Biological Control 66 (2013) 65–71

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Biological Control

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

Samuel Julio Martins, Flavio Henrique Vasconcelos de Medeiros ⇑, Ricardo Magela de Souza,Mário Lúcio Vilela de Resende, Pedro Martins Ribeiro JuniorUniversidade Federal de Lavras, Departamento de Fitopatologia, 37200-000 Lavras, MG, Brazil

h i g h l i g h t s

� Four PGPR strains reduced bacterialwilt severity on common bean byseed treatment.� The four PGPR also increased plant

growth promotion.� PGPR led to a significant increase in

the phenolics’ content and ligninaccumulation.� Cff seams to block plant response

defense by decreasing PAL activity.

1049-9644/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.biocontrol.2013.03.009

⇑ Corresponding author. Address: Department oUniversitário, Universidade Federal de Lavras, CP30Brazil. Fax: +55 35 38291283.

E-mail addresses: samueljmt@yahoo.com.br (S.J. Mufla.br (F.H.V. de Medeiros), rmagelas@dfp.ufla.br (Rufla.br (M.L.V. de Resende), ribeirojuniorpm@yahoo.c

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 2012Accepted 20 March 2013Available online 4 April 2013

Keywords:Seed treatmentResistance suppressionPGPRISRPOXPAL

a b s t r a c t

Bacterial wilt (BW) caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is an emerging, seed-transmitted disease of common bean (Phaseolus vulgaris) in Brazil, and plant growth-promoting rhizobac-teria (PGPR) have the potential to be used in disease management. The present work aimed at determin-ing the potential of selected PGPR on the biological control of BW through seed treatment, growthpromotion and induced resistance. Bean seeds cv. ‘Pérola’ were artificially inoculated with Cff, immersedin a PGPR suspension, and sown in 4 L pots containing a soil: sand mixture (2:1). Plants were assessed forseedling 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 ofCff. The disease control ranged from 42% to 76%, respectively, for Bacillus subtilis UFLA285 and ALB629compared to the untreated control. PGPR treatments also increased RGI, SDW, and RDW. Upon Cff inoc-ulation, UFLA285 increased phenolics’ content and ALB629 in the lignin accumulation compared to theuntreated control. Without the pathogen inoculation, both PGPR promoted an increase in phenylalanineammonia lyase activity and total phenolics content and UFLA285 in the lignin accumulation. Our findingsdemonstrated the potential of selected PGPR for disease control, enhancement of the RGI and biomassaccumulation. Surprisingly, instead of a priming effect of PGPR, Cff apparently blocks the defenseresponse development although the overall phenotype is disease control, suggesting there is a comple-mentary and/or compensatory mode of action involved.

� 2013 Elsevier Inc. All rights reserved.

ll rights reserved.

f Plant Pathology, Campus37, 37200-000 Lavras, MG,

artins), flaviomedeiros@dfp..M. de Souza), mlucio@dfp.

om.br (P.M. Ribeiro Junior).

1. Introduction

Among the common bean diseases, bacterial wilt, caused byCurtobacterium flaccumfaciens pv. flaccumfaciens (Cff) (Hedges) Col-lins and Jones (Hedges, 1922, 1926) is considered an emergingmenace. Cff was first reported in South Dakota (Hedges, 1922)and although the pathogen still is considered a quarantine microbe

66 S.J. Martins et al. / Biological Control 66 (2013) 65–71

in many countries it has been found in diverse geographical areasaround the world (Harveson and Schwartz, 2007; Hedges, 1926;Hsieh et al., 2004; Krause et al., 2009a). In Brazil, the disease wasfirst reported in 1995 (Maringoni and Rosa, 1997) and, thereafterit became of emerging importance to common beans in differentregions (Herbes et al., 2008).

The bacterium is seed- or soil-borne and causes reduction inseed germination and seedling height emergence, as well as wiltof infected plants (Hall, 1994). Once it enters the plant, the bacte-rium colonizes the vascular tissue and causes wilting (Hedges,1926; Souza and Maringoni, 2008).

