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Impact of a new biopesticide produced byPaenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities

Impact of a new biopesticide produced byPaenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities
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  Pest Management Science Pest Manag Sci   63 :269–275 (2007) Impact of a new biopesticide producedby   Paenibacillus  sp. strain B2 on thegenetic structure and density of soil bacterialcommunities Sameh Selim, 1 Fabrice Martin-Laurent, 2 Nadine Rouard, 2 Silvio Gianinazzi 1 andDiederik van Tuinen 1 ∗ 1 UMR INRA 1088/CNRS 5184/Universit ´ e de Bourgogne, Plante-Microbe-Environnement CMSE-INRA, 17 rue Sully, BP 86510, 21065Dijon Cedex, France 2 CMSE, UMR 1229 INRA/Universit ´ e de Bourgogne, Microbiologie et G´ eochimie des Sols, 17 rue Sully, BP 86510, 21065 Dijon Cedex,France Abstract: The effect of paenimyxin, a new biopesticide produced by  Paenibacillus  sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and  nirK   quantitative polymerasechain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number,which was significantly reduced 2 and 4days after treatment. The effect of paenimyxin on cultivatable bacteriawas negligible 7days after treatment. Approximately 10 7 16S rDNA sequences per gram of soil (dry weight) weredetected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition, RISA fingerprinting showed that the genetic structure of the bacterial communities was significantly modified 2days after paenimyxin application at 50 µ  M  and 4days aftertreatment at lower concentrations (0.5 and 5 µ  M ). The impact of paenimyxin treatment on the genetic structureof soil bacterial communities was transient, as no effect could be observed after 7, 14 and 28days when comparedwith the untreated control. ©  2007 Society of Chemical Industry Keywords:  biopesticide; paenimyxin; soil DNA; quantitative PCR  1 INTRODUCTION Soil health refers to the biological, chemical andphysical features of soil that are essential to long-term,sustainable agricultural productivity with minimalenvironmental impact. 1 The average losses in cropproductivity owing to biotic agents (pests, diseases,weeds) is about 40%. 2 The control of plantpests, diseases and weeds is achieved mainly byspraying crops with synthetic chemical pesticides. 2 , 3 Although pesticide use permits a certain qualityof agricultural production, intense use is knownto contribute to the contamination of soil andwater resources at concentrations often exceeding theEuropean limit of 0 . 1 µ gL  − 1 in drinking water. 4 Inaddition, the heavy use of chemicals in agriculturereduces soil biodiversity, leading to soil compactionand other disturbances. This in turn leads toadverse ecological alterations, resulting in a lossof agricultural productivity. 1 The development of new practices will improve agricultural sustainability.An alternative option for maintaining crop healthand yields is to develop biological products basedon beneficial microorganisms. 5 Microbial pesticidesare being introduced as part of this new scenarioof crop protection. Currently, several beneficialmicroorganisms are the active ingredients of a newgeneration of microbial pesticides or the basis of manynatural antagonistic products of microbial srcin. 6 Although more than 500 antimicrobial peptideshave been described, 7 and a relatively high numberpatented as biopesticides, only a few have beenregisteredforagriculturaluse.Theexcessivespecificityof most biopesticides and their biosafety in termsof impact on the soil microflora and environmentare major factors limiting their use in agriculture. 6 Notably, the impact of biopesticide applicationon the environment needs to be evaluated, and,more particularly, the effect on soil microorganisms,which are among the most diverse components of terrestrial ecosystems, responsible for organic mattertransformation and contributing to the C and Nratio.No studies of the impact of purified antimicrobialcompounds on soil microbial diversity on the basis of a culture-independent approach have been reportedto date. The present authors recently identified a ∗ Correspondence to: Diederik van Tuinen, UMR INRA 1088/CNRS 5184/Universit´ e de Bourgogne, Plante-Microbe-Environnement CMSE-INRA, 17 rue Sully,BP 86510, 21065 Dijon Cedex, FranceE-mail:  Received 15 March 2006; revised version received 29 August 2006; accepted 12 October 2006  )Published online 23 January 2007 ;  DOI: 10.1002/ps.1335 © 2007 Society of Chemical Industry.  