In£uence of transgenic T4-lysozyme-producing potato plants onpotentially bene¢cial plant-associated bacteria

Jana Lottmann a, Holger Heuer b, Kornelia Smalla b, Gabriele Berg a;*a Department of Biology, Microbiology, University of Rostock, Gertrudenstrasse 11a, D-18055 Rostock, Germany

b Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Virology, Microbiology and Biosafety,Messeweg 11/12, D-38104 Braunschweig, Germany

Received 3 February 1999; revised 21 April 1999; accepted 21 April 1999

Abstract

A field release of genetically engineered potato plants that produce bacteriophage T4-lysozyme for enhanced bacterialresistance was monitored for changes in plant-associated bacterial populations, in the functions of potentially beneficialbacteria and in the diversity of antagonistic bacterial species. These parameters have been analyzed for two T4-lysozyme-expressing lines, a transgenic control and a non-transgenic line, over a period of 2 years at different stages of plant developmentand at two different locations. Two microenvironments, the rhizo- and geocaulosphere, were investigated. No significantdifferences in aerobic plate counts were observed between the four plant lines. In addition, no significant differences in thefunctions of potentially beneficial bacteria (percentage of auxin [indole-3-acetic acid = IAA]-producing isolates) andantagonistic bacteria (antagonists to Erwinia carotovora and Verticillium dahliae) were found. The diversity of antagonisticspecies isolated from each plant line and microenvironment was investigated to determine if the diversity and composition ofpotentially beneficial bacteria were influenced. Altogether, 28 different potato-associated species with antagonistic effects tophytopathogens were detected. Antagonistic strains of seven species were found only on control plants. The observed effect wasminor relative to the natural variability observed during the monitoring period. This is the first study including plant-associatedbacteria responsible for plant growth and health and provides an example for performing risk assessment studies for transgenicplants under a variety of environmental conditions. ß 1999 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Risk assessment; Transgenic potato; T4-lysozyme; Potentially bene¢cial plant-associated bacteria

1. Introduction

In recent years, molecular techniques for modify-ing the genome of a plant have become common, but

little is known of the consequences of these geneti-cally engineered plants on plant-associated bacteria.There have been numerous ¢eld releases of transgen-ic plants but the majority of these ¢eld tests havebeen evaluated only for e¤cacy of the plant engi-neered traits or outcrossing e¡ects. Practical ap-proaches for testing the e¡ects of transgenic plantson key groups of soil microorganisms have been

0168-6496 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 9 6 ( 9 9 ) 0 0 0 3 2 - X

* Corresponding author. Tel. : +49 (381) 494 2049;Fax: +49 (381) 494 2244;E-mail: gabriele.berg@biologie.uni-rostock.de

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suggested [1,2]. Risk assessments for transgenicplants with respect to plant-associated microorgan-isms have been reported in the last few years. Wilsonet al. [3] analyzed the colonization of mannitylopine-producing transgenic tobacco plants with amannityl opine-catabolizing strain of Pseudomonassyringae. They observed enhanced colonization ofthe phyllosphere by the catabolizing strain comparedto that of the near isogenic (non-catabolizing) strainof P. syringae. Opine-producing potato plants ex-erted an in£uence on the naturally occurring root-associated micro£ora [4] and on introduced bacteria[5]. Donegan et al. [6] observed an in£uence of cot-ton plants which were genetically engineered to pro-duce the Bacillus thuringiensis (Bt) endotoxin, onboth abundance and diversity of indigenous soil bac-teria and fungi. The authors suggested that the ob-served e¡ects may not have resulted from the foreigngene product, but from a change in plant character-istics due to the genetic manipulation. In contrast,the environmental release of Bt-producing potatoplants had no signi¢cant or persistent impact onthe microbial species associated with plant leaves[7]. Minor di¡erences in the species composition ofbacteria in the phyllosphere of T4-lysozyme-produc-ing potatoes and control plants were detected byHeuer and Smalla [8].

Potato plants were engineered to produce T4-lyso-zyme to enhance their resistance to Erwinia caroto-vora ssp. atroseptica. This is a broad-host-rangepathogen causing seed piece decay, blackleg and aer-ial soft rot, especially of potatoes [9]. Lysozymes area widespread family of enzymes. Most of the inves-tigated soil- and plant-associated bacteria such asPseudomonas stutzeri, Acinetobacter calcoaceticus,Bacillus subtilis and Rhizobium spp. were susceptibleto T4-lysozyme at very low concentrations [10]. Inthese experiments the sensitivity of bacteria was alsoobserved in the absence of the chelating agent EDTA[10]. Bacteriophage T4-lysozyme is the most activemember of a class of bacteriolytic enzymes, whichhave been detected in several plant species [11,12].The secretion of foreign T4-lysozyme into the inter-cellular spaces of transgenic potato plants a¡ects re-sistance against Erwinia carotovora already at rela-tively low levels of expression [13]. T4-lysozyme wasthe ¢rst trait to be exploited to produce plants withresistance against bacteria. Recently, this strategy

was also applied for other plants, e.g. oilseed rapeand tomato [14].

Plant-associated bacteria are subdivided into ben-e¢cial, deleterious and neutral groups on the basis oftheir e¡ects on plant growth and health [15]. Bene¢-cial bacteria are responsible for plant growth promo-tion and biological control of soil-borne pathogens[15,16]. If this group of bacteria is disturbed due tothe in£uence of T4-lysozyme, the positive e¡ect ofthe constructed plant would be cancelled. Alterna-tively, if bene¢cial bacteria were not in£uenced, asynergistic e¡ect could occur. Due to the non-selec-tive e¡ects of T4-lysozyme, it is necessary to assessthe in£uence on the potato-associated bene¢cial bac-teria. For this study we investigated the e¡ects onbacteria in the rhizosphere [15,17] and in the geo-caulosphere (tuber surface) [18].

The objective of our investigations was to evaluatethe e¡ects of transgenic T4-lysozyme-producing po-tatoes on plant-associated bacteria, and especially onspecies that are potentially bene¢cial bacteria in therhizo- and geocaulosphere, under ¢eld conditions.These potentially bene¢cial bacteria were character-ized according to their ability to produce indole-3-acetic acid (IAA); to show antagonistic activity tothe bacterial target pathogen E. carotovora ssp. atro-septica ; or antagonistic activity to the phytopatho-genic fungus Verticillium dahliae. All of the activestrains were identi¢ed to monitor the possible in£u-ence of T4-lysozyme on the diversity of species withantagonistic properties.

2. Materials and methods

2.1. Plants and experimental design

The transgenic potato lines, DL4, DL5 constitu-tively expressing and secreting T4-lysozyme, and atransgenic control line, DC1, carrying the same con-struct [13], including the nptII gene, but without T4-lysozyme gene were derived from variety Desiree. Allplant lines, including the wild-type DESI, were pro-vided by Dr. K. Du«ring (Federal Centre for BreedingResearch on Cultivated Plants [BAZ], Quedlinburg,Germany). In 1996 and 1997 potato plants weregrown in ¢eld trials in GroM Lu«sewitz (GL) andQuedlinburg (QB) (Germany, areas of the BAZ).

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Trials at both sites consisted of 32 completelyrandomized plots; eight plots were planted withDESI, eight plots were planted with DC1, eight plotswere planted with DL4, and eight plots were plantedwith DL5. Each of the 32 plots contained 15 plants.Soil parameters at both locations were analyzed bythe Institute for Agricultural Analysis and Research(LUFA) Rostock (Germany) and are described inTable 1. The potatoes were planted as seed pieceson 28 May 1996 and harvested on 1 October 1996.In the second year the potatoes were planted on16 April 1997 and harvested on 8 September 1997.Samples were collected on three occasions during thevegetation period corresponding to the growth stagesof plants: young plants (growth stages 11^19); £ow-ering plants (growth stages 61^69); senescent plants(growth stages 91^97), according to Hack et al. [19].For sampling the potato plants, di¡erent combina-tions of experiments were performed as described inTable 2.

2.2. Determination of culturable bacterial counts

Plant roots with adhering soil taken from ¢veplants and from one plot were aseptically sampledinto sterile Stomacher bags and treated as one sam-ple. Prior to cell extraction, 5 g of each pooled sam-ple was transferred into a new Stomacher bag. Sam-ples were extracted in a Stomacher laboratoryblender (BagMixer, Interscience, St Nom, France)with di¡erent sterile solutions: (a) 20 ml PBS con-tained the following constituents per liter demineral-ized water: 8 g NaCl, 0.2 g KCl, 1.4 g Na2HPO4,0.24 g KH2PO4 (all from Merck, Darmstadt, Ger-many); (b) 15 ml SDP contained per liter deminer-alized water: 1 g sodium deoxycholate (Merck), 25 gpolyethylene glycol 6000 (Merck); (c) 0.5 g Chelex100 resin (Bio-Rad, Hercules, CA, USA); (d) 15 mldemineralized water. The extraction protocol wasbased on the method of Herron and Wellington[20]. Five tubers from ¢ve plants from one plotwere sampled into sterile Stomacher bags and treatedas one sample. From each tuber surface, a thin layerwas rasped under aseptic conditions with a commer-cial grater and 5 g of fresh tuber material was ex-tracted in a Stomacher blender as described above.For each sample the three combined solutions ( = 50ml) were serially diluted and plated on R2A medium

(Difco Laboratories, Detroit, MI, USA). Plates wereincubated for 5 days at 20³C and bacterial colonieswere counted to calculate the means of colonies(log10 CFU) based on fresh weight. Bacterial platecounts of the rhizosphere population were analyzedto determine di¡erences between transgenic andnon-transgenic plants and temporal e¡ects over thegrowing season. The aerobic plate counts, for thegeocaulosphere populations, were also analyzed fordi¡erences between the four plant lines and geocau-losphere populations from di¡erent soil types werecompared. For further testing, bacterial isolates ateach sampling time and microenvironment wererandomly selected and subcultured in culture tubeson nutrient agar (Gibco, Paisley, UK) resulting in2304 rhizosphere isolates and 1344 geocaulosphereisolates (Table 3). The culture tubes were stored at4³C.