Although, some works have been published currently regardingdifferences in levels of resistance to Cff (Conner et al., 2008; Huanget al., 2007; Krause et al., 2009a,b; Schwartz et al., 2010), there areno commercial genetic cultivars or chemical treatments availablein Brazil against this disease. Currently, recommended manage-ments for bacterial wilt rely on pathogen-free seeds and crop rota-tion with non-host crops (Mohan and Hagedorn, 1989). Therefore,other methods such as biological control might have potential formanagement of pathogenic bacteria. A promising disease controlstrategy 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 indi-rectly by suppressing plant pathogens and their harm (Hahmet al., 2012). Plant pathogen suppression by PGPR may occurthrough a combination of mechanisms including induced resis-tance (Sundaramoorthy et al., 2012) and a priming effect, i.e., theantagonist sets the plant in an ‘‘alert’’ state to pathogen detectionwith the response occurring faster and/or stronger compared toplants not previously exposed to the priming stimulus (Junget al., 2012).

Crop losses due to bacterial wilt can be severe, especially wheninfection occurs early in the crop season (Mohan and Hagedorn,1989). Thus, control strategies undertaken at early crop develop-ment may be more efficient especially for a seed-transmitted path-ogen. Considering the importance of seeds in the transmission ofpathogens and the need to reduce fungicide loads in the environ-ment, PGPR seed treatment may result in a practical and cost-effec-tive strategy to reduce seed-borne pathogens (Machado, 2000)such as Cff (Hsieh et al., 2003).

The present study was aimed at investigating the potential ofselected 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 eval-uate the biochemical plant defense responses in the presence orabsence of Cff.

2. Materials and methods

2.1. Artificial seed inoculation

The Cff isolate used for this study was the yellow variant of Cfffrom Santa Catarina State, Brazil (Cff SC – Feij-2928, isolated inMarch 23rd 2003 at Campos Novos, Santa Catarina State, Brazilfrom common bean Phaseolus vulgaris cv. Pérola), which was ob-tained from the culture collection of the plant bacteriology labora-tory at the Universidade Estadual Paulista (Botucatu, Brazil),preserved for long-term in peptone-glycerol and for short-termin dried leaves from where it was recovered before eachexperiment.

The pathogen isolated from the dried leaves was grown on 523medium (Kado and Heskett, 1970) in Petri dishes and incubated atroom temperature (28 �C) for 48 h. Seeds of cv. ‘Pérola’ were ini-tially disinfested in a series of 70% ethanol for 30 s, sodium hypo-

chlorite (0.5% active chloride) for 10 min, and in sterile distilledwater (SDW). Seeds were then air-dried in a flow cabinet for 8 h.Disinfested bean seeds were artificially inoculated with Cff by thephysiological conditioning technique (Deuner et al., 2011).

2.2. Screening test

Eight PGPR strains were used for the initial screening test: Pae-nibacillus lentimorbus MEN2, Bacillus subtilis ALB629, B. subtilisUFLA285, B. subtilis sp. UFLA168�, B. subtilis UFLA246, B. subtilisUFLA373, B. subtilis UFLA116, and B. subtilis UFLA29 which wereobtained from rhizosphere soil and endophytics of roots of field-cultivated cotton plant or donated by research centers (Medeiroset al., 2008, 2009). The screening tests were carried out undergreenhouse conditions (Temperature ca.30 �C, relative humidityca.63% and light intensity ca.1000 lmol m�2 s�1). The experimentwas repeated with the most efficient strains.

2.3. Biological seed treatment with PGPR

Selected PGPR were preserved in peptone glycerol at �80 �Cand before every experiment, they were cultivated on agar nutrientmedium in Petri dishes and incubated at room temperature (28 �C)for 48 h. Cells were transferred to the nutrient-broth medium andcultivated for 48 h on a shaker at 150 rpm at room temperature(28 �C). The endospore concentration was adjusted in a Neubauerchamber to 1 � 108 CFU ml�1 and used to treat seeds by soakingthem for 30 min in the antagonist’s suspension (2 ml g�1 seed)108 CFU ml�1, fungicide copper oxychloride or water(2 g seeds L�1). They were dried overnight and sown in pots of5 L containing a mixture of soil and sand (2:1), and with 10 seedsper pot. Plants were kept under greenhouse conditions as de-scribed previously and watered to field capacity.

2.4. Biocontrol of bacterial wilt

Selected PGPR from the screening test were tested under green-house conditions as described previously for their effectiveness tocontrol common bean artificially inoculated with Cff to measurethe percentage seedling emergence (PSE), speed emergence index(SEI), relative growth index (RGI), root dry weight (RDW), as wellas shoot dry weight (SDW).

A total of four replicates for each treatment were used and ar-ranged in a randomized block design. The experiment was per-formed three times.

2.5. Assessment of the analyzed variables

Seedling emergence from the 5th to the 12th day after sowing(DAS) was recorded daily and used to calculate the speed emer-gence index (SEI) according to Teixeira and Machado (2003), aswell as percentage of seedling emergence (PSE) from the last eval-uated period.

At 12, 15, 18, 21, 24 DAS, plants were assessed for disease sever-ity of bacterial wilt with disease scores ranging from 0 to 5, where0 = no wilt symptoms; 1 = wilt on one of the primary leaves;2 = wilt on both primary leaves but not on the first trifoliolate;3 = wilt on the first trifoliolate; 4 = death of seedling after develop-ment of primary leaves and 5 = unemerged seedling or death ofseedling before development of primary leaves. With the valuesof this scale, AUDPC was calculated (Hsieh et al., 2003).

At the same day of disease evaluations, plant height was also re-corded by measuring the distance from the cotyledon insertion tothe apical bud and the obtained data was used to calculate the rel-ative growth index (RGI) as RGI = (LnP2�LnP1)/(T2�T1), where

S.J. Martins et al. / Biological Control 66 (2013) 65–71 67

Ln = natural logarithm, P2 and P1 = seedlings height which have gotin the times T2 (end time) and T1 (initial time).

At 24 DAS, all plants were harvested and the shoot was sepa-rated from roots. Roots were thoroughly washed in tap waterand both plant parts were wrapped up, dried in an oven at 70 �Cfor 72 h until constant weight to obtain shoot (SDW) and rootdry weight (RDW). The experiment was performed three times.

2.6. Characterization of biochemical mechanisms involved in thedefense responses

In order to study the biochemical mechanisms involved inPGPR-mediated plant defense, an experiment was carried out sim-ilar to the one used to confirm efficacy of the PGPR strains, usingthe two more promising strains, a positive control, (treated withacibenzolar-S-methyl – ASM at 0.01%), and a negative one treatedwith 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 first trifoliolateand V4 – fully expanded third trifoliolate. In each sampling, threeleaves that had reached the fully expanded leaf stage were excised,immediately wrapped in aluminum foil dipped in liquid nitrogenand then kept in the freezer at �80 �C where samples were storeduntil the beginning of the tests. For total phenol and lignin con-tents, only the last sampling time period was considered. A totalof three replicates for each treatment were used and arranged ina randomized block design with 3 seeds per pot.

2.7. Biochemical assays

2.7.1. Enzyme activitiesThe total soluble protein concentration was measured using a

standard curve of bovine serum albumin according to the methodadopted by Bradford (1976) and calculated as peroxidase (POX)and phenylalanine ammonium lyase (PAL) activities.

POX extraction and assay were carried out according to Urba-nek et al. (1991). For PAL, the procedures followed the one de-scribed by Mori et al. (2001).

2.7.2. Total phenol and lignin contentsAbsorbance values of the total phenol reaction were determined

at 725 nm in a spectrophotometer and calculated based on cate-chol curve (0–100 lg lg ml�1). The total phenolic compoundswere expressed in equivalent lg of catechol per mg of dry weightaccording to the method described by Spanos and Wrolstad (1990).

For lignin content, the absorbance was determined in a spectro-photometer at 280 nm, and the values calculated based on lignincurve (0–100 lg ml�1) and expressed in lg of soluble lignin permg of dry matter Doster and Bostock (1988).

Assays were done in triplicate.

Table 1Effects of seed treatments on bean artificially inoculated with Cff by percentageseedling emergence (PSE) and speed emergence index (SEI).

Treatments PSE SEI

Cff 53.58b 3.60bCff + Copper oxychloride 41.34b 2.71bCff + UFLA285 46.22b 2.96bCff + UFLA168* 51.82b 2.94bCff + ALB629 57.73b 3.27bCff + MEN2 60.30b 3.66bWater (control) 78.30a 4.95aCV (%) 22.23 22.64

* Means followed by the same letter do not differ significantly according to Tukey’stest (P 6 0.05). Means of three experiments of four replicates of ten seedlings each.CV: coefficient of variation.

2.8. Experimental design and statistical analysis

All experiments were performed under greenhouse conditionsat the Universidade Federal de Lavras (UFLA), in Lavras, Minas Ger-ais, Brazil (915 m altitude, 21�130340 0S and 44�580310 0W). Theexperimental design was in randomized blocks, data were submit-ted to one-way variance analysis (ANOVA) and for significantmeans Tukey’s multiple range tests (P = 0.05) were applied. Forall analyses, the assumptions of normality and homogeneity of var-iance were checked and no transformation was necessary. Sisvar(Build 72) Copyrigth Daniel Furtado Ferreira 1999–2007 computersoftware 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) orthe percentage seedling emergence (PSE). However, there was adecrease for PSE and SEI after pathogen inoculation when com-pared with the non-treated control (P < 0.001) (Table 1).

All PGPR strains reduced disease severity by 42–76% of the areaunder the disease progress curve (AUDPC) when means were com-pared by the Tukey’s test. There was no significant interaction be-tween treatments and time when the experiment was carried out(P = 0.13), but there was a significant effect for treatments whenthe experiment was carried out (P < 0.001) (Fig. 1).

Regarding RGI, which was recorded at the same period as dis-ease severity, all PGPR strains statistically differed from the othertreatments with highest means when compared to the untreatedcontrol or fungicide (P < 0.001) (Fig. 2).

When plants were artificially inoculated with Cff and treatedwith PGPR, it was found that both shoot and root dry weight signif-icantly 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 contentWithout inoculation, both PGPR promoted an increase in the to-

tal soluble phenolics and UFLA285 promoted a content higher thanthe ASM treatment. This last one was higher than the control(water) while in the presence of the pathogen, UFLA285 promotedthat increase (Fig. 4).

Without inoculation, ASM induced lignin accumulation, fol-lowed by UFLA285. In the presence of the pathogen ASM andALB629 induced that accumulation when compared to the control(water) (Fig. 5).

3.2.2. Enzyme activitiesBean plants with PGPR seed treatment with ALB629 or UFLA285

showed that UFLA285 and ASM without pathogen inoculation pro-moted an increase in PAL activity. Upon inoculation both PGPR iso-lates promoted a PAL increase at the second phenological stage(VE), however, with disease development the enzyme content de-creased with no difference at the last considered stage (V4) (Fig. 6).

In regard to POX, upon Cff inoculation PGPR treatment inducedan accumulation higher than the control at V1 (ALB629 strain) andat V2, V3, and V4 phenological stages (ALB629 and UFLA285strains). For plants not inoculated, PGPR induced an accumulationhigher than the control at V3 (UFLA285) and V4 (ALB629 andUFLA285 strains) (Fig. 7). The only increase in this enzyme activitywas observed for plants treated with ASM at the last sampling timepoint for plants that were not inoculated with the pathogen.

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

Fig. 2. Effect of seed treatment with PGPR, copper oxychloride and water onrelative growth index (RGI) CV = 13.50. Bars headed with the same letter are similarat the 5% level according to Tukey’s test.

