(2.0)souto y col 2004 identification bacillus iturin j.appl.microbiol

Genetic and functional characterization of a Bacillus sp. strainexcreting surfactin and antifungal metabolites partiallyidentified as iturin-like compounds

G.I. Souto, O.S. Correa, M.S. Montecchia, N.L. Kerber, N.L. Pucheu, M. Bachurand A.F. GarcıaCatedra de Microbiologıa, Facultad de Agronomıa, UBA and Instituto de Investigaciones Bioquımicas y Fisiologicas (IBYF-CONICET),

Ciudad Autonoma de Buenos Aires, Argentina

2004/0328: received 23 March 2004, revised 28 June 2004 and accepted 29 June 2004

ABSTRACT

G. I . SOUTO, O.S . CORREA, M.S. MONTECCHIA, N.L . KERBER, N .L . PUCHEU, M. BACHUR AND

A.F . GARC IA . 2004.

Aims: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium,

Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean.

Methods and Results: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were

resistant over a wide pH range and to hydrolytic enzymes. By 1H-NMR and thin-layer chromatography analyses

surfactin and iturin-like compounds were partially identified. Moreover, soya bean seeds bacterization with BNM

122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene.According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis.PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic

fingerprinting revealed a close genetic relationship to B. amyloliquefaciens. However, by physiological character-

ization using API tests, this strain resembled more B. subtilis.Conclusions: This is the first report describing the co-production of surfactin and iturin-like compounds by a

putative strain of B. amyloliquefaciens. The synergistic effect of both lipopetides is a remarkable trait for a candidate

biocontrol agent.

Significance and Impact of the Study: This kind of research has relevance in order to minimize the use of

synthetic fungicides and surfactants, contributing to the preservation of the environment.

Keywords: Bacillus amyloliquefaciens, biological control, compost-based formulation, fungal phytopathogens,

surfactin and iturin co-production.

INTRODUCTION

Plant fungal diseases reduce yield and productivity of several

economical crops all over the world. Resistant plant

cultivars, cultural practices and chemical applications are

routinely used to provide disease control. However, resistant

cultivars for every disease are not available and cultural

practices are not always economically or technologically

feasible. Moreover, available chemical fungicides are often

expensive and also have adverse effects on human beings.

Therefore, biological control appears to constitute an

alternative strategy for controlling diseases, perhaps as part

of an integrated control system, thus reducing the use of

chemical products and contributing to the preservation of

the environment.

Correspondence to: Olga S. Correa, Catedra de Microbiologıa, Facultad de

Agronomıa, UBA and Instituto de Investigaciones Bioquımicas y Fisiologicas

(IBYF-CONICET), Av. San Martın 4453, C1417DSE, Ciudad Autonoma de

Buenos Aires, Argentina (e-mail: [email protected]).

ª 2004 The Society for Applied Microbiology

Journal of Applied Microbiology 2004, 97, 1247–1256 doi:10.1111/j.1365-2672.2004.02408.x

The use of bacteria as biocontrol agents has been

extensively studied (Expert and Digat 1995; Asaka and

Shoda 1996; Podile and Prakash 1996; Kim et al. 1997; Mao

et al. 1997; Singh et al. 1998; de Vrije et al. 2001). The heat-and desiccation-resistant structures of spore-producing

Gram-positive bacteria can be readily formulated into stable

products (Handelsman and Stabb 1996). In particular,

different Bacillus species excrete peptides and lipopeptides

to the culture medium, such as fungicine, iturin, bacillom-

icine and others, having antifungal properties (Katz and

Demain 1977; Jacques et al. 1993; Zuber et al. 1993;

Lebbadi et al. 1994; Eshita et al. 1995; Kajimura et al.1995; Yakimov et al. 1995; Yu et al. 2002; Chitarra et al.2003; Cho et al. 2003). These antifungal peptides inhibit thegrowth of a large number of fungi, including Aspergillus,Penicillium and Fusarium species (Munimbazi and Buller-

man 1998), as well as some yeasts (Thimon et al. 1995). Inthis paper, we show that a Bacillus sp. strain, designated as

BNM 122, was effective in vitro against several plant fungal

pathogens. We also investigated the mechanisms of biolo-

gical activity and we tested a compost-based formulation of

BNM 122 against soya bean damping-off caused by

Rhizoctonia solani. Furthermore, BNM 122 has been

characterized by means of its phenotypic characteristics as

well as by sequencing its 16S rDNA, PCR analysis of the

16S-23S rRNA intergenic spacer region (IGS-PCR) and

repetitive sequence-based PCR (rep-PCR) genomic finger-

printing.

MATERIALS AND METHODS

Bacterial strains and culture conditions

The Bacillus strain used in this paper was isolated from a

sclerotium of Sclerotinia sclerotiorum withdrawn from a

sunflower (Heliantus annus L.) capitulum. The sclerotium

was superficially disinfected with 2% (v/v) NaClO for

5 min and exhaustively washed with sterile distilled water. It

was placed on potato glucose agar (PDA) and incubated at

30�C for 5 days. There was an abundant growth of mycelia

from the sclerotium except in a region of the plate where

bacterial growth was evident. A Gram-positive, aerobic,

endospore-forming, rod-shaped bacterium was isolated from

that plate and it was designated as BNM 122.

