J.

Phytopathology 153, 120123 (2005) 2005 Blackwell Verlag, Berlin

Plant Pathology Department, Federal University of Vicosa, Vicosa, MG, Brazil

Macromolecules Released by a Plant Growth-promoting Rhizobacterium as Elicitors of Systemic Resistance in Tomato to Bacterial and Fungal Pathogens
R. S.Romeiro , R.LannaFilho1, J. R. Vieira Junior1, H. S. A. Silva2, M. C. Baracat-Pereira3 and M. G. Carvalho1 Authors addresses: 1Plant Pathology Department, Federal University of Vicosa, 36571-000 Vicosa, MG, Brazil; 2Embrapa Meio Ambiente, Caixa Postal 69, 13820-000 Jaguariuna, SP, Brazil; 3Biochemistry Department, Federal University of Vicosa, 36571-000 Vicosa, MG, Brazil (correspondence to R. da Silva Romeiro. E-mail: rromeiro@ufv.br) Received September 2, 2004; accepted December 16, 2004 Keywords: Pseudomonas syringae pv., Alternaria solani, Xanthomonas campestris pv., Bacillus cereus, biological control, rhizobacteria, induced systemic resistance, elicitors, macromolecules, tomato
1

Abstract
One of 500 rhizobacteria isolated from soil, rhizosphere and rhizoplane of healthy tomato plants was previously selected in laboratory, greenhouse and eld tests as a good inducer of systemic resistance. This plant growth-promoting rhizobacterium (PGPR) was identied as Bacillus cereus by fatty-acid analysis. Bacillus cereus bacterial cells were removed from liquid culture by centrifugation and the supernatant repeatedly dialyzed (cut-o 12 000 daltons) against distilled water. Dialysates applied to roots protected tomato plants against leaf fungal and bacterial pathogens, evidence that macromolecules synthesized by the PGPR and released into the environment act as elicitors of systemic resistance.

Introduction
It is well-known that some plant growth-promoting rhizobacteria (PGPR) induce systemic resistance to diseases after colonizing plant roots and that this resistance may be expressed both locally and far from the pathogen site of contact. Consequently, the next question is how they act as elicitors. A logical assumption is that some sort of molecule having a bacterial origin ought to trigger such a biochemical mechanism. Bacterial molecules possibly originated in either of two ways on the surface layers of the bacterial cell or molecules synthesized within the rhizobacterium cell and then liberated into the environment. Such molecules, in both cases, were named bacterial determinants by Loon et al. (1998a,b). In regard to bacterial cell constituents, several works focused on the lipopolysaccharide (LPS) of the outer membrane (Dow et al., 2000; Leeman et al., 1995; Romeiro and Kimura, 1997; Reitz et al., 2000). Additionally, there are several reports on small molecules liberated by rhizobacteria into the environment that might act as induced systemic resistance (ISR) elicitors

such as salicylic acid (Loon et al., 1998a,b; Meyer et al., 1998; Chen et al., 1999), siderophores (Thomashow, 1996; Loon et al., 1998a; Ramamoorthy et al., 2001) and phenazines (Chin-A-Woeng et al., 1998, 2000, 2001). On the contrary, no consistent report could be found up to now on macromolecules synthesized and given o in the environment by rhizobacteria that might be able to act as elicitors of ISR in plants. Nevertheless, after the discovery of harpins (Wei et al., 1992; Wei and Beer, 1993) and of secretory systems in bacteria (Madigan et al., 2003) this hypothesis seems to deserve further attention. We present here evidence that a rhizobacterium (Bacillus cereus, isolate UFV-101) previously tested and selected as a good biocontrol agent against tomato diseases (Silva, 2002; Silva et al., 2004) synthesizes macromolecules that act as inducers of systemic resistance.

Materials and Methods

Experiments were carried out using the facilities of the Laboratory Plant Bacteriology and Biological Control of the Department of Plant Pathology of the Federal University of Vicosa, Minas Gerais, Brazil. Santa Cruz Tomato plants cv. Kada were grown in non-sterilized soil, in a greenhouse with temperature and relative humidity partially controlled.
Microorganisms, their origin and cultivation

Unless otherwise indicated, bacteria were grown in medium 523 (Kado and Heskett, 1970) and fungi in potato dextrose agar (PDA; Tuite, 1969). Cultures of tomato pathogens were obtained from the collection of the Department of Plant Pathology of the Universidade Federal de Vicosa, Brazil. Culture UFV-101, iden tied as B. cereus by fatty-acid analysis, was isolated from a soil sample collected from a tomato plantation in a farm located at Coimbra, Minas Gerais State, www.blackwell-synergy.com

Plant Growth-promoting Rhizobacterium

121

Brazil and selected as a good inducer of ISR in previous works (Silva et al., 2003, 2004).
PGPR cultivation in minimal medium

For this experiment, isolate UFV-101 of the PGPR B. cereus was grown in modied Simmons medium (Schaad et al., 2001) in which citrate was replaced by 0.1% (w/v) glucose as the sole carbon source by Gijsegem et al. (1995, 2000) point out that detection of macromolecules given o by bacteria in supernatant is easier after their cultivation in minimal culture media. The turbidity of liquid cultures in side-arm asks under continuous shacking, at room temperature (26 4C), was recorded at dierent time intervals to determine the inection point in the exponential phase of the growth curve.
Preparation of crude dialysates

for 2 h in the crude dialysate at room temperature. Dialysed sterile culture media, saline and a suspension of live PGPR cells were used as controls. After exposure, plants were replaced in plastic pots containing non-sterile soil and kept in the greenhouse.
Inoculation of challenging pathogens

PGPR cells grown in modied Simmons liquid culture medium up to the inection point of the exponential growth phase were removed from suspension by one centrifugation at 10 000 g for 15 min in a RC-5B Sorvall refrigerated centrifuge (Kendro Laboratory Products, San Diego, CA, USA). The supernatant solution was sterilized by ltration through a 0.45 l-pore membrane, transferred into dialysis bags (cut-o 12 kDa) and dialyzed against 400 volumes of distilled water, at 4C, under continuous stirring, with several changes of water over a 48-h period. Dialysates were lyophilized and stored at )80C for later usage.
Analytical methods

Three days after exposure of root systems to dialysates, water or PGPR alive cells, plants were inoculated with Pseudomonas syringae pv. tomato by spraying with an inoculum suspension (108 cfu/ml). Other phylloplane pathogens (Alternaria solani, Xanthomonas campestris pv. vesicatoria, and Corynespora cassiicola) were also tested. Plant pathogenic bacteria were inoculated by spraying an inoculum suspension (108 cfu/ml), and Cor. cassiicola was inoculated in the same way by using an inoculum suspension previously adjusted to 2.0 104 conidia/ml and A. solani to 9.5 105 conidia/ml, according to procedures described by Silva et al. (2003). In all cases, plants were kept in a moist chamber for 24 h before and after inoculation. Inoculated plants were maintained in a greenhouse and lesions counted after disease symptoms had fully developed. For each challenging pathogen test, there were used 10 plants per treatment, with one plant per pot considered one replicate. Statistical analysis was performed according the Tukeys test.

Results
As expected, B. cereus grew slower in modied Simmons than in 523 medium (Kado and Heskett, 1970). The inection point for the exponential growth phase for the former took place after approximately 16 h, while for the latter it was observed nearly 5 h after starting the culture (Fig. 1). The colourless dialysate turned into a white powder during lyophilization and, just before freeze-drying, contained 42.07 lg of bovine serum albumin (BSA) equivalents per ml and 2.77 lg of glucose equivalents per ml. No detectable inhibition haloes were spotted on the overlay in the bioassay plates when several tomato pathogens (A. solani, P. syringae pv. tomato, Cor. cassiicola, X. campestris pv. vesicatoria, P. syringae pv. syringae,

As a reference, protein was quantied in crude dialysates as described by Bradford (1976) and results expressed as bovine serum albumin equivalents. The anthrone method (Gerhardt, 1994) was used to estimate total hexoses and results were expressed as glucose equivalents.
In vitro bioassays

To full one of the criteria suggested by Steiner and Schonbeck (1995), the putative toxic activity of crude dialysates against tomato pathogenic fungi and bacteria was veried by the overlay diusion method. Melted semisolid culture media [0.8% (w/v) agar, 48C] containing propagules of every pathogen, was poured into Petri dishes containing a basic layer of solid water-agar, in an amount enough for a 1 mm-thick overlay. After solidication, a cork borer was used to produce a 1 cm2 well in the central part of the overlay and 100 lL of the crude dialysate placed in the cavity. Bioassay plates were moved to an incubator (28C) and checked daily for presence of inhibition haloes for a week.
Exposure of plants to crude dialysates

Soil from 40-day-old tomato plants, grown in plastic pots in a greenhouse whose temperature was partially controlled, was carefully removed from roots under running tap water. The naked roots were maintained

Fig. 1 Growth curve of Bacillus cereus, isolate UFV-101, on liquidmodied Simmons medium at 28C

122

Romeiro et al.

