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
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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
1250 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
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).
GENETIC AND FUNCTIONAL CHARACTERIZATION OF A BACILLUS SP. 1251
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 1247–1256, doi:10.1111/j.1365-2672.2004.02408.x
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
1252 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
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
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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.
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