Pest Manag Sci   1526–498X/2007/$30.00  S Selim  et al  . new group of biopesticides produced by the gram-positive bacterium  Paenibacillus  sp. strain B2 andknown as paenimyxin. 8 , 9 Paenimyxin is a mixture of three active biopesticides, lipopeptides with a mass of approximately 1kDa. The structure is similar to thatof polymyxin B, with a cyclic heptapeptide moietyattached to a tripeptide side chain and a fatty acylresidue. They all contain threonine, phenylalanine,leucine and 2,4-diaminobutyric acid residues. Theprincipal peptide of paenimyxin contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of thepolymyxin side chain. It has an antagonistic activityagainst gram-positive and gram-negative bacteria,and also against many important soil-borne fungalpathogens. 8 The aim of the present study was to assess indifferent ways the impact of paenimyxin on the soilmicroflora:1. The cultivatable soil bacteria were enumerated byplate counting.2. The total number of 16S rDNA sequenceswas estimated by quantitative polymerase chainreaction (PCR).3. The genetic structure of the soil microbial com-munities was determined by ribosomal intergenicspacer analysis (RISA).4. The density of the  nirK   gene, which codes akey enzyme of the respiratory chain transformingnitrite into nitrous oxide (NO), was measured byquantitative PCR on soil DNA extracts. 2 MATERIALS AND METHODS2.1 Soil The soil used in this study was collected from thetop 20cm layer of an agricultural field at the InstitutNational de la Recherche Agronomique domain of Epoisses, France. This soil is an eutric calcariccambisoil (granulometric composition: clay 48%, silt44.6%, sand 7.2%, organic carbon 20%, pH 7.5).The soil moisture content was measured and sampleswere then sieved to 5mm and stored at 4 ◦ C untilfurther use. 2.2 Bacterial strain The  Paenibacillus  sp. strain B2 used in this study hadbeen isolated from the mycorrhizosphere of   Sorghumbicolor   L. var. Esquirol inoculated with  Glomus mosseae (Nicol & Gerdemann) Gerdemann & Trappe BEG12. 9 2.3 Preparation of paenimyxin Bacterial growth conditions and purification of the Paenibacillus  sp. strain B2 antagonistic compound(paenimyxin) were as described previously. 8 2.4 Soil treatment Soil samples (10g dry weight) were moistened to25% of the water-holding capacity and treated withdifferent concentrations of paenimyxin of 0, 0.5, 5 and50 µ M  (corresponding respectively to 0, 0.06, 0.6 and6mgg − 1 soil) and incubated in the dark at 20 ◦ C for28days. Control soil was treated with sterile water.Soil samples were collected 2, 4, 7, 14 and 28daysafter treatment. Each time point was triplicated andtreated separately. 2.5 DNA extraction DNA was extracted from 250mg (dry weight)aliquots of soil samples. 10 Briefly, the samples werehomogenized in 1mL of extraction buffer for 30s at1600rpm in a mini beadbeater cell disrupter (Mikro-DismembratorS; B. Braun Biotech International,Germany). Soil and cell debris were eliminated bycentrifugation (14000 ×  g   for 5min at 4 ◦ C). Sodiumacetate (5  M ) was then added to remove the proteins,and the nucleic acids were precipitated using ice-cold isopropanol. The DNA was washed with 70%ethanol and purified through a Sepharose 4B spincolumn. It was then quantified by spectrophotometryat 260nm using a BioPhotometer (Eppendorf,Hamburg, Germany) and compared with standardDNA using 10gL  − 1 agarose gel electrophoresis. 2.6 Enumeration of total cultivatable bacteriafrom soil A quantity of 1g of soil was added to 9mL of sterile water and mixed vigorously. The resulting soilsuspension was serially diluted tenfold in sterile waterand 100 µ L of the serial dilutions (10 − 4  –10 − 6 ) wereplated on nutrient agar medium (2.3g nutrient Sigmaagar and 15g agar L  − 1 ) containing cycloheximide(100mg L  − 1 ), a fungicide preventing the growth of fungi on this medium. The total cultivatable bacteriawere counted after 2days of incubation at 28 ◦ C. Eachdilution was triplicated. 2.7 Quantification of 16S rDNA and  nirK   genesequences from soil samples Quantitative PCR was carried out with an ABI Prism7900 (Applied Biosystem ® ) using SYBR Green asthe detection system in a reaction mixture of 25 µ L containing: 0 . 5 µ M  of each primer [358for (5 ′ -CCTACG GGA GGC AGC AG-3 ′ ) and 517rev (5 ′ -ATT CCG CGG CTG CTG GCA-3 ′ ) 11 for 16SrDNA, and nirK876 (ATY GGC GGV AYG GCGA) and nirK1040 (GCC TCG ATC AGR TTR TGGTT) for  nirK   gene], 12 12 . 