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Biological Control
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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
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
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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
67
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.
68
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
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.
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.
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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.
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