Polymerase Chain Reaction(PCR) AmplificationThe total DNA was extracted according to the protocolof Burgmann et al. (2001). In brief, 5 g soil (stored at−20 ◦C) and 2.5 g glass beads were suspended in a washTENP buffer (50 mM Tris, 20 mM eathylene diaminetetraacetic acid (EDTA), 100 mM NaCl, 0.01 g/mlPolyvinylpolypyrrolidone (PVPP), pH 10) and washed

three times by vortexing for 10 min each time. Thedeposits were mixed with 13.5 ml of DNA extractionbuffer (100 mM Tris–HCl, 100 mM sodium EDTA,100 mM sodium phosphate pH 8.0, 1.5 M NaCl, 1%hexadecyltrimethylammonium bromide (CTAB)) and 50µl of proteinase K (20 mg/ml) in centrifuge tubes byshaking at 160 rpm for 30 min at 37 ◦C. Then 1.5 ml of20% Sodium dodecyl sulfate (SDS) was added, and thesamples were incubated at 65 ◦C in a water bath for 2 hwith gentle mixing every 15 min. The supernatants werecollected after centrifugation at 12,000 g for 5 min atroom temperature and transferred into a 50 ml centrifugetube. The nucleic acids were purified with an equal vol-ume of phenol:chloroform:isoamyl (25:24:1) for 15 minand centrifuged for 5 min (10,000 g) at room temperature.The recovered supernatant was further extracted withchloroform:isoamyl (24:1) two times and centrifuged asbefore. The aqueous phase was recovered by centrifu-gation and precipitated with two volumes of water-freeethanol at −20 ◦C for 2 h. The total DNA was obtainedby centrifugation at 16,000 g for 20 min and washed withcold 70% ethanol. The pellet was air dried, resuspendedin 1 ml of TE buffer (10 mM Tris–HCl, 1 mM EDTA,pH 8), and stored at −20 ◦C for further analysis.

For 16S rDNA amplification, the fluorescently FAM-labeled forward primer 27f (5′-AGA GTT TGA TCMTGG CTC AG-3′) and unlabeled reverse primer 1495r (5′-CTA CGG CTA CCT TGT TAC GA-3′; Barberio et al.,2001) were used. PCR reactions were carried out in 25µl volume containing 2.5 µl of 10× PCR buffer (20 mMTris–HCl, 25 mM KCl, 1.5 mM MgCl2, 0.5% Tween 20,and 100 mg of bovine serum albumin per ml), 0.5 µl of2.5 mM deoxynucleoside triphosphates, 0.5 µl of eachprimer (10 pmol/µl), 19.7 µl sterile water, 0.3 µl of TaqDNA polymerase (5 U/µl; TAKARA), and 1 µl of tem-plate (DNA samples were diluted 1:50 in sterile distilledwater). The cycling program for 16S rDNA consisted ofa 5-min initial denaturation step at 95 ◦C followed by fivecycles of 95 ◦C for 30 s, 60 ◦C for 30 s, and 72 ◦C for2 min; and five cycles of 95 ◦C for 30 s, 55 ◦C for 30s, and 72 ◦C for 2 min; and then 25 cycles of 95 ◦C for30 s, 50 ◦C for 30 s, and 72 ◦C for 2 min, a 10-min finalextension step at 72 ◦C (Di cello et al., 1997).

The amoA genes were amplified by using a nestedPCR approach, with aliquots of the first-round PCR prod-ucts used as templates in the second round of PCR. Thefirst-round PCR primers were amoA-2F/5R (Webster et al.,2002) and the second-round PCR primers were amoA-1F/2R (Rotthauwe et al., 1997). The amoA-1F primer wasalso labeled with carboxyfluorescein (6-FAM) for furthervisualization of the terminal restriction fragments (TRFs).For amoA gene amplification, touchdown PCR (Websteret al., 2002) was used for the first round, and the condi-tions for second round PCR were as follows: 95 ◦C for

214 Chapter 20 Influence of Intercropping and Intercropping plus Rhizobial Inoculation

5 min followed by 40 cycles of 95 ◦C for 30 s, 60 ◦C for30 s, and 72 ◦C for 40 s and a 10-min final extension stepat 72 ◦C, as described by Rotthauwe et al. (1997). Theamplified PCR products were then further purified usinga QIA quick PCR purification kit (TIANGEN, China),according to the manufacturer’s recommendations.