The reference strains used in the present study were:

Bacillus amyloliquefaciens DSM 7T, B. amyloliquefaciensDSM 1060, B. subtilis subsp. subtilis DSM 10, B. subtilisDSM 1088, B. licheniformis DSM 1913 and Bacillus sp.

DSM 1325, from the Deutsche Sammlung von Mikroor-

ganismen und Zellkulturen GmbH (DSMZ). All the strains

were routinely grown aerobically at 28�C in nutrient broth

(NB) or nutrient agar (NA). All the strains were stored at

)50�C in NB with 30% glycerol.

Fungus isolation and culture conditions

The fungal strains were obtained from strain collection of

the Banco Nacional de Microorganismos, Catedra de

Microbiologıa Agrıcola, Facultad de Agronomıa, Universi-

dad de Buenos Aires, Argentina. These fungi were routinely

grown on PDA at 28�C and stored on the same medium at

4�C.

Antifungal activity of the whole cultureof Bacillus sp. BNM 122

A bacterial culture grown in NB to a concentration

of ca 108 CFU ml)1 was streaked in a straight line on one

side of a Petri dish (3 cm from the centre) containing NA,

PDA or a mix (1 : 1, v/v) of NA : PDA. Simultaneously a

9 mm diameter agar plug containing fungal mycelium of

Fusarium oxysporum f. sp. lycopersici, F. solani, R. solani, orS. sclerotiorum, grown in PDA for 48 h, was placed in the

centre of the plate. After 7 days at 28�C the inhibitory effect

on fungal growth was evaluated. All in vitro antagonism

assays were made in triplicate.

Activity of cell-free supernatants onS. sclerotiorum ascospore germination

Cell-free BNM 122 cultures grown for 72 h were concen-

trated by freeze-drying to recover the excreted antifungal

metabolites. The lyophilized metabolites were dissolved in

distilled water to obtain a fivefold concentrated preparation

(5·) and were sterilized by filtration (0Æ2 lm pore-size

membrane). The evaluation of their activity on the germi-

nation of S. sclerotiorum ascospores was performed by

mixing on microslides 25 ll of the concentrate preparation

with 25 ll of ascospores suspension (2 · 108 asco-

spores ml)1 in 13% sucrose in sterile distilled water).

Controls consisted of 25 ll of NB filter-sterilized, plus 25 llof the same ascospores suspension. After incubation for 16 h

at 28�C in a humidity chamber, germination of the

ascospores was microscopically evaluated. All assays were

performed in triplicate.

Stability of cell-free supernatants

The concentrated preparation of antifungal metabolites

was tested for resistance to temperature, pH and hydro-

lytic enzymes. All assays were performed as described by

Lebbadi et al. (1994). To evaluate the residual antifungal

activity after each treatment, 50 or 100 ll of the treated

antifungal solutions were placed into wells made in

opposite sides of PDA plates while, in the centre of each

plate, a plug of actively growing fungal mycelium

was simultaneously inoculated. Inhibition strength was

1248 G. I . SOUTO ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 1247–1256, doi:10.1111/j.1365-2672.2004.02408.x

assumed to be proportional to the clear, mycelia-free

zones, appearing around the wells after 5 days at 28�C.All assays were performed in triplicate against all fungal

pathogens.

Preparation of antifungal metabolites frombacterial cultures

BNM 122 was grown in NB for 72 h. Cell-free supernatant

(15 000 g for 15 min at 4�C) was precipitated at 80%

ammonium sulphate saturation and the precipitate was

dissolved in distilled water. The remaining ammonium

sulphate was removed by extensive dialysis against distilled

water (MWCO 12 KDa, cellulose dialysis tubing from

Sigma Chemical Co.). The solution was freeze-dried and

stored at )20�C.

1H-NMR analysis

A freeze-dried sample of antifungal metabolites was dis-

solved in acetone-d6 and1H-NMR, correlated spectroscopy

(COSY90), total correlation spectroscopy (TOCSY) and

heteronuclear correlations (HMQC) were recorded on a

Bruker Avance DPX 400 spectrometer operating at

400 MHz (Bruker BioSpin, Ontario, Canada). The spectra

were compared with those produced by commercially

available surfactin from B. subtilis (Sigma Chemical Co.).

Thin-layer chromatography analysis (TLC)

Cell-free supernatants from 72 h grown bacterial cultures

were precipitated with 70 mMM MnCl2 as described by

Feignier et al. (1995). The pellet was dialysed against

distilled water and freeze-dried. Antifungal metabolites were

extracted from the lyophilized material with chloro-

form : methanol 2 : 1 (v/v) and the extract was applied to

silica gel 60 TLC plates (Merck, Darmstadt, Germany) and

run in chloroform : methanol : water 65 : 25 : 4 (v/v/v) as

described by Sandrin et al. (1990). The Rf of the compounds

were compared with those of pure lipopeptides surfactin and

iturin A from B. subtilis (Sigma).

Soya bean seed bacterization

BNM 122 cultures grown in NB for 72 h were harvested by

centrifugation and the pellet was lyophilized (LP) and kept

at room temperature pending its use. Soya bean seeds were

surface disinfected 2 min with 2% NaClO and exhaustively

washed with sterile tap water. The bacterization was made

by mixing seeds with a suspension of LP in sterile distilled

water plus 0Æ5% carboxy-methyl-cellulose (CMC). The

mean number of BNM 122 adhering to the seeds

(4 · 107 CFU per seed) was determined by dilution plating.

