Diversity of Paenibacillus polymyxa strains isolated from ...ainfo.cnptia.embrapa.br/.../item/45203/1/Diversity-Paenibacillus.pdf · Diversity of Paenibacillus polymyxa strains isolated

Diversity of Paenibacillus polymyxa strains isolated from the rhizosphereof maize planted in Cerrado soil

Irene von der Weida, Edilson Paivab, Alberto Nóbregaa, Jan Dirk van Elsasc, Lucy Seldina*

a Laboratório de Genética Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo deGóes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

b EMBRAPA-CNPMS, Sete Lagoas, MG, Brazilc IPO-DLO, Wageningen, The Netherlands

Received 16 July 1999; accepted 15 February 2000

Abstract — Paenibacillus polymyxa populations present in the rhizosphere of maize (cultivar BR-201) plantedin Cerrado soil were investigated in order to assess their diversity at four stages of plant growth. A total of67 strains were isolated and all strains were identified as P. polymyxa by classical biochemical tests, API 50CHtests and a set of species-specific primers based on the 23S rDNA sequence. To compare the isolated strains,phenotypic characteristics (utilization of different carbohydrates, resistance to antibiotics and production ofantimicrobial substances) and genetic approaches (hybridization with a Klebsiella pneumoniae nifKDH probeand BOX-PCR) were used. Fermentation of glycerol, arabinose, xylose and rhamnose varied among theisolates and these data divided the strains into five groups. Fifty strains (75%) showed homology to plasmidpSA30 (containing the nifKDH genes) resulting in five different hybridization patterns. Using BOX-PCR, 18groups were observed. Phenetic analyses were applied based on the unweighted pair group method witharithmetic means using the phenotypic and genetic data, separately. All P. polymyxa isolates could be dividedinto two main clusters at approximately 52% and into 18 groups at approximately 89% of similarity, whenphenotypic data were used. Also, two main clusters were formed at 65% of similarity when genetic data wereused. In this dendrogram, clusters were further split into 10 and 22 groups, at about 88 and 97% of similarity,respectively. Finally, all phenotypic and genetic data, or just the genetic data, were used in a multivariateanalysis of variance (MANOVA) in order to address the heterogeneity among P. polymyxa populations duringthe different stages of maize growth. The resulting data showed that strains isolated 10, 30, 60 and 90 daysafter maize sowing were statistically different. © 2000 Éditions scientifiques et médicales Elsevier SAS

Paenibacillus polymyxa / rhizosphere of maize / genetic diversity / phenotypic diversity

1. Introduction

It has been extensively demonstrated thatplant growth-promoting rhizobacteria (PGPR)can colonize the rhizosphere and enhance thegrowth of many plant species [11, 17, 25, 31]. Ithas also been established that bacterial adapta-

tion to a specific, heterogeneous and fluctuatingenvironment such as the plant rhizosphere isdependent on the genetic diversity of the bac-terial population [32, 49]. The diversity of rhizo-bacteria with the ability to promote plant growthis affected by the amount and composition oforganic materials excreted by plant roots [4]which may vary during plant development andin different plant species and cultivars [8, 37,38]. Different phenotypic and genetic methodsare available for a sound estimation of soilbacterial diversity [61]. These methods provideessential knowledge for the selection of poten-tial inoculant strains for use in agriculture.

* Correspondence and [email protected]: PGPR, plant growth-promoting rhizobacte-rium; MANOVA, multivariate analysis of variance; TB,thiamine-biotin; UPGMA, unweighted pair group methodwith arithmetic mean.

Res. Microbiol. 151 (2000) 369–381© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0923250800001601/FLA

Many different PGPRs have already beendescribed, including symbiotic ones, such as theroot-nodulating Rhizobium spp. [14, 31] andfree-living rhizobacteria, such as Azospirillum[15], Bacillus and Paenibacillus spp. [7, 9, 46, 48,53, 57].

Strains belonging to the species P. polymyxawere described as plant growth-promotingrhizobacteria when crested wheatgrass andwhite clover responded positively to inocula-tion with this bacterium [7, 23]. Thereafter,different studies have described the coloniza-tion of important crop species by P. polymyxa[17, 22, 23, 27, 32, 33, 36, 46, 59]. A wide range ofplants was studied in these reports. Further-more, most studies focussed on strains andplants from temperate regions [2, 12]. It hasbeen shown that the soil in which the crop isgrown can affect the population structure of thebacterium studied [2, 57]. Also, P. polymyxastrains can utilize various substrates released byplant roots [32, 52], and nitrogen fixation hasbeen reported in many strains [19, 52]. Thesestrains also produce a wide variety of secondarymetabolites, including plant growth-regulatingsubstances [23, 26] and antibiotic compounds[33, 44, 47, 58, 64]. In addition, they can resist arange of environmental stress conditions due totheir capability to form endospores [45, 52].