20.2.6 Analysis of BacterialCommunityThe bacterial community in soils was estimated bythe terminal restriction fragment length polymorphism(T-RFLP) method (see Chapters 5 and 9) in which thepurified fluorescent-labeled PCR products were digestedseparately with MspI, HhaI, and HaeIII at 37 ◦C for16S rDNA; and with TaqI at 65 ◦C for the amoA gene.The digestion mixture contained 10 µl of purified PCRproduct, 2 µl of enzyme buffer, 7 µl of sterile water,and 1 µl of 10 U restriction endonuclease. All digestionswere carried out for 3 h. The digested products werepurified and 2 µl of digests were mixed with 2 µl ofdeionized formamide, 0.5 µl of ROX-labeled GS500internal size standard (Applied Biosystems), and 0.5 µlof loading buffer. Each sample was then denatured byheating at 95 ◦C for 5 min, and afterwards immediatelytransferred to ice. The samples were electrophoresedusing an ABI3730 genetic analyzer (Applied Biosystems).The size of the TRFs was estimated by reference to theinternal size standard (GS500, Applied Biosystems). TheT-RFLP profiles were analyzed using GENEMAKERsoftware (version 1.5.1, Applied Biosystems). The termi-nal fragments between 50 bp and 500 bp were chosenfor further analysis. Community composition basedon relative areas of TRFs was analyzed by principalcomponent analysis (PCA), using Canoco for Windows4.5. Then, the relative position of each sample alongthe principal component axes was used to describethe community-level similarity between samples. Assig-nation of TRF signals to bacterial taxa was performed

using the National Center for Biotechnology Informationdatabase (The National Center for Biotechnology Infor-mation (NCBI), http://trflp.limnology.wisc.edu/index.jsp;Gil et al., 2006).

20.2.7 Statistical AnalysesAll experimental data were processed by Microsoft Excel2003. The effect of intercropping and intercropping withrhizobial inoculation on soil variables was determined byone-way analysis of SPSS 13.0 Systems. The least signif-icant difference (LSD) was used to test the significancebetween means.

20.3 RESULTS

20.3.1 Plant YieldsGrain yields of alfalfa and Siberian wildrye were mea-sured in 2007 and 2008, which were the second and thirdyears after sowing (Table 20.1). In 2 years the intercrop-ping markedly increased the yields of alfalfa comparedto monocropping, and the treatment of intercropping withrhizobial inoculation seemed even better than intercrop-ping alone in 2007. Both treatments also slightly improvedthe yields of Siberian wildrye.

20.3.2 Microbial Biomass CThe contents of soil MBC in the rhizosphere of mono-cultured alfalfa and Siberian wildrye differed from eachother. The MBC in the rhizosphere of Siberian wildryewas significantly higher than that of alfalfa in monocul-ture. Intercropping and intercropping–rhizobial inocula-tion significantly increased MBC in the rhizosphere ofalfalfa. The soil MBC of intercropping with inoculationwas higher than that in intercropping alone, though thedifference was not significant. The contents of soil MBCincreased from 118.3 mg C/kg soil in the rhizosphere

Table 20.1 Grain yields of alfalfa and Siberian wildrye in different treatments

Crop Treatments2007

Yield (kg/20 m2)Yield Increase

(%)2008

Yield (kg 20/m2)Yield Increase

(%)

Monoculture 10.2 ± 0.9 a — 11.7 ± 1.4 a —Alfalfa Intercropa 18.2 ± 0.7 c 78.4 21.1 ± 4.2 b 80.3

Intercrop with inoculationb 26.1 ± 3.7 d 155.8 20.7 ± 1.7 b 76.9Monoculture 12.5 ± 0.6 ab — 10.3 ± 1.3 a —

Siberian wildrye Intercrop 17.2 ± 0.6 bc 37.6 22.1 ± 2.6 b 114.6Intercrop with inoculation 14.2 ± 0.9 abc 13.6 12.3 ± 3.1 a 19.4

Means of three replicates ± standard error. Values in the same column followed by different letters are significantly different (P < 0.05).a Intercrop: alfalfa with Siberian wildrye intercropping.b Intercrop with inoculation: alfalfa with Siberian wildrye intercropping with rhizobial inoculation in the rhizosphere of alfalfa.

20.3 Results 215

Table 20.2 Microbial biomass C and soil pH in the rhizosphere of alfalfa and Siberian wildrye in different treatmentsin 2007

Crop Treatments MBC (mg C/kg soil) pH

Monoculture 118.3 ± 8.0 a 8.28 ± 0.03 aAlfalfa Intercropa 189.0 ± 7.9 bc 8.28 ± 0.03 a

Intercrop with inoculationb 212.2 ± 7.8 c 8.28 ± 0.03 aMonoculture 189.4 ± 6.3 bc 8.38 ± 0.03 b

Siberian wildrye Intercrop 179.6 ± 12.7 b 8.42 ± 0.03 bIntercrop with inoculation 166.8 ± 6.7 b 8.45 ± 0.03 b

Means of three replicates ± standard error. Values in the same column followed by different letters are significantly different (P < 0.05).a Intercrop: alfalfa with Siberian wildrye intercropping.b Intercrop with inoculation: alfalfa with Siberian wildrye intercropping with rhizobial inoculation in the rhizosphere of alfalfa.

of monocultured alfalfa to 212.2 mg C/kg soil in therhizosphere of alfalfa in intercropping with inoculation(Table 20.2). In Siberian wildrye, the soil MBC exhibitedsimilar levels in different treatments.

20.3.3 Enzyme AssaysIntercropping and intercropping–rhizobial inoculationshowed certain differences in their effects on enzy-matic activities in rhizosphere soils of the two forages(Fig. 20.1). Both treatments noticeably enhanced theurease activity and invertase activity in alfalfa rhi-zosphere compared to the monoculture: 10.56% and15.65% for urease and 16.27% and 19.34% for invertase,respectively. The activity of alkaline phosphatase alsoshowed an increasing trend and the difference wassignificant in intercropping with inoculation treatment,but no pronounced treatment effects on acid phosphataseactivity were found. In the rhizosphere soil of Siberianwildrye, soil invertase and alkaline phosphatase activ-ities were significantly reduced in intercropping andintercropping–rhizobial inoculation; the activity of acidphosphatase did not vary much in intercropping–rhizobialinoculation, but was significantly decreased in inter-cropping; soil urease activity in the two treatments wassimilar to that in monoculture.

20.3.4 Bacterial CommunityCompositionThe 16S rDNA gene T-RFLP fingerprints generatedfrom three separate restriction digests, HhaI, HaeIII, andMspI, showed consistent patterns and only the results ofHaeIII are shown as an example (Fig. 20.2). In PCA,the first principal component explained 37.4% of thevariance and the second principal component explained25.4% of the total variance (Fig. 20.2). Clear differencesin the community structure between the plants weredisplayed. The similarities among the different treatments

in alfalfa rhizosphere soils were relatively higher thanthose in Siberian wildrye, and no distinct differencewas found among the treatments of intercropping,intercropping–rhizobial inoculation, and monoculture.For Siberian wildrye, intercropping and monoculturewere clustered together, but intercropping with inocula-tion seemed to have a relatively strong impact on thecommunity profile of Siberian wildrye rhizosphere soil.

