See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/226718509

A PCR-based method for the screening of bacterial strains with antifungal

activity in suppressive soybean rhizosphere

Article  in  World Journal of Microbiology and Biotechnology · February 2001

DOI: 10.1023/A:1016610610294

CITATIONS READS

28 88

5 authors, including:

Mónica N Giacomodonato Maria Julia Pettinari

Universidad de Buenos Aires Universidad de Buenos Aires
21 PUBLICATIONS   264 CITATIONS    118 PUBLICATIONS   1,371 CITATIONS   

SEE PROFILE SEE PROFILE

Beatriz S Méndez Nancy I López

National Scientific and Technical Research Council Universidad de Buenos Aires
76 PUBLICATIONS   1,447 CITATIONS    76 PUBLICATIONS   1,611 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Manipulation of global regulators in Escherichia coli View project

Glycerol utilization View project

All content following this page was uploaded by Maria Julia Pettinari on 15 June 2017.

The user has requested enhancement of the downloaded file.

 World Journal of Microbiology & Biotechnology 17: 51±55, 2001. 51
Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.

A PCR-based method for the screening of bacterial strains with antifungal

activity in suppressive soybean rhizosphere

MoÂnica N. Giacomodonato1, M. Julia Pettinari1, Guadalupe I. Souto3, Beatriz S. MeÂndez1

and Nancy I. LoÂpez1,2,*
1
Dpto. de QuõÂmica BioloÂgica and 2Dpto. de Ciencias BioloÂgicas, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, 1428-Buenos Aires, Argentina
3
CaÂtedra de MicrobiologõÂa, Facultad de AgronomõÂa, Universidad de Buenos Aires, Av. San MartõÂn 4453,
1417-Buenos Aires, Argentina
*Author for correspondence: 4to. Piso, PabelloÂn II, 1428-Buenos Aires, Argentina. Tel.: 54-11-4576-3334,
Fax: 54-11-4576-3342, E-mail: nan@qb.fcen.uba.ar

Received 8 September 2000; accepted 8 December 2000

Keywords: Antifungal activity, Bacillus sp., PCR, peptide synthetase genes, rhizosphere, suppressive soybean soils

Summary

Selection and evaluation of microbial strains for their antifungal activity in natural environments is time- and
energy-consuming. We have adapted a PCR-based method to avoid these inconveniences. Soils that are naturally
suppressive to plant disease were chosen as a source of antibiotic-producing bacteria. The screening was performed
by means of PCR ampli®cation using degenerate primers corresponding to peptide synthetase genes. Ampli®cation
fragments were obtained using template DNA from the rhizosphere of three di€erent soybean ®elds. In order to
assay their potential utility in pathogen control, several Bacillus strains were analysed for their in vitro antifungal
activity by testing growth inhibition of Sclerotinia sclerotiorum. Four Bacillus sp. isolates gave a positive
ampli®cation signal, and three of them had an inhibitory e€ect on S. sclerotiorum growth, whereas two strains that
failed to give an ampli®cation signal did not inhibit fungal growth. These results show that PCR-based techniques
could be useful to assess the presence of strains with potential use as biocontrol agents.

Introduction synthetases. These peptides possess a varied range of

The rhizosphere provides a front-line defence for roots
against attack by pathogens (Weller 1988), and bacteria
present in it probably play a role in natural biological
control due to production of antibiotic substances. In
addition, the production of antagonist substances may
confer on these microorganisms a selective advantage in
competition for nutrients and space within their ecolog-
ical niches (Fravel 1988). Isolation of bacterial strains
directly from suppressive soybean soils could be a good
alternative for the selection of biocontrol agents, since
they are already well adapted to survive in the ®eld, in
contrast to laboratory modi®ed strains.
Most of the sporulating members of the Bacillus genus
are antibiotic-producing microorganisms whose heat-
and desiccation-resistant spores can be readily formu-
lated into stable products to be applied in biocontrol
(Emmert & Handelsman 1999). In spite of this, Bacillus
spp. have received less attention than ¯uorescent pseu-
domonads as biocontrol agents. The majority of the
antibiotics from Bacillus sp. are low molecular weight
peptides, produced via the nonribosomal biosynthetic
pathway, which involves speci®c enzymes called peptide
remarkable biological activities, including antimicrobial,
antiviral and antitumoral activities. Peptide synthetases
are formed by repetitive and coordinated groups of
active sites called modules, in which each module is
responsible for the catalysis of one complete polypeptide
chain elongation and associated functional group mod-
i®cations (Cane et al. 1998).
The complete sequences of several bacterial operons,
including grs, srfA, tyc, bac, fen, and lic for the
biosynthesis of the peptide antibiotics gramicidin S,
surfactin, tyrocidine, bacitracin, fengycin and lichenysin,
respectively, have recently been determined (Marahiel
et al. 1997; Konz et al. 1999; Lin et al. 1999). These
operons span regions of 18±45 kb and encode several
peptide synthetases comprising one to six modules. A
minimal module contains an adenylation and a thiola-
tion domain. The adenylation domains of di€erent
peptide synthetase modules have been previously used as
targets for PCR ampli®cation (Marahiel et al. 1997).
The use of degenerate primers designed from these
conserved sequences to amplify putative peptide syn-
thetase genes from soil DNA and bacterial isolates,
could constitute a good approach to detect the presence
52
of strains from natural environments that could be
useful as biocontrol agents.
The objective of this work was to assess the use of a
PCR-based technique in the preliminary screening of
antibiotic peptide-producing microorganisms. The ra-
tionale to achieve this objective was the use of primers
from the conserved published peptide synthetase se-
quences and the selection of soils suppressive to fungal
diseases for the screening. In addition, the antibiotic
activity of the selected strains was demonstrated by
growth inhibition of Sclerotinia sclerotiorum, a wide-
spread soybean pathogen in Argentina (Wrather et al.
1997).
Materials and Methods
Enumeration of heat resistant bacteria
Three di€erent soybean ®elds were selected from Junin
(Buenos Aires province). All three were suppressive to
Sclerotinia stem rot of soybean. Samples were taken
monthly along the di€erent stages of plant growth from
December 1998 to May 1999. At each sampling event,
six randomly selected soybean plants were harvested
from each ®eld and used to enumerate and to isolate
bacterial strains and for DNA extraction. Three soybean
roots of each ®eld were cut, placed in three Erlenmeyer
¯asks containing 100 ml of sterile 0.0125 M phosphate
bu€er and shaken for 30 min at 30 °C using the Kim
et al. (1997) protocol with slight modi®cations. Aliquots
(100 ll) from serial dilutions were spread onto triplicate
nutrient agar (Merck) plates and heat-treated at 80 °C
for 10 min according to the conventional Bohall & Vary
(1986) method. Plates were incubated at 30 °C for 72 h.
Heat-resistant aerobic bacteria in the bulk soil were
counted from 15 g of soil after soybean harvest using the
same procedure.
Bacterial strains
The strains used in this study were Bacillus subtilis
ATCC 33677 and six natural isolates called UBA30-30,
UBA30-31, UBA30-32, UBA30-33, UBA30-34 and
UBA30-35. These strains were chosen at random from
the heat-resistant aerobic bacteria obtained after plat-