The application of P. polymyxa strains to seedsto enhance seedling growth and to control soil-borne pathogens could be an ideal situation intropical conditions where unfavorable climaticconditions are prevalent. However, it has beenshown that survival of introduced bacteria asvegetative cells is often low [62]. The use ofbacterial species with which the plant coexistedpreviously [7, 8] or the stimulation of an indig-enous bacterial species in the soil in which theplant is to be grown might offer the best per-spectives. Thus, in this study, we assessed thediversity of P. polymyxa strains associated withthe maize rhizosphere during the growth ofmaize plants in a tropical soil, i.e. Cerrado soil.All extreme conditions of a tropical region (hightemperatures, soil drought and/or heavy rainfalls) occurred during this season. To addressthis diversity from different angles, both genetic

and phenotypic characteristics were determined.Specifically, we used a nif KDH probe in hybrid-ization experiments and a primer correspond-ing to ubiquitous repetitive DNA sequences –154-bp BOX element [30] – that could be used to‘fingerprint’ the different P. polymyxa strains.Also, all strains were studied with respect totheir biochemical profiles, as to their capabilityto produce antimicrobial substances and theirresistance to different antibiotics. Theseapproaches will lead to a better understandingof the diversity of P. polymyxa populations dur-ing a maize growth season in a tropical soil.

2. Materials and methods

2.1. Experimental conditions and isolationof P. polymyxa from the rhizosphere of maize

A well-characterized maize genotype, BR-201(Zea mays L. BR-201, single cross hybrid), wasplanted in Cerrado soil in Sete Lagoas, Brazil(EMBRAPA-CNPMS, National Center ofResearch in Maize and Sorghum). This soilcovers 22% of the tropical belt of the world andrepresents 205 million hectares in Brazil, ofwhich 112 million are suitable for agriculturalproduction. Cerrado soil is a dark-red distrophiclatosol with clay-like texture; it is stronglyweathered and generally acidic. P, Co, Mg andZn deficiencies are common, and Al toxicity isusually high [29]. Untreated maize seeds weresown in this soil and 80 kg of nitrogen per hawere supplied after 40 days. Strains of P. poly-myxa were isolated from the rhizosphere ofmaize plants 10, 30, 60 and 90 days after sowing,by using a modification of the method of Baldaniand Döbereiner [1], described in detail by Seldinet al. [57]. Three plants were harvested, andtheir roots were shaken to remove the looselyattached soil. One gram of the adhering soil,considered the rhizosphere, was mixed with9 mL of distilled water. Suspensions of rhizo-sphere soil were pasteurized (10 min, 80 °C) andtwo-fold serial dilutions were plated ontothiamine-biotin (TB) agar [53] and incubatedanaerobically in Gaspak jars (80% N2, 10% CO2and 10% H2) for 3 days. Further isolation and

370 I. von der Weid et al. / Res. Microbiol. 151 (2000) 369–381

purification were performed as described before[52]. All presumptive P. polymyxa strains (brightyellow, convex and mucous colonies) werestored aerobically at room temperature on glu-cose broth (GB) [52] agar slants supplementedwith 1% CaCO3.

2.2. Phenotypic features

Identification of strains isolated from themaize rhizosphere was performed by usingcultural and biochemical tests proposed by Gor-don et al. [18]. Cellular morphology, form andposition of spores, and swelling of the sporan-gia were observed by microscopy using crystalviolet-stained smears. The fermentation pat-terns were compared among all strains testedand strains were grouped with regard to thefermentation of four carbohydrates (glycerol,D(+)-xylose, L(+)-arabinose and L(+)-rhamnose).API (Appareils et Procédés d’Ídentification,bioMérieux sa, Lyon, France) 50CH kits (#50430) were used as described in Seldin andPenido [55]. The API test galleries were pre-pared according to the API 50CH manual andwere incubated at 30 °C for 2 days.

2.3. Assay for antimicrobial substance production

The method described by Rosado and Seldin[47] was used to assay for antimicrobial sub-stances produced by P. polymyxa strains. Thestrain to be tested was spot-inoculated (5 µL) onthe surface of GB agar plates and, after incuba-tion for 48 h at 30 °C, killed by exposure tochloroform vapors for 15 min. After leaving for15 min to allow evaporation of the chloroform,the plates were flooded with the indicator strain(Micrococcus sp.), and incubated for 18 h at30 °C. Antimicrobial substance production wasindicated by zones of growth inhibition aroundthe spot.

