Biological Control 66 (2013) 6571

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Biological control of bacterial wilt of common bean by plant growth-promoting

rhizobacteria
Samuel Julio Martins, Flavio Henrique Vasconcelos de Medeiros , Ricardo Magela de Souza,
Mrio Lcio Vilela de Resende, Pedro Martins Ribeiro Junior
Universidade Federal de Lavras, Departamento de Fitopatologia, 37200-000 Lavras, MG, Brazil

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Four PGPR strains reduced bacterial

wilt severity on common bean by

seed treatment.
 The four PGPR also increased plant
growth promotion.
 PGPR led to a signicant increase in
the phenolics content and lignin
accumulation.
 Cff seams to block plant response
defense by decreasing PAL activity.

a r t i c l e

i n f o

Article history:
Received 14 November 2012
Accepted 20 March 2013
Available online 4 April 2013
Keywords:
Seed treatment
Resistance suppression
PGPR
ISR
POX
PAL

a b s t r a c t
Bacterial wilt (BW) caused by Curtobacterium accumfaciens pv. accumfaciens (Cff) is an emerging, seedtransmitted disease of common bean (Phaseolus vulgaris) in Brazil, and plant growth-promoting rhizobacteria (PGPR) have the potential to be used in disease management. The present work aimed at determining the potential of selected PGPR on the biological control of BW through seed treatment, growth
promotion and induced resistance. Bean seeds cv. Prola were articially inoculated with Cff, immersed
in a PGPR suspension, and sown in 4 L pots containing a soil: sand mixture (2:1). Plants were assessed for
seedling emergence (SE), speed emergence index (SEI), relative growth index (RGI), root dry weight
(RDW), shoot dry weight (SDW), as well as biochemical plant responses in the presence or absence of
Cff. The disease control ranged from 42% to 76%, respectively, for Bacillus subtilis UFLA285 and ALB629
compared to the untreated control. PGPR treatments also increased RGI, SDW, and RDW. Upon Cff inoculation, UFLA285 increased phenolics content and ALB629 in the lignin accumulation compared to the
untreated control. Without the pathogen inoculation, both PGPR promoted an increase in phenylalanine
ammonia lyase activity and total phenolics content and UFLA285 in the lignin accumulation. Our ndings
demonstrated the potential of selected PGPR for disease control, enhancement of the RGI and biomass
accumulation. Surprisingly, instead of a priming effect of PGPR, Cff apparently blocks the defense
response development although the overall phenotype is disease control, suggesting there is a complementary and/or compensatory mode of action involved.
2013 Elsevier Inc. All rights reserved.

1. Introduction
Corresponding author. Address: Department of Plant Pathology, Campus
Universitrio, Universidade Federal de Lavras, CP3037, 37200-000 Lavras, MG,
Brazil. Fax: +55 35 38291283.
E-mail addresses: samueljmt@yahoo.com.br (S.J. Martins), aviomedeiros@dfp.
ua.br (F.H.V. de Medeiros), rmagelas@dfp.ua.br (R.M. de Souza), mlucio@dfp.
ua.br (M.L.V. de Resende), ribeirojuniorpm@yahoo.com.br (P.M. Ribeiro Junior).
1049-9644/$ - see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biocontrol.2013.03.009

Among the common bean diseases, bacterial wilt, caused by

Curtobacterium accumfaciens pv. accumfaciens (Cff) (Hedges) Collins and Jones (Hedges, 1922, 1926) is considered an emerging
menace. Cff was rst reported in South Dakota (Hedges, 1922)
and although the pathogen still is considered a quarantine microbe

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S.J. Martins et al. / Biological Control 66 (2013) 6571