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4. Discussion

A growing demand for sustainable disease control supports thesearch for a safe and efficient control strategy, and we showed that

Fig. 3. Effect of seed treatment with PGPR, copper oxychloride and water on the shoot dcollection. CV = 22.91 and 28.71, respectively for SDW and RDW. For each variable, bars hTukey’s test.

endospore-forming strains have the potential for disease control(Fig. 1). The most promising strain was B. subtilis ALB629, whichhas also shown a consistent disease control even at different tem-peratures (Martins et al., in press). Besides disease control, thePGPR strains did not cause phytotoxicity to the bean plants butpromoted plant growth as expressed by RGI as well as by inducedshoot and root dry weight in the presence of the pathogen, assur-ing a similar level of plant growth in the absence of the pathogen.Further experiments will be carried out to determine if these PGPRisolates are also able to increase plant growth promotion withoutthe pathogen inoculation. The growth promotion in inoculatedplants may be related to a compensatory effect (Ribeiro et al.,2004; Shimada et al., 2000), where plants inoculated with Cff andtreated with PGPR could grow more than control treatment (water)(Fig. 2) and exhibit a higher final dry matter (Fig. 3). In relation tocopper fungicide it was not effective for control of bacterial wiltand did not increase the PSE and SEI or SDW and RDW.

The PGPR strains tested did not keep germination of the patho-gen-free seeds. Similar responses were observed by Hsieh et al.(2006), where seeds infected with Cff have been shown to reduceseedling emergence. Therefore, the preventive-basis of bacterialcontrol would be more efficient than the curative one.

In addition, this study indicated that the biological seed treat-ment with the selected PGPR strains led to a significant increasein the phenolics content and lignin accumulation both for infectedand non-infected bean plants, suggesting that these parameters areimplicated in the defense response as already reported for someother pathosystems (Abo-Elyousr et al., 2009; Kandan et al.,2002; Mandal, 2010; Zhang et al., 2011). These defense responsesmay include the elaboration of cell wall thickenings usually accom-panied by the deposition of lignin, a polymer of aromatic phenolics(Fattah et al., 2011). This cell wall providing structural support anda passive barrier against invading pathogens (Carpita and McCann,2000) and could play a role as a physical barrier to stop Cff spreadthrough the plant. Hernández-Blanco et al. (2007) found that analteration of secondary cell wall integrity by inhibiting cellulosesynthesis has lead to specific activation of plant defense responseagainst 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-activitywas activated by two of the selected PGPR strains. In the inoculatedtreatment, the highest activation appeared to be related to the sec-ond phenological stage (V2) when compared to the non-inoculatedone which reached the highest activation at V4. Surprisingly, withdisease progress, instead of a priming effect (Jung et al., 2012) bythe PGPR the enzyme content decreased with no difference atthe last stage. It appears that the pathogen blocked the plant

ry weight (SDW) and root dry weight (RDW) on plants 24 days old at time of dataeaded by the same letters are not significantly different at the 5% level according to

Fig. 4. Total phenol at V1, V2, V3, and V4 bean plant phenological in the presence (left) or absence (right) of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) inoculum.Bars headed with the same letter are similar at the 5% level according to Tukey’s 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 flaccumfaciens pv. flaccumfaciens (Cff)inoculum. Bars headed with the same letter are similar at the 5% level according to Tukey’s test. Bars headed with the same letter are similar at the 5% level according toTukey’s 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 Curtobacteriumflaccumfaciens pv. flaccumfaciens (Cff). The line on each bar represents ±SE.

S.J. Martins et al. / Biological Control 66 (2013) 65–71 69

defense response development as already reported (Aslam et al.,2008; Cooper et al., 2008). Considering that the overall phenotypeis disease control, other modes of action should be involved. POX ispromptly triggered and this induction in PGPR-treated plants isfaster than the chemical inducer (ASM), and this seems to be a con-sensus for PGPR treatment (Ishida et al., 2008). The enzyme is acti-vated 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 theASM-treated, especially at V2.

In conclusion, these results open new prospects of bacterial wiltcontrol. Since a broad spectrum activity and a wide host range arecrucial for a successful biocontrol agent (Nagorska et al., 2007),UFLA285 strain reduces post-emergence damping-off in cotton

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

70 S.J. Martins et al. / Biological Control 66 (2013) 65–71

when used as a seed treatment in a 2-year field trial (Medeiroset al., 2008) and ALB629 used to increase grafting capacity of cacaoplantlets (Alan Pomella, personal communication) making thempromising biological control agents also for bacterial wilt of com-mon bean in Brazil.

Acknowledgments

We thank Conselho Nacional de Desenvolvimento CientificoCultural (CNPq), Fundação de Apoio à Pesquisa do Estado de MinasGerais (FAPEMIG) and Programa de Apoio a Primeiros Projetos(PAPP/UFLA) for providing the financial support necessary for thedevelopment of this work. And we thank CNPq for providing assis-tantships for the first, third and fourth authors.

References

Abo-Elyousr, K.A.M., Hashem, M., Ali, E.H., 2009. Integrated control of cotton rootrot disease by mixing fungal biocontrol agents and resistance inducers. CropProt. 28, 295–301.

Aslam, S.N., Newman, M.A., Erbs, G., Morrissey, K.L., Chinchilla, D., Boller, T., Jensen,T.T., De Castro, C., Ierano, T., Molinaro, A., Jackson, R.W., Knight, M.R., Cooper,R.M., 2008. Bacterial polysaccharides suppress induced innate immunity bycalcium chelation. Curr. Biol. 18, 1078–1083.

Bradford, M.M., 1976. A rapid sensitive method for the quantification of microgramquantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72, 248–254.

Carpita, N.C., McCann, M., 2000. The Cell Wall. In: Buchanan, B., Gruissem, W., Jones,R.L. (Eds.), Biochemistry and Molecular Biology of Plants. American Society ofPlant Physiologists, Rockville, MD, pp. 52–108.

Conner, R.L., Balasubramanian, P., Erickson, R.S., Huang, H.C., Mündel, H.H., 2008.Bacterial wilt resistance in kidney beans. Can. J. Plant Sci. 8, 1109–1113.

Cooper, A.J., Latunde-Dada, A.O., Woods-Tör, A., Lynn, J., Lucas, J.A., Crute, I.R., Holub,E.B., 2008. Basic compatibility of Albugo candida in Arabidopsis thaliana andBrassica juncea causes broad-spectrum suppression of innate immunity. Mol.Plant Microbe Interact. 21, 745–756.

Deuner, C.C., Souza, R.M., Ishida, A.K.N., Zacaroni, A.B., Von Pinho, E.V.R., Machado,J.C., Camera, J.N., 2011. Inoculation of dry bean seeds with Curtobacteriumflaccumfaciens pv. flaccumfaciens (Cff) using the physiological conditioningtechnique. Rev. Bras. Sementes 33, 009–020.

Doster, M.A., Bostock, R.M., 1988. Quantification of lignin formation in almond barkin response to wounding and infection by Phytophthora species. Phytopathology78, 473–477.

El Modafar, C., Elgadda, M., El Boutachfaiti, R., Abouraicha, E., Zehhar, N., Petit, E., ElAlaoui-Talibi, Z., Courtois, B., Courtois, J., 2012. Induction of natural defenceaccompanied by salicylic acid-dependant systemic acquired resistance intomato seedlings in response to bioelicitors isolated from green algae. Sci.Hortic-Amsterdam 138, 55–63.

Fattah, G.M.A., El-Haddad, S.A., Hafez, E.E., Rashad, Y.M., 2011. Induction of defenseresponses in common bean plants by arbuscular mycorrhizal fungi. Microbiol.Res. 166, 268–281.

Ferreira, D.F., 2011. Program SISVAR. A computer statistical analysis system. Ciênc.Agrotec. 35, 1039–1042.

Hahm, M.S., Sumayo, M., Hwang, Y.J., Jeon, S.A., Park, S.J., Lee, J.Y., Ahn, J.H., Kim, B.S.,Ryu, C.M., Ghim, S.Y., 2012. Biological control and plant growth promotingcapacity of rhizobacteria on pepper under greenhouse and field conditions. J.Microbiol. 50, 380–385.

Hall, R., 1994. Compendium of Bean Diseases. American Phytopathological Society,St. Paul, Minnesota, p. 73.

Harveson, R.M., Schwartz, H.F., 2007. Bacterial diseases of dry edible beans in thecentral high plains. Plant Health Prog.. http://dx.doi.org/10.1094/PHP-2007-0125-01-DG.

Hedges, F., 1922. A bacterial wilt of the bean caused by Bacterium flaccumfaciensnov. sp. Science 55, 433–434.