2.4. Resistance to antibiotics

Resistance to antibiotics was determined byspot-inoculating 18-h-old cultures (10 µL) ofP. polymyxa strains on TBN agar plates supple-mented with erythromycin, tetracycline,

chloramphenicol, rifampicin, streptomycin andkanamycin (all at 10 µg·mL–1).

2.5. Phage propagation

Phage IPy1 was propagated on P. polymyxaLoutit [28] as described in Seldin et al. [54] usingGB agar (1.5%) and soft agar (0.75%) supple-mented or not with 25 mM CaCl2 and 10 µMMnCl2.

2.6. Extraction of plasmid and phage DNAs

Plasmid DNA from Escherichia coli HB101containing the nifKDH genes from Klebsiellapneumoniae [5] was isolated by the alkaline lysismethod described by Birnboim and Doly [3].The preparations were purified in CsCl-EtBrgradients as described by Sambrook et al. [51].Phage DNA was prepared by phenol extractionaccording to Sambrook et al. [51] for the purifi-cation of phage lambda.

2.7. Preparation of genomic DNAs

Total DNAs were extracted using the methoddescribed by Pitcher et al. [43]. DNAs werequantified spectrophotometrically (GeneQuantapparatus, Pharmacia). Chromosomal DNA wasdigested with 10–20 U of EcoRI per µg of DNAfor 20 h at 37 °C. Agarose gel electrophoresis ofrestricted DNA samples was performed in 0.8%agarose (w/v) in TBE buffer [51] at 2 V·cm–1, for16 h at room temperature. Bacteriophage lambdaDNA digested with HindIII and labeled withdigoxigenin (Boehringer Mannheim Biochemi-cals, BMB) was routinely used as the molecularweight marker.

2.8. Preparation of probes, blottingand hybridization procedure

The plasmid and phage DNA probes wererandom-primer labeled with digoxigenin (DIG)by using a DIG labeling kit (BMB). Chromo-somal DNAs were blotted from gels to posi-tively charged nylon membranes (BMB) asdescribed by Sambrook et al. [51]. Prehybridiza-tion and hybridization conditions using DIG-

I. von der Weid et al. / Res. Microbiol. 151 (2000) 369–381 371

labeled probes were those described by theBMB manual for the DIG nucleic acid detectionkit.

2.9. PCR amplifications

Amplification reactions using primerBOXA1R [30] were performed as described byVersalovic et al. [63]. The cycle applied was: 1 ×[90 s 94 °C, 3 min 53 °C, 3 min 72 °C], 34 × [30s 94 °C, 30 s 53 °C, 1 min 72 °C], 10 min 72 °C.PCR amplifications of 23S rDNA fragmentsusing the P. polymyxa-specific primers Bac 11and POL were performed as described byPetersen et al. [41, 42]. Thermal cycling con-sisted of an initial denaturing period [4 min 94°C], 35 × [20 s 94 °C, 20 s 54 °C, 45 s 72 °C], 5 min72 °C. Agarose gel electrophoresis of PCR prod-ucts was performed in 1.6% agarose in TBEbuffer at 10 V·cm–1, for 2 h at room temperature.

2.10. Phenetic analysis

The analysis of similarities among P. polymyxastrains was based on the presence or absence ofbands in hybridization experiments using plas-mid pSA30 as a probe, as well as on the pres-ence or absence of specific bands in BOX-PCRanalyses. A similarity matrix was obtained bycomparisons between pairs of strains using thesimple matching coefficient. For these analyses,the NTSYS software package (version 1.8, ExeterSoftware, Setauket, New York) was used. Adendrogram was constructed using theunweighted pair group method with arithmeticmean (UPGMA). A symmetrical matrix of cophe-netic similarity values based on UPGMA wasalso obtained using phenotypic features. Theresults of phenotypic and genetic analyses werecollected into a matrix and data were analyzedfor principal components with Euclidean dis-tance. The computational calculations of princi-pal components and cluster analyses were per-formed with routines written by A. Nobregawith the software MATHEMATICA (WolframResearch Inc., Champaign, IL). Multivariateanalysis of variance (MANOVA) of phenotypicand/or genetic data was also performed byNTSYS.

3. Results

3.1. Phenotypic features

Sixty-seven strains were isolated from therhizosphere of maize planted in Cerrado soilafter 10, 30, 60 and 90 days of maize sowing(table I) and were identified as P. polymyxa. Allstrains were Gram-positive and their sporeswere ellipsoidal, distending the sporangia. Colo-nies were translucent to white, convex andmucoid when grown in GB agar and werebright yellow in TBN agar. All strains werefacultatively anaerobic, catalase-positive, able togrow at pH 5.7, as well as to hydrolyze starchand decompose casein. Furthermore, they wereVoges-Proskauer (acetoin fermentation) positiveand reduced nitrate to nitrite. None of thestrains was able to grow in 5% NaCl and to formcrystalline dextrin and indole from tryptophane.Production of acid from glucose and mannitolwas observed in all isolates; however, variableresults were obtained when arabinose and xylosewere tested (table II).