Control seeds were treated in the same way that those

bacterized but without LP. Seeds were pregerminated in

moist chambers at 25�C for 48 h.

Plant growth chamber assays

The soil used was a commercial mix (7% organic matter,

13Æ9 C/N, pH 5Æ0). Soil was artificially infested by

R. solani to evaluate the potential biocontrol of BNM 122.

Fungal inoculum was prepared from R. solani grown for

2 weeks at 28�C on autoclaved wheat seeds. Sterilized soil

by tyndallization was infested with 0Æ5% fungal inoculum.

Ten pregerminated seeds were planted in 1 l plastic pots

filled with the soil mix with or without fungal inoculum.

Plants were conducted in plant growth chamber at 25�Cand 12 h light (15 000 lux) and maintained at field

capacity with tap water. Treatments were: (i) nonbacter-

ized seeds in soil without R. solani; (ii) bacterized seeds in

soil with 0Æ5% fungal inoculum; (iii) nonbacterized seeds

in soil with 0Æ5% fungal inoculum. Ten replicates of each

treatment were performed in a completely randomized

block design. Growth chamber assays were repeated three

times.

Greenhouse experiment

Greenhouse experiments were conducted under natural

temperature and light conditions. Seeds were surface

disinfected in the same way as above and were sown

immediately after treatments (not pregerminated). Com-

mercial compost was sterilized by autoclaving in polypro-

pylene bags. An LP suspension in sterile distilled water was

added to 200 g of compost and kept 1 week at 28�C. Thisformulated product with a BNM 122 concentration of

1 · 109 CFU g)1 was mixed with soya bean seeds plus a

water suspension of 0Æ5% CMC as adhesive. Before sowing

the mean number of BNM 122 adhering to the seeds

(5 · 107 CFU per seed) was determined on NA.

Treatments were: (i) seeds coated with sterile compost

and sowed in soil without R. solani; (ii) seeds coated with the

BNM 122 compost-based formulation and sowed in soil

without fungal inoculum; (iii) seeds coated with the BNM

122 compost-based formulation in soil with 0Æ5% fungal

inoculum; (iv) seeds coated with sterile compost sowed in

soil with 0Æ5% R. solani containing 500 ll of pentachloro-nitrobenzene (PCNB) water suspension (0Æ5%, w/v) applied

to soil where seed was located; (v) seeds coated with sterile

compost in soil with 0Æ5% R. solani seeds coated with sterile

compost in soil with 0Æ5% R. solani. Ten seeds were sowed

in 1 l plastic pots filled with the soil mix. Pots were watered

with tap water and maintained at field capacity. Ten

replicates of each treatment were performed in a completely

randomized block design.

GENETIC AND FUNCTIONAL CHARACTERIZATION OF A BACILLUS SP. 1249


Statistical analysis

Analysis of variance was performed using the general linear

models procedure of SAS and mean values were compared

using Fisher’s protected least significant difference (SAS

Institute, Cary, NC, USA).

Bacterial identification

Identification of BNM 122 strain was carried out using the

API 50CHB and API 20E tests (bioMerieux, Marcy l’Etoile,

France) as recommended by the manufacturer and the

sequence of its 16S rDNA was determined. These studies

were complemented by 16S-23S rRNA IGS-PCR analysis

and rep-PCR genomic fingerprinting (Jensen et al. 1993;

Versalovic et al. 1994).The determination of the 16S rRNAgene sequence of strain

BNM 122 was performed by MIDI Labs (Newark, DE,

USA). For PCR fingerprinting, total genomic DNA of the

bacteria was prepared from NB cultures using the Wizard

genomic DNA purification kit (Promega Inc., Madison, WI,

USA) and adjusted to a concentration of 50 ng ll)1. All DNA

preparations were stored at 4�C. To amplify the 16S-23S

rDNA intergenic spacer region, primers FGPS1490 and

FGPS132¢ were used (Laguerre et al. 1996). The reactions

were carried out in a total volume of 50 ll containing 50 ng of

DNA, 5% dimethyl sulphoxide, 1Æ5 mMM MgCl2, 0Æ2 mMM of

each dNTP, 0Æ3 lMMof each primer, 1Æ25 U ofTaq polymerase

(Promega) and the buffer provided with the enzyme.

Amplifications were carried out in an MJ Research PTC-

100 thermocycler with the following temperature programme:

initial denaturation for 5 min at 95�C followed by 30 cycles

each consistingof denaturation (94�C,1 min), annealing (55�C,40 s) and extension (72�C, 2 min)with a final extension step at

72�C for 8 min. Five ll of the PCR products were loaded onto

10 cm-long 2% Metaphor agarose gels (FMC Bioproducts,

Rockland, ME, USA) and run at room temperature in TBE

buffer (89 mMMTris, 89 mMMBoric acid, 2 mMMEDTA, pH 8Æ0)at 5 V cm)1 for 2Æ5 h. As size control, a 100 bp DNA ladder

(Promega) was included. Rep-PCR genomic fingerprinting

was performed with BOXA1R, REP (REP1R-I and REP2-I)

and ERIC (ERIC1R and ERIC2) primers, as previously

described by Versalovic et al. (1994). Eight ll of the PCR pro-

ductswere run in 1Æ5%agarose gels inTBEbuffer at 5 V cm)1

for 2 h. As reference, 1 kb DNA ladder (Promega) was used.