in many countries it has been found in diverse geographical areas

around the world (Harveson and Schwartz, 2007; Hedges, 1926;
Hsieh et al., 2004; Krause et al., 2009a). In Brazil, the disease was
rst reported in 1995 (Maringoni and Rosa, 1997) and, thereafter
it became of emerging importance to common beans in different
regions (Herbes et al., 2008).
The bacterium is seed- or soil-borne and causes reduction in
seed germination and seedling height emergence, as well as wilt
of infected plants (Hall, 1994). Once it enters the plant, the bacterium colonizes the vascular tissue and causes wilting (Hedges,
1926; Souza and Maringoni, 2008).
Although, some works have been published currently regarding
differences in levels of resistance to Cff (Conner et al., 2008; Huang
et al., 2007; Krause et al., 2009a,b; Schwartz et al., 2010), there are
no commercial genetic cultivars or chemical treatments available
in Brazil against this disease. Currently, recommended managements for bacterial wilt rely on pathogen-free seeds and crop rotation with non-host crops (Mohan and Hagedorn, 1989). Therefore,
other methods such as biological control might have potential for
management of pathogenic bacteria. A promising disease control
strategy is the use of plant growth-promoting rhizobacteria (PGPR)
(Kloepper et al., 1989; Shankar et al., 2009).
Besides disease control, PGPR may promote plant growth indirectly by suppressing plant pathogens and their harm (Hahm
et al., 2012). Plant pathogen suppression by PGPR may occur
through a combination of mechanisms including induced resistance (Sundaramoorthy et al., 2012) and a priming effect, i.e., the
antagonist sets the plant in an alert state to pathogen detection
with the response occurring faster and/or stronger compared to
plants not previously exposed to the priming stimulus (Jung
et al., 2012).
Crop losses due to bacterial wilt can be severe, especially when
infection occurs early in the crop season (Mohan and Hagedorn,
1989). Thus, control strategies undertaken at early crop development may be more efcient especially for a seed-transmitted pathogen. Considering the importance of seeds in the transmission of
pathogens and the need to reduce fungicide loads in the environment, PGPR seed treatment may result in a practical and cost-effective strategy to reduce seed-borne pathogens (Machado, 2000)
such as Cff (Hsieh et al., 2003).
The present study was aimed at investigating the potential of
selected PGPR for biological treatment of Cff contaminated seeds,
to evaluate its potential to increase percentage seedling emergence
(PSE), speed emergence index (SEI), relative growth index (RGI),
root dry weight (RDW), shoot dry weight (SDW) as well as to evaluate the biochemical plant defense responses in the presence or
absence of Cff.

chlorite (0.5% active chloride) for 10 min, and in sterile distilled

water (SDW). Seeds were then air-dried in a ow cabinet for 8 h.
Disinfested bean seeds were articially inoculated with Cff by the
physiological conditioning technique (Deuner et al., 2011).
2.2. Screening test
Eight PGPR strains were used for the initial screening test: Paenibacillus lentimorbus MEN2, Bacillus subtilis ALB629, B. subtilis
UFLA285, B. subtilis sp. UFLA168, B. subtilis UFLA246, B. subtilis
UFLA373, B. subtilis UFLA116, and B. subtilis UFLA29 which were
obtained from rhizosphere soil and endophytics of roots of eldcultivated cotton plant or donated by research centers (Medeiros
et al., 2008, 2009). The screening tests were carried out under
greenhouse conditions (Temperature ca.30 C, relative humidity
ca.63% and light intensity ca.1000 lmol m2 s1). The experiment
was repeated with the most efcient strains.
2.3. Biological seed treatment with PGPR
Selected PGPR were preserved in peptone glycerol at 80 C
and before every experiment, they were cultivated on agar nutrient
medium in Petri dishes and incubated at room temperature (28 C)
for 48 h. Cells were transferred to the nutrient-broth medium and
cultivated for 48 h on a shaker at 150 rpm at room temperature
(28 C). The endospore concentration was adjusted in a Neubauer
chamber to 1  108 CFU ml1 and used to treat seeds by soaking
them for 30 min in the antagonists suspension (2 ml g1 seed)
108 CFU ml1,
fungicide
copper
oxychloride
or
water
(2 g seeds L1). They were dried overnight and sown in pots of
5 L containing a mixture of soil and sand (2:1), and with 10 seeds
per pot. Plants were kept under greenhouse conditions as described previously and watered to eld capacity.
2.4. Biocontrol of bacterial wilt
Selected PGPR from the screening test were tested under greenhouse conditions as described previously for their effectiveness to
control common bean articially inoculated with Cff to measure
the percentage seedling emergence (PSE), speed emergence index
(SEI), relative growth index (RGI), root dry weight (RDW), as well
as shoot dry weight (SDW).
A total of four replicates for each treatment were used and arranged in a randomized block design. The experiment was performed three times.
2.5. Assessment of the analyzed variables

2. Materials and methods

2.1. Articial seed inoculation
The Cff isolate used for this study was the yellow variant of Cff
from Santa Catarina State, Brazil (Cff SC Feij-2928, isolated in
March 23rd 2003 at Campos Novos, Santa Catarina State, Brazil
from common bean Phaseolus vulgaris cv. Prola), which was obtained from the culture collection of the plant bacteriology laboratory at the Universidade Estadual Paulista (Botucatu, Brazil),
preserved for long-term in peptone-glycerol and for short-term
in dried leaves from where it was recovered before each
experiment.
The pathogen isolated from the dried leaves was grown on 523
medium (Kado and Heskett, 1970) in Petri dishes and incubated at
room temperature (28 C) for 48 h. Seeds of cv. Prola were initially disinfested in a series of 70% ethanol for 30 s, sodium hypo-