As the capability to produce acid from thesefour carbohydrates is considered to be charac-teristic for P. polymyxa [18, 52] and divergentresults were obtained, fermentation of two othercarbohydrates (glycerol and rhamnose) wasincluded in this study. Forty-five strains (67%)produced acid from glycerol and 11 strains(16%) showed production of acid from rham-nose (table II). The utilization of this novelcombination of four carbohydrates, i.e. glycerol,rhamnose, arabinose and xylose, varied amongthe strains isolated in this study; these resultswere thus used to divide all strains into fivegroups (I–V, table II). The largest group (group I,encompassing 60% of the isolates) was formedby strains able to utilize glycerol, xylose andarabinose but not rhamnose.

As variable fermentation patterns wereobserved among the isolates, the 22 glycerol-negative strains were tested using the API 50CHkit to elucidate their taxonomic position. Thepercentage of strains that utilized each carbonsource is presented in table III, together withthat of P. polymyxa (as given by the manufac-


turer). All strains tested produced acid fromribose, galactose, glucose, fructose, mannose,mannitol, amygdalin, arbutin, esculin, salicin,

cellobiose, maltose, lactose, melibiose, sucrose,raffinose, starch and glycogen. None of the 22glycerol-negative strains was able to utilize 21

Table I. Strains of P. polymyxa isolated from rhizosphere of maize at different stages of growth in Cerrado soil*.

Sampling (number of days after planting) Number of isolates Designation of strains

Sa 1 (10) 9 1, 7, 8, 13, 14, 20, 22, 23, 24Sa 2 (30) 20 51, 52, 53, 55, 56, 57, 58, 62, 65, 66, 67, 68,

69, 70, 71, 72, 73, 75, 76, 79Sa 3 (60) 18 101, 102, 103, 105, 106, 107, 108, 109, 110,

111, 112, 113, 115, 116, 117, 118, 119, 120Sa 4 (90) 20 151, 153, 154, 155, 156, 157, 158, 159, 160,

163, 164, 167, 168, 170, 171, 172, 173, 174,175, 177

* Strains numbered from 1 to 50 were isolated from rhizosphere of maize after 10 days of growth; numbers from 51 to 100,strains isolated from rhizosphere of maize after 30 days of growth; numbers from 101 to 150, strains isolated fromrhizosphere of maize after 60 days of growth; numbers from 151 to 200, strains isolated from rhizosphere of maize after90 days of growth.

Table II. Phenotypic characteristics of P. polymyxa strains isolated from rhizosphere of maize at different stages of growth in Cerradosoil.

Strains Groupa Pheneticgroupb

Acid from: Abilityto

produce

Resistance to antibioticd

glycerol rhamnose arabinose xylose anti-microbial

substancec

Rif Km Sm Tc

51, 52, 53, 55, 56, 58,66, 67, 68, 70, 71, 75,76, 107, 110, 113, 118

I A1 + – + + + – – – –

57, 62, 69, 79, 103, 116,117, 120, 151, 153, 163,167, 170, 171

I A2 + – + + + – – – +

13 I A3 + – + + + + + – +14 I A4 + – + + + – + – +72, 73, 112 I A5 + – + + + – + – –101 I A6 + – + + + – – + +168 I A7 + – + + + + – – +111, 119 I A8 + – + + + – – + –20 II A9 + + + + + + + – +106 II A10 + + + + + – – – +102, 109, 175 II A11 + + + + + – – – –108 III A12 – + + + + – – – +115 III A13 – + + + – – – – –156, 157, 160, 173 III A14 – + + + + – – – –1 IV B1 – – – – + + – – –7, 8, 22, 23, 65, 105,158, 172, 174

IV B2 – – – – + – – – +

159 IV B3 – – – – + – – – –24, 154, 155, 164, 177 V B4 – – – + + – – – +

a Groups were formed based on fermentation of carbohydrates; b phenetic groups were obtained using all phenotypicfeatures presented in this table on a symmetrical matrix of cophenetic similarity values based on UPGMA; c growthinhibition of Micrococcus sp.; d Rif: rifampicin, Km: kanamycin, Sm: streptomycin, Tc: tetracycline.


carbohydrates. Fermentation of the remainingten carbohydrates varied between the 22 strains.

Two other approaches were included to assistin the identification of the 22 glycerol negativestrains: a) hybridization with DIG-labeled DNAfrom phage IPy1 (specific for P. polymyxa strains[16, 54], b) PCR amplification of 23S rDNAusing primers Bac 11 and POL, species-specificfor P. polymyxa [41, 42]. Genomic EcoRI frag-ments from all 22 isolates hybridized with phageIPy1 as a probe, and PCR products of thepredicted size, i.e. 632 bp, were also observed inthese strains (data not shown). These resultswere also obtained with the other 45 (glycerol-positive) strains.