Gels were stained with ethidium bromide (0Æ6 lg ml)1), and

photographed with a Polaroid type 667 film.

Nucleotide sequence accession number

The 16S rDNA sequence determined for strain BNM 122

was submitted to the GeneBank database under accession

number AF411118.

RESULTS

Antifungal activity of Bacillus sp. BNM 122

Mycelia growth of F. oxysporum f. sp. lycopersici, F. solani,R. solani and S. sclerotiorum was inhibited using the dual

culture technique in all media tested. Figure 1 shows the

myceliar growth and sclerotia production of S. sclerotiorum

Fig. 1 Effect of Bacillus sp. BNM 122 on S. sclerotiorum growth and

sclerotia production in dual culture. Plate A shows a pure culture of

S. sclerotiorum and its abundant sclerotia formation. Plate B shows the

fungal growth inhibition by BNM 122 (PDA : NA medium) displaying

a scarce sclerotia production. An arrow points to a clear zone

showing growth inhibition of mycelia



in PDA : NA medium (Fig. 1A). When S. sclerotiorum was

challenged with the bacterial culture (Fig. 1B) growth

inhibition of mycelia occurred accompanied by a decreased

sclerotia production.

Effect of cell-free supernatants on S. sclerotiorumascospores germination

The germination of ascospores and the inhibitory effect of

supernatant are shown in Fig. 2. There was total inhibition

on S. sclerotiorum ascospores germination using the bacterial

culture supernatant of 72 h (Fig. 2). We observed inhibition

of ascospores germination also with 12-, 24- and 48-h

supernatant (data not shown).

Stability of excreted antifungal metabolites

The effects of autoclaving, pH and hydrolytic enzymes upon

the antifungal activity of cell-free supernatants are shown in

Table 1. No significant difference in the size of the inhibited

zone was found comparing the control and the treatment at

121�C for 20 min. Treatments at pH from 4Æ0 to 10Æ0 did

not affect the activity but it was completely lost at pH 2Æ0.Proteinase K, trypsin and lipase A had no effect on the

antifungal activity. The same results were observed with the

precipitated metabolites (data not shown).

The antifungal activity of freeze-dried extracts was also

stable when dissolved in chloroform : methanol 2 : 1 (v/v)

or 90% acetone.

1H-NMR analysis

1H-NMR spectra of B. subtilis surfactin and the metabolites

excreted to the culture medium by Bacillus sp. BNM 122

were identical, and in addition an unidentified minor

compound was also detected in our preparation (Fig. 3).

By HMQC, peak position of the seven amino acids of

commercial surfactin was compared with the main metabo-

lite of BNM 122, showing complete identity between both

samples (data not shown).

Analysis of antifungal components by TLC

The TLC profiles of the lyophilized organic extract revealed

two main fractions, one showing an identical chromato-

Fig. 2 S. sclerotiorum ascospores germination and their inhibition by

antifungal metabolites excreted by Bacillus sp. BNM 122. Photomi-

crographs (40·) of ascospores germination in the presence of NB

(control) and total inhibition by BNM 122 excreted metabolites (72 h

supernatant). Bars ¼ 1Æ1 lm

Table 1 Stability of the antifungal metabolites excreted by Bacillus sp.

BNM 122 to heat, hydrolytic enzymes and pH, tested against

Sclerotinia sclerotiorum

Treatments Inhibition zone diameter* (mm)

Temperature

Control 30Æ0 ± 0Æ2121�C for 20 min. 29Æ5 ± 0Æ4

Enzymes

Control 27Æ0 ± 0Æ3Proteinase K 26Æ5 ± 0Æ5Tripsin 26Æ2 ± 0Æ6Lipase A 27Æ2 ± 0Æ3

pH

Control 27Æ5 ± 0Æ3pH 2Æ0 NI

pH 4Æ0 27Æ3 ± 0Æ2pH 8Æ0 27Æ3 ± 0Æ4pH 10Æ0 27Æ0 ± 0Æ3

*Diameter of mycelia-free zone (mm) around the wells inoculated with

excreted antifungal metabolites.

NI, no inhibition. Values are mean ± S.D.S.D. (n ¼ 3).



graphic mobility to B. subtilis surfactin (Rf 0Æ68) and the

other one to iturin A (Rf 0Æ51). Furthermore, two additional

compounds (Rf 0Æ41 and Rf 0Æ38) were detected, whereas anintensely pigmented fraction remained at the origin (data not

shown).

Biological control on soya bean seeds

Assays in plant growth chamber. The number of plants

per pot was recorded 15 days after sowing. Table 2 shows

the results obtained in a representative growth chamber

experiment. Soya bean seed treatments with strain BNM

122 (biocontrol treatment) resulted in a significant

(P £ 0Æ05) increase in mean stand per pot compared with

the pathogen check. However, the mean stand per pot from

biocontrol treatment was significantly lower (P £ 0Æ05) thanthat of the healthy check. A reinforcement of bacterial

treatment through irrigation applied to the shoot base,

immediately after emergence, did not increase significantly

the number of plants per pot (data not shown).