ing samples from the three ®elds. All six strains were
spore-forming, Gram positive, rod-shaped aerobic bac-
teria, as expected, and will be therefore referred to as
Bacillus sp.
DNA recovery
Chromosomal DNA extraction from bacterial cultures
was performed using standard extraction methods
(Sambrook et al. 1982). Rhizosphere DNA extraction
was performed with a soil DNA puri®cation kit (Mo Bio
Laboratories, Inc) on 0.25 g of soil obtained by gentle
shaking of the soybean roots. Soil samples were taken
M.N. Giacomodonato et al.
after soybean harvest and DNA was extracted using the
same procedure as with rhizosphere soils.
PCR ampli®cation
PCR ampli®cation of the peptide synthetase genes was
done using two sets of degenerate synthetic oligonucle-
otides. The ®rst set of primers (TGD/LGG) was
designed by Turgay & Marahiel (1994) and ampli®ed a
fragment around 500 bp from Bacillus licheniformis
spanning the adenylation and thiolation domains.
The second set of degenerate primers (Psup/Pslow)
was designed for this work using the most conserved
region included in the adenylation domain correspond-
ing to 11 data base available sequences (bacitracin,
gramicidin, surfactin, lichenysin, tyrocidine and fengy-
cin synthetase). The expected ampli®cation fragment
size is about 750 bp. The nucleotide sequences were:
Psup 50 -GCNTAYNTNRTNTAYACNW
SNGGNWSNACNGG-30
Pslow 50 -CKNAYYTTNACYTGNTBRTC
DWTNCKNCC-30
Ampli®cations were done using standard protocols. For
the Ps primers the annealing temperature was 40 °C and
the elongation temperature 72 °C for 35 cycles. For
TGD and LGG primers, the annealing temperature for
the ®rst ®ve cycles was 45 °C and 50 °C for the next 30
cycles (Turgay & Marahiel 1994). Booster PCR was
used to amplify DNA extracted from rhizosphere. A
negative control using sterile water instead of template
DNA was run for every reaction. The PCR-ampli®ed
DNAs were visualized by electrophoresis in horizontal
1% agarose gels stained with ethidium bromide.
Antagonism assays in vitro
Bacterial isolates previously analysed by PCR ampli®-
cation were tested for antifungal activity. This activity
was monitored by screening for growth inhibition of S.
sclerotiorum. A bacterial culture grown in nutrient broth
at a density of 108 cfu ml)1 was streaked in a line on one
side of a Petri dish containing 1:1 nutrient agar:potato
dextrose agar (PDA). A 9 mm agar plug of S. sclero-
tiorum grown on PDA for 48 h was placed in the centre
of the agar plates. Bacteria and fungus were inoculated
simultaneously, and plates were incubated at 28 °C
during 7 days before evaluating growth inhibition. Each
bacterial isolate was replicated four times and the entire
experiment was repeated three times.
Statistical analysis
Bacterial numbers of all the plate counts were log10
transformed prior to statistical analysis. We used two-
way analysis of variance to determine di€erences in
Screening of antifungal bacterial strains
bacterial number between the three soybean ®elds
studied throughout the soybean growth season.
Results
Enumeration of heat-resistant aerobic bacteria
in the soybean rhizosphere
In order to have a preliminary idea of the variations in
the indigenous heat-resistant aerobic bacteria, their
number was determined between December 1998, shortly
after the time of soybean sowing, and its harvest, in May
1999 (Figure 1). Three di€erent soybean ®elds from
Junin (Buenos Aires province) that are natural disease-
suppressive soils were studied. All three were suppressive
to Sclerotinia stem rot of soybean. No signi®cant
di€erences (P > 0.05) in the number of heat-resistant
aerobic bacteria were observed between the three soy-
bean ®elds analysed. However, signi®cant di€erences
were obtained along the time (P < 0.05). Counts ranged
between 8 á 105 and 3 á 107 cfu g)1 dry weight of root.
The number of heat-resistant aerobic bacteria was higher
in soybean rhizosphere compared to bulk soil after
soybean harvest in the same ®elds (data not shown).
PCR ampli®cation
DNA obtained from six Bacillus strains isolated from
soybean rhizosphere and B. subtilis ATCC 33677 was
used for PCR ampli®cation with primers TGD and
LGG. Bacillus subtilis and four of the Bacillus strains
isolated from the rhizosphere (UBA30-30, UBA30-31,
UBA30-32 and UBA30-33) gave ampli®cation frag-
ments of the predicted size, 500 bp. No bands were
obtained for isolates UBA30-34 and UBA30-35
(Figure 2). Faint bands of a larger size were also