The strains isolated were also tested for theircapacity to produce antimicrobial substancesand for their resistance to different antibiotics.Among the isolates, 66 (99%) were able toinhibit the growth of the indicator strain (Micro-coccus sp.). None of the strains tested was ableto grow in media containing chloramphenicolor erythromycin. Their spectra of resistance torifampicin, kanamycin, streptomycin and tetra-cycline are shown in table II.

A symmetrical matrix of cophenetic similarityvalues based on UPGMA was obtained using

these phenotypic features (fermentation of car-bohydrates, antimicrobial substance productionand resistance to antibiotics). The 67 strainscould be divided into two main clusters atabout 52% of similarity (data not shown). Strainsincapable of metabolizing either glycerol orarabinose and/or xylose were grouped in onecluster which was further split into four sub-groups (table II, B1–B4) with increasing levels ofsimilarity. Two of these subgroups consisted ofa single isolate (B1 and B3), while the other twosubgroups consisted of nine and five isolates,respectively (table II). The other main clusterwas formed by strains that showed negative orpositive results in glycerol utilization but posi-tive results in the arabinose and xylose tests. Atabout 89% of similarity, this major cluster wassplit into 14 different subgroups (A1–A14). Thenumber of isolates in each subgroup variedfrom one to 17 strains (table II). Subgroupscontaining more than one strain were usuallyformed by strains isolated during the differentstages of maize growth.

3.2. Genetic features

All strains were tested for the presence ofsequences homologous to the K. pneumoniae

Table III. Comparison of P. polymyxa (A) and the 22 glycerol-negative strains (B) usingAPI 50CH tests.

Percentage of positive results for:

Aa Bb A B A B

Glycerol 97 0 Inositol 14 27 Inulin 76 27Erythritol 4 0 Mannitol 100 100 Melezitose 61 0D-arabinose 0 0 Sorbitol 1 0 Raffinose 100 100L-arabinose 100 27 Methyl-D-mannoside 14 0 Starch 100 100Ribose 100 100 Methyl-D-glucoside 100 77 Glycogen 100 100D-xylose 100 50 N acetyl glucosamine 4 0 Xylitol 9 0L-xylose 0 0 Amygdalin 100 100 Gentibiose 80 82Adonitol 0 0 Arbutin 100 100 D-turanose 100 64Methyl-xyloside 90 23 Esculin 100 100 D-lyxose 0 0Galactose 100 100 Salicin 95 100 D-tagatose 0 0Glucose 100 100 Cellobiose 95 100 D-fucose 0 0Fructose 100 100 Maltose 95 100 L-fucose 0 0Mannose 100 100 Lactose 100 100 D-arabitol 0 0Sorbose 0 0 Melibiose 100 100 L-arabitol 0 0Rhamnose 19 27 Sucrose 100 100 Gluconate 33 0Dulcitol 4 0 Trehalose 90 64 2 keto-gluconate 0 0

5 keto-gluconate 4 0

a Data from the API 50 CH (# 50300) identification table; b data obtained in this study.


nifKDH gene cluster (nitrogenase structuralgenes cloned in plasmid pACYC184) by South-ern hybridization. Total genomic DNA from 50strains (75%) digested with EcoRI showedhomology to plasmid pSA30 but not to thecloning vector. Their hybridization patternsallowed the separation of these strains into fivedifferent groups, N1–N5 (table IV, figure 1). Thesixth group formed (N6) encompasses theremaining 17 strains without homology topSA30 (table IV). In 46 strains, a 1.1-kb fragmenthybridized with the probe for the structural nifgenes. The largest group (N3) was formed by 21strains isolated 30 and 60 days after maizesowing. Nitrogen-fixing strains isolated 10 daysafter maize sowing were all grouped together ingroup N5.

Using the optimized parameters for BOX-PCR described previously [48], amplification oftemplate DNA of each of the 67 isolates wasachieved with the repetitive sequence primerBOXA1R. Figure 2 and table V show the differentgroups formed based on this PCR pattern. Fromthe variation in the number and size of bands, itwas possible to identify similarities amongststrains, resulting in 18 groups. Group a wasformed by 31% of the strains. Three bands, ofapproximately 0.4, 0.6 and 0.8 kb, were presentin 82, 86.5 and 99% of the isolates, respectively.Strain 120 (group j) was the only one in whichthe 0.8-kb band could not be observed (table V,figure 2). The remaining strains (69%) wereallocated in the other 17 groups. No correlationbetween sampling time and PCR pattern couldbe observed.