Greenhouse assay. In greenhouse, soya bean seed treat-

ment with the compost-based formulation resulted in a

mean stand per pot that was without significant difference

(P £ 0Æ01) with the healthy check and it was as effective as

the PCNB treatment (Table 2). Soya bean seed treatment

with BNM 122 together with the pathogen resulted in mean

plant weight and mean plant height that were significantly

greater (P £ 0Æ05) than the pathogenic check but lesser in

weight than the healthy and bacterial check (Table 2). On

the other hand, there was no evidence of phytotoxicity to

soya bean due to seed treatment with BNM 122 (bacterial

check). The smaller stand of plants per pot obtained in

greenhouse assay compared with the stand in growth

chamber (Table 2) was because of the use of non-preger-

minated seeds.

Identification of strain BNM 122

Molecular and biochemical assays were used to identify the

strain BNM 122. The biochemical profiling obtained by

using the API systems did not produce conclusive results.

By using API 50CHB only 47% identity (Id) with B. subtilisand 36% Id with B. licheniformis were obtained. By

combining the results of that kit with those produced using

API 20E, our isolate showed 76Æ7% Id with B. subtilis,18Æ1% Id with B. licheniformis and 3Æ7% Id with

B. amyloliquefaciens.The almost complete 16S rRNA gene sequence deter-

mined for strain BNM 122 consisted of 1545 nucleotides.

On the basis of 16S rDNA sequence analysis, this strain

appeared to belong to the genus Bacillus, �B. subtilis group�,being closely related to B. subtilis and B. amyloliquefaciens.These species show a very high similarity level of their 16S

rDNA sequences and are characterized by a strict phylo-

genetic relationship (Ash et al. 1991).The assessment of a more variable region of the rRNA

operon, enabling the differentiation of these closely related

Bacillus species, revealed identical IGS-PCR patterns for

strain BNM 122 and also for two reference strains of

B. amyloliquefaciens, and differing, from the patterns dis-

played by B. subtilis, B. licheniformis and Bacillus sp. (Fig. 4).Therefore, BNM 122 and the B. amyloliquefaciens strains

5 4 3 2 1 0

p.p.m.

p.p.m.

5 4 3 2 1 0

–3·7

64–4

·015 –2

·221

–1·9

11

–1·3

01

–0·8

68

–2·0

75

–2·8

30

–0·9

86

–2·7

91

–2·2

26–2

·075

–1·9

11

–1·3

08 –0·8

74–0

·986

(a)

(b)

Fig. 3 1H-NMR spectra of B. subtilis surfactin (a) and the antifungal

metabolites excreted to the culture medium by the strain BNM 122 (b).

The bar points to the main difference between both spectra



were analysed by rep-PCR in order to further determine the

identity of the biocontrol strain and to distinguish those

strains from each other. Figure 5 shows the fingerprints

obtained with BOX, REP and ERIC primers. The strain

DSM 1060 had unique genomic fingerprints with each

primer set, but strains BNM 122 and DSM 7T showed

identical BOX and REP-PCR fingerprints, indicating their

closely genetic relationship. However, strain BNM 122

could be distinguished from B. amyloliquefaciens DSM 7T

by using ERIC primers (Fig. 5).

DISCUSSION

Bacillus strain BNM 122 isolated in our laboratory excreted

metabolites with antifungal activity against mycelia growth

of F. oxysporum f. sp. lycopercisi, F. solani, R. solani and

S. sclerotiorum. Those compounds efficiently inhibited

in vitro ascospore germination and sclerotia production of

S. sclerotiorum.The antifungal activity was resistant to high temperature,

a wide range of pH and the action of many hydrolytic

Table 2 Biocontrol of damping-off of soya

bean caused by Rhizoctonia solani with Bacil-

lus sp. BNM 122

Treatments

Growth

chamber Greenhouse

Weight* (mg) % Height* (cm) %Stand* % Stand* %

Healthy check 9Æ8a 100 8Æ1a 100 157a 100 9Æ5a 100

Bacterial check n.d. 8Æ2a 101 154a 98 9Æ2a 97

BNM 122 + R. solani 8Æ2b 83 8Æ3a 102 134b 85 10Æ2a 107

PCNB + R. solani n.d. 8Æ2a 101 127b 80 9Æ3a 98

Pathogenic check 6Æ4c 65 5Æ5b 68 65c 41 7Æ5b 79

*Mean values (n ¼ 100) followed by the same letter in each column are not significantly different

(P £ 0Æ05). Stand: mean plant per pot, weight: mean plant dry weight per pot, height: mean plant

height per pot.

n.d., not determined.

