Plant Physiology and Biochemistry 41 (2003) 277281 www.elsevier.

com/locate/plaphy

Original article

Three newly isolated plant growth-promoting bacilli facilitate the seedling growth of canola, Brassica campestris
Sibdas Ghosh a,*, Jon N. Penterman b, Rebecca D. Little a, Rocky Chavez a, Bernard R. Glick c
a

Department of Natural Sciences and Mathematics, Dominican University of California, San Rafael CA 94901, USA b Department of Plant Microbial Biology, University of California-Berkeley, Berkeley, CA 94720, USA c Department of Biology, University of Waterloo, Waterloo, Ont., N2L 3G1, Canada Received 14 May 2002; accepted 12 November 2002

Abstract Three strains of plant growth-promoting bacteria were isolated from southeastern Wisconsin soils, based upon the ability to utilize the compound 1-aminocyclopropane-1-carboxylic acid (ACC) as a sole nitrogen source. These novel bacteria have been identied as Bacillus circulans DUC1, Bacillus rmus DUC2, and Bacillus globisporus DUC3. Each strain displayed similar levels of ACC deaminase activity (EC 4.1.99.4) and stimulated root elongation in canola (Brassica campestris) seedlings under gnotobiotic conditions. Soil inoculations with respective bacterial strains increased the root and shoot lengths and fresh and dry weights of potted canola plants. Similarly, a soil inoculation with B. globisporus DUC3 promoted root and shoot growth of plants subjected to a diurnal temperature regime. This is the rst report of plant growth-promoting bacilli with the ability to catabolize ACC. 2003 ditions scientiques et mdicales Elsevier SAS. All rights reserved.
Keywords: Bacillus circulans DUC1; Bacillus rmus DUC2; Bacillus globisporus DUC3

1. Introduction In soil, the greatest concentrations of microbes exist in the immediate area surrounding plant roots, forming complex biological aggregates [28,35]. This is due to the extensive exudation of organic compