soil metagenomics reveals differences under conventional crop rotation

Applied Soil Ecology 72 (2013) 49 61

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Applied Soil Ecology

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Soil metagenomics reveals differences under conno-tillage with crop rotation or succession

Renata C ezaMarco Ana Embrapa Sojab Universidade ilc Embrapa Sund Laboratrio N e Jane

a r t i c

Article history:Received 18 FeReceived in reAccepted 28 M

Keywords:Environmental shotgun sequencing (ESS)Soil microbial biodiversitySoil metagenomicsSoil managementCrop managemNo-tillage

al fonvestear

tional tillage (CT) with plowing and disking, or no-tillage (NT) with direct sowing into the residues ofprevious crops, in a crop succession [soybean (summer)/wheat (winter)] or rotation [soybean/maize(summer)/wheat/lupine/oat (winter)]. About 1 million reads per treatment revealed very high levels ofdiversity. The majority of the sequences were attributed to the Bacteria (54%), and 0.3% and 0.2% ttedinto Archaea and Eucarya domains, respectively; 46% showed no similarity with any known sequences.Major differences were associated with tillage and, to a lesser extent, with crop management. Statistically

1. Introdu

A recentshows that oerately degrpoor agricutilizers andto prevent ties participcycles. Howtionality anresulting in(Brown et a

CorresponParan, Brazil.

E-mail [email protected](M. Hungria).

0929-1393/$ http://dx.doi.oent signicant higher abundances with CT encompassed microorganisms associated with residue decompo-sition, carbon and nitrogen cycling, and xenobiosis. Eucarya were also more abundant with CT, possiblyrelated to higher tolerance of environmental stresses. In contrast, NT showed higher abundances par-ticularly of nitrogen-xing Rhizobiales and Archaea that inhabit environments rich in organic matter.

2013 Elsevier B.V. All rights reserved.

ction

assessment of the state of the planets land resourcesverall, 25% and 8% are considered to be highly and mod-aded, respectively; soil degradation is mostly related toltural management practices and improper use of fer-

pesticides (FAO, 2012). Biotic activity is fundamentalor reverse soil degradation, since microbial communi-ate in critical processes, such as the biogeochemicalever, anthropogenic activities can affect both the func-d the biodiversity of microbial communities, potentially

curtailment of microbial functions and loss of speciesl., 2002; FAO, 2012). Therefore, the global adoption of

ding author at: Embrapa Soja, Caixa Postal 231, 86001-970 Londrina, Tel.: +55 43 33716206; fax: +55 43 33716100.resses: [email protected] (R.C. Souza),[email protected] (M.E. Canto), [email protected] (A.T.R. Vasconcelos),[email protected] (M.A. Nogueira), [email protected],pq.br, [email protected], [email protected]

soil conservation practices in agriculture is critical to help to reversesoil degradation, and to maintain soil fertility and soil biodiver-sity.

In contrast to conventional agricultural practices of soil manage-ment using plowing (020 cm) and disking (08 cm), the systemknown as no-tillage is characterized by sowing directly into theresidues of previous crops. It has been broadly shown that no-tillagecan contribute signicantly to soil conservation, mainly by thepresence of crop residues on the surface, ameliorating adverse tem-perature and moisture conditions (Derpsch et al., 1991), increasingsoil organic matter content, and improving soil physical properties(Calegari et al., 2008; Castro Filho et al., 2002; Derpsch et al., 1991;FAO, 2012; Franchini et al., 2007; Lal et al., 2007). Most microbialactivity occurs within a few centimeters of the soil surface (Babujiaet al., 2010); therefore, an increasing number of reports shows thatlack of soil disruption and coverage by plant residues in the no-tillage system also favor increased microbial biomass and activity(Babujia et al., 2010; Franchini et al., 2007; Kaschuk et al., 2010;Pereira et al., 2007; Silva et al., 2010, 2013). Estimates indicatethat no-tillage systems are practiced on over 117 million hectaresworldwide, 48% of which in South America (FAO, 2012). Brazil isa world leader with over 25.5 million hectares under no-tillage,

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.apsoil.2013.05.021arolini Souzaa,b, Mauricio Egdio Cantoc, Ana Tertonio Nogueiraa,b, Mariangela Hungriaa,b,

, Cx. Postal 231, 86001-970 Londrina, Paran, Brazil Estadual de Londrina, Dept. Microbiologia, Cx. Postal 60001, 86051-990 Londrina, Brazos e Aves, Cx. Postal 21, 89700-000 Concrdia, Santa Catarina, Brazilacional de Computac o Cientca, Rua Getlio Vargas 333, 25651-071 Petrpolis, Rio d

l e i n f o

bruary 2013vised form 27 May 2013ay 2013

a b s t r a c t

Soil conservation practices are criticsequencing approach was used to icrop-management practices in a 13-y/ locate /apsoi l

ventional and

Ribeiro Vasconcelosd,

iro, Brazil

r agricultural sustainability, and in this study the shotgunigate the effects on soil biodiversity of different soil- andeld trial in southern Brazil. Treatments consisted of conven-

50 R.C. Souza et al. / Applied Soil Ecology 72 (2013) 49 61

which is expected to increase to 33 million hectares in the nextyears (FEBRAPDP, 2012).

Avoiding monoculture is another practice critical to diminish-ment of soil degradation, and rotations/associations should involveat least three different crops (FAO, 2012). Unfortunately, monocul-tures or sucincluding thorganic maincrease wwith green ical and chebeen showDerpsch et ity of resulunexpectedity in someet al., 2007shows the how microb

In the laadvances inmicrobial ctrophoresis2001; Muyztures (Hedl2001). Signianalyses, wRoesch et aronmental analysis of rRNA are

Our undcropped aroffer the pragement syadopted fortional tillagrotation (vtrial in Braz

2. Materia

2.1. Descrip

The studlished in thEmbrapa Solongitude 5Latossolo Vdox (US taxgiven in Ta(Silva et al.,

The treatillage (CT)max L. Merthe winter]mays L.) inL.) and oat ments are dsuccession mentary TaThe trial hreplicates.

Supplemonline vers

2.2. Soil sampling and processing

Sampling was performed in early November of 2010, immedi-ately before sowing of soybean, and 3 weeks after harvesting thewinter crop

soil tem.squaes wken fere pimesly discreted our pled, thve bal an

omog

onst su

(Silvs soflgori, as dweenot shonsid

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tagen 45at thwww

fragments

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uenc2011cessions involving only two crops prevail worldwide e southern region of South America and can depletetter content, result in predisposition to diseases, andeed infestation. In contrast, crop rotations, especiallymanures, break pathogen cycles and improve soil phys-mical properties, including soil organic matter, as has

n in Brazil (Boddey et al., 2010; Calegari et al., 2008;al., 1991; Franchini et al., 2007). However, the complex-ts obtained with various crop rotations, including the

absence of differences in microbial biomass and activ- reports in Brazil (Balota et al., 1998, 2004; Franchini; Hungria et al., 2009; Silva et al., 2010, 2013), clearlyneed for more research to improve understanding ofial communities are affected by crops.st decade, a variety of molecular tools has fostered

our knowledge of diversity and composition of soilommunities, including denaturing gradient gel elec-

(DGGE) (Helgason et al., 2010; Kozdrj and van Elsas,er et al., 1993; Peixoto et al., 2006) and fatty acid signa-und, 2002; Helgason et al., 2010; Kozdrj and van Elsas,cant advances are now possible with DNA-sequencingith emphasis on metagenomics (Delmont et al., 2012;l., 2007). However, studies of metagenomes using envi-shotgun sequencing (ESS) instead of the sequencingcloned libraries or specic genes such as that for 16Sstill rare.erstanding of microbial communities in intensively

eas of South America is still poor, and metagenomicsomise of revealing diversity under varied soil and man-stems. In this study, a metagenomic approach was

examination of an Oxisol under no-tillage or conven-e conditions with crop succession (soybean/wheat) ore crops in a 5-year period) practices in a long-term eldil.

ls and methods

tion of the eld trial and treatments

y was performed in a long-term experiment, estab-e summer of 1997/1998 at the experimental station ofja, in Londrina, State of Paran, Brazil, latitude 2311 S,111 W, and elevation of 620 m. The soil is classied asermelho Eutrofrrico (Brazilian system), Rhodic Eutru-onomy) and main chemical and physical properties areble 1. Climate conditions have been given elsewhere

2010).tments consisted of no-tillage (NT) and conventional, each under a crop succession (CS) [soybean (Glyciner.) in the summer and wheat (Triticum aestivum L.) in

or a crop rotation (CR) with soybean and maize (Zea the summer and wheat, lupine (Lupinus angustifolius(Avena strigosa Schreb.) in the winter. The four treat-esignated as NTS, NTR, CTS and CTR. Crops grown in

and rotation for the last 7 years are shown in Supple-ble S1. Each plot measured 8 m width 15 m length.ad a completely randomized block design, with four

entary material related to this article found, in theion, at http://dx.doi.org/10.1016/j.apsoil.2013.05.021.

to theNT sysmetal residuwas taples weight tspatialThe diconsistwith foremovmm siephysic

2.3. H

To cwas rbeforeumericwith amatrixity bet(data nwere cfor the

2.4. D

MereplicaLaboraquantiter at 2by ele50 ng/

Mein theence), http://domlyFragm(Jones,accordspheresubmiport Pi2009).

To r(Niu etorder tcates sHolmeset wathe sub

2.5. D

Seqet al., , wheat. Crop residues of the wheat were incorporatedin the CT system and left on the soil surface in the

An area of 0.4 m2 was delineated on each plot with are and the surface 010-cm layer was determined. Cropere removed and a soil sample of approximately 300 grom the middle of each square using an auger. The sam-laced in labeled bags and the procedure was repeated

in each of the four replications in the eld, at pointsstributed as representative of the area of each replicate.e soil samples were combined so that each replicationf a composite sample of approximately 2.5 kg per plot,ots per treatment. In the laboratory, plant residues weree samples were homogenized and passed through a 2-efore analysis. Subsamples were taken for chemical andalyses, and the remaining was kept at 20 C.

eneity among replicates

rm homogeneity for microbial DNA, each eld replicatebmitted to 16S rRNA analysis by PCR-DGGE, as describeda et al., 2013). The DGGE gels were analyzed using Bion-tware (Applied Mathematics, Kortrijk, Belgium, v.4.6),thm UPGMA and tolerance of 5% to create a distanceened before (Silva et al., 2013). The level of similar-

replicates in each treatment ranged from 98.8 to 100%own). Therefore, the four replicates of each treatmentered homogeneous and were pooled to extract the DNAagenomic analysis.

traction and pyrosequencing analysis

omic DNA was extracted by using 10 g of each soilnd the PowerMaxTM Soil DNA Isolation Kit (MoBios), following the manufacturers procedures. DNA wasnd purity was veried in a NanoDrop spectrophotome-nd 280 nm. DNA purity and quantity were also veriedhoresis in 1% agarose gels and samples adjusted to

omic DNA was submitted to sequencing analysis4 platform (GS-FLX Titanium Roche Applied Sci-e Labinfo of LNCC (Petrpolis, Rio de Janeiro, Brazil.labinfo.br). For the pyrosequencing, DNA was ran-ented by nebulization with compressed nitrogen gas.

of 300800 bp were selected (Bioanalyzer DNAChip)0). Fragments were then linked to adaptors A and B,o the manufacturers procedure and linked to the micro-he emulsion PCR (Margulies et al., 2005). Samples wereo sequence analysis with the Titanium kit, and the sup-erPlate (Roche Applied Science) (Imelfort and Edwards,

ve low-quality sequences and articial duplicated reads010) lters were applied to the sequencing analysis. Inove articial reads and low-quality sequences, Repli-

are (Gomez-Alvarez et al., 2009) and Lucy (Chou and01) were applied to the sequences obtained. The dataosited in the NCBI-SRA (Sequence Read Archive) with

sion Accession Number SRA050780.

nalyses

es were analyzed with the MEGAN4 software (Huson), based on the comparison of BLASTX with all the

R.C. Souza et al. / Applied Soil Ecology 72 (2013) 49 61 51

Table 1Soil chemical and granulometrica properties in the 010 cm layer.

Soil managementb Crop managementc pH Al H + Al K Ca Mg SBd TCECd P C N BSd

CaCl (cmol dm3) (mg dm3) (g dm3) (g dm3) %

NT 4 NT 0 CT 1 CT 5

a Granulomb Conventioc Crop succe soybe

described in Td SB, sum of ase sae Data repre

sequences ithe hits andlowest comthe softwar10. Data we

Data wesoftware (Ponomical dpairs of treaFishers testcomparisonestimated w

3. Results

3.1. Sequencurves

About 1four treatm1,050,712 scession (CTrotation (Nsuccession (with the Ne51, the bigg1055 bp; NT

As the nwere normaparisons. Nchosen by dbetween treeach treatmby the MEGFigure S1. saturated, iHavelsrud ein CaliforniaCanada and53,533 read

Supplemonline versi

3.2. Total so

It is imstudy werements and Therefore, rstudies basfour genom

sultsces d 0.2tivelyhandX) ws of r meperibtainr et f thesest Delmtion unbamesces cns, bu

cteri

hin tant psses ), De.55%)rotebacthizoblly i. Wiodopen uundse so

n emriodsult a

the2 c

CS 5.39e 0 4.67 0.53 4.03 1.2CR 5.68 0 3.95 0.58 4.50 1.2CS 5.23 0 4.77 0.46 3.73 1.0CR 5.32 0 3.91 0.47 4.06 1.1

etric composition: 710 g kg1 of clay, 82 g kg1 of silt and 208 g kg1 of sand.nal tillage (CT), no-tillage (NT).ssion (CS) with soybean (summer) and wheat (winter) and crop rotation (CR) withable 1S.

bases (Ca + Mg + K); TCEC, total cation exchange capacity (H + Al + Ca + Mg + K); BS, bsent the means of four eld replicates.

n the NCBI-NR databank. In MEGAN, the comparison of attribution of a taxon for each sequence is based on themon ancestor algorithm (LCA). The parameters used ine were the min-support: 5; min-score: 60; top-percent:re also normalized by using the MEGAN software.re also submitted to statistical analysis with the STAMParks and Beiko, 2010), to identify differences in tax-istribution in the comparison of all combinations oftments. Statistical signicance was estimated with the

for p 0.05, using Storeys FDR correction method for of pairs of treatments. The condence interval wasith the method of NewcombeWilson.

and discussion

cing analysis, data normalization, and rarefaction

million sequences were generated for each of theents: conventional tillage with crop rotation (CTR),equences, average size of 304 bp; CT with crop suc-S), 1,080,923 sequences of 307 bp; no-tillage with cropTR), 913,435 sequences of 306 bp; no-tillage with cropNTS), 1,034,153 sequences of 311 bp. In the assemblagewbler software, very few contigs were generated: CTR,est with 1323 bp; CTS, 83, with 1287 bp; NTR, 31, withS, 58, with 1505 bp.

umber of reads was similar among the treatments, datalized for a set of 100,000 sequences, to facilitate com-

ormalization means that the 100,000 sequences wereenition of the software to represent a common numberatments, but it refers to the 1,000,000 sequences froment that were analyzed. Rarefaction curves generatedAN for the species level are shown in SupplementaryEven with 1 million sequences, the curves were notndicating a high level of diversity. As a comparison,t al. (2011) obtained 459,262 reads form sea sediments, USA, and in the comparison with four soils from USA,

Brazil, Roesch et al. (2007) analyzed a maximum ofs per site.

the resequen0.3% anrespecother (BLASTportionanothested Exwere o(Meye34.5% ohad clotively (proporbe the of genosequendomai

3.3. Ba

Witdomining cla(20.8%ria (8Alphapxing Bradyrculturacyclingwas Rhhas becompoobic, thwith aing pecan resleast inentary material related to this article found, in theon, at http://dx.doi.org/10.1016/j.apsoil.2013.05.021.

il microbial diversity

portant to highlight that the results shown in our obtained with a shotgun approach, reading all frag-without a previous amplication of ribosomal genes.esults should represent a much broader diversity thaned on ribosomal genes. A comparative tree of thees was generated with the MEGAN, and Fig. 1 shows

Sphingoorder of Alpgenus Sphinact in the mour experimheavily appabundant gbon cycling(Marks et lales (Fig. 2contributio5.79 10.47 66.0 24.0 2.6 55.006.28 10.22 55.5 25.8 2.8 61.245.20 9.97 14.8 19.2 1,6 52.115.68 9.60 20.6 19.4 1.8 59.22

an and maize (summer), and wheat, lupine, and oat in the winter, as

turation (SB/TCEC) 100.

obtained at the Phylum level. The majority of thewere attributed to the Bacteria domain (53.5%), while% were attributed to the Archaea and Eucarya domains,, whereas viruses represented only 0.0001%. On the, 46.1% of the sequences did not show any similarityith any sequence of the NCBI-NR (Fig. 1). Similar pro-microorganisms in the three domains were reported intagenome study of a natural grassland soil at Rotham-mental Station, Harpenden, UK: over 12.5 million readsed, and, based on functional classication of MG-RAST

al., 2008) an even more restricted database only reads were annotated, of which 88.6%, 0.91% and 1.41%homologies with Bacteria, Eucarya and Archaea, respec-ont et al., 2012). One possible explanation for the low

of Eucarya and Archaea in our study, and in others, maylanced sequence database, i.e. the much larger number

and sequences of Bacteria. It is possible that most of thelassied as no hits belong to the Eukcarya and Archaet this remains to be conrmed.

a, Archaea and Eucarya domains, and virus

he Bacteria domain, the Proteobacteria represented thehylum in all four treatments - 41.5% (Fig. 1). The prevail-were Alphaproteobacteria (51.1%), Betaproteobacterialtaproteobacteria (19.6%) and Gammaproteobacte-

(Fig. 2A). The order Rhizobiales dominated theobacteria (Fig. 2B), with numerous symbiotic nitrogen-eria, including the genera Rhizobium, Sinorhizobium,ium and Mesorhizobium. Within this order, another agri-mportant genus was Nitrobacter, critical for nitrogenthin the genus Rhodopseudomonas, one relevant speciesseudomonas palustris, a phototrophic bacterium that

sed as a model for anaerobic degradation of aromatic (Perrotta and Harwood, 1994). Although mostly aer-ils in our study are rich in clay (Table 1), and pores phasis on micropores can become anaerobic for vary-

of time, mainly after rain; furthermore, anaerobiosislso from soil compaction caused by excessive trafc, at

topsoil.

monadales represented the second most abundanthaproteobacteria (Fig. 2B), with predominance of thegomonas. Among other functions, these bacteria mayineralization of herbicides (Srensen et al., 2001) and inent, in both CT and NT systems, herbicides have beenlied. In the order Caulobacterales (Fig. 2B), the mostenus was Caulobacter, including bacteria involved in car-

and capable of surviving in oligotrophic environmentsal., 2010). Other hits t into the order Rhodospiril-B), including Azospirillum, which may give importantns to plant growth due both to nitrogen xation and


Fig. 1. Compasuccession (S)

plant-growet al., 2010;

The Betathe Proteoborder Bukhrative tree generated by the MEGAN software of four metagenomes of an Oxisol unde or rotation (R).

th promotion via the biosynthesis of hormones (Hungria Steenhoudt and Vanderleyden, 2000).proteobacteria class was the second most abundant ofacteria (Fig. 2A), with the great majority of hits in theolderiales, followed by Neisseriales, Nitrosomonadales

and Rhodocin soils, incof xenobiotmotion (Es(Gyaneshwr 13 years of no-tillage (NT) or conventional tillage (CT) with crop

yclales (Fig. 2C). Burkholderia has a variety of functionsluding biological control of pathogens, bioremediationics (Coenye and Vandamme, 2003), plant-growth pro-trada-De Los Santos et al., 2001), and nitrogen xationar et al., 2011). The order Nitrosomonadales included


Fig. 2. Abunda(D) Deltaproteor rotation (R)

several cheNitrosospiraet al., 2006)

The numof Betaprotorders partnce estimates generated by the MEGAN software of (A) classes of the Phylum Bacteria, anobacteria, (E) Gammaproteobacteria in four metagenomes of an Oxisol under 13 years o.

molithotrophic genera, including Nitrosomonas and, related to nitrication (Schmidt and Bock, 1997; Shaw.ber of hits with Deltaproteobacteria was similar to thateobacteria (Fig. 2D), and bacteria belonging to theseicipate in iron and sulfate reduction (Foti et al., 2007;

Hori et al., 2of these nuin the Gammorders weradales and Pseudomond orders of (B) the class Alphaproteobacteria, (C) Betaproteobacteria,f no-tillage (NT) or conventional tillage (CT) with crop succession (S)

010), and may have important roles in the availabilitytrients for both plants and soil microorganisms. Finally,

aproteobacteria class (Fig. 2A) the four most abundante Xanthomonadales, Pseudomonadales, Alteromon-Chromatiales (Fig. 2E), with the genera Xanthomonas,as, Marinobacter and Nitrosococcus, respectively.


Fig. 3. Abundmetagenomes

Xanthomonunder limitsoils (Habteterized by participate cycling, degplant-grow

Actinobaria (24.0%) Solirubrobaetales are of antibiotidant in ournitrogen-xrina (Chaia most abundAcidobacterVerrucomic(2.98%), Bac(2.48%), animportant from a varie2002). The runderstoodet al., 2007ance estimates generated by the MEGAN software of (A) phylum Actinobacteria, (B) o of an Oxisol under 13 years of no-tillage (NT) or conventional tillage (CT) with crop succ

as spp. are usually plant pathogens capable of survivinging nutrient conditions, being strong competitors in

and Alexander, 1975). Pseudomonas spp. are charac-high versatility in nutrient requirements; the speciesin several important processes, including nitrogenradation of aromatic compounds (Palleroni, 2009), andth promotion (Saharan and Nehra, 2011).cteria was the second most abundant phylum of bacte-(Fig. 1), and the main orders were Actinomycetales,cterales and Rubrobacterales (Fig. 3A). The Actinomyc-biotechnologically important due to the productioncs by Streptomyces (Schlatter et al., 2009), also abun-

study. Another genus with several hits was Frankia, aing symbiont with actinorhizal genera Alnus and Casua-et al., 2010). The phylum Acidobacteria was the thirdant (12.7%) (Fig. 1), with the orders Solibacterales andiales (Fig. 3B). Other phyla with several hits were therobia (3.45%), Choloexi (3.40%), Gemmatimonadetesteroidetes (2.60%), Planctomycetes (2.60%), Firmicutesd Cyanobacteria (1.50%) (Fig. 1). Verrucomicrobia aremembers of the rhizosphere, and have been isolatedty of plant species, e.g. from Pinus contorta (Chow et al.,ole of these microorganisms in the rhizosphere is poorly, but there are reports of methane oxidation (Duneld; Pol et al., 2007). Choloexi include bacteria with an

important et al., 2005was Gemmber of this polyphosphBrazilian Ocontents inaurantiaca teroidetes iand celluloamong othare capablestability (Istion, they met al., 2010lites (Ccerbiodegradaas plant-grincluded Banitrite oxidtomycetes Sagulenko,

Indepenhave shownteria, Actinrder Acidobacteria, (C) classes of the phylum Euryarchaeota in fouression (S) or rotation (R).

role in the decomposition of organic matter (Yamada). In the Gemmatimonadetes the only species detectedatimonas aurantiaca (data not shown); the rst mem-phylum was recently described and recognized as aate-accumulating microorganism (Zhang et al., 2003).xisols are usually limited by phosphorus and the P

our experiment were not high (Table 1); therefore, G.might be important in tropical soils. The phylum Bac-ncluded plant-growth promoting (Soltani et al., 2010)se-decomposing (Verkhovtseva et al., 2007) bacteria,er functions. Cyanobacteria are photosynthetic; some

of xing nitrogen and others improve soil-aggregationsa et al., 2007), a key aspect of soil conservation. In addi-ay contribute to pathogen suppression (Domracheva

), and bioremediation of pesticides and toxic metabo-es et al., 2008). Some Firmicutes are involved in thetion of hydrocarbons (Das and Mukherjee, 2007), as wellowth promotion (Lima et al., 2011), and in our studycillus and Clostridium. The phylum Nitrospirae hosts

izers (Attard et al., 2010; Lcker et al., 2010), and Planc-participate in the oxidation of ammonia (Fuerst and2011).dently on the method of analysis, soil metagenomes

predominance of the phyla Proteobacteria, Acidobac-obacteria, Verrucomicrobia, Bacteroidetes, Chloroexi,


Fig. 4. Cluster h the of no-tillage (N

Planctomycemphasis o2006; Roeslar results inin the less-aof our studabundance whereas Ve

Few seqfollowing mchaeota andMethanomi(Fig. 3C). Mgeneral preet al., 2010)marchaeotanitrogen by2011). The nronments ribut their fu

Not manand the kin(Fig. 1). In thcota, with with plant rorder Sordaomycetes), Petro et al., 1bon compoand Sordariest (NowroEurotiomycter (Omem1988). In Oxdescribed a

Viral seq(Fig. 1). Thebacteria andsition of bac

The numpared betwdistance, esas a phylothe NT anmicrobial-cpresence ofmanagemen

n anemee so

ng thanalhylu

to troteocs notatisig. 5acteitrosmonbunudo

to bacte

ain d

ughinop reineraions,inerarogeers oCT sanic sis o

xen analysis based on Euclidean distance and generated with the MEGAN software witT) or conventional tillage (CT) with crop succession (S) or rotation (R).

etes, Gemmatimonadetes and Firmicutes, with ann the Proteobacteria (Delmont et al., 2012; Janssen,ch et al., 2007; Yin et al., 2010). Our data show simi-

a Brazilian Oxisol, but some differences were detectedbundant phyla. For example, in contrast to the resultsy, pyrosequencing of the 16S rRNA indicated greaterof Bacteroidetes in four soils, including one from Brazil,rrucomicrobia were not detected (Roesch et al., 2007).uences were classied in the Archaea domain, with theain orders: Euryarchaeota, Thaumarchaeota, Crenar-

Korarchaeota (Fig. 1). In the Euryarchaeota, the classescrobia, Halobacteria and Thermococci predominatedethanomicrobia includes methanogenic archaeans, insent in environments rich in organic matter (Pazinato; the other phyla were less abundant. Within the Thau-

are microorganisms that participate in the cycling of nitrication, and of carbon by CO2 xation (Pester et al.,on-thermophile Crenarchaeota are also found in envi-ch in organic matter and some colonize roots of plants,nction is not well understood (Simon et al., 2000).y sequences were attributed to the Eucarya domain,

gdoms Fungi, Metazoa and Viridiplantae predominatede Fungi, the most abundant phylum was of the Ascomy-a variety of saprophytes and endophytes associatedoots. There were sequences of species belonging to theriales, including Chaetomium globosum (class Sordari-an antibiotic producer that suppresses pathogens (Di992), Podospora anserina, which degrades complex car-

unds, such as lignin and cellulose (Espagne et al., 2008),a macrospora, a model fungus of biotechnological inter-usian et al., 2010). Another genus was Aspergillus (classetes), which includes decomposers of soil organic mat-

rotatiomanagthan th

Usiin the dant prelatedDeltapstatistiwere stion (FCaulobales, NXanthomore ae.g. PseparisonActino

3.4. M

Plorate crand mconditand mand nitAll ordin the of orgemphation ofu et al., 2005) and phosphate solubilizers (Asea et al.,isols of the Brazilian Cerrados, Castro et al. (2008) alson abundance of Sordariomycetes.uences were few, and t into the order Caudoviralesse are phages with double-stranded DNA that infect

some archaeans, affecting the abundance and compo-terial communities (Swanson et al., 2009).ber of sequences attributed to each taxon was com-een the treatments based on a Euclidean matrix oftimated with the UPGMA algorithm and representedgenetic tree. Two main clusters were observed, ford the CT systems, clearly indicating differences inommunity structure resulting from the absence or

plowing and disking practices (Fig. 4). Within each soil-t system, two subclusters were formed, related to crop

communityresidues thcrop seasonshown a rafresh plant Lundquist ethat the adcorrelated what wouldalso rememin the CT (B2007; Hungmicroorgansources arewith a prevUPGMA algorithm for four metagenomes of an Oxisol under 13 years

d crop succession (Fig. 4). Therefore, an effect of cropnt on diversity was also conrmed, but it was weakeril management effect.e STAMP software, the majority of differences observedyses with MEGAN4 were conrmed. The most abun-m Proteobacteria was statistically larger under CT,he Alpha and Betaproteobacteria classes, whereas thebacteria were more abundant in the NT system (Fig. 2A,t shown). Among the main orders of Proteobacteria thattically more abundant with CT, both under crop rota-) and succession (Fig. 6), were the Sphingomonadales,rales (Alphaproteobacteria), Burkholderales, Neisseri-omonadales, Rhodocyclales (Betaproteobacteria), andadales (Gammaproteobacteria). Some orders weredant with CT, but only under rotation or succession,monadales (Gammaproteobacteria) in the CTS in com-the NTS. The order Solirubrobacterales of the phylumria was also more abundant under CT (Figs. 5 and 6).

ifferences associated with soil and crop management

g and disking in the CT system break up and incorpo-sidues into the surface layers of the soil. Decompositionlization are then accelarated, especially under tropical

resulting in prompt increases in microbial respirationlization, and following decreases in microbial carbonn contents (Franchini et al., 2007; Hungria et al., 2009).f Proteobacteria that were statistically more abundantystem are functionally related to the decompositionmatter, and carbon and nitrogen cycling with ann mineralization and denitrication and degrada-obiotics. It is, therefore, plausible that the microbial has been selected to decompose and mineralize theat are incorporated into the soil at the end of each. It should be remembered that several studies have

pid increase of bacterial biomass after incorporation ofresidues (e.g. Fierer et al., 2007; Franchini et al., 2007;t al., 1999); specically Fierer et al. (2007) have showndition of easily degradable organic C was signicantlywith the abundance of Alpha and Betaproteobacteria,

support the results from our study. However, we mustber that with time, carbon and nitrogen stocks decreaseabujia et al., 2010; Derpsch et al., 1991; Franchini et al.,ria et al., 2009; Lal et al., 2007; Wright et al., 2008), andisms that can utilize more effectively a variety of carbon

selected. Interestingly, these results are in agreementious study from our group performed in vitro, in which


Fig. 5. Statistically signicant differences between orders of microorganisms in an Oxisol under 13 years of no-tillage (NT) or conventional tillage (CT) with crop rotation.The graphic, obtained with the STAMP software, shows the differences between the proportions of sequences in each treatment with a condence interval of 95%.


Fig. 6. Statistically signicant differences between orders of microorganisms in an Oxisol under 13 years of no-tillage (NT) or conventional tillage (CT) with crop succession.The graphic, obtained with the STAMP software, shows the differences between the proportions of sequences in each treatment with a condence interval of 95%.


Fig. 7. Statisti(B) conventionsequences in e

rhizobial stto utilize a bsystem (HustatisticallyNT reportedarea (e.g. Ba2009; Pereipathogens bacteria of observed inwe also shomore toleraand salinitadapted to 1991; Hungorders iden

One surpdomain weFungi. It is gNT because(e.g. Castroto draw conmetagenomno hits was cally signicant differences between orders of microorganisms in an Oxisol under 13 yeaal tillage with crop rotation (CTR) or succession (CTS). The graphic, obtained with the ach treatment with a condence interval of 95%.

rains isolated from the CT system had higher capacityroader range of carbon sources than those from the NT

ngria et al., 2001). It is also noteworthy that, despite the lower microbial biomass with CT in comparison with

in several studies performed by our group in the samebujia et al., 2010; Franchini et al., 2007; Hungria et al.,ra et al., 2007; Silva et al., 2010), the prevalence of plantis higher in the former, what could be attributed tothe orders Xanthomonadales and Pseudomonadales, as

this metagenome study. Finally, in our previous studywed that rhizobia isolated from the CT system werent of environmental stresses such as high temperaturey (Hungria et al., 2001), indicating that they werethe stressful conditions of the CT system (Derpsch et al.,ria and Vargas, 2000). Accordingly, the most abundanttied in this study are also tolerant of stresses.rising result was that microorganisms of the Eucarya

re more abundant in the CT system, mainly the kingdomeneral believed that the fungi are more abundant with

conventional agricultural practices can disrupt hyphae et al., 2008). As we mentioned before, it is prematureclusions about the fungal community, as the number ofic reads of fungi was low. Furthermore, the number ofhigher under NT, thus the unknown sequences may hide

a large popabundanceto environm

In the Nzobiales should bation is a kcan fullll Previous recommon becated that the rates of(Ferreira etand higher nomics appxation untal conditioVargas, 200

All ordeDesulfuromSyntrophob(Figs. 5 andococcales, ahigher orgars of (A) no-tillage with crop rotation (NTR) or succession (NTS), andSTAMP software, shows the differences between the proportions of

ulation of fungi. For now, we propose that the greater of fungi observed with CT is related to higher toleranceental stresses, typical of CT systems.

T system, the statistically higher abundance of the Rhi-composed of several nitrogen-xing rhizobial speciese emphasized (Figs. 5 and 6). Biological nitrogen x-ey process in Brazilian areas planted to soybean andmost of that legumes N needs (Hungria et al., 2006).ports from our group with microsymbionts both froman (Phaseolus vulgaris L.) and from soybean have indi-the number of cells, rhizobial genetic diversity, and

nitrogen xation were higher under NT than with CT al., 2000; Kaschuk et al., 2006; Pereira et al., 2007),abundance has now been conrmed using the metage-roach. Increased contributions from biological nitrogender NT have been attributed to better environmen-ns and improvement of soil aggregation (Hungria and0).rs of the Deltaproteobacteria class Myxococcales,onadales, Desulfovibrionales, Desulfobacterales, andacterales were also more abundant in the NT system

6). A higher number of hits was attributed to the Myx-nd it is possible that the bacteria were favored by thenic matter content with NT (Lueders et al., 2006).


Our knowledge about the Archaea domain needs improvement,particularly since the majority of the available information hascome from studies of extremophiles. Intriguingly, Archaea wereconsistently more abundant under NT (Figs. 3C, 5 and 6). Thedominant pmarchaeotarich in orgaabundance organic-mative to CT-mfrom our stquality.

Differento crop maHowever, mdifferent inActinomyce(Fig. 7A), worders (Fig.several prevthe lack of stion in parastocks of ca2007; Hungsider that thhigher thanbial diversiimportant bcycle.

Althoughmicrobial bnumber of rthat our unnities remacrop-managciated withbe to explor

Acknowled

R.C. Sou(Coordenac A.T.R. Vascoresearch feand TechnoManagemening and comicrobiologsuggestionsby PROBIO I(470515/20and approvas manuscr

References

Asea, P.E.A., Kuby two Pe459464.

Attard, E., PolRoux, X.L.,ers underlpractices.

Babujia, L.C., Hand activdecades o2174218

Balota, E.L., Colozzi Filho, A., Andrade, D.S., Dick, R.P., 2004. Long-term tillage andcrop rotation effects on microbial biomass and C and N mineralization in aBrazilian Oxisol. Soil Till. Res. 77, 137145.

Balota, E.L., Colozzi Filho, A., Andrade, D.S., Hungria, M., 1998. Biomassa microbiana esua atividade em solos sob diferentes sistemas de preparo e sucesso de culturas.

Bras. R.M.,kow, iaga, ropica.G., H

nical wultur

T., Mmiphoobiol., A., Hauimaragemn. J. 1., Quiil funs. Arcilho, Cy undna, Br., Wallphyte.-H., Hoval. B.L., Ra

charaospheraphi

T., VapyingMukhllus suontamt, T.O.,irsch, cture,

J. 6 (, R., Ril: Sistolo. Dborn, , A., Guced t of dheva,

and a, P.F.,Zhou,lier, P

n extr 879, E., Leury, J.in, E.

, A., Beanchirre, Bgenomome BDe Loholdetal anod anre a

://ww010).P (Fevada r /?i1RQBSessed, M.C.,ge meadyrh., Bradbacter

Soroken, J.cing b093hylum was the Euryarchaeota, followed by the Thau- and Crenarchaeota, all of which inhabit environmentsnic matter. It remains to be determined if the greaterof Archaea in the NT system results from the higher soiltter content, or if these microorganisms are more sensi-anagement practices. In any case, one important resultudy is that the Archaea are strong bioindicators of soil

ces in metagenomic composition were also attributednagement, but to a lesser extent than tillage practice.icroorganisms that beneted from crop rotation were

the NT and CT systems. Under NT, the abundance oftales (Actinobacteria) was reduced under crop rotationhereas with CT there was a decrease in seven other

7B), with no clear pattern of associated functionality. Inious studies from our group, we have been surprised bytatistical differences between crop succession and rota-meters such as microbial biomass and activity and soilrbon and nitrogen (Balota et al., 1998; Franchini et al.,ria et al., 2009; Silva et al., 2010, 2013). We might con-e number of plant species in the rotation was not much

in the succession, with only a slight impact on micro-ty. And certainly, we must keep in mind that the mostenet of crop rotation is to break the plantpathogen

our study has greatly improved our knowledge ofiodiversity in tropical-subtropical Oxisols, the higheads without any known hit is suprising and conrmsderstanding of the structure of soil microbial commu-ins poor. Differences were attributed to the soil- andement systems, and indications of microrganisms asso-

higher sustainability were obtained. Our next step wille the functionality of soil microorganims.

gments

za acknowledges an MSc fellowship from CAPESo de Aperfeic oamento de Pessoal de Nvel Superior);ncelos, M.A. Nogueira and M. Hungria are grateful forllowships from CNPq (National Council for Scienticlogical Development). Our special thanks to the Soilt Team of Embrapa Soja for their efforts in establish-

nducting long-term experiments that are crucial forical comparisons, and to Dr. Allan R. J. Eaglesham for

on the manuscript. The project was partially nancedI, CNPq-Repensa (562008/2010-1) and CNPq-Universal12-0). Prior to submission the manuscript was analyzeded for publication by the Editorial Board of Embrapa Sojaipt 05/2012.

cey, R.M.N., Stewart, J.W.B., 1988. Inorganic phosphate solubilizationnicillium species in solution culture and soil. Soil Biol. Biochem. 20,

y, F., Commeaux, C., Laurent, F., Terada, A., Smets, B.F., Recous, S., 2010. Shifts between Nitrospira- and Nitrobacter-like nitrite oxidiz-ie the response of soil potential nitrite oxidation to changes in tillageEnviron. Microbiol. 12, 315326.ungria, M., Franchini, J.C., Brookes, P.C., 2010. Microbial biomass

ity at various soil depths in a Brazilian Oxisol after twof no-tillage and conventional tillage. Soil Biol. Biochem. 42,

1.

Rev.Boddey,

DiecUrqusubt

Brown, Gtechagric

Cceres,fenaMicr

CalegariS., GmanAgro

Castro, Aof soeld

Castro FbilitPara

Chaia, Eendo

Chou, Hrem

Chow, Mularrhizgeog

Coenye,occu

Das, K., Bacioil c

DelmonE., HStruISME

DerpschBrasdo sEsch

Di Petroprodagen

Domracteria

DuneldJ.H., Bodeby a450,

EspagneA., ACoppdaisP., DLucaThe Gen

Estrada-Burkmen

FAO (Focultuhttp02/2

FEBRAPDcultiorg.be46A(Acc

FerreiraTillaof br

Fierer, Nsoil

Foti, M.,Kuenredu73, 2Ci. Solo 22, 641649. Jantalia, C.P., Conceic o, P.C., Zanatta, J.A., Bayer, C., Mielniczuk, J.,J., Dos Santos, H.P., Denardin, J.E., Aita, C., Giacomini, S.J., Alves, B.J.R.,S., 2010. Carbon accumulation at depth in Ferralsols under zero-tilll agriculture. Glob. Change Biol. 16, 784795.ungria, M., Oliveira, l.J., Bunning, S., Montanez, A., 2002. Internationalorkshop on biological management of soil ecosystems for sustainable

e: Program, abstracts and related documents. Embrapa/FAO, Londrina.egharaj, M., Naidu, R., 2008. Biodegradation of the pesticides by ten different species of green algae and cyanobacteria. Curr.

57, 643646.rgrove, W.L., Rheinheimer, D.D.S., Ralisch, R., Tessier, D., Tourdonnet,es, M.F., 2008. Impact of long-term no-tillage and cropping systement on soil organic carbon in an Oxisol: A model for sustainability.00, 10131019.rino, B., Pappas, G., Kurokawa, A., Neto, E., Krger, R., 2008. Diversitygal communities of Cerrado and its closely surrounding agricultureh. Microbiol. 190, 129139.., Lourenc o, A., Guimares, M.F., Fonseca, I.C.B., 2002. Aggregate sta-er different soil management systems in a red latosol in the state ofazil. Soil Till. Res. 65, 4551., L., Huss-Danell, K., 2010. Life in soil by the actinorhizal root nodule

Frankia: A review. Symbiosis 51, 201226.olmes, M.H., 2001. DNA sequence quality trimming and vectorioinformatics 17, 10931104.domski, C.C., McDermott, J.M., Davies, J., Axelrood, P.E., 2002. Molec-cterization of bacterial diversity in Lodgepole pine (Pinus contorta)re soils from British Columbia forest soils differing in disturbance andc source. FEMS Microbiol. Ecol. 42, 347357.ndamme, P., 2003. Diversity and signicance of Burkholderia species

diverse ecological niches. Environ. Microbiol. 5, 719729.erjee, A.K., 2007. Crude petroleum-oil biodegradation efciency ofbtilis and Pseudomonas aeruginosa strains isolated from a petroleum-inated soil from North-East India. Biores. Technol. 98, 13391345.

Prestat, E., Keegan, K.P., Faubladier, M., Robe, P., Clark, I.M., Pelletier,P.R., Meyer, F., Gilbert, J.A., Le Paslier, D., Simonet, P., Vogel, T.M., 2012.

uctuation and magnitude of a natural grassland soil metagenome.9), 16771687.oth, C.H., Sidiras, N., Kopke, U., 1991. Controle da eroso no Paran,emas de cobertura do solo plantio direto e preparo conservacionistaeutsche Gesellschaft fr Technissche Zusammenarbeit (GTZ)/IAPAR,Germany/Londrina, Brazil, pp. 272 (Sonderpublikation der GTZ 245).ut-Rella, M., Pachlatko, J.P., Schwinn, F.J., 1992. Role of antibioticsby Chaetomium globosum in biocontrol of Pythium ultimum, a causalamping-off. Phytopathology 82, 131135.L., Shirokikh, I., Fokina, A., 2010. Anti-Fusarium activity of cyanobac-ctinomycetes in soil and rhizosphere. Microbiology 79, 871876.

Yuryev, A., Senin, P., Smirnova, A.V., Stott, M.B., Hou, S., Ly, B., Saw, Z., Ren, Y., Wang, J., Mountain, B.W., Crowe, M.A., Weatherby, T.M.,.L.E., Liesack, W., Feng, L., Wang, L., Alam, M., 2007. Methane oxidationemely acidophilic bacterium of the phylum Verrucomicrobia. Nature882.spinet, O., Malagnac, F., Da Silva, C., Jaillon, O., Porcel, B., Couloux,-M., Segurens, B., Poulain, J., Anthouard, V., Grossetete, S., Khalili, H.,, Dequard-Chablat, M., Picard, M., Contamine, V., Arnaise, S., Bour-rteaux-Lecellier, V., Gautheret, D., de Vries, R., Battaglia, E., Coutinho,n, E., Henrissat, B., Khoury, R., Sainsard-Chanet, A., Boivin, A., Pinan-., Sellem, C., Debuchy, R., Wincker, P., Weissenbach, J., Silar, P., 2008.e sequence of the model ascomycete fungus Podospora anserina.

iol. 9, R77.s Santos, P., Bustillos-Cristales, R.O., Caballero-Mellado, J., 2001.ria a genus rich in plant-associated nitrogen xers with wide environ-d geographic distribution. Appl. Environ. Microbiol. 67, 27902798.d Agriculture Organization of the United Nations, 2012. Agri-

nd consumer protection department. Conservation agriculture,w.fao.org/nr/cgrfa/cthemes/cgrfa-micro-organisms/en/ (accessed

derac o Brasileira de Plantio Direto na Palha), 2012. Evoluc o da reano sistema de plantio direto na palha Brasil, http://www.febrapdp.=34eAcoBnLhRWY05WYsBXYlJXYa12&i2=4b8QYlJXYfde&i3=ZkBSYlJXwece&i4=&i5=34eAcoBnLhRWY05WYsBXYlJXYa12&m=1

02/2010). Andrade, D.S., Chueire, L.M.O., Takemura, S.M., Hungria, M., 2000.thod and crop rotation effects on the population sizes and diversityizobia nodulating soybean. Soil Biol. Biochem. 32, 627637.ford, M.A., Jackson, R.B., 2007. Toward an ecological classication ofia. Ecology 88, 13541364.in, D.Y., Lomans, B., Mussman, M., Zacharova, E.E., Pimenov, N.V.,G., Muyzer, G., 2007. Diversity, activity, and abundance of sulfate-acteria in saline and hypersaline Soda Lakes. Appl. Environ. Microbiol.2100.


Franchini, J.C., Crispino, C.C., Souza, R.A., Torres, E., Hungria, M., 2007. Microbiologicalparameters as indicators of soil quality under various soil management and croprotation systems in southern Brazil. Soil Till. Res. 92, 1829.

Fuerst, J.A., Sagulenko, E., 2011. Beyond the bacterium: planctomycetes challengeour concepts of microbial structure and function. Nat. Rev. Microbiol. 9, 403413.

Gomez-Alvaremetageno

Gyaneshwar, Estrada-deE.K., 2011future pro

Habte, M.A., AXanthomo

Havelsrud, O.metagenosediments

Hedlund, K., 2managem

Helgason, B.L.,microbial cosystems

Hori, T., Mulleiron-reducISME J. 4, 2

Hungria, M., Vgrain legu151164.

Hungria, M., Cstrains of wheat in B

Hungria, M., Ction of fasSoil Biol. B

Hungria, M., CManero, Ffast-growi343356.

Hungria, M., Fmicrobial soil-tillage

Huson, D.H., Mtive analy1552156

Imelfort, M., Edgeneration

Issa, O.M., DfaNordenbeon the mi290, 2092

Janssen, P.H., 2rRNA and

Jones., W., 201944952.

Kaschuk, G., Alstudies in tions for im

Kaschuk, G., Hmon beansouthern B

Kozdrj, J., vaarable soilprinting an

Lal, R., Reicoskand the ra

Lima, A.S.T.d.,M.V.B., 20Bradyrhizo

Lcker, S., WaDamst, J.Silluminatebacteria. P

Lueders, T., Kinof bacteriaEnviron. M

Lundquist, E.J.and commporation o221236.

Margulies, M.,BravermanGodwin, BJarvie, T.P.S.M., Lei, MM.P., MyerG.T., SarkisA., Vogt, K

R.F., Rothberg, J.M., 2005. Genome sequencing in microfabricated high-densitypicolitre reactors. Nature 437, 376380.

Marks, M.E., Castro-Rojas, C.M., Teiling, C., Du, L., Kapatral, V., Walunas, T.L., Crosson,S., 2010. The genetic basis of laboratory adaptation in Caulobacter crescentus. J.Bacteriol. 192, 36783688.

F., Paariguezics RAtional

G., deulationn reac957u, L., Ss of msian, Mmpkemith, b eukel org, A.M.,hes bechnoi, N.J.,dbook.H., Beageno, J.M.,acterire-de

R.S., s, J.D., cture aRes. 9A.A., Hes, E.,xac oolo 31, J.A.1-carbopseu., Scheir pheijma. Met

874L.F.Wub, S.nume, B.S., ew. Lifr, D., Fndme

Micro, I., BosomoJ., Nic

can pobiol.P., Baass u

expe., Babdiversuther.M., D

plantA.-A., , 2010ted frn, S.R.rizatioroturoudt, Oeriumcts. FE, M.Mses inl. Biol.tsevastructuA.L., Hity anl. Soil , T., Selocalizz, V., Teal, T.K., Schmidt, T.M., 2009. Systematic artifacts inmes from complex microbial communities. ISME J. 3, 13141317.P., Hirsch, A.M., Moulin, L., Chen, W.-M., Elliott, G.N., Bontemps, C.,

los Santos, P., Gross, E., dos Reis, F.B., Sprent, J.I., Young, J.P.W., James,. Legume-nodulating betaproteobacteria: diversity, host range, andspects. Mol. PlantMicrobe Interact. 24, 12761288.lexander, M., 1975. Protozoa as agents responsible for the decline ofnas campestris in soil. Appl. Microbiol. 29, 159164., Haverkamp, T., Kristensen, T., Jakobsen, K., Rike, A., 2011. Amic study of methanotrophic microorganisms in coal oil point seep. BMC Microbiol. 11, 221.002. Soil microbial community structure in relation to vegetationent on former agricultural land. Soil Biol. Biochem. 34, 12991307.

Walley, F.L., Germida, J.J., 2010. Long-term no-till management affectsbiomass but not community composition in Canadian prairie agroe-. Soil Biol. Biochem. 42, 21922202.r, A., Igarashi, Y., Conrad, R., Friedrich, M.W., 2010. Identication ofing microorganisms in anoxic rice paddy soil by 13C-acetate probing.67278.argas, M.A.T., 2000. Environmental factors affecting N2 xation inmes in the tropics, with an emphasis on Brazil. Field Crops Res. 65,

ampo, R.J., Souza, E.M., Pedrosa, F.O., 2010. Inoculation with selectedAzospirillum brasilense and A. lipoferum improves yields of maize andrazil. Plant Soil 331, 413425.hueire, L.M.O., Coca, R.G., Megas, M., 2001. Preliminary characteriza-t growing rhizobial strains isolated from soyabean nodules in Brazil.iochem. 33, 13491361.hueire, L.M.O., Megas, M., Lamrabet, Y., Probanza, A., Guttierrez-., Campo, R.J., 2006. Genetic diversity of indigenous tropicalng rhizobia isolated from soybean nodules. Plant Soil 288,

ranchini, J.C., Brando-Junior, O., Kaschuk, G., Souza, R.A., 2009. Soilactivity and crop sustainability in a long-term experiment with three

and two crop-rotation systems. Appl. Soil Ecol. 42, 288296.itra, S., Ruscheweyh, H.-J., Weber, N., Schuster, S.C., 2011. Integra-

sis of environmental sequences using MEGAN4. Genome Res. 21,0.wards, D., 2009. De novo sequencing of plant genomes using second-

technologies. Brief. Bioinf. 10, 609618.rge, C., Le Bissonnais, Y., Marin, B., Duval, O., Bruand, A., DAcqui, L.P.,

rg, S., Annerman, M., 2007. Effects of the inoculation of cyanobacteriacrostructure and the structural stability of a tropical soil. Plant Soil19.006. Identifying the dominant soil bacterial taxa in libraries of 16S

16S rRNA genes. Appl. Environ. Microbiol. 72, 17191728.0. High-throughput sequencing and metagenomics. Estuar. Coasts 33,

berton, O., Hungria, M., 2010. Three decades of soil microbial biomassBrazilian ecosystems: lessons learned about soil quality and indica-proving sustainability. Soil Biol. Biochem. 42, 113.

ungria, M., Santos, J.C.P., Berton-Junior, J.F., 2006. Differences in com- rhizobial populations associated with soil tillage management inrazil. Soil Till. Res. 87, 205217.

n Elsas, J.D., 2001. Structural diversity of microbial communities ins of a heavily industrialised area determined by PCR-DGGE nger-d FAME proling. Appl. Soil Ecol. 17, 3142.y, D.C., Hanson, J.D., 2007. Evolution of the plow over 10,000 yearstionale for no-till farming. Soil Till. Res. 93, 112.

Barreto, M.d.C.S., Arajo, J.M., Seldin, L., Burity, H.A., Figueiredo,11. Sinergismo Bacillus, Brevibacillus e, ou, Paenibacillus na simbiosebium-caupi. Rev. Bras. Ci. Solo 35, 713721.gner, M., Maixner, F., Pelletier, E., Koch, H., Vacherie, B., Rattei, T.,.S., Spieck, E., Le Paslier, D., Daims, H., 2010. A Nitrospira metagenomes the physiology and evolution of globally important nitrite-oxidizingroc. Natl. Acad. Sci. U S A 107, 1347913484.dler, R., Miltner, A., Friedrich, M.W., Kaestner, M., 2006. Identicationl micropredators distinctively active in a soil microbial food web. Appl.icrobiol. 72, 53425348.

, Jackson, L.E., Scow, K.M., Hsu, C., 1999. Changes in microbial biomassunity composition and soil carbon and nitrogen pools after incor-f rye into three California agricultural soils. Soil Biol. Biochem. 31,

Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J.,, M.S., Chen, Y.-J., Chen, Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V.,

.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L.I.,, Jirage, K.B., Kim, J.-B., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz,., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., McKenna,s, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., Ronan, M.T., Roth,, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., Tartaro, K.R., Tomasz,.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P., Begley,

Meyer, Rodnomfunc

Muyzer,popchai59, 6

Niu, B., Fread

NowrouJ., KeS., S40 mmod

OmemustarcBiot

PalleronHan

Parks, Dmet

Pazinatocharcultu

Peixoto,ElsastruTill.