Figure 1. Enumeration of heat-resistant aerobic bacteria in the rhiz-
osphere between sowing and harvest of soybean. Values represent
mean (‹SD) of log transformed data for all three ®elds.
53
Figure 2. Agarose gel electrophoresis of the PCR products using TGD
and LGG primers and DNA from di€erent bacteria and rhizosphere
samples as template. Lane: (1) 100 bp marker (BRL); (2) Bacillus
subtilis ATCC 33677; (3) sterile water; (4) strain UBA30-30; (5) strain
UBA30-31; (6) strain UBA30-32; (7) strain UBA30-33; (8) strain
UBA30-34; (9) strain UBA30-35; (10) rhizosphere sample from the
®eld 1; (11) rhizosphere sample from the ®eld 2; (12) rhizosphere
sample from the ®eld 3.
observed in the case of B. subtilis ATCC 33677 and
isolates UBA30-30, UBA30-32 and UBA30-33. Positive
ampli®cation signals were obtained from DNA extract-
ed from 0.25 g rhizosphere soil sample from the three
soybean ®elds studied after a second ampli®cation
reaction (Figure 2) with the same primers. No ampli®-
cation bands were obtained for DNA extracted from
soil after soybean harvest (data not shown).
When primers Psup and Pslow were used, an ampli-
®cation signal of the expected size (750 bp) was obtained
for Bacillus isolates UBA30-30, UBA30-31, UBA30-32
and UBA30-33 and for Bacillus subtilis ATCC 33677.
A smaller faint band was observed for strain number
UBA30-31 (Figure 3). These primers, designed in this
work, corresponding to the sequence of the most
conserved region of 11 peptide synthetase genes, were
not suitable for the detection of ampli®cation fragments
in environmental samples (Figure 3). This could be due
to the high degree of degeneration in the primers
combined with the low speci®c DNA template concen-
tration in rhizosphere samples. However, they allowed
the ampli®cation of DNA from the same bacterial
isolates that gave a positive ampli®cation signal with the
®rst set of primers.
Evaluation of antagonism in vitro
Selected bacterial isolates were characterized for in vitro
inhibition of S. sclerotiorum to correlate the PCR
ampli®cation signal with antifungal activity. Growth
54
Figure 3. Agarose gel electrophoresis of the PCR products using Psup
and Pslow primers and DNA from di€erent bacteria and rhizosphere
samples as template. Lane: (1) 100 bp marker (BRL); (2) Bacillus
subtilis ATCC 33677; (3) sterile water; (4) strain UBA30-30; (5) strain
UBA30-31; (6) strain UBA30-32; (7) strain UBA30-33; (8) strain
UBA30-34; (9) strain UBA30-35; (10) rhizosphere sample from the
®eld 1; (11) rhizosphere sample from the ®eld 2; (12) rhizosphere
sample from the ®eld 3.
inhibition assays indicated that Bacillus sp. strains
UBA30-31, UBA30-32 and UBA30-33, which gave a
positive PCR ampli®cation signal, inhibited mycelial
growth of S. sclerotiorum on PDA (Table 1, Figure 4).
In contrast, no inhibitory e€ect on S. sclerotiorum
Figure 4. Growth inhibition of Sclerotinia sclerotiorum by UBA30-30, UBA30-31, UBA30-32 and UBA30-33 strains.
M.N. Giacomodonato et al.
Table 1. In vitro inhibition of Sclerotinia sclerotiorum by isolated
soybean rhizosphere bacterial strains.
Bacillus strain Inhibition
UBA30-30 )
UBA30-31 ++
UBA30-32 ++
UBA30-33 ++
UBA30-34 )
UBA30-35 )
): No zone of inhibition, the fungus overgrew the bacterial growth.
+: A distinct zone of inhibition, with fungal growth inhibited less
than 5 mm from the point where the bacteria were streaked.
++: A distinct zone of inhibition, with fungal growth inhibited
5±10 mm from the point where the bacteria were streaked.
+++: A distinct zone of inhibition, with fungal growth inhibited
>10 mm from the point where the bacteria were streaked.
growth was observed for strains UBA30-30, UBA30-34
and UBA30-35 (Table 1).
Discussion
In order to screen for antibiotic-producing bacterial
strains that could be used as putative biocontrol agents
of soybean fungal diseases, DNA extracted from rhiz-
osphere samples and from several Bacillus isolates was
used as template for PCR ampli®cation using two sets of
primers corresponding to peptide synthetase genes. Both
sets of degenerate primers used in this study were
suitable for the ampli®cation of template DNA from
Bacillus subtilis ATCC 33677 laboratory strain and
rhizosphere isolates UBA30-30, UBA30-31, UBA30-32
and UBA30-33.
 Screening of antifungal bacterial strains
In this work, primers TGD and LGG, that have been
previously used to amplify peptide synthetases genes
from a Bacillus licheniformis laboratory strain (Turgay
& Marahiel 1994), were also suitable for the ampli®ca-