A phenetic analysis was applied based onUPGMA using these genetic data (BOX-PCRand nifKDH DNA hybridization) to address the

Table IV. Grouping of P. polymyxa isolates by their hybridization patterns obtained using K. pneumoniae nifKDH genes as a probe.

Group Strainsa Size of fragments (kb)b

N1 109 6.6 4.0 1.1N2 57, 62, 69, 116 6.6 2.2N3 51, 52, 53, 55, 56, 58, 66, 67, 68, 70, 71, 72, 73, 75,

76, 110, 111, 112, 113, 118, 1196.6 1.2 1.1

N4 102, 108, 115, 156, 157, 160, 173, 175 4.0 1.7 1.1N5 1, 7, 8, 22, 23, 24, 65, 105, 154, 155, 158, 159, 164,

172, 174, 1772.7 1.7 1.1

N6 13, 14, 20, 79, 101, 103, 106, 107, 117, 120, 151,153, 163, 167, 168, 170, 171

–c – –

a Underlined strains are representatives of each group in figure 1; b fragments originated from the digestion of P. polymyxaDNA with EcoRI; c no hybridization.

Figure 1. Southern hybridization of DNAs of P. polymyxa strainswith nifKDH from K. pneumoniae. Only one representative strainof each group is presented here. The arrows indicate the 1.1-kbfragment. Lanes 1, 3 and 5, pattern of phage lambda DNAdigested with HindIII; lane 2, group N1; lane 4, group N2; lane 6,group N3; lane 7, group N4; lane 8, group N5.


diversity among all P. polymyxa isolates (figure3). At about 65% of similarity, two main clusters(I and II) were visible. Cluster II was formed byfour groups at about 92% of similarity (G4, G5,G8 and G10), whereas cluster I was formed bythe remaining 18 groups (figure 3). Groups form-ing cluster II encompassed the glycerol-,arabinose- and/or xylose-negative strains. Clus-ter I was further split into two major subgroups(a and b) at about 70% similarity. Strains with nohybridization with nifKDH were spread overnine groups (G3, G9, G11, G12, G13, G14, G17,G19 and G22). Groups G15 and G20 were formedby strains showing negative tests for glycerolbut positive tests for arabinose and xylose uti-lization. At about 88% similarity, ten groupscould be distinguished, whereas at about 97% ofsimilarity, a total of 22 different groups wasobtained. Some of these groups consisted of asingle isolate, whereas others encompassed twoto 20 strains (figure 3). This last group (G1) wasexclusively formed by strains isolated after 30and 60 days of maize sowing and corresponded

Figure 2. Grouping of P. polymyxa strains by their amplification patterns by PCR with BOX1AR primer. Lanes on gel correspond to eachgroup presented in table IV.

Table V. Grouping of P. polymyxa strains by their amplificationpatterns by PCR with BOX1AR primer; strains in each group arenoted.

Groups Strains

a 51, 52, 53, 55, 56, 57, 58, 66, 67, 68, 70, 71, 72, 73,75, 76, 110, 111, 112, 113, 119

b 107c 22, 172, 174d 8, 65, 154e 62, 69, 116, 118f 1, 7, 105, 155, 158, 159, 164g 13, 101, 151, 153, 167h 23, 24, 177i 20, 79, 106j 120k 103, 117l 14m 108, 115n 109o 168, 171, 175p 163q 156, 157, 160, 173r 102, 170

The underlined strains correspond to the samples on thegel (figure 2).


to group N3 (with the exception of strain 118) inthe nifKDH hybridization studies (table IV).

An overall analysis was performed to addressthe heterogeneity among P. polymyxa popula-tions during the different stages of maizegrowth. First, all the 67 strains were classifiedinto groups according to different samplingscarried out 10, 30, 60 and 90 days after sowing(samplings Sa1, Sa2, Sa3 and Sa4). Either allphenotypic and genetic data or just the geneticfeatures were used for MANOVA. When bothgenetic and phenotypic data sets or only genetic

data were used, 67 × 46 or 67 × 37 matrix datawere obtained, respectively. Because of the largenumber of parameters, the matrix data werefirst transformed according to principal compo-nent analysis, such that only the more impor-tant factors were retained for MANOVA. In ourcase, for both genetic and phenotypic data, thefirst three factors represented 70% of the vari-ance, and the first ten factors represented 89% ofthe variance. For genetic data only, the firstthree factors represented 71% of the variance,and the first ten factors 92%. The factors used in