Pereira, Torrna Ci. S

Perrottaene-Rhod

Pester, Mof th

Pol, A., H2007450,

Roesch, Daroing e

Saharanrevi

Schlatteamesoil.

SchmidtNitro

Shaw, L.spp.Micr

Silva, A.biomterm

Silva, A.Pbial of so

Simon, Htrial

Soltani, A.H.isola

Srenseacteisop

Steenhobactaspe

SwansonViruApp

Verkhovthe

Wright, activApp

Yamadasity rmann, D., DSouza, M., Olson, R., Glass, E., Kubal, M., Paczian, T.,, A., Stevens, R., Wilke, A., Wilkening, J., Edwards, R., 2008. The metage-ST server a public resource for the automatic phylogenetic and

analysis of metagenomes. BMC Bioinform. 9, 386. Waal, E.C., Uitterlinden, A.G., 1993. Proling of complex microbials by denaturing gradient gel electrophoresis analysis of polymerasetion-amplied genes coding for 16S rRNA. Appl. Environ. Microbiol.00.un, S., Li, W., 2010. Articial and natural duplicates in pyrosequencingetagenomic data. BMC Bioinform. 11, 187.., Stajich, J.E., Chu, M., Engh, I., Espagne, E., Halliday, K., Kamerewerd,

n, F., Knab, B., Kuo, H.-C., Osiewacz, H.D., Pggeler, S., Read, N.D., Seiler,K.M., Zickler, D., Kck, U., Freitag, M., 2010. De novo assembly of aaryotic genome from short sequence reads: Sordaria macrospora, aanism for fungal morphogenesis. PLoS Genet. 6, e1000891.

Akpan, I., Bankole, M.O., Teniola, O.D., 2005. Hydrolysis of raw tubery amylase of Aspergillus niger AM07 isolated from the soil. Afr. J.l. 4, 1925.

2009. The Genus Pseudomonas. In: Goldman, E., Green, L.H. (Eds.), of Microbiology. , 2nd ed. CRC Press, pp. 231242.iko, R.G., 2010. Identifying biologically relevant differences betweenmic communities. Bioinformatics 26, 715721.

Paulo, E.N., Mendes, L.W., Vazoller, R.F., Tsai, S.M., 2010. Molecularzation of the archaeal community in an Amazonian wetland soil andpendent isolation of methanogenic Archaea. Diversity 2, 10261047.Coutinho, H.L.C., Madari, B., Machado, P.L.O.A., Rumjanek, N.G., VanSeldin, L., Rosado, A.S., 2006. Soil aggregation and bacterial communitys affected by tillage and cover cropping in the Brazilian Cerrados. Soil0, 1628.ungria, M., Franchini, J.C., Kaschuk, G., Chueire, L.M.O., Campo, R.J.,

2007. Variac es qualitativas e quantitativas na microbiota do solo e biolgica do nitrognio sob diferentes manejos com soja. Rev. Bras., 13971412., Harwood, C.S., 1994. Anaerobic metabolism of cyclohex-1-oxylate a proposed intermediate of benzoate degradation, bydomonas palustris. Appl. Environ. Microbiol. 60, 17751782.leper, C., Wagner, M., 2011. The Thaumarchaeota: an emerging viewylogeny and ecophysiology. Curr. Opin. Microbiol. 14, 300306.ns, K., Harhangi, H.R., Tedesco, D., Jetten, M.S.M., Op den Camp, H.J.M.,hanotrophy below pH 1 by a new Verrucomicrobia species. Nature878.., Fulthorpe, R.R., Riva, A., Casella, G., Hadwin, A.K.M., Kent, A.D.,H., Camargo, F.A.O., Farmerie, W.G., Triplett, E.W., 2007. Pyrosequenc-rates and contrasts soil microbial diversity. ISME J. 1, 283290.Nehra, V., 2011. Plant growth promoting rhizobacteria: a criticale Sci. Med. Res. LSMR-21, 130.ubuh, A., Xiao, K., Hernandez, D., Hobbie, S., Kinkel, L., 2009. Resourcents inuence density and competitive phenotypes of Streptomyces inb. Ecol. 57, 413420.ck, E., 1997. Anaerobic ammonia oxidation with nitrogen dioxide bynas eutropha. Arch. Microbiol. 167, 106111.ol, G.W., Smith, Z., Fear, J., Prosser, J.I., Baggs, E.M., 2006. Nitrosospiraroduce nitrous oxide via a nitrier denitrication pathway. Environ.

8, 214222.bujia, L.C., Franchini, J.C., Souza, R.A., Hungria, M., 2010. Microbialnder various soil- and crop-management systems in short- and long-riments in Brazil. Field Crops Res. 119, 2026.ujia, L.C., Matsumoto, L.S., Guimares, M.F., Hungria, M., 2013. Micro-ity under different soil tillage and crop rotation systems in an oxisoln Brazil. Open Agric. J. 7 (Suppl 1-M6), 4047.odsworth, J.A., Goodman, R.M., 2000. Crenarchaeota colonize terres-

roots. Environ. Microbiol. 2, 495505.Khavazi, K., Asadi-Rahmani, H., Omidvari, M., Dahaji, P., Mirhoseyni,. Plant growth promoting characteristics in some Flavobacterium spp.om soils of Iran. J. Agric. Sci. 2, 106115., Ronen, Z., Aamand, J., 2001. Isolation from agricultural soil and char-n of a Sphingomonas sp. able to mineralize the phenylurea herbiciden. Appl. Environ. Microbiol 67, 54035409.., Vanderleyden, J., 2000. Azospirillum, a free-living nitrogen-xing

closely associated with grasses: genetic, biochemical and ecologicalMS Microbiol. Rev. 24, 487506.., Fraser, G., Daniell, T.J., Torrance, L., Gregory, P.J., Taliansky, M., 2009.

soils: morphological diversity and abundance in the rhizosphere. Ann. 155, 5160., N., Kubarev, E., Mineev, V., 2007. Agrochemical agents in maintainingre of the soil microbial community. Russ. Agric. Sci. 33, 100102.ons, F.M., Lemon, R.G., McFarland, M.L., Nichols, R.L., 2008. Microbiald soil C sequestration for reduced and conventional tillage cotton.Ecol. 38, 168173.kiguchi, Y., Imachi, H., Kamagata, Y., Ohashi, A., Harada, H., 2005. Diver-ation, and physiological properties of lamentous microbes belonging


to chloroexi sub phylum in mesophilic and thermophilic methanogenic sludgegranules. Appl. Environ. Microbiol. 71, 74937503.

Yin, C., Jones, K.L., Peterson, D.E., Garrett, K.A., Hulbert, S.H., Paulitz, T.C., 2010. Mem-bers of soil bacterial communities sensitive to tillage and crop rotation. Soil Biol.Biochem. 42, 21112118.

Zhang, H., Sekiguchi, Y., Hanada, S., Hugenholtz, P., Kim, H., Kamagata, Y., Nakamura,K., 2003. Gemmatimonas aurantiaca gen. nov. sp. nov., a Gram-negative, aerobic,polyphosphate-accumulating micro-organism, the rst cultured representativeof the new bacterial phylum Gemmatimonadetes phyl nov. Int. J. Syst. Evol.Microbiol. 53, 11551163.

Soil metagenomics reveals differences under conventional and no-tillage with crop rotation or succession1 Introduction2 Materials and methods2.1 Description of the field trial and treatments2.2 Soil sampling and processing2.3 Homogeneity among replicates2.4 DNA extraction and pyrosequencing analysis2.5 Data analyses

3 Results and discussion3.1 Sequencing analysis, data normalization, and rarefaction curves3.2 Total soil microbial diversity3.3 Bacteria, Archaea and Eucarya domains, and virus3.4 Main differences associated with soil and crop management

AcknowledgmentsReferences

soil metagenomics reveals differences under conventional crop rotation

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