tion of rhizosphere DNA, giving an ampli®cation
fragment of the expected size.
No ampli®cation signal was obtained from the soil
samples after soybean harvest. This could indicate that
the presence of antibiotic-producing strains is associated
with the plants, but this result could also be due to the
increase in total heat-resistant aerobic bacteria.
Four out of six Bacillus sp. isolates gave a positive
ampli®cation signal with the primers designed for the
ampli®cation of peptide synthetases, and three of them
had an inhibitory e€ect on S. sclerotiorum growth.
Neither of the two isolates that gave no ampli®cation
signal had antifungal activity. These results suggest that
the presence of these ampli®cation fragments could be a
good indicator of antifungal activity, and indicate that
this approach is useful to assess the presence of strains
with potential use as biocontrol agents.
We have shown that the presence of strains with
antibiotic activity can be detected by PCR analysis in
the natural rhizosphere bacterial community of sup-
pressive soils. The use of direct PCR could improve the
search for strains that are more e€ective in speci®c crop-
pathogen interactions and are better adapted to soil
conditions than laboratory strains. A similar approach
has been used to check strains of antibiotic-producing
¯uorescent Pseudomonas spp. in soils, detected by
colony hybridization (Raaijmakers et al. 1997).
In view of the results obtained in this work, we
propose the use of a rapid method based on the
ampli®cation of total rhizosphere DNA as a ®rst
screening procedure to assay the possibilities of isolating
useful strains with antifungal activity. In this way, the
analysis of samples which are unlikely to contain
antibiotic-producing strains with the time-consuming
plating method is avoided. In addition, our results show
that ampli®cation fragments were obtained only in the
presence of the soybean plants, so we suggest that the
screening should be performed in the natural bacterial
community of the rhizosphere of suppressive soils.
View publication stats
55
Acknowledgements
We are grateful to Manuel Vadillo, Alejandro Navone,
Javier Alasia and Martin Rolandi for help with soybean
samples. This work was supported by grants from
University of Buenos Aires to NIL and CABBIO to
BSM. BSM and NIL are career investigator from
CONICET.
References
Bohall, J.R. & Vary, P.S. 1986 Transposition of Tn917 in Bacillus
megaterium. Journal of Bacteriology 167, 716±718.
Cane, D.E., Walsh, C.T. & Khosla, C. 1998 Harnessing the biosyn-
thetic code: combinations, permutations, and mutations. Science
282, 63±68.
Emmert, E.A.B. & Handelsman, J. 1999 Biocontrol of plant disease; a
(Gram-) positive perspective. FEMS Microbiology Letters 171, 1±9.
Fravel, D.R. 1988 Role of antibiosis in the biocontrol of plant diseases.
Annual Review of Phytopathology 26, 75±91.
Kim, D.-S., Weller, D.M. & Cook, R.J. 1997 Population dynamics of
Bacillus sp. L324-92R12 Pseudomonas ¯uorescens 2-79RN10 in the
rhizosphere of wheat. Biological Control 87, 559±564.
Konz, D., Doekel, S. & Marahiel, M.A. 1999 Molecular and
biochemical characterization of the protein template controlling
biosynthesis of the lipopeptide lichenysin. Journal of Bacteriology
181, 133±140.
Lin, T.-P., Chen, C.-l., Chang, L.-K., Tschen, J.S.-M. & Liu, S.-T.
1999 Functional and trancriptional analyses of fengycin synthetase
gene, fenC, from Bacillus subtilis. Journal of Bacteriology 181,
5060±5067.
Marahiel, M.A., Stachelhaus, T. & Mootz, H.D. 1997 Modular
peptide synthetases involved in nonribosomal peptide synthesis.
Chemical Reviews 97, 2651±2673.
Raaijmakers, J.M., Weller, D.M. & Thomashow, L.S. 1997 Frequency
of antibiotic-producing Pseudomonas spp. in natural environments.
Applied and Environmental Microbiology 63, 881±887.
Sambrook, J., Frisch, E.F. & Maniatis, T. 1982 Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY. ISBN 0-87969-136-0.
Turgay, K. & Marahiel, M.A. 1994 A general approach for identifying
and cloning peptide synthetase genes. Peptide Research 7, 238±241.
Weller, D.M. 1988 Biological control of soilborne plant pathogens in
the rhizosphere with bacteria. Annual Review of Phytopathology 26,
379±407.
Wrather, J.A., Anderson, T.R., Arsyad, D.M., Gai, J., Ploper, L.D.,
Porta-Puglia, A., Ram, H.H. & Yorinoti, J.T. 1997 Soybean
disease loss estimates for the top 10 soybean producing countries in
1994. Plant Disease 81, 107±110.