2.3. Screening for bacteria isolated from therhizo- and geocaulosphere antagonistic againstE. carotovora and V. dahliae

The in vitro inhibition of E. carotovora was deter-mined in a dual culture assay on Luria-Bertani-agar(LB, Difco). 100 Wl of an overnight culture of E.carotovora ssp. atroseptica was plated on LB agarand bacteria were streaked as broad bands. Zonesof inhibition were measured after incubation at20³C at 24 and 48 h. Two di¡erent test strainswere investigated: E. carotovora ssp. atrosepticaDSM 30168 and E. carotovora ssp. atroseptica (strainno. 459, National Collection of Plant PathogenicBacteria, Harpenden, UK).

The in vitro inhibition of V. dahliae was deter-mined in a dual culture assay on Waksman agar(WA), according to Berg and Ballin [21]. Zones ofinhibition were determined after 5 days of incu-bation at 20³C. The strain used, V. dahliae V16 (cul-ture collection of the University of Rostock, Ger-many), was originally isolated from Solanumtuberosum.

2.4. Screening for IAA-producing bacteria isolatedfrom the rhizo- and geocaulosphere

The microplate method used was a modi¢cation ofthe method developed by Sawar and Kremer [22].

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All isolates were grown on half-strength tryptic soyagar (TSA, Gibco, Paisley, UK). For the colorimet-ric IAA assay, a 24-h culture of each isolate wassuspended in sterile PBS to an optical density ofapproximately 0.5 at 500 nm. The suspension(500 Wl) was added to 5 ml of growth medium, whichcontained the following per liter demineralizedwater: 5 g glucose, 0.025 g yeast extract, 0.204 gL-tryptophan. Controls were prepared by substitut-ing bacterial suspension with sterile water. Tubeswere incubated in the dark at 20³C for 72 h. Priorto analyses for auxin (IAA equivalents), 1 ml ofgrowth medium of each isolate was centrifuged at4³C for 10 min. The supernatant of each sample(150 Wl) was dispensed into wells of 96-well micro-plates followed by addition of 100 Wl of Salkowskireagent (2 ml 0.5 M FeCl3 mixed with 98 ml 35%perchloric acid). Following reaction for 30 min, thecolor intensity was measured at 530 nm on a micro-plate reader (Spectramax-250, Molecular Devices,Sunnyvale, CA, USA).

2.5. Identi¢cation of bacterial antagonists

All isolates with antagonistic properties againstE. carotovora and/or V. dahliae were identi¢ed onthe basis of whole-cell cellular fatty acids, derivatizedto methyl esters, i.e., FAME (fatty acid methyl es-ters), and analyzed by gas chromatography using theMIDI system (Microbial Identi¢cation System, Inc.,Newark, NJ, USA). Strains which could not be iden-ti¢ed by FAME analysis were additionally tested byBiolog (Biolog, Hayward, CA, USA) or using theanalytical pro¢le index API 20E or 20NE (bio-Merieux, France).

2.6. Determination of root biomass

At the end of the growing season, the dry weightof roots was determined for all plant lines. For thisanalysis, eight replicates of pooled root samples(based on three roots) were taken for each plantline (DESI, DC1, DL4 and DL5), weighed and driedfor 24 h at 105³C. For each plant line, the data ofthe calculated mean per root were analyzed and theresults were tested for signi¢cances between the fourlines.

2.7. Statistics

All statistical analyses were performed at P6 0.05level with the SAS software package (Proc GLM orProc GENMOD in SAS release 6.12; SAS Institute,Cary, NC, USA). Bacterial counts were transformedto log10 values before two-factor analysis of variance(rhizosphere: plant lines and growth stages [samplingtimes] ; geocaulosphere: plant lines and ¢eld sites[GL, QB]). Similarly, both the percentages of IAA-producing bacteria and of antagonistic bacteria werestudied by two-factor analysis of variance. Root dryweight data of DESI, DC1, DL4 and DL5 were an-alyzed by multiple comparison of the means of oneroot per plant line.

3. Results

3.1. Determination of culturable bacterial counts

The rhizosphere was sampled at three di¡erenttimes during the monitoring period according to dif-ferent growth stages of potato plants. The number ofaerobic bacteria that could be recovered from therhizosphere on R2A was not signi¢cantly di¡erentbetween the four plant lines and remained at levelsof ca. log10 7.7 CFU g31 fresh weight (fw) of root(Fig. 1). In the second year (1997) signi¢cantly morebacteria could be recovered at the second (£oweringplants) and third (senescent plants) sampling time forall four plant lines.

The geocaulosphere was only sampled at the endof growing season (senescent potato plants) whentubers were fully developed. The number of aerobic

Table 1Characteristics of soils used in this study; GL (GroM Lu«sewitz),QB (Quedlinburg)

Parametera GL QB

Texture sandy loam silt loampH 5.9 5.9Organic matter (%) 1.1 2.2P2O5 (mg 100 g31 soil) 20 36K2O (mg 100 g31 soil) 14 31Mg (mg 100 g31 soil) 10 15

aAnalyzed by the Institute for Agricultural Analysis and Re-search (LUFA), Rostock (Germany).