Fig. 2 Severity of bacterial speck (Pseudomonas syringae pv. tomato), articially inoculated in plants whose roots were previously exposed to live plant growth-promoting rhizobacterium (PGPR) cells, to the crude dialysate, uninoculated and dialysed Simmons medium or to water (LSD (least signicant dierence), Tukey 0.05)

Fig. 4 Severity of late blight (Alternaria solani) articially inoculated into plants whose roots were previously exposed to alive plant growth-promoting rhizobacterium (PGPR) cells, to the crude dialysate or to water (MSD, Tukey 0.05)

Fig. 3 Severity of bacterial blight (Xanthomonas campestris pv. vesicatoria), articially inoculated into plants whose roots were previously exposed to live plant growth-promoting rhizobacterium (PGPR) cells, to the crude dialysate or to water (MSD, Tukey 0.05)

Fig. 5 Severity of bacterial target spot (Corynespora cassiicola) articially inoculated into plants whose roots were previously exposed to live plant growth-promoting rhizobacterium (PGPR) cells, to the crude dialysate or to water (MSD, Tukey 0.05)

Pse. corrugata, Ralstonia solanacearum and Clavibacter michiganensis pv. michiganensis) were used as indicators. Plants whose naked root had been exposed to the crude dialysate and were inoculated next with the challenging pathogens showed fewer lesions than the unexposed plants (see Figs 25).

Discussion and Conclusions

Experiments with cultures harvested at the inection point of the exponential phase rest on the assumption that, before this point, the target macromolecules might not have been synthesized by and exported from the bacterial cells, whereas after it, although export processes might have occurred, cells could have used these molecules as carbon and nitrogen sources, as they were growing in a minimal medium (Madigan et al., 2003). A basal minimal medium was selected tentatively for this work, based on the observation by Gijsegem et al. (1995) that macromolecules synthesized by bacteria and exported into the environment are easily detected in the supernatants of a poor culture medium.

As shown in Figs 25, initial exposure of tomato roots to the crude dialysate resulted in less disease by a challenging pathogen in comparison with the water controls, and this was similar to the eect when instead whole living PGPR cells were used. At this point, we cannot put too much restriction to the statement that a macromolecule or several macromolecules present in the dialysate-induced resistance in the plants and that this resistance had a systemic nature. There is no need to meet all criteria proposed by Steiner and Schonbeck (1995) to validate the assumption that the dialysate acted as a true elicitor of ISR sensu Sticher et al. (1997) and Loon et al. (1998a). We have here presented evidence that the dialysate elicited ISR. Initially, the dialysate was applied to roots, this was followed by each challenger inoculated on leaves, where resistance to the challenging pathogen increased signicantly, far from the site of exposure. Secondly, this fact clearly implied a time lag between that of exposure and when resistance was expressed. Thirdly, the crude dialysate showed no direct toxic activity in the in vitro bioassay, at least within the limits of the adopted technique. Fourthly, the tomato plants were simultaneously and somewhat unspecically protected