Using the NCBI databases, the presence and relativeabundances of the main bacterial groups in the soil sam-ples were estimated from the T-RFLP profiles obtainedin the HhaI, HaeIII, and MspI digests (Fig. 20.3). Somediscrepancies between the three digests were indicated inthe following. In the rhizosphere of alfalfa, intercroppingincreased the relative abundances of Betaproteobacteria,Deltaproteobacteria, Actinobacteria, and Bacteroidetes;whereas relative abundances of Gammaproteobacteria,Firmicutes, and some uncultured bacteria were decreased.In the rhizosphere of intercropping–rhizobial inoculation,the relative abundances of Alphaproteobacteria, Betapro-teobacteria, and Bacteroidetes were increased, but therelative abundances of Gammaproteobacteria, Deltapro-teobacteria, Firmicutes, and Actinobacteria were slightlydecreased. In the case of Siberian wildrye, the relativeabundances of Betaproteobacteria and Deltaproteobacte-ria were increased while those of Alphaproteobacteria,Firmicutes, and Bacteroidetes were decreased in inter-cropping and intercropping–rhizobial inoculation. Otherdiscrepancies also existed: intercropping increasedthe relative abundances of Gammaproteobacteria andActinobacteria, and intercropping with inoculationrepresented inverse results.

In this study, fragments of amoA were successfullyamplified from all soil DNA samples. Table 20.3 displayedTF sizes and their corresponding AOB groups based on T-RFLP profile of amoA gene with TaqI restriction enzyme(Park and Noguera, 2004; Siripong and Rittmann, 2007).T-RFLP analysis with TaqI generated several commonpeaks such as 48, 219, 283, and 491 bp in all treatments,

216 Chapter 20 Influence of Intercropping and Intercropping plus Rhizobial Inoculation

(a)

0

a

MA IA IAI MW IW IWI

b b bcc

b

50

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ase

activ

ity(m

g N

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il/d)

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aa c

abbc

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Add phosphataseAllcaline phosphatase

a

a

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a

MA IA IAI MW IW IWI

Figure 20.1 Soil urease activity (a), invertase activity (b), andphosphatase activity (c) under different treatments. Bars withdifferent letters indicate significant differences at p < 0.05. MA,alfalfa in monoculture; IA, alfalfa in intercrop; IAI, alfalfa inintercrop with inoculation; MW, Siberian wildrye in monoculture;IW, Siberian wildrye in intercrop; IWI, Siberian wildrye in intercropwith inoculated alfalfa.

which indicated the presence of Nitrosomonas andNitrosospira in the rhizosphere soil of Siberian wildryeand alfalfa (Fig. 20.4). Figure 20.4 also showed that therelative abundance of 283 bp TF was higher than thatof other TFs. Therefore, AOB belonging to Nitrosospiraspp. were estimated to be the predominant groups irre-spective of treatments. Intercropping and intercropping

PCA 1 (37.4%)

MA MW

IWIWI

IA

IAIP

CA

2 (

25.4

%)

–0.5

–1.0 1.0

1.5

Figure 20.2 PCA was performed using the 16S rRNA gene TRF(HaeIII) relative abundance data obtained from the rhizosphere ofalfalfa and Siberian wildrye in different cropping systems. MA,alfalfa in monoculture; IA, alfalfa in intercrop; IAI, alfalfa inintercrop with inoculation; MW, Siberian wildrye in monoculture;IW, Siberian wildrye in intercrop; IWI, Siberian wildrye in intercropwith inoculated alfalfa.

Apb Bpb0

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35

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ativ

e ab

unda

nce

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Gpb Dpb fir act bac nns

MA

MWIWIWI

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Figure 20.3 Relative abundances of specific bacterial groups inT-RFLP profiles obtained from different treatments. Apb,Alphaproteobacteria; Bpb, Betaproteobacteria; Gpb,Gammaproteobacteria; Dpb, Deltaproteobacteria; fir, Firmicutes;act, Actinobacteria; bct: Bacteroides; uns, clones, strains, anduncultured microorganisms. Other groups were <2%. MA, alfalfa inmonoculture; IA, alfalfa in intercrop; IAI, alfalfa in intercrop withinoculation; MW, Siberian wildrye in monoculture; IW, Siberianwildrye in intercrop; IWI, Siberian wildrye in intercrop withinoculated alfalfa.