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bacteria that could be recovered from the geocaulo-sphere (tuber surface) on R2A was not signi¢cantlydi¡erent between the four plant lines and remainedat ca. log10 7.1 CFU g31 fresh weight of tuber (Fig.2). Signi¢cantly fewer (P6 0.0001) bacteria could bedetected from the caulosphere from plants from

Quedlinburg (log10 6.93 CFU g31 tuber fw) thanfrom the ¢eld site of GroM Lu«sewitz (log10 7.37CFU g31 tuber fw). Overall, during the monitoringperiod, the number of culturable bacteria from therhizosphere was signi¢cantly larger (P = 0.0001) thanfrom the geocaulosphere.

Fig. 2. Mean values þ S.E.M. of bacterial plate counts of the geocaulosphere for the ¢eld sites in GroM Lu«sewitz and Quedlinburg. Log10

of colony-forming units (CFU) per gram of tuber fresh weight of geocaulosphere population densities of the four plant lines DESI (non-transgenic), DC1 (transgenic control), DL4 and DL5 (transgenic test lines). No signi¢cant di¡erence was observed between the means ofthe four plant lines over both years and locations (P = 0.6007). Signi¢cant di¡erences in bacterial plate counts in the geocaulosphere weredetermined between the means of both locations over all plant lines and both years (P6 0.0001).

Fig. 1. Mean values þ S.E.M. of bacterial plate counts of the rhizosphere for the ¢eld site in GroM Lu«sewitz. Log10 of colony-formingunits (CFU) per gram of root fresh weight of rhizosphere population densities of the four plant lines DESI (non-transgenic), DC1 (trans-genic control), DL4 and DL5 (transgenic test lines). Populations at each sampling time (1 and 4: young plants; 2 and 5: £owering plants;3 and 6: senescent plants) represent the average bacterial population densities of eight replicates. No signi¢cant di¡erence was observedbetween the means of the four plant lines (P = 0.7704) over all sampling times. Signi¢cant di¡erences (P6 0.0001) in bacterial plate countsin the rhizosphere were determined between the means of the di¡erent stages of plant development over all plant lines in 1997.

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3.2. Screening for bacteria isolated from therhizo- and geocaulosphere antagonistic againstE. carotovora and V. dahliae

A total of 3648 isolates (Table 3) were analyzedfor their ability to suppress the two phytopathogensE. carotovora and V. dahliae in a dual culture assay(Table 4). Ninety-six bacterial isolates (Table 3) wererandomly picked and investigated per plant line andsampling time. The percentage of antifungal isolatesin the rhizosphere of all plant lines varied stronglybetween the di¡erent samplings and ranged from 1

to 11. There were no signi¢cant di¡erences in thepercentage of antifungal bacteria isolated from therhizosphere between the four plant lines at any of thesamplings (Table 4). No antibacterial isolate was ob-served from the rhizosphere in 1996, whilst 40 wereisolated in 1997, suggesting temporal variation. In1997 the percentage of rhizosphere isolates with anti-bacterial activity ranged from 0 to 7.

In the geocaulosphere, antifungal bacteria repre-sented up to 8% of the population (Table 4). Signi¢-cant di¡erences between the lines were found in 1997for the ¢eld site in GL. On average 8% of the bac-

Fig. 3. Mean values þ S.E.M. of IAA-producing bacteria in the rhizosphere for the ¢eld site in GroM Lu«sewitz. Percentage of IAA-produc-ing bacteria in the rhizosphere of potato (mean þ S.E.M.) of the four plant lines DESI (non-transgenic), DC1 (transgenic control), DL4and DL5 (transgenic test lines). In the rhizosphere no signi¢cant di¡erence in IAA-producing bacteria was observed among the means ofthe four plant lines over all sampling times (P = 0.9475). Statistical signi¢cances were determined between the means of the six samplingtimes (1 and 4: young plants; 2 and 5: £owering plants; 3 and 6: senescent plants) over all plant lines (P6 0.0001).

Table 2Monitoring experiments to evaluate the e¡ects of T4-lysozyme-producing potato plants on plant-associated bacteria and root biomass

Sampling times 1996 and 1997 Sample material Monitoring experiments

1: young plants fresh roots CFUa of culturable bacteria2: £owering plants screening for bacteria antagonistic against E. c. a.b

screening for bacteria antagonistic against V. d.b

screening for IAA-producing bacteriac

identi¢cation of antagonistsd

3: senescent plants fresh roots and tubers CFUa of culturable bacteriascreening for bacteria antagonistic against E. c. a.b

screening for antagonistic bacteria against V. d.b

screening for IAA-producing bacteriac

identi¢cation of antagonistsd

whole fresh roots determination of root dry weight per plant

aColony-forming units (CFU) based on the total number of culturable bacteria on R2A medium containing cycloheximide (100 mg l31).bDual culture assay on Luria-Bertani agar (E. carotovora ssp. atroseptica) and Waksman agar (V. dahliae).cMicroplate method according to Sawar and Kremer [22].dFatty acid methyl ester (FAME) pro¢les, Biolog (Biolog, Hayward, CA), API 20E/API 20NE (bioMerieux, France).