5 µ L of SYBR Green PCR master mix (QuantiTect ™ SYBR  ® Green PCR Kit,QIAGEN, France), 1 µ L of (T4 Gene 32 product),0.5ng of soil DNA and Rnase-free water up to 25 µ L.The conditions for 16S quantitative PCR were asfollows: 600s at 95 ◦ C, 40 cycles of 15s at 95 ◦ Cfor denaturation, 30s at 60 ◦ C for annealing, 30sat 72 ◦ C for extension and 15s at 81 ◦ C for a finaldata acquisition step. One last step from 60 to 95 ◦ Cwith an increase of 0.2 ◦ C s − 1 was added to obtain aspecific denaturation curve. The conditions used for nirK   quantitative PCR were as described previously. 12 270  Pest Manag Sci   63 :269–275 (2007)DOI: 10.1002/ps  Impact of paenimyxin on soil bacteria Purity of the amplified products was checked by theobservation of a single melting peak. The 16S rDNAand  nirK   gene quantitative PCR was calibrated from10 2 to 10 8 copies by serial dilution of the appropriatecloned target sequence. The calibration curve relatingthe log of the copy number of the target sequence tocycle threshold (Ct) was  y =− 3 . 5318 x + 31 . 981 for16S rDNA and  y =− 3 . 4745 x + 30 . 948 for  nirK  . 2.8 Ribosomal intergenic spacer analysis The 16S–23S intergenic spacer of the bacterial rDNAwas amplified in a final volume of 100 µ L from 1ngof soil DNA with 1 µ M  of 38r (5 ′ -CCG GGT TTCCCC ATT CGG-3 ′ ) and 72f (5 ′ -TGC GGC TGGATC TCC TT-3 ′ ) universal primers 13 using 2.5 Uof Taq DNA polymerase (Appligene Oncor, France).PCRs were carried out in a PTC 200 gradient cycler(MJ Research, Waltham, MA) with the followingconditions: 5min at 94 ◦ C, 35 cycles of 1min at94 ◦ C, 1min at 55 ◦ C and 2min at 72 ◦ C, plus anadditional 15min cycle at 72 ◦ C. Aliquots (3 µ L)were separated by electrophoresis on a native 6%acrylamide gel run for 16h at 8mA. Gels were stainedwith SYBR Green II (Molecular Probes, Leiden, TheNetherlands). RISA profiles were analysed with theOne-D Scan 2.03 program (Scanalytics program)to develop matrices (presence–absence and relativeintensity of each band). Principal component analysis(PCA) was performed on the covariance matrix usingADE-4 software. 14 2.9 Statistical analysis The abundance of 16S rDNA and of   nirK   wascompared between the different soil samples usinga one-way analysis of variance. Means were comparedusing the least significant difference (LSD) test at P   <  0 . 05. 3 RESULTS3.1 Total cultivatable bacteria enumeration The impact of paenimyxin was determined byenumerating the total cultivatable soil bacteria, whichwas around 7 × 10 6 colony-forming units (CFUs)g − 1 soil. A significant reduction (60–70%) of theCFUs was observed 2days after treatment (Fig. 1)in soil samples treated with paenimyxin, irrespectiveof the concentration applied. Only those soil samplestreated with high concentrations of paenimyxin (5 and50 µ M ) presented a significant decrease in CFUs whencompared with the control 4days after treatment.The same CFU counts were obtained in controland paenimyxin-treated soil samples after 7days of treatment. 3.2 Quantitative PCR assay  The impact of paenimyxin on bacterial number wasassessed by determining the 16S rDNA copy numberin the total DNA from each of the soil samples. Thetotal amount of DNA extracted from each soil sampleranged from 0 . 2 µ gg − 1 soil at  T  0  to 0 . 8 µ gg − 1 soilafter 28days of treatment, and the number of 16SrDNA sequences ranged from 2 × 10 7 to 9 × 10 7 copies g − 1 soil. The statistical analysis did not revealany significant differences in the number of bacteria,estimated by 16S rDNA quantitative PCR, betweencontrol and paenimyxin-treated soil. The impact of paenimyxin on denitrifying bacteria was evaluatedby determining the  nirK   copy number in all thesoil samples. The  nirK   copy number ranged from1 . 6 × 10 4 to5 × 10 4 copiesg − 1 soil.Statisticalanalysisrevealed that the number of denitrifying bacteria inthe soil samples was not significantly affected by theapplication of paenimyxin. 3.3 Structure of soil microbial communities The structure of the soil microbial communities wasinvestigated by ribosomal intergenic spacer analysis(RISA) on DNA extracted directly from the soil.RISArevealsthelengthpolymorphismofthe16S–23Sintergenic spacer in the bacterial ribosomal operon.RISAconducted onDNAextracteddirectlyfromeachsoil sample, treated or not treated with paenimyxin(0.5, 5 or 50 µ M ), produced relatively complexfingerprints (approximately 35 bands per lane). Inmost cases, the fingerprints for the replicates of a 0,5 µ M5 µ M50 µ M Paenimyxin; 020406080100120140024714    C   F   U   % Control a a a aab b baabbaaaaaaa aa aa 28 aba aa aa aDays after treatment Figure 1.  Ratio of the number of colony-forming units (CFUs) from soil samples treated with different paenimyxin concentrations (0.5, 5 or 50 µ M  ) tothe number of CFUs from control samples collected after 2, 4, 7, 14 and 28days of incubation. Values followed by the same letter do not differstatistically (   n = 9,  P  <  0 . 