20.4 Discussion 217

Table 20.3 The TF sizes and their corresponding AOBgroups based on T-RFLP of amoA gene with TaqIrestriction enzyme (Park and Noguera, 2004; Siripongand Rittmann, 2007)

Nitrifier Group TRF Size

Nitrosomonas europaea/eutropha lineage 219/491Nitrosomonas oligotropha lineage 48/354/491Nitrosomonas cryotolerans lineage 48/354/441/491Nitrosomonas marina lineage 48/491Nitrosomonas communis lineage 491Nitrosospira lineage 283

MA

0

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ativ

e ab

unda

nce

(%)

20

40

60

80

100

IA IAI MW IW IWI

T-RFS(bp)

48219283354441491

Figure 20.4 Relative abundances of specific AOB in T-RFLPprofiles obtained from different treatments. Assignations wereobtained from T-RFLP profiles of amoA gene digested with TaqI.MA, alfalfa in monoculture; IA, alfalfa in intercrop; IAI, alfalfa inintercrop with inoculation; MW, Siberian wildrye in monoculture;IW, Siberian wildrye in intercrop; IWI, Siberian wildrye in intercropwith inoculated alfalfa.

with inoculation tended to increase the diversity of amoAbecause an additional 441 bp fragment was observed inalfalfa intercropping and intercropping–rhizobial inocu-lation treatments. In the rhizosphere of Siberian wildrye,a 354 bp fragment was detected, although its relativeabundance was low. Among these TFs, the 283 bp TFcorresponded to Nitrosospira cluster and others belongedto Nitrosomonas-like organisms. Although the relativeabundance of the ammonia oxidizers varied amongtreatments, we could find that the relative abundanceof Nitrosomonas increased and that of Nitrosospiradecreased in intercropping with inoculation treatment.

20.4 DISCUSSION

Most of the previous studies have indicated that legumeand cereal intercropping are profitable for improving soil

fertility and increasing land productivity (Li et al., 2007;Zhang et al., 2004). In our study, the yield data clearlydemonstrate the superiority of the integrated use of inter-cropping and inoculating rhizobium especially in 2007.This beneficial effect may be attributed to the maintenanceand improvement of soil nutrient status (Li et al., 2007)as well as microbial activity and community composition(Bastida et al., 2008).

MBC reflects the size of the soil microbial communityand is believed to be an indicator of microbiological prop-erties and soil fertility (Bastida et al., 2008). In the rhi-zosphere of alfalfa, intercropping and intercropping withrhizobial inoculation significantly enhanced the microbialbiomass, which was beneficial in improving crop yields.Meanwhile, the better growth of plants in intercroppingcould improve the quality and quantity of root exudatesand the turnover of root biomass to the soil, which subse-quently benefits the microbial growth in rhizosphere (Yanget al., 2007; Bastida et al., 2008; Chu et al., 2007). Theincrease in the yield of Siberian wildrye was not as pro-nounced as that of alfalfa and that was in accordance withthe absence of significant changes in microbial biomassin both treatments.

Soil enzymes are derived primarily from soil fungi,bacteria, plant roots, and plant and animal residues (Yanget al., 2008), and their activities can reflect the strengthof biochemical processes in soil (Liu et al., 2008). Theincrease of urease, invertase, and alkaline phosphataseactivities in the rhizosphere of alfalfa in intercroppingand intercropping plus rhizobial inoculation was similar tothat seen in some previous studies on intercropping withthe same or different plants (Kandeler et al., 1999; Starket al., 2008; Yang et al., 2007). The increase demonstratedthat these two treatments could stimulate the growth ofmicroorganisms and the biochemical cycles of N, C, andP in alfalfa rhizosphere that might improve the nutrientsupply and then the yields of plants. Acid phosphatasewas usually active in acid soils (Iyyemperumal and Shi,2008) and the soil pH was around 8.0 in the present study,which could be the explanation why no difference wasfound in acid phosphatase activity. In the rhizosphere ofSiberian wildrye, the changes in enzyme activities weredifferent from those in the rhizosphere of alfalfa. Thedifference in microbial community compositions mighthelp interpret the dissimilarities of enzyme activities inSiberian wildrye rhizophere between the intercropping andintercropping–rhizobial inoculation treatments.