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teria isolated from the transgenic line DL5 were an-tagonistic against fungi whilst from the controlplants only 0^2% of bacteria were antifungal. Butthese di¡erences were not observed either in the oth-er samplings or for the ¢eld site in QB. The percent-age of antibacterial isolates ranged from 0 to 2. Onlyone isolate of the geocaulosphere with antibacterialproperties against E. carotovora ssp. atroseptica wasisolated in the ¢rst year.

Overall, the percentage of antifungal bacteria inthe rhizosphere (GL: 5.8%) was signi¢cantly higher(P = 0.0187) than in the geocaulosphere (GL: 3.4%,and QB: 4.0%) over all plant lines. In general, sig-ni¢cantly more antifungal isolates were found in therhizo- and geocaulosphere than isolates with antibac-terial activity.

3.3. Screening for IAA-producing bacteria isolatedfrom the rhizo- and geocaulosphere

The same 3648 isolates (Table 3) from the twoinvestigated microenvironments were analyzed fortheir ability to produce the plant growth hormoneauxin (IAA equivalents) in vitro. A large numberof bacterial isolates from both microenvironmentswere able to excrete this plant growth hormone.The percentage of positive isolates in the rhizosphere

Table 3Composition of selected bacteria for investigating IAA-producingbacteria and antagonists

Number of rhizospherea isolates

Plant line Sampling times1996

Sampling times1997

Total

1 2 3 4 5 6

DESI 96 96 96 96 96 96 576DC1 96 96 96 96 96 96 576DL4 96 96 96 96 96 96 576DL5 96 96 96 96 96 96 576

Number of geocaulospherea;b isolates

Plant line Sampling time1996

Sampling time1997

Total

GL QB GL QBc

DESI 96 96 96 48 336DC1 96 96 96 48 336DL4 96 96 96 48 336DL5 96 96 96 48 336

aIsolates were randomly selected from R2A and subcultured inculture tubes on nutrient agar. The culture tubes were stored at4³C.bInvestigated only at the end of the growing season at both ¢eldsites (GL, GroM Lu«sewitz; QB, Quedlinburg).cReduced sample size due to partial destruction of the ¢eld.

Fig. 4. Mean values þ S.E.M. of IAA-producing bacteria in the geocaulosphere for the ¢eld sites in GroM Lu«sewitz and Quedlinburg. Per-centage of IAA-producing bacteria in the geocaulosphere of potato (mean þ S.E.M.) of the four plant lines DESI (non-transgenic), DC1(transgenic control), DL4 and DL5 (transgenic test lines). In the geocaulosphere no signi¢cant di¡erence in IAA-producing bacteria wasobserved among the means of the four plant lines over both locations (P = 0.9204). No signi¢cant di¡erence was found between the meansof the two locations (P = 0.5262) over all plant lines.

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Tab

le4

Per

cent

age

ofba

cter

iaan

tago

nist

icag

ains

tV

.da

hlia

ean

dE

.ca

roto

vora

inth

erh

izos

pher

ean

dge

ocau

losp

here

oftr

ansg

enic

and

non-

tran

sgen

icpo

tato

es

Rhi

zosp

here

Geo

caul

osph

ere

Lin

eSa

mpl

ing

tim

es19

96Sa

mpl

ing

tim

es19

97L

ine

GroM

Lu «s

ewit

zQ

uedl

inbu

rg

12

34

56

1996

1997

1996

1997

Per

cent

age

ofba

cter

iaan

tago

nist

icag

ains

tV

.da

hlia

eD

ESI

6.25

þ5.

988.

33þ

17.8

19.

37þ

12.1

42.

08þ

3.60

8.33

þ9.

313.

12þ

4.03

DE

SI6.

25þ

5.89

02.

08þ

3.85

6.25

þ7.

14D

C1

5.20

þ8.

832.

38þ

4.06

7.14

þ10

.12

5.20

þ8.

268.

33þ

11.0

29.

37þ

8.77

DC

14.

16þ

11.7

82.

08þ

3.60

4.16

þ6.

296.

25þ

5.51

DL

46.

25þ

7.38

4.16

þ6.

291.

04þ

2.94

8.33

þ7.

216.

25þ

5.51

4.16

þ5.

89D

L4

2.08

þ3.

852.

08þ

5.51

4.16

þ6.

294.

16þ

5.79

DL

56.

25þ

9.70

2.08

þ3.

854.

16þ

6.29

2.08

þ3.

6011

.45

þ7.

147.

29þ

6.50

DL

52.

08þ

3.85

8.33

þ9.

311.

04þ

2.94

4.16

þ5.

89P

erce

ntag

eof

bact

eria

anta

goni

stic

agai

nst

E.

caro

tovo

raD

ESI

00

00

3.12

þ5.

791.

04þ

2.75

DE

SI0

00

2.08

þ3.

60D

C1

00

03.

12þ

5.79

3.12

þ5.

796.

25þ

5.51

DC

10

00

0D

L4

00

02.

08þ

3.60

5.20

þ10

.97

4.16

þ8.

33D

L4

00

00

DL

50

00

2.08

þ3.

604.

16þ

5.89

7.29

þ8.

77D

L5

01.

04þ

2.75

02.