Figure 3. Dendrogram (UPGMA) based on data from hybridization studies using plasmid pSA30 as a probe and from BOX-PCRanalyses, showing the similarities between 67 P. polymyxa strains isolated from rhizosphere of maize planted in Cerrado soil. Strains ineach group are as follows: G1: 51, 52, 53, 55, 56, 58, 66, 67, 68, 70, 71, 72, 73, 75, 76, 110, 111, 112, 113, 119; G2: 57; G3: 107; G4:22, 172, 174; G5: 8, 65, 154; G6: 62, 69, 116; G7: 118; G8: 1, 7, 105, 155, 158, 159, 164; G9: 13, 101, 151, 153, 167; G10: 23, 24, 177;G11: 20, 79, 106; G12: 120; G13: 103, 117; G14: 14; G15: 108, 115; G16: 109; G17: 168, 171; G18: 175; G19: 163; G20: 156, 157,160, 173; G21: 102; G22: 170.


MANOVA were a heterogeneous weighted aver-age of the original variables; such that dominat-ing variables responsible for the discriminationof the test could not be demonstrated. Usingthese ten factors and only the genetic features,the scores of comparison between the Sa1, Sa2,Sa3 and Sa4 groups were: multivariate test ofsignificance (Wilk’s lambda value) = 0.17996; Fstatistic = 4.211 and P (probability of groups tobe statistically the same) = 1.5 × 10–9. The scoresof comparison between groups using pheno-typic and genetic features (using only ten andthree factors) are shown in table VI. Both analy-ses clearly showed that the P. polymyxa strainsisolated from the rhizosphere of maize plantedin Cerrado soil were significantly related tosampling time (10, 30, 60 and 90 days aftermaize sowing). Strains from Sa1 and Sa4 andstrains from Sa2 and Sa3 appeared to be morehomogeneous, when they were compared to thewhole population (P10 = 16 and 5.3%, respec-tively). Moreover, all three-by-three groupingsthat included Sa2 strains showed the most sig-nificant difference by MANOVA (P10 varyingfrom 10–8 to 10–13).

4. Discussion

Studies using P. polymyxa strains isolated fromthe rhizosphere of different crops as a PGPR

have been extensively developed in temperateregions [17, 22, 23, 27, 32, 33, 36, 46, 59]. How-ever, P. polymyxa strains isolated from maizecultivated in tropical environments have neverbeen studied before. Therefore, in this study,strains of P. polymyxa were isolated from therhizospheres of maize plantlets 10, 30, 60 and 90days after maize sowing during one season.

The analyses performed on the phenotypicfeatures showed that 67% of the isolates werecapable of metabolizing glycerol, arabinose andxylose, as already described for P. polymyxaspecies [18]. Glycerol fermentation has not tra-ditionally been used for P. polymyxa identifica-tion but it was tested here based on the recog-nition of P. peoriae as a new species [35],separating the strains with negative tests onglycerol utilization from P. polymyxa. In thisstudy, different strains showing variable resultsfor arabinose and xylose metabolism and anegative test for glycerol fermentation wereisolated and identified as P. polymyxa because oftheir morphology, their biochemical profile withother carbohydrates (including the API 50CH),the presence of sequences homologous to the P.polymyxa-specific phage IPy1 [16, 54] and theamplification with P. polymyxa-specific primers[41, 42]. Shishido et al. [59] have also isolatedstrains from roots of lodgepole pine seedlingsthat they could not assign to species, and the

Table VI. MANOVA of 67 P. polymyxa strains isolated after 10 (Sa1), 30 (Sa2), 60 (Sa3) and 90 (Sa4) days of maize sowing in Cerradosoil, using phenotypic and genetic data.

Canonical variate analysis P10 P3

Sa1 × Sa2 8.6 × 10–6 2.0 × 10–7

Sa1 × Sa3 1.3 × 10–3 7.2 × 10–4

Sa1 × Sa4 1.6 × 10–1 2.2 × 10–1

Sa2 × Sa3 5.3 × 10–2 3.9 × 10–3

Sa2 × Sa4 1.4 × 10–12 1.2 × 10–12

Sa3 × Sa4 3.5 × 10–3 9.6 × 10–3

Sa1 × Sa2 × Sa3 7.2 × 10–8 3.5 × 10–8

Sa1 × Sa2 × Sa4 1.1 × 10–13 4.7 × 10–16

Sa2 × Sa3 × Sa4 4.7 × 10–8 1.6 × 10–8

Sa1 × Sa3 × Sa4 1.6 × 10–3 1.0 × 10–3

Sa1 × Sa2 × Sa3 × Sa4 5.9 × 10–10 2.5 × 10–11

P10, probability of different groups being statistically the same, considering ten factors in principal component analysis(representing 89% of variance); P3, probability of different groups being statistically the same, considering three factorsin principal component analysis (representing 70% of variance).


identification of these strains as P. polymyxa wasconsidered tentative.