Fig. 4 IGS-PCR fingerprinting of Bacillus strains. Lane: M, 100 bp

DNA ladder; lane 1, B. amyloliquefaciens DSM 1060; lane 2, B.

amyloliquefaciens DSM 7T; lane 3, B. licheniformis DSM 1930; lane 4,

Bacillus sp. BNM 122; lane 5, B subtilis subsp. subtilis DSM 10; lane 6,

B. subtilis DSM 1088; lane 7, Bacillus sp. DSM 1025

Fig. 5 rep-PCR-generated genomic fingerprints of Bacillus sp. BNM

122 and B. amyloliquefaciens strains. Lane: M, 1 kb DNA ladder; lanes

1–3, BOX-PCR; lanes 4–6, ERIC-PCR; lanes 7–9, REP-PCR of

B. amyloliquefaciens DSM 1060, B. amyloliquefaciens DSM 7T and

Bacillus sp. BNM 122 respectively



enzymes. These characteristics indicate that the antifungal

compounds may belong to the iturin group of antibiotics

(Chitarra et al. 2003). Bacteria of the genus Bacillus are

known as producers of a number of peptides with antibiotic

properties effective against bacteria, fungi and yeasts (Katz

and Demain 1977) and also with a high stability attributable

to their structure. They are small or cyclic lipopeptides

having uncommon amino acids as constituents, such as

ornithine or DD-amino acids (Lebbadi et al. 1994; Munimbazi

and Bullerman 1998). B. subtilis is considered the major

producer of those antibiotic peptides and a B. subtilis strainproducing iturin A and surfactin was shown to be effective

for the control of damping-off caused by R. solani in tomato

plants (Asaka and Shoda 1996). These previous results

prompted us to search for small lipopeptides as responsible

for the high antifungal activity of cell-free extracts obtained

from Bacillus sp. BNM 122.1H-NMR analysis pointed to the presence of surfactin as a

main product excreted to the medium by that strain, while

the presence of some minor compounds was also evident.

When we analysed these excreted metabolites using TLC,

the presence of one compound with the same Rf of iturin A

was revealed. In addition, two additional compounds were

detected showing Rf values identical with those of iturin B

and C from B. subtilis as determined by Peypoux et al.(1973). We assayed the pure lipopeptides, iturin A and

surfactin, an inhibitory effect against fungal mycelium

growth was produced only by iturin A (data not shown).

The co-production of surfactin, which has surfactant

properties, and iturin, with antifungal activity, by the same

bacterial strain could be advantageous as a synergistic effect

of surfactin on the activity of iturin A was earlier

demonstrated (Thimon et al. 1992).This study suggests that antibiotic production was

involved in the disease-suppression by BNM 122. Soya

bean seed-coating with BNM 122 induced significant

protection against R. solani, under growth chamber and

greenhouse conditions. Moreover, a compost-based formu-

lation delivered to soya bean seeds was as effective as soil

application of the fungicide PCNB in controlling Rhizocto-nia damping-off.

Bacillus sp. BNM 122 was characterized by various

phenotypic and genotypic methods. With the API identifi-

cation systems, strain BNM 122 showed the highest

percentage of identification (76% Id) with B. subtilis and

only 3Æ7% Id with B. amyloliquefaciens. However, compar-

ison of 16S-23S rDNA IGS patterns generated by PCR

suggests that strain BNM 122 could be assigned to

B. amyloliquefaciens species, as the IGS-PCR fingerprints

are unique for each species among members of the B. subtilisgroup (Wunschel et al. 1994; Daffonchio et al. 1998a,b).

Moreover, rep-PCR fingerprinting was a different source of

molecular evidence of the close genetic relatedness existing

between strains BNM 122 and B. amyloliquefaciens DSM

7T, supporting the assignation of strain BNM 122 to

B. amyloliquefaciens. However, DNA-DNA hybridization

data are required to definitely assign strain BNM 122 as

belonging to that species.

The antifungal activity of B. amyloliquefaciens DSM 7T

was confirmed in in vitro assays and the same excreted

compounds were revealed by TLC when DSM 7T and

BNM 122 extracts were run on the same plate (de Estrada

2003).

The commercial use of micro-organisms as biocontrol

agents requires physiological and molecular fingerprints for

characterization, registration, patenting and identification of

introduced biocontrol strains from native microbial popu-

lations. Rep-PCR has been successfully used to identify and

differentiate among different strains of the genus Bacillus(Herman et al. 1998; da Silva et al. 1999; Herman and

Heyndrickx 2000; Marten et al. 2000), thus ERIC-PCR

fingerprinting could be confidently used for the genotyping

of the strain BNM 122.

The characterization of strain BNM 122 showed that it

lied genetically closer to B. amyloliquefaciens than to

B. subtilis although it physiologically resembled more the

latter species. Our results support recent evidence that

B. amyloliquefaciens strains produce iturins (Yoshida et al.2001; Yu et al. 2002). We also determined the synthesis of a

surfactant compound that was identified as surfactin.

Although the iturins production by B. amyloliquefacienshas been reported, the co-production of surfactin and iturins

has been reported only in B. subtilis strains (Sandrin et al.1990; Thimon et al. 1992; Asaka and Shoda 1996; Ahimou

et al. 2000). This two species are closely related and it may

be that some B. amyloliquefaciens strains were declassified as

B. subtilis. The results obtained with the type strain of

B. amyloliquefaciens species permit us to speculate that the

co-production of surfactin and iturins-like compounds could

not be an uncommon trait among B. amyloliquefaciensstrains. The co-production is an interesting characteristic

with potential practical applications.