05). Pest Manag Sci   63 :269–275 (2007)  271 DOI: 10.1002/ps  S Selim  et al  . III 0 0.5 5 50 T0 PC1 27.47% 34579   2 5   50 -404040 PC2 23.58% 0 µM0.5 µM5 µM50 µM 30.5-40 500050.5 900612501404320242 B 1234   6789   10 24 -202020 PC2 22.50% 1234   6789   10-20 0 µM0.5 µM5 µM50 µM 50050.500.5550 T4 A 0 0.5 5 50900612501404320242 Figure 2.  Panels I, RISA fingerprints of PCR products amplified with 16S–23S rDNA universal primers (38r and 72f) from DNA extracts of soilsamples treated or not treated with different concentrations of paenimyxin (0.5, 5, 50 µ M  ): panel IA, soil samples before treatment (  T  0  ); panel IB, soilsamples after 4days of incubation (  T  4  ). Panel IIA, PCA ordination of the genetic structure of the whole soil bacterial communities found in soilsamples before treatment (  T  0  ); panel IIB, soil samples after 4days of incubation (  T  4  ). given treatment were similar, illustrating the relativelygood reproducibility of DNA extraction, amplificationand separation (Fig. 2, panels IA, IB, IIA and IIB).Although differences in the RISA fingerprints betweenthe control and paenimyxin-treated soil samples couldnot be visually distinguished, numerical analysis of these fingerprints followed by pairwise analysis usingprincipal component analysis made it possible toordinatethemicrobialcommunitiesonaplanedefinedby the first two principal components, and to comparethe magnitude of the changes induced by paenimyxintreatment (Fig. 2, panels IIA and IIB).At T  0  thefirstprincipalcomponentexplained27.5%ofthevariancesinthedata,andthesecondcomponent23.6% (Fig. 2, panel IIA). The factorial map showedthatordinationonthefirstprincipalcomponent(PC1)did not allow discrimination between the differentsoil samples. Two days after paenimyxin treatment,PC1 explained 30.7% of the data variance, and thesecond component (PC2) 23% (data not shown).The factorial map showed that ordination on PC1only permitted discrimination between the microbialcommunities found in the soil samples treated with50 µ M  and the other treatments (0, 0.5 and 5 µ M ).Four days after treatment, PC1 explained 31.9% of the data variance, and PC2 22.5% (Fig. 2, panel IIB).The factorial map showed that ordination on PC2permitteddifferentiationofthemicrobialcommunitiesaccording to the paenimyxin treatment applied tothe soil. No such discrimination was possible fromnumerical analysis of the RISA patterns in the soilsamples collected 7, 14 and 28days after treatment. 4 DISCUSSION Plant protection against pathogens, pests and weedshas been progressively reoriented from a systematicapproach to a rational use of chemical pesticides inwhich consumer health and environmental preserva-tion prevail over any other productive or economicconsiderations. 6 Social and political concerns haveinfluenced the practice of crop protection, leadingprogressivelytoareductioninthenumberofregisteredactive ingredients to those apparently unavoidable, 272  Pest Manag Sci   63 :269–275 (2007)DOI: 10.1002/ps  Impact of paenimyxin on soil bacteria more selective, less toxic and with lower negativeenvironmental impact. 15 , 16 In addition, an alternativeto chemical pesticides could be the use of natu-ral molecules exhibiting pesticide activity, known asbiopesticides, which havethe advantageof being easilydegraded by the soil microflora since they alreadyexistintheenvironment.Inthiscontext,thepresentauthorsrecently isolated and characterized a novel lipopeptidebiopesticide, paenimyxin, produced by  Paenibacillus sp. strain B2. This biopesticide exhibits antagonisticactivity against several bacterial and fungal strains. 8 Before using it as a biopesticide for crop production,the impact produced by its application to bare soil wasevaluated by analysing the structure and density of thesoil bacterial communities.The total number of cultivatable bacteria in soilsamples treated or not treated with paenimyxin(concentration ranging from 0.5 to 50 µ M ) wasdetermined by the plate counting method. This rangeisuptotenfoldoftheminimalinhibitoryconcentrationof paenimyxin against gram-negative bacteria. 