Hedges, F., 1926. Bacterial wilt of bean (Bacterial flaccumfaciens Hedges), includingcomparisons with Bacterial phaseoli. Phytopathology 16, 1–22.

Herbes, D.H., Theodoro, G.F., Maringoni, A.C., dal Piva, C.A., de Abreu, L., 2008.Detection of Curtobacterium flaccumfaciens pv. flaccumfaciens inseeds of common bean produced in Santa Catarina. Trop. Plant Pathol. 33, 53–156.

Hernández-Blanco, C., Feng, D.X., Hu, J., Sánchez-Vallet, A., Deslandes, L., Llorente, F.,Berrocal-Lobo, M., Keller, H., Barlet, X., Sánchez-Rodríguez, C., Anderson, L.K.,Somerville, S., Marco, Y., Molina, A., 2007. Impairment of cellulose synthasesrequired for arabidopsis secondary cell wall formation enhances diseaseresistance. Plant Cell 19, 890–903.

Hsieh, T.F., Huang, H.C., Mundel, H.H., Erickson, R.S., 2003. A rapid indoor techniquefor screening common bean (Phaseolus vulgaris L.) for resistance to bacterial wilt[Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges) Collins and Jones].Rev. Mex. Fitopatol. 21, 370–374.

Hsieh, T.F., Huang, H.C., Erickson, R.S., 2006. Bacterial wilt of common bean: effect ofseedborne inoculum on disease incidence and seedling vigour. Seed Sci.Technol. 34, 57–67.

Hsieh, T.F., Huang, H.C., Conner, R.L., 2004. Bacterial wilt of bean: current status andprospects. Recent Res. Dev. Plant Sci. 2, 181–206.

Huang, H.C., Erickson, R.S., Hsieh, T.F., 2007. Control of bacterial wilt of bean(Curtobacterium flaccumfaciens pv. flaccumfaciens) by seed treatment withRhizobium leguminosarum. Crop Prot. 26, 1055–1061.

Ishida, A.K.N., Souza, R.M., Resende, M.L.V., Cavalcanti, F.R., Oliveira, D.L., Pozza, E.A.,2008. Rhizobacterium and acibenzolar-S-methyl (ASM) in resistance inductionagainst bacterial blight and expression of defense responses in cotton. Trop.Plant Pathol. 33, 027–034.

Jung, S.C., Martinez-Medina, A., Lopez-Raez, J.A., Pozo, M.J., 2012. Mycorrhiza-induced resistance and priming of plant defenses. J. Chem. Ecol. 38, 651–664.

Kado, C.I., Heskett, M.G., 1970. Selective media for isolation of Agrobacterium,Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60,969–976.

Kandan, A., Commare, R.R., Nandakumar, R., Ramiaii, M., Raguchander, T.,Samiyappan, R., 2002. Induction of phenylpropanoid metabolism byPseudomonas fluorescens against tomato spotted wilt virus in tomato. FoliaMicrobiol. 47, 121–129.

Kloepper, J.W., Lifshitz, R., Zablotowicz, R.M., 1989. Free-living bacterial inocula forenhancing crop productivity. Trends Biotechnol. 7, 39–44.

Krause, W., Rodrigues, R., Gonçalves, L.S.A., Neto, F.V.B., Leal, N.R., 2009a. Geneticdivergence in snap bean on agronomic traits and resistance to bacterial wilt.Crop Breed. Appl. Biotechnol. 9, 246–252.

Krause, W., Rodrigues, R., Leal, N.R., 2009b. Identification of resistance sources andevaluation of Curtobacterium flaccumfaciens pv. flacumfacceins inoculationmethods in snap bean. Ciênc. Agrotec. 33, 1901–1907.

S.J. Martins et al. / Biological Control 66 (2013) 65–71 71

Machado, J.C., 2000. Tratamento de Sementes no Controle de Doenças, 1st ed.Lavras, Brazil.

Mandal, S., 2010. Induction of phenolics, lignin and key defense enzymes ineggplant (Solanum melongena L.) roots in response to elicitors. Afr. J. Biotechnol.9, 8038–8047.