ACKNOWLEDGEMENTS

AFG wishes to acknowledge support from the Program of

SETCIP: Cooperation Argentina-Germany. The authors

wish to acknowledge financial support from the Centro

Argentino-Brasileno de Biotecnologıa (CABBIO), Grant

13AR-07BR.

REFERENCES

Ahimou, F., Jacques, P. and Deleu, M. (2000) Surfactin and iturin A

effects on Bacillus subtilis surface hydrophobicity. Enzyme Microbio-

logy Technology 27, 749–754.



Asaka, O. and Shoda, M. (1996) Biocontrol of Rhizoctonia solani

damping-off of tomato with Bacillus subtilis RB14. Applied Environ-

mental and Microbiology 62, 4081–4085.

Ash, C., Farrow, J.A.E., Wallbanks, S. and Collins, M.D. (1991)

Phylogenetic heterogeneity of the genus Bacillus revealed by

comparative analysis of small-subunit-ribosomal RNA sequences.

Letters of Applied Microbiology 13, 202–206.

Chitarra, G.S., Breeuwer, P., Nout, M.J.R., van Aelst, A.C., Rombouts,

F.M. and Abee, T. (2003) An antifungal compound produced by

Bacillus subtilisYM10–20 inhibits germination ofPenicillium roqueforti

conidiospores. Journal of Applied Microbiology 94, 159–166.

Cho, S-J., Lee, S.K., Cha, B.J., Kim, Y.H. and Shin, K-S. (2003)

Detection and characterization of the Gloeosporium gloeosporioides

growth inhibitory compound iturin A from Bacillus subtilis strain

KS03. FEMS Microbiology Letter 223, 47–51.

Daffonchio, D., Borin, S., Consolandi, A., Mora, D., Manachini, P. L.

and Sorlini, C. (1998a) 16S-23S rRNA internal transcribed spacers

as molecular markers for the species of the 16S rRNA group I of the

genus Bacillus. FEMS Microbiology Letters 163, 229–236.

Daffonchio, D., Borin, S., Frova, G., Manachini, P.L. and Sorlini, C.

(1998b) PCR fingerprinting of whole genomes: the spacer between

the 16S and 23S rRNA genes and of intergenic tRNA gene regions

reveal a different intraspecific genomic variability of Bacillus cereus

and Bacillus licheniformis. International Journal of Systematic Bac-

teriology 48, 107–116.

Eshita, S.M., Roberto, N.H., Beale, J.M., Mamiya, B.M. and

Workman, R.F. (1995) Bacillomycin Lc, a new antibiotic of iturin

group: isolation, structures and antifungal activities of the congeners.

Journal of Antibiotics 48, 1240–1247.

de Estrada, M. (2003) Mecanismos involucrados en la actividad

antifungica de Bacillus amyloliquefaciens DSM7T. Thesis, Catedra de

Microbiologıa Agrıcola, Facultad de Agronomıa, UBA, Buenos

Aires.

Expert, J.M. and Digat, B. (1995) Biocontrol of Sclerotinia wilt of

sunflower by Pseudomonas fluorescens and Pseudomonas putida strains.

Canadian Journal of Microbiology 41, 685–691.

Feignier, C., Besson, F. and Michel, G. (1995) Studies on lipopeptide

biosynthesis by Bacillus subtilis: isolation and characterization of

iturin), surfactin+ mutants. FEMS Microbiology Letters 127, 11–15.

Handelsman, J. and Stabb, E.V. (1996) Biocontrol of soilborne plant

pathogens. Plant Cell 8, 1855–1869.

Herman, L. and Heyndrickx, M. (2000) The presence of intragenically

located rep-like elements in Bacillus sporothermodurans is sufficient

for rep-PCR typing. Research in Microbiology 151, 255–261.

Herman, L., Heyndrickx, M. and Waes, G. (1998) Typing of Bacillus

sporothermodurans and other Bacillus species isolated from milk by

repetitive element sequence based PCR. Letters in Applied Microbio-

logy 26, 183–188.

Jacques, Ph., Hbid, C., Vanhentenryck, F., Destain, J., Bare, G.,

Razafindralambo, H., Paquot, M. and Thonart, Ph. (1993) Quan-

titative and qualitative study of the production of broad-spectrum

antifungal lipopeptide from Bacillus subtilis S499. In Proceedings of

the 6th European Congress on Biotechnology, Florence, Italy, Vol. 9 ed.

Albergina, L., Frontali, L. and Sensi, P. pp. 1067–1070. Amsterdam,

the Netherlands: Elsevier Science.

Jensen, M.A., Webster, J.A. and Straus, N. (1993) Rapid identification

of bacteria on the basis of polymerase chain reaction-amplified

ribosomal DNA spacer polymorphisms. Applied and Environmental

Microbiology 59, 945–952.

Kajimura, Y., Sugiyama, M. and Kaneda, M. (1995) Bacillopeptins,

new cyclic lipopeptide antibiotics from Bacillus subtilis FR-2. Journal

of Antibiotics 48, 1095–1103.

Katz, E. and Demain, A.L. (1977) The peptide antibiotics of Bacillus:

chemistry, biogenesis, and possible functions. Bacteriological Review

41, 449–474.

Kim, D., Cook, R.J. and Weller, D. (1997) Bacillus sp. L324-92 for

biological control of three root diseases of wheat grown with reduced

tillage. Phytopathology 87, 551–558.