Plant Growth-promoting Rhizobacterium

123 (eds), Pathogenesis and Host Specicity in Plant Diseases, Kidlington, UK, Elsevier Science, 1995. Gijsegem F, Vasse J, Camus JC, Marenda M, Boucher C, Gijsegem F. (2000) Ralstonia solanacearum produces Hrp-dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells. Mol Microbiol 36:249260. Kado CI, Heskett MG. (1970) Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60:969979. Leeman M, Pelt JA, Ouden FM et al. (1995) Induction of systemic resistance against fusarium wilt of radish by lipopolysaccharides of Pseudomonas uorescens. Phytopathology 85:10211027. Loon LC, Bakker P, Pieterse CMJ. (1998a) Systemic resistance induced by rhizosphere bacteria. Ann Rev Phytopathol 36:453483. Loon LC, Bakker P, Pieterse CMJ, Duy B, Rosenberger U, Defago G. (1998b) Induction and expression of PGPR-mediated induced resistance against pathogens. Bull OILB SROP 21:103110. Madigan MM, Martinko J, Parker JE. Brock Biology of Microorganisms. New York, USA, Prentice Hall, 2003. Meyer G, Hofte M, Duy B, Rosenberger U, Defago G. (1998) Induction of systemic resistance by the rhizobacterium Pseudomonas aeruginosa 7NSK2 is a salicylic acid dependent phenomenon in tobacco. Bull OILB SROP 21:117121. Ramamoorthy V, Viswanathan R, Raguchander T, Prakasam V, Samiyappan R. (2001) Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pests and diseases. Crop Prot 20:111. Reitz M, Rudolph K, Schroeder I, Homann HS, Hallmann J, Sikora RA. (2000) Lipopolysaccharides of Rhizobium etli strain G12 act in potato roots as an inducing agent of systemic resistance to infection by the cyst nematode Globodera pallida. Appl Environ Microbiol 66:35153518. Romeiro RS, Kimura O. (1997) Induced resistance in pepper leaves inltrated with puried elicitors from Xanthomonas campestris pv. vesicatoria. J Phytopathol 145:495498. Schaad NW, Jones JB, Chun W. Laboratory Guide for Identication of Plant Pathogenic Bacteria, 3rd edn. Saint Paul, Minnesota, USA, The American Phytopathological Society, 2001. Silva HSA. Rizobacterias como indutoras de resistencia sistemica a patogenos foliares e como promotoras de crescimento em tomateiro. PhD Dissertation. Universidade Federal de Vicosa, Departa mento de Fitopatologia, 2002, 108 pp. Silva HSA, Romeiro RS, Mounteer A. (2003) Development of a root colonization bioassay for rapid screening of rhizobacteria for potential biocontrol agents. J Phytopathol 151:4246. Silva HSA, Romeiro RS, Macagnan D, Halfeld-Vieira BA, Pereira MCB, Mounteer A. (2004) Rhizobacterial induction of systemic resistance in tomato plants: non-specic protection and increase in enzyme activities. Biol Control 29:288295. Steiner U, Schonbeck F. Induced disease resistance in monocots. In: Hammerschmidt R, Kuc J (eds), Induced Resistance to Disease in Plants (Developments in Plant Pathology, Vol 4), Dordrecht, The Netherlands, Kluwer Academic Pub., 1995. Sticher L, Mauch-Mani B, Metraux JP. (1997) Systemic acquired resistance. Ann Rev Phytopathol 35:235270. Thomashow LS. (1996) Biological control of plant root pathogens. Curr Opin Biotechnol 7:343347. Tuite J. Plant Pathological Methods. Minneapolis, USA, Burgess Pub. Co., 1969. Wei ZM, Beer SV. (1993) HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family. J Bacteriol 175:79587967. Wei ZM, Laby RJ, Zumo CH et al. (1992) Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257:8588.

against distinct fungal and bacterial pathogens. Last, but not least, the crude dialysate aorded similar protection to that provided by living PGPR. The aforementioned provide sound evidence that the PGPR under investigation synthesizes and exports into the environment one or several kinds of macromolecules that act as elicitors of ISR in tomato. How precisely the PGPR does so is unknown, but it is quite possible that one or more secretory systems are involved in their production. Provided that our data substantiate a scientic fact, additional aspects now need to be claried. At present, we know essentially nothing about the chemical nature and properties of these macromolecules; it is not known if they are proteins, polysaccharides, or glycoproteins, as we know only that their molecular mass is larger than 12 kDa, or whether the elicitor molecules are a single substance or a group, although sharing similar biological properties. In the Laboratory of Plant Bacteriology and Biological Control in the Universidade Federal de Vicosa, a research group is seeking answers to these questions related to this new nding. Data presented in this paper should be a contribution to a better understanding of how PGPR induce ISR in plants and they suggest lines of research that may eventually lead to the development of bioproducts similar to the well-known harpins.
References
Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254. Chen C, Belanger RR, Benhamou N, Paulitz TC. (1999) Role of salicylic acid in systemic resistance induced by Pseudomonas spp. against Pythium aphanidermatumin cucumber roots. Eur J Plant Pathol 105:477486. Chin-A-Woeng TFC, Bloemberg GV, Van der Bij AJ et al. (1998) Biocontrol by phenazine-1-carboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by Fusarium oxysporum f. sp. radicislycopersici. Mol Plant-Microbe Interact 11:10691077. Chin-A-Woeng TFC, Bloemberg GV, Mulders IHM, Dekkers LC, Lugtenberg BJJ. (2000) Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol PlantMicrobe Interact 13:13401345. Chin-A-Woeng TFC, Broek D, Voer D et al. (2001) Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium. Mol Plant-Microbe Interact 14:969979. Dow M, Newman MA, Roepenack E. (2000) The induction and modulation of plant defense responses by bacterial lipopolysaccharides. Ann Rev Phytopathology 38:241261. Gerhardt PE. (ed.), Methods for General and Molecular Bacteriology. Washington, USA, American Society for Microbiology, 1994. Gijsegem F, Genin S, Boucher C. Hrp and avr genes, key determinants controlling the interaction between plants and gram-negative phytopathogenic bacteria. In: Singh US, Singh RP, Kohmoto K