All strains were capable of metabolizing starchand, as proposed previously for P. azotofixans[57], starch metabolism might play an importantrole in the establishment of rhizobacteria in thevicinity of maize roots. Mavingui et al. [32]suggested the same for sorbitol metabolism inwheat roots; we tested our strains for thisfeature, but found that none of them was able tometabolize this sugar (not shown).

Sixty-six strains showed the capacity to pro-duce antimicrobial substances against the indi-cator strain (table II). The use of PGPR that cansuppress or prevent damage to plants by nutri-ent stress or pathogens is currently being devel-oped in several laboratories [6, 60]. A range ofnovel antimicrobial substances produced by P.polymyxa strains has recently been character-ized. These strains show a broad activity spec-trum against Gram-positive and Gram-negativebacterial as well as fungal species [33, 44, 47, 58,64]. The fact that almost all P. polymyxa strainsisolated here are capable of producing antimi-crobial substances suggests an important rolefor these bacteria as biocontrol agents againstphytopathogens.

Table II also shows resistances to tetracycline,rifampicin, kanamycin and streptomycin in thestrains studied. These characteristics may beimportant for the establishment of these strainsin Cerrado soil. For example, streptomycin-resistant strains were good survivors in Cerradosoil, in which the presence of a high number ofStreptomyces spp. has been described [10, 24].Furthermore, the capability of Paenibacillus spe-cies to form endospores resistant to stress con-ditions usually observed in tropical conditionsis key to their survival.

Nitrogen fixation has been used as a criterionto screen for a high level of growth promotionactivity [21] and in the selection of inoculantsfor rice [40] and maize [2]. Also, recombinantDNA probes containing Klebsiella pneumoniaestructural nif genes have been used to identifyrelated sequences from several nitrogen-fixingorganisms [48, 50, 56, 57]. In the current study,75% of the isolates showed conservation of

nucleotide sequences coding for nitrogenaseand different hybridization patterns wereobserved among these strains. A previous studyhas also demonstrated the presence of differenthybridization patterns in P. polymyxa [39].

P. polymyxa strains were analyzed by BOX-PCR as another tool to check the genetic vari-ability of the isolates. A high degree of diversitywithin the isolates was observed, yielding atotal of 18 distinct groups (table V, figure 2). TheBOX groups obtained also revealed a higherdegree of diversity among P. polymyxa strainsthan the phenotypic and nif homology results.The same observation was described amongP. azotofixans strains isolated from maize [57].

Data from the genetic features were used toconstruct a dendrogram. All strains could bedistributed in 22 groups (figure 3). Some groupscontained more than one strain, indicating thatthese are genetically closely related and theymay have originated from a common ancestoror be a co-isolate. In a previous report, Seldin etal. [57] demonstrated that P. azotofixans strainsisolated from maize planted in Cerrado soilwere very homogeneous. In this study, the P.polymyxa strains were revealed to be more het-erogeneous. However, the differences amongthe hybridization and BOX patterns were notlarge and common fragments could be detectedamong P. polymyxa isolates in nif hybridizationstudies and in BOX-PCR. This might indicatethat we are dealing with a diverse group ofclosely related strains.

The data from genetic features and the com-bination of phenotypic and genetic data wereused for principal component analysis and fac-tors representing more than 70% of the variancewere used for MANOVA. These analyses wereable to link plant development to the diversityof P. polymyxa populations associated with maizeplanted in Cerrado soil. Diversity associatedwith plant growth has been demonstrated inmaize by Di Cello et al. [13] and McArthur et al.[34], working with Burkholderia cepacia, and bySeldin et al. [57] working with P. azotofixans.

One possible explanation for the difference inP. polymyxa populations observed in this studyis the change of the production and diffusion


levels of root exudates during the plant lifecycle. As these exudates represent nutritionalsources for rhizosphere microorganisms, theymay directly influence the microbial populationas suggested by Hamlen et al. [20]. We couldobserve a more homogeneous P. polymyxa popu-lation during the middle stages of maize growth(30 and 60 days after sowing) and also in thefirst stage (10 days) and after 90 days of maizegrowth. This could be explained by the morestable ecosystem of growing plants compared tothe unstable ecosystem of very young or declin-ing plants.

The intraspecific diversity observed in thisstudy may be important to provide the plantwith a variety of strains capable of coping withthe different stresses caused by tropical condi-tions. This fact should be taken into account inthe selection of P. polymyxa strains for use asinoculants of maize in Cerrado soil. Additionaldata are necessary to determine whether thesame results would be obtained using differentsoils and maize genotypes.

Acknowledgments

This work was supported by FINEP and bythe National Research Council of Brazil (CNPq).Thanks are due to colleagues from EMBRAPA-CNPMS who provided maize samples.

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