Seedling emergence from the 5th to the 12th day after sowing
(DAS) was recorded daily and used to calculate the speed emergence index (SEI) according to Teixeira and Machado (2003), as
well as percentage of seedling emergence (PSE) from the last evaluated period.
At 12, 15, 18, 21, 24 DAS, plants were assessed for disease severity of bacterial wilt with disease scores ranging from 0 to 5, where
0 = no wilt symptoms; 1 = wilt on one of the primary leaves;
2 = wilt on both primary leaves but not on the rst trifoliolate;
3 = wilt on the rst trifoliolate; 4 = death of seedling after development of primary leaves and 5 = unemerged seedling or death of
seedling before development of primary leaves. With the values
of this scale, AUDPC was calculated (Hsieh et al., 2003).
At the same day of disease evaluations, plant height was also recorded by measuring the distance from the cotyledon insertion to
the apical bud and the obtained data was used to calculate the relative growth index (RGI) as RGI = (LnP2LnP1)/(T2T1), where

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S.J. Martins et al. / Biological Control 66 (2013) 6571

Ln = natural logarithm, P2 and P1 = seedlings height which have got

in the times T2 (end time) and T1 (initial time).
At 24 DAS, all plants were harvested and the shoot was separated from roots. Roots were thoroughly washed in tap water
and both plant parts were wrapped up, dried in an oven at 70 C
for 72 h until constant weight to obtain shoot (SDW) and root
dry weight (RDW). The experiment was performed three times.
2.6. Characterization of biochemical mechanisms involved in the
defense responses
In order to study the biochemical mechanisms involved in
PGPR-mediated plant defense, an experiment was carried out similar to the one used to conrm efcacy of the PGPR strains, using
the two more promising strains, a positive control, (treated with
acibenzolar-S-methyl ASM at 0.01%), and a negative one treated
with water. Plants were sampled at different growth stages: V1
emergence (cotyledons above the ground), V2 primary leaves
(two fully expanded primary leaves), V3 onset of rst trifoliolate
and V4 fully expanded third trifoliolate. In each sampling, three
leaves that had reached the fully expanded leaf stage were excised,
immediately wrapped in aluminum foil dipped in liquid nitrogen
and then kept in the freezer at 80 C where samples were stored
until the beginning of the tests. For total phenol and lignin contents, only the last sampling time period was considered. A total
of three replicates for each treatment were used and arranged in
a randomized block design with 3 seeds per pot.
2.7. Biochemical assays
2.7.1. Enzyme activities
The total soluble protein concentration was measured using a
standard curve of bovine serum albumin according to the method
adopted by Bradford (1976) and calculated as peroxidase (POX)
and phenylalanine ammonium lyase (PAL) activities.
POX extraction and assay were carried out according to Urbanek et al. (1991). For PAL, the procedures followed the one described by Mori et al. (2001).
2.7.2. Total phenol and lignin contents
Absorbance values of the total phenol reaction were determined
at 725 nm in a spectrophotometer and calculated based on catechol curve (0100 lg lg ml1). The total phenolic compounds
were expressed in equivalent lg of catechol per mg of dry weight
according to the method described by Spanos and Wrolstad (1990).
For lignin content, the absorbance was determined in a spectrophotometer at 280 nm, and the values calculated based on lignin
curve (0100 lg ml1) and expressed in lg of soluble lignin per
mg of dry matter Doster and Bostock (1988).
Assays were done in triplicate.
2.8. Experimental design and statistical analysis
All experiments were performed under greenhouse conditions
at the Universidade Federal de Lavras (UFLA), in Lavras, Minas Gerais, Brazil (915 m altitude, 21130 340 0 S and 44580 3100 W). The
experimental design was in randomized blocks, data were submitted to one-way variance analysis (ANOVA) and for signicant
means Tukeys multiple range tests (P = 0.05) were applied. For
all analyses, the assumptions of normality and homogeneity of variance were checked and no transformation was necessary. Sisvar
(Build 72) Copyrigth Daniel Furtado Ferreira 19992007 computer
software version 5.1 was used for statistical analyses (Ferreira,
2011).