Maringoni, A.C., Rosa, E.F., 1997. Ocorrência de Curtobacterium flaccumfaciens pv.flaccumfaciens em feijoeiro no Estado de São Paulo. Summa Phytopathol. 23,160–162.

Medeiros, F.H.V., Moraes, I.S.F., Neto, E.B.S., Mariano, R.L.R., 2009. Management ofmelon bacterial blotch by plant beneficial bactéria. Phytoparasitica 37, 453–460.

Medeiros, F.H.V., Souza, R.M., Ferro, H.M., Medeiros, F.C.L., Pomela, A.W.V.,Machado, J.C., Santos Neto, H., Soares, D.A., Pare, P.W., 2008. Bacillus spp. tomanage seed-born Colletotrichum gossypii var. cephalosporioides damping-off.Phytopathology 98, S102–S103.

Mohan, S.K., Hagedorn, D.J., 1989. Additional Bacterial Diseases. In: Schwartz, H.F.,Pastor-Corrales, M.A. (Eds.), Bean Production Problems in the Tropics, 2nd ed.Centro Internacional de Agricultura Tropical, Colombia, CA, USA, pp. 303–319.

Mori, T., Sakurai, M., Sakuta, M., 2001. Effects of conditioned médium on activitieson PAL, CHS, DAHP synthase (DS-Co and DS-Mn) and anthocyanin production insuspension cultures of Fragaria ananassa. Plant Sci. 160, 355–360.

Nagorska, K., Bikowski, M., Obuchowskii, M., 2007. Multicellular behavior andproduction of a wide variety of toxic substances support usage of Bacillus subtilisas a powerful biocontrol agent. Acta Biochim. Pol. 54, 495–508.

Ribeiro, N.D., Cargnelutti Filho, A., Hoffmann Júnior, L., Possebon, S.B., 2004.Experimental precision in the evaluation of beans cultivars of different habits ofgrowth. Cienc. Rural 34, 1371–1377.

Schwartz, H.F., Brick, M.A., Otto, K., Ogg, J.B., 2010. Germplasm evaluation forresistance to bacterial wilt in common bean, 2008–2009. APS Plant DreamixManagement Reports 4:v125, p. 2.

Shankar, A.C.U., Nayaka, S.C., Niranjan-Raj, S., Kumar, H.B., Reddy, M.S., Niranjana,S.R., Prakash, H.S., 2009. Rhizobacteria-mediated resistance against the blackeye cowpea mosaic strain of bean common mosaic virus in cowpea (Vignaunguiculata). Pest Manag. Sci. 65, 1059–1064.

Shimada, M.M., Arf, O., Sá, M.E., 2000. Yield Components and crop growth ofcommon bean under different plant densities. Bragantia 59, 181–187.

Souza, V.L., Maringoni, A.C., 2008. Análise ultraestrutural da interação deCurtobacterium flaccumfaciens pv. flaccumfaciens em genótipos de feijoeiro.Summa Phytopathol. 34, 318–320.

Spanos, G.A., Wrolstad, R.E., 1990. Influence of processing and storage on thephenolic composition of Thompson seedless grape juice. J. Agric. Food Chem. 38,1565–1571.

Sundaramoorthy, S., Raguchander, T., Ragupathi, N., Samiyappan, R., 2012.Combinatorial effect of endophytic and plant growth promoting rhizobacteriaagainst wilt disease of Capsicum annum L. caused by Fusarium solani. Biol.Control 60, 59–67.

Teixeira, H., Machado, J.C., 2003. Transmissibility and effect of Acremonium strictumin maize seeds. Ciênc. Agrotec. 27, 1045–1052.

Urbanek, H., Kuzniak-Gebarowska, E., Herka, H., 1991. Elicitation of defenceresponses in bean leaves by Botrytis cinerea polygalacturonase. Acta Physiol.Plant 13, 43–50.

Zhang, W., Yang, X., Qiu, D., Guo, L., Zeng, H., Mao, J., Gao, Q., 2011. PeaT1-inducedsystemic acquired resistance in tobacco follows salicylic acid-dependentpathway. Mol. Biol. Rep. 38, 2549–2556.