08þ

3.60

Bac

teri

aan

tago

nist

icag

ains

tV

.da

hlia

ein

the

rhiz

osph

ere

ofpo

tato

.D

ESI

(non

-tra

nsge

nic)

,D

C1

(tra

nsge

nic

cont

rol)

,D

L4

and

DL

5(t

rans

geni

cte

stlin

es).

On

the

basi

sof

3648

test

edba

cter

ial

isol

ates

(199

6:

1^3

;19

97:

4^6)

the

aver

age

ofan

tifu

ngal

bact

eria

was

dete

rmin

ed.

Inth

erh

izos

pher

eno

sign

i¢ca

ntdi

¡er

ence

inan

tifu

ngal

bact

eria

was

obse

rved

betw

een

the

mea

nsof

the

four

plan

tlin

es(P

=0.

8642

).N

ost

atis

tica

lsi

gni¢

canc

ew

asde

term

ined

betw

een

the

mea

nsof

the

six

sam

plin

gti

mes

over

all

lines

(P=

0.34

84).

Inth

ege

ocau

losp

here

nosi

gni¢

cant

di¡

eren

cein

anti

fung

alba

cter

iaw

asob

serv

edbe

twee

nth

em

eans

ofth

efo

urpl

ant

lines

(P=

0.93

53)

and

betw

een

the

mea

nsof

the

two

loca

tion

s(P

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FEMSEC 1045 23-7-99

J. Lottmann et al. / FEMS Microbiology Ecology 29 (1999) 365^377372

ranged from 8.4 to 67.7 (Fig. 3). In 1996, for allplant lines, the percentage of IAA-producing bacte-ria in the rhizosphere decreased from young to £ow-ering plants, and increased from £owering to senes-cent plants. No signi¢cant di¡erence could bedetected between the four plant lines DESI, DC1,DL4 and DL5 at any of the sampling times. In1997 £owering and senescent plants had signi¢cantlyhigher levels of IAA-producing bacteria than youngplants (Fig. 3).

In the geocaulosphere the percentage of positiveisolates ranged from 10.4 to 45.8. In 1996 higherlevels of IAA-producing bacteria in the geocaulo-sphere were found in the QB ¢eld, whereas in thesecond year the reverse result was obtained withhigher levels found at the GL site (Fig. 4).

In the rhizosphere signi¢cantly (P = 0.0035) higherpercentages of IAA-producing isolates (32.6%) werefound than in the geocaulosphere (GL: 29.9%, QB:24.0%) for all four plant lines.

3.4. Identi¢cation of bacterial antagonists

All isolates with antagonistic properties against E.carotovora and/or V. dahliae were characterized ac-cording to their fatty acid patterns (FAME). Isolateswhich could not be identi¢ed by FAME analysiswere additionally tested by Biolog and/or API. Therelative abundances of the common, recurrently iso-lated bacterial antagonists are shown in Table 5.From the 124 antagonistic bacteria characterizedfrom the six sampling dates, 28 species could be

Table 5Biodiversity of antagonistic bacteria associated with transgenic and non-transgenic potatoes

Species Number of isolates identi¢eda

rhizosphere geocaulosphere

DESI DC1 DL4 DL5 DESI DC1 DL4 DL5

Actinobacillus lignieresii ^ 1 ^ ^ ^ ^ ^ ^Agrobacterium tumefaciens 1 ^ ^ ^ ^ ^ 1 1Bacillus amyloliquefaciens ^ ^ ^ ^ ^ 1 ^ ^Bacillus cereus ^ ^ ^ ^ ^ ^ 1 ^Bacillus megaterium ^ ^ ^ ^ ^ 1 ^ ^Bacillus mycoides ^ ^ ^ ^ ^ ^ 1 ^Bacillus thuringiensis ^ ^ ^ ^ ^ ^ 1 ^Burkholderia cepacia 1 1 ^ ^ 1 ^ ^ ^Comamonas acidovorans ^ ^ ^ ^ 1 2 1 ^Enterobacter intermedius ^ ^ ^ ^ ^ ^ 1 ^Francisella philomiragia ^ 1 ^ ^ ^ ^ ^ ^Paenibacillus macerans ^ ^ ^ ^ ^ ^ ^ 1Paenibacillus polymyxa ^ ^ ^ 1 ^ ^ ^ ^Pantoea agglomerans ^ 2 3 3 ^ ^ ^ ^Proteus vulgaris ^ ^ 1 ^ ^ ^ ^ ^Pseudomonas chlororaphis 5 3 3 5 1 1 ^ ^Pseudomonas corrugata 1 ^ ^ 1 ^ ^ ^ ^Pseudomonas £uorescens 5 4 3 2 1 1 ^ 2Pseudomonas faecalis ^ ^ ^ 1 ^ ^ ^ ^Pseudomonas marginalis 2 2 ^ ^ ^ 1 1 ^Pseudomonas putida 1 7 2 4 3 4 2 5Pseudomonas savastoni ^ ^ 1 ^ ^ ^ ^ 1Pseudomonas syringae 2 3 1 1 ^ ^ ^ ^Serratia grimesii 3 1 ^ ^ ^ ^ ^ ^Serratia plymuthica ^ 1 ^ 1 ^ ^ ^ ^Serratia proteamaculans ^ ^ ^ 1 ^ ^ ^ ^Sphingomonas paucimobilis ^ ^ ^ ^ ^ 1 ^ ^Stenotrophomonas maltophilia 1 ^ ^ ^ 1 ^ 1 2

aIdenti¢cation of 28 species based on FAME analysis or API 20E/20NE and Biolog.