3. Results
3.1. Bacterial wilt pathogen and biocontrol agents
No PGPR isolate increased the speed emergence index (SEI) or
the percentage seedling emergence (PSE). However, there was a
decrease for PSE and SEI after pathogen inoculation when compared with the non-treated control (P < 0.001) (Table 1).
All PGPR strains reduced disease severity by 4276% of the area
under the disease progress curve (AUDPC) when means were compared by the Tukeys test. There was no signicant interaction between treatments and time when the experiment was carried out
(P = 0.13), but there was a signicant effect for treatments when
the experiment was carried out (P < 0.001) (Fig. 1).
Regarding RGI, which was recorded at the same period as disease severity, all PGPR strains statistically differed from the other
treatments with highest means when compared to the untreated
control or fungicide (P < 0.001) (Fig. 2).
When plants were articially inoculated with Cff and treated
with PGPR, it was found that both shoot and root dry weight significantly increased when compared with the non-treated control
(P < 0.001) (Fig. 3).
3.2. Analysis of biochemical responses
3.2.1. Estimation of total phenol and lignin content
Without inoculation, both PGPR promoted an increase in the total soluble phenolics and UFLA285 promoted a content higher than
the ASM treatment. This last one was higher than the control
(water) while in the presence of the pathogen, UFLA285 promoted
that increase (Fig. 4).
Without inoculation, ASM induced lignin accumulation, followed by UFLA285. In the presence of the pathogen ASM and
ALB629 induced that accumulation when compared to the control
(water) (Fig. 5).
3.2.2. Enzyme activities
Bean plants with PGPR seed treatment with ALB629 or UFLA285
showed that UFLA285 and ASM without pathogen inoculation promoted an increase in PAL activity. Upon inoculation both PGPR isolates promoted a PAL increase at the second phenological stage
(VE), however, with disease development the enzyme content decreased with no difference at the last considered stage (V4) (Fig. 6).
In regard to POX, upon Cff inoculation PGPR treatment induced
an accumulation higher than the control at V1 (ALB629 strain) and
at V2, V3, and V4 phenological stages (ALB629 and UFLA285
strains). For plants not inoculated, PGPR induced an accumulation
higher than the control at V3 (UFLA285) and V4 (ALB629 and
UFLA285 strains) (Fig. 7). The only increase in this enzyme activity
was observed for plants treated with ASM at the last sampling time
point for plants that were not inoculated with the pathogen.
Table 1
Effects of seed treatments on bean articially inoculated with Cff by percentage
seedling emergence (PSE) and speed emergence index (SEI).
Treatments

PSE

SEI

Cff
Cff + Copper oxychloride
Cff + UFLA285
Cff + UFLA168*
Cff + ALB629
Cff + MEN2
Water (control)
CV (%)

53.58b
41.34b
46.22b
51.82b
57.73b
60.30b
78.30a
22.23

3.60b
2.71b
2.96b
2.94b
3.27b
3.66b
4.95a
22.64

*
Means followed by the same letter do not differ signicantly according to Tukeys
test (P 6 0.05). Means of three experiments of four replicates of ten seedlings each.
CV: coefcient of variation.

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S.J. Martins et al. / Biological Control 66 (2013) 6571

Fig. 1. Effect of seed treatment with PGPR, copper oxychloride and water on the
area under the disease progress curve (AUDPC) CV = 20.95. Bars headed with the
same letter are similar at the 5% level according to Tukeys test.

Fig. 2. Effect of seed treatment with PGPR, copper oxychloride and water on
relative growth index (RGI) CV = 13.50. Bars headed with the same letter are similar
at the 5% level according to Tukeys test.

4. Discussion
A growing demand for sustainable disease control supports the
search for a safe and efcient control strategy, and we showed that