8 Thebiopesticide significantly reduced the total numberof cultivatable bacteria, but only during the first4days following its application. The total numbersof cultivatable bacteria in the control and paenimyxin-treated soil samples did not differ significantly 7, 14and28daysaftertreatment.Theseresultsdemonstratethatpaenimyxinproducedonlyatransitorytoxiceffecton cultivatable soil microorganisms, and illustrate theresilience of the soil microflora. This transitory effectcould be due either to adsorption of the biopesticideto soil particles, thus reducing its bioavailability, orto its rapid biodegradation by microbial extracellularenzymes and/or soil microbial populations.Although the plate counting method has theadvantages of being relatively rapid and inexpensive,and provides information on the active cultivatablecomponent of the microflora, its limitations includethe difficulty in dislodging bacteria or spores fromsoil particles or biofilms and specific selection by thegrowth medium. 17 During the last decade, it has beensuggested that at least 99% of the bacteria observedunder a microscope cannot be cultured by commonlaboratorytechniques. 18–20 Recentapplicationsofnewmolecular tools based primarily on amplification bypolymerase chain reaction of the DNA extracteddirectly from soil have provided an alternative to theconventional culture-based microbiological methodsand a unique insight into the composition, richnessand structure of microbial communities. 10 Althoughthe molecular techniques are very powerful, becauseof the remarkable diversity of soil microbiota, nosingle method can give a complete image of theimpact of xenobiotics on soil microbiota. Thistechnical limit can be partially overcome by thecombination of a global and a more targetedapproach. The recent development of quantitativePCR allows quantification of the total number of bacteria or functional bacterial communities from soilDNA extracts and avoids the bias resulting fromthe non-cultivatability of many microorganisms byconventional methods such as MPN. QuantitativePCR can be applied even in samples from complexenvironments to yield precise and reproducibleestimates of the number of targets. 21 A quantitativePCR assay has been used to estimate the number of 16SrDNAsequencesinsoilsamplesandtoenumeratesoil bacterial populations. It should be noted that thefinal determination of bacterial load by quantitativePCR in a multispecies population will be influencedby the variation in the number of rRNA operons in agiven species. 22 However, the numbers of 16S rDNAsequences found in control and paenimyxin-treatedsoilsdidnotdiffersignificantly,irrespectiveofthetimeof sampling or the paenimyxin concentration underconsideration. The 16S rDNA quantitative PCR results contradicted the conventional counts of totalcultivatable bacteria on nutrient agar, which impliedthat paenimyxin caused a significant reduction inCFUs in the period following biopesticide application.It is most likely that the fraction of cultivatablemicroorganisms sensitive to paenimyxin revealed byconventional microbiological methods could not bedetected by quantitative PCR owing to the very smallsize of this fraction compared with the large numberof 16S rDNA sequences present in the soil DNAextracts. Quantitative PCR was also used to estimatethe number of   nirK   sequences in the soil samplesand to quantify the nitrite reductase denitrifiers. Aspreviously observed for 16S rDNA quantification, thenumbers of   nirK   sequences found in control andpaenimyxin-treated soils did not differ significantly,whatever the time of sampling. It should be notedthat the average density of nitrite reductase denitrifierswas 10 4 bacteria g − 1 soil, which is in accordancewith a previous study in which  nirK   sequences inseveral soils ranged from 10 4 to 10 6 bacteria g − 1 soil. 12 All together, the quantitative analyses of 16SrDNA and  nirK   sequences tend to indicate thatthe application of paenimyxin did not produce anysignificant modifications in the densities of either thetotal bacteria or of the nitrite reductase denitrifyingcommunity.The impact of paenimyxin on the structure of thesoil microflora was also estimated by applying anRISA to soil DNA extracts. RISA has already beendemonstrated to be relevant and sensitive enough tostudy bacterial communities associated with differentmicroscale environments 23 or vegetation cover 24 andthe impact of the introduction of a bacterium-producing biopesticide on the microbial communitiesof the rhizosphere. 25 The RISA fingerprints generatedfrom soil DNA were relatively complex and showedgoodreproducibilitybetweenreplicates,indicatinglowvariability in the soil samples and/or DNA extractsstudied and low PCR amplification bias. 10 RISAanalysesrevealed that the structure of the soil bacterialcommunity was modified by paenimyxin application.The strongest structural alterations were recordedduring the first 4days after paenimyxin treatment. Pest Manag Sci   63 :269–275 (2007)  273 DOI: 10.1002/ps
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