In the present study, all soil samples were treatedunder the same PCR conditions to eliminate the effects ofpotential PCR bias. Therefore, the T-RFLP analysis couldreflect the relative composition of 16S rRNA genes in amodel community (Schwarz et al., 2007). The bacterialcommunity composition in the rhizosphere of alfalfa wasdiscriminated from the communities in the rhizosphere

218 Chapter 20 Influence of Intercropping and Intercropping plus Rhizobial Inoculation

of Siberian wildrye (Fig. 20.2). Several studies haveindicated the marked relation between soil microbialcommunities and plant species (Song et al., 2007; Wardleet al., 1999; Yang et al., 2007). In the rhizosphere ofalfalfa, the bacterial community compositions in rhizo-sphere soils of monoculture, intercropping, and intercrop-ping with rhizobial inoculation were very similar, indicat-ing that the treatments had little impact in this case. Thatderived from the effect of key plant species on microbialcommunities, which was important to consider because theabundance, activity, and community compositions oftenvaried considerably with plant species (Habekost et al.,2008). The high similarities between the Siberian wildryerhizosphere samples in intercropping and monocultureindicated a slight effect of intercropping on the rhizo-sphere bacterial community of this plant. However, theinoculation of alfalfa with rhizobium caused a remarkabledrift of the bacterial community composition in the rhizo-sphere of intercropped Siberian wildrye, which indicatedan important integrated role of intercropping and rhizobialinoculation in shaping the soil microbial communities.

In our study, the similarity was higher in species com-position among treatments in alfalfa rhizosphere, but weobserved the shifts in the relative abundance of majorphylogenetic groups of bacteria in intercropping and inter-cropping with rhizobial inoculation treatments (Fig. 20.3).For Siberian wildrye, some treatment effects were alsofound; the relative abundance of main bacterial groupsalso showed some changes that displayed the effects ofintercropping and intercropping with rhizobial inoculationon soil microbes on the other hand.

Changes in the community composition of AOBinduced by intercropping and intercropping with inoc-ulation were also detected by the presence/absenceof TRFs and their relative abundance. Our study alsoverified that the Nitrosospira lineage was the dominantAOB in the soil samples, which was consistent withprevious findings (Phillips et al., 2000; Kowalchucket al., 2000). The appearance of certain “new” fragmentsthat were absent in controls suggested that certaingroups of AOB were stimulated by intercropping andrhizobial inoculation. Bartelt-Rysera et al., 2005 alsoobserved that in the short term, soil carry-over effectsof plant diversity are mediated by a general stimulationof soil microbes. Changes in relative abundance ofdifferent TRFs also demonstrated the important effects ofintercropping, especially intercropping with inoculation,on the community composition of AOB. On the basisof the earlier discussion, it is clear that intercroppingand rhizobial inoculation affected the composition of keymicrobial functional groups driving N dynamics, and thechanges in diversity and relative abundance might play

important roles in organic N degradation, mineralization,and immobilization, which benefit the nitrogen uptake byplants.

In conclusion, this study displayed the predominanteffects of intercropping, especially intercropping with rhi-zobial inoculation, on increasing plant yields in 2007.Intercropping and intercropping with rhizobial inoculationshowed important effects on microbial activities as well ascommunity structure. Our experiments also revealed theinteractions between above- and belowground terrestrialecosystems.

ACKNOWLEDGMENTS

This study was supported by China State Program forBasic Research “973” (2006CB100206) and Natural Sci-ence Foundation of China (30670071).

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