endospore-forming strains have the potential for disease control

(Fig. 1). The most promising strain was B. subtilis ALB629, which
has also shown a consistent disease control even at different temperatures (Martins et al., in press). Besides disease control, the
PGPR strains did not cause phytotoxicity to the bean plants but
promoted plant growth as expressed by RGI as well as by induced
shoot and root dry weight in the presence of the pathogen, assuring a similar level of plant growth in the absence of the pathogen.
Further experiments will be carried out to determine if these PGPR
isolates are also able to increase plant growth promotion without
the pathogen inoculation. The growth promotion in inoculated
plants may be related to a compensatory effect (Ribeiro et al.,
2004; Shimada et al., 2000), where plants inoculated with Cff and
treated with PGPR could grow more than control treatment (water)
(Fig. 2) and exhibit a higher nal dry matter (Fig. 3). In relation to
copper fungicide it was not effective for control of bacterial wilt
and did not increase the PSE and SEI or SDW and RDW.
The PGPR strains tested did not keep germination of the pathogen-free seeds. Similar responses were observed by Hsieh et al.
(2006), where seeds infected with Cff have been shown to reduce
seedling emergence. Therefore, the preventive-basis of bacterial
control would be more efcient than the curative one.
In addition, this study indicated that the biological seed treatment with the selected PGPR strains led to a signicant increase
in the phenolics content and lignin accumulation both for infected
and non-infected bean plants, suggesting that these parameters are
implicated in the defense response as already reported for some
other pathosystems (Abo-Elyousr et al., 2009; Kandan et al.,
2002; Mandal, 2010; Zhang et al., 2011). These defense responses
may include the elaboration of cell wall thickenings usually accompanied by the deposition of lignin, a polymer of aromatic phenolics
(Fattah et al., 2011). This cell wall providing structural support and
a passive barrier against invading pathogens (Carpita and McCann,
2000) and could play a role as a physical barrier to stop Cff spread
through the plant. Hernndez-Blanco et al. (2007) found that an
alteration of secondary cell wall integrity by inhibiting cellulose
synthesis has lead to specic activation of plant defense response
against the soil-borne bacterium Ralstonia solanacearum. Generally,
major accumulations of PAL coincides with the disease reduction
(El Modafar et al., 2012). In the present work, the PAL-activity
was activated by two of the selected PGPR strains. In the inoculated
treatment, the highest activation appeared to be related to the second phenological stage (V2) when compared to the non-inoculated
one which reached the highest activation at V4. Surprisingly, with
disease progress, instead of a priming effect (Jung et al., 2012) by
the PGPR the enzyme content decreased with no difference at
the last stage. It appears that the pathogen blocked the plant

Fig. 3. Effect of seed treatment with PGPR, copper oxychloride and water on the shoot dry weight (SDW) and root dry weight (RDW) on plants 24 days old at time of data
collection. CV = 22.91 and 28.71, respectively for SDW and RDW. For each variable, bars headed by the same letters are not signicantly different at the 5% level according to
Tukeys test.

S.J. Martins et al. / Biological Control 66 (2013) 6571

69

Fig. 4. Total phenol at V1, V2, V3, and V4 bean plant phenological in the presence (left) or absence (right) of Curtobacterium accumfaciens pv. accumfaciens (Cff) inoculum.
Bars headed with the same letter are similar at the 5% level according to Tukeys test. CV = 2.76 and 7.97, respectively, for left and right graphics.

Fig. 5. Lignin content at V1, V2, V3, and V4 bean plant phenological stages at the presence (left) or absence (right) of Curtobacterium accumfaciens pv. accumfaciens (Cff)
inoculum. Bars headed with the same letter are similar at the 5% level according to Tukeys test. Bars headed with the same letter are similar at the 5% level according to
Tukeys test. CV = 18.30 and 2.90, respectively, for left and right graphics.

Fig. 6. Phenylalanine ammonium lyase (PAL) activity at V1, V2, V3, and V4 phenological stages of bean plants at the presence (left) and absence (right) of Curtobacterium
accumfaciens pv. accumfaciens (Cff). The line on each bar represents SE.

defense response development as already reported (Aslam et al.,

2008; Cooper et al., 2008). Considering that the overall phenotype
is disease control, other modes of action should be involved. POX is
promptly triggered and this induction in PGPR-treated plants is
faster than the chemical inducer (ASM), and this seems to be a consensus for PGPR treatment (Ishida et al., 2008). The enzyme is activated faster to contain the reactive oxygen species produced by

host response to stop the pathogen growth. Without the pathogen,

its activity is reduced for the PGPR-treated plants compared to the
ASM-treated, especially at V2.
In conclusion, these results open new prospects of bacterial wilt
control. Since a broad spectrum activity and a wide host range are
crucial for a successful biocontrol agent (Nagorska et al., 2007),
UFLA285 strain reduces post-emergence damping-off in cotton

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S.J. Martins et al. / Biological Control 66 (2013) 6571

Fig. 7. Peroxidase analysis at V1, V2, V3, and V4 phenological stages of bean plants at the presence (left) and absence (right) of Curtobacterium accumfaciens pv.
accumfaciens (Cff). The line on each bar represents SE.

when used as a seed treatment in a 2-year eld trial (Medeiros

et al., 2008) and ALB629 used to increase grafting capacity of cacao
plantlets (Alan Pomella, personal communication) making them
promising biological control agents also for bacterial wilt of common bean in Brazil.
Acknowledgments
We thank Conselho Nacional de Desenvolvimento Cientico
Cultural (CNPq), Fundao de Apoio Pesquisa do Estado de Minas
Gerais (FAPEMIG) and Programa de Apoio a Primeiros Projetos
(PAPP/UFLA) for providing the nancial support necessary for the
development of this work. And we thank CNPq for providing assistantships for the rst, third and fourth authors.
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