Laguerre, G., Mavingui, P., Allard, M.R., Charnay, M.P., Louvrier,

P., Mazurier, S.I., Rigottier-Gois, L. and Amarger, N. (1996)

Typing of rhizobia by PCR DNA fingerprinting and PCR-restric-

tion fragment length polymorphism analysis of chromosomal and

symbiotic gene regions: application to Rhizobium leguminosarum and

its different biovars. Applied and Environmental Microbiology 62,

2029–2036.

Lebbadi, M., Galvez, A., Maqueda, M., Martınez-Bueno, M. and

Valdivia, E. (1994) Fungicin M4: a narrow spectrum peptide

antibiotic from Bacillus licheniformis M-4. Journal of Applied

Bacteriology 77, 49–53.

Mao, W., Lewis, J.A., Hebbar, P.K. and Lumsden, R.D. (1997) Seed

treatment with fungal or a bacterial antagonist for reducing corn

damping-off caused by species of Pythium and Fusarium. Plant

Disease 81, 450–454.

Marten, P., Smalla, K. and Berg, G. (2000) Genotypic and phenotypic

differentiation of an antifungal biocontrol strain belonging to Bacillus

subtilis. Journal of Applied Microbiolology 89, 463–471.

Munimbazi, C. and Bullerman, L.B. (1998) Isolation and partial

characterization of antifungal metabolites of Bacillus pumilus. Journal

of Applied Microbiology 84, 959–968.

Peypoux, F., Guinand, M., et Michel, G. (1973) Isolement de l’acide

3-amin 12-methytetradecanoique a partir de l’iturine, antibiotique de

Bacillus subtilis. Tetrahedron 29, 3455–3459.

Podile, A.R. and Prakash, A.P. (1996) Lysis and biological control of

Aspergillus niger by Bacillus subtilis AF1. Canadian Journal of

Microbiology 42, 533–538.

Sandrin, C., Peypoux, F. and Michel, G. (1990) Co-production of

surfactin and iturin A, lipopeptides with surfactant and antifungal

properties, by Bacillus subtilis. Biotechnology and Applied Biochemistry

12, 370–375.

da Silva, K.R., Rabinovitch, L. and Seldin, L. (1999) Phenotypic and

genetic diversity among Bacillus sphaericus strains isolated in Brazil,

potentially useful as biological control agents against mosquito

larvae. Research Microbiology 150, 153–160.

Singh, P.P., Shin, Y.C, Park, C.S. and Ching, Y.R. (1998) Biological

control of Fusarium wilt of cucumber by chitinolytic bacteria.

Phytophatology 89, 92–99.

Thimon, L., Peypoux, F., Maget-Dana, R., Roux, B. and Michel, G.

(1992) Interactions of bioactive lipopeptides, iturin A and surfactin

from Bacillus subtilis. Biotechnology and Applied Biochemistry 16, 144–

151.

Thimon, L., Peypoux, F., Wallach, J. and Michel, G. (1995) Effect of

lipopeptide antibiotic, iturin A, on morphology and membrane

ultrastructure of yeast cells. FEMS Microbiology Letters 128, 101–

106.



Versalovic, J., Schneider, M., de Bruijn, F.J. and Lupski, J.R. (1994)

Genomic fingerprinting of bacteria using repetitive sequence-based

polymerase chain reaction. Methods of Molecular and Cell Biology 5,

25–40.

de Vrije, T., Antoine, N., Buitelaar, R.M., Bruckner, S., Dissevelet,

M., Durand, A., Gerlagh, M., Jones, E.E. et al. (2001) The fungal

biocontrol agent Coniothyrium minitans: production by solid-state

fermentation, application and marketing. Applied Microbiology and

Biotechnology 56, 58–68.

Wunschel, D., Fox, K.F., Black, G.E. and Fox, A. (1994) Discrim-

ination among the B. cereus group, in comparison to B. subtilis, by

structural carbohydrate profiles and ribosomal RNA spacer region

PCR. Systematic of Applied Microbiology 17, 625–635.

Yakimov, M.M., Timmis, K.N., Wray, V. and Fredrickson, H.L.

(1995) Characterization of a new lipopeptide surfactant produced by

thermotolerant and halotolerant subsurface Bacillus licheniformis

BAS50. Applied and Environmental Microbiology 61, 1706–1713.

Yoshida, S., Hiradate, S., Tsukamoto, T., Hatakeda, K. and Shirata,

A. (2001) Antimicrobial activity of culture filtrate of Bacillus

amyloliquefaciens RC-2 isolated from mulberry leaves. Phytopathol-

ogy 91, 181–187.

Yu, G.Y., Sinclair, J.B., Hartman, G.L. and Bertagnolli, B.L. (2002)

Production of iturin A by Bacillus amyloliquefaciens suppressing

Rhizoctonia solani. Soil Biology and Biochemistry 34, 955–963.

Zuber, P., Nakano, M. and Marahiel, M.A. (1993) Peptide antibiotics:

Bacillus subtilis and other Gram-positive bacteria. In Biochemistry,

Physiology, and Molecular Genetics ed. Sonenshein, A.L., Hoch, J.A.

and Losick, R. pp. 897–916. Washington, DC: American Society of

Microbiology.



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