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identi¢ed. Most (71.4%) of the identi¢ed species wereGram-negative, belonging to the group of £uorescentpseudomonads. Pseudomonas putida was the mostcommon species comprising 22.6% of all antagonisticisolates. Pseudomonas chlororaphis and P. £uorescenseach comprised another 14.5% of the antagonisticisolates. Non-£uorescent pseudomonads which couldbe identi¢ed were Burkholderia cepacia, Sphingomo-nas paucimobilis or Stenotrophomonas maltophilia. Incontrast, only 6.5% of the antagonistic bacteria(28.6% of the identi¢ed species) were Gram-positive.All of them belonged to the genera Bacillus or Pae-nibacillus. For 14 other species, only one antagonisticisolate could be detected.

Seven species, Actinobacillus lignieresii, Bacillusamyloliquefaciens, Bacillus megaterium, Burkholderiacepacia, Francisella philomiragia, Serratia grimesiiand Sphingomonas paucimobilis, were isolated onlyfrom control plants (DESI, DC1). These isolates rep-resented 9.7% of all identi¢ed antagonistic isolates inthis work.

3.5. Determination of root biomass

At the end of the growing season the dry weight ofroots was determined. Over the monitoring period,the root biomass of the transgenic line DL4 wassigni¢cantly (P = 0.0001) lower than the biomassesof the three other lines DESI, DC1 and DL5 (Table

6). No signi¢cant di¡erence was found between theroot dry weights of these three plant lines. The meandry weight of DESI, DC1 and DL5 was 4.7 g perroot.

4. Discussion

The secretion of the T4-lysozyme into the intercel-lular spaces changes the composition of commonroot exudates or of common compounds of thewhole plant tissues of transgenic potato plants.Over the period of ¢eld experiments in 1996 and1997, expression of the foreign T4-lysozyme genewas monitored by Western blotting analysis (datanot shown) using an a¤nity-puri¢ed polyclonalanti-T4-lysozyme antibody from rabbit [13]. T4-lyso-zyme could be detected in protein extracts in eachsample (leaves) over the two growing seasons. Thepercentage levels of total soluble protein varied from3 to 6U1034 (1996) and 7 to 10U1034 (1997) [23].The expression level of T4-lysozyme was higher forthe transgenic line DL4 than for the line DL5 [23].

The plant species-dependent [24] or cultivar-de-pendent [25] colonization of the rhizosphere by bac-teria has been reported. Oger et al. [4] demonstratedthat the increased number of mannopine (producedby transgenic potatoes)-utilizing bacteria detected inthe rhizosphere of transgenic plants appears to berelated to the production of mannopine by theplants. All these reports support the thesis of Rovira[26] that root exudates play a key role in the selectivestimulation of microorganisms. If there is any e¡ectof T4-lysozyme in potato, it should be detected uponinvestigation of root- and tuber-associated bacteria.The results obtained from all the experiments in thisstudy indicated that T4-lysozyme produced by trans-genic potatoes did not cause any detectable e¡ect onthe counts of culturable aerobic bacteria. There wasno signi¢cant di¡erence in total bacterial counts be-tween the four plant lines in both microenviron-ments. The detected aerobic plate counts in the rhi-zosphere (log10 7.7 CFU g31 root fw) werecomparable with counts determined on other hostplants, e.g. log10 7^8 CFU g31 fw for barley andwheat [27], log10 8 CFU g31 fw for maize [28], andlog10 8.17 CFU g31 fw for oilseed rape [29].Although only 1^10% of bacteria can be recovered

Table 6Biomass of roots of the four plant lines at the end of growingseason at the ¢eld sites of GroM Lu«sewitz (GL) and Quedlinburg(QB)

Line Root dry weight (g root31)a

1996 1997

GL QB GL QBb

DESI 4.4 4.4 5.9 n.i.DC1 3.6 4.1 5.5 n.i.DL4 2.8c 2.3c 2.9c n.i.DL5 4.0 3.8 6.4 n.i.

aThree roots from one plot were sampled into Cryovac bags(Grace Multi£ex GmbH, Flensburg, Germany) and treated asone sample; samples (eight replicates per plant line) wereweighed and dried for 24 h at 105³C.bNot investigated (n.i.).cSigni¢cantly lower than root dry weights of the other four plantlines.

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[30], the widely used methodology (dilution plates),despite its faults and problems, is still much used[15]. However, the e¡ect on the larger group of un-culturable bacteria could not be detected with thismethod. Therefore, culture-independent methods,such as temperature or denaturing gradient gel elec-trophoresis, should be used [31,32].

No di¡erence in the percentage of potentially ben-e¢cial bacteria between di¡erent plant lines was ob-served. With respect to the distribution of bene¢cialbacteria, the abundance of IAA-producing isolateswas signi¢cantly higher than that of isolates whichwere antagonistic. These two parameters, IAA pro-duction and antagonistic activity, were chosen tomonitor the main functions of potentially bene¢cialbacteria. Microbially derived IAA has been impli-cated in the stimulation of growth or pathogenesisof plants [33]. Since auxins are also produced byplants, one way to prove the involvement of bacterialIAA in the stimulation of plant growth would be totest either Tn5 mutants de¢cient in IAA productionor mutants overproducing these substances. Barbieriand Galli [34] showed that mutants of Azospirillumbrasiliense de¢cient in IAA production did not pro-mote the formation of lateral roots as compared tothe wild-type. Conversely, an IAA-overproducingmutant of P. £uorescens stimulated the root develop-ment of cherry [35]. These results indicate a concen-tration-dependent e¡ect of IAA. Concentrations ofIAA produced by the investigated isolates were com-parable with those of other plant growth-promotingrhizobacteria [21,36]. Only a small number of bacte-ria were antagonistic to our model pathogens. Theproportion of the isolates which were antagonisticranged from 1 to 10%. Hebbar et al. [37] reportedthat 1^7% of the rhizobacteria of maize were anti-fungal. Kremer et al. [38], who studied the ecology ofthe rhizosphere of di¡erent weeds, reported thatantifungal bacteria represented up to 18% of cultur-able bacteria.

The results do not indicate any in£uence of trans-genic T4-lysozyme on the function of potentiallybene¢cial bacteria. However, it is possible that thisfunction was performed by other bacterial speciesbecause of the varying sensitivity of rhizobacteriato T4-lysozyme [10]. To analyze the compositionand biodiversity of these bene¢cial bacteria the ac-tive isolates were identi¢ed by various methods. The

species composition of the antagonistic bacteria al-lowed us to estimate potential changes in the bacte-rial populations between transgenic and non-trans-genic plants. Antagonistic strains of seven specieswere only found on control plants. Most of themwere found once, with the exception of Serratia gri-mesii which was found four times. However, thecomposition of the bacterial community in the rhizo-sphere is in£uenced by various abiotic and bioticconditions and the speci¢city of each plant is alsovery high [28]. Considering this, the di¡erences foundwere relatively low. Further approaches, with theinoculation of seed potatoes with S. grimesii strainsor other T4-lysozyme-sensitive isolates, could clarifypossible e¡ects of transgenic T4-lysozyme. The re-lease of both a modi¢ed P. £uorescens and thewild-type resulted in signi¢cant, but transient, per-turbations of some of the culturable componentsof the indigenous microbial community of wheat[39,40].

Twenty-eight species with antagonistic e¡ects wereidenti¢ed. The group of £uorescent pseudomonadsassociated with potatoes included P. chlororaphis,P. £uorescens and P. putida. They were reported tobe the most bene¢cial and antifungal rhizobacteriaof potato [41,42]. The non-£uorescent pseudomo-nads, e.g., Stenotrophomonas maltophilia or Burkhol-deria cepacia, were also reported as members of thenaturally occurring rhizosphere community [43,44].Other qualitatively important species were membersof the genus Serratia. They were also isolated asantagonistic strains from roots [45], but the detectedspecies S. grimesii has not been previously reportedas a plant-associated bacterium.

Determination of the root weight allows indirectconclusions concerning the in£uence of T4-lysozymeproduced by transgenic potato plants on plantgrowth-promoting rhizobacteria. If the root develop-ment is disturbed, the plant growth and health maybe clearly reduced. Di¡erences in root dry weightwere observed for both years among the transgenicline DL4 and the three other lines (DESI, DC1 andDL5). The reason for this di¡erence is unclear. Thetwo transgenic T4-lysozyme-expressing lines di¡er inexpression level to the e¡ect that DL4 shows higherlevels than DL5 [23]. In several laboratory and ¢eldstudies there have been unintentional changes inplant characteristics due to genetic manipulation

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and position e¡ects from insertion site(s) [6]. Wesuppose the phenotypic deviation of DL4 is causedby a genotypic position e¡ect or by a somaclonalvariation.

The results suggest that the methods used weresuitable because they were statistically valid: e.g.,signi¢cant di¡erences in culturable geocaulospherebacteria between di¡erent soil types, in the percent-age of strains antagonistic against V. dahliae betweenthe two locations, and in the percentage of IAA-pro-ducing strains between the sampling times.

The monitoring of plant-associated bacteria andcomparison of the collected data with establishedinformation should be included as a risk assessmentprocedure regarding plant-associated bacteria [7].Our results provide an example for performing riskassessment studies for transgenic plants and forstudying the e¡ects on potentially bene¢cial bacteriaunder a variety of environmental conditions, e.g.,di¡erent soil types.

Acknowledgements

The authors thank Kerstin Schmidt (Fa. BioMath,Rostock, Germany) for ¢eld design and statisticalanalysis, Prof. Kroppenstedt and his group for helpin performing the FAME analysis, Hella Goschkeand Henrike Westphal for excellent technical assis-tance. The research was supported by Grant0311299A from the Bundesministerium fu«r Bildung,Wissenschaft, Forschung und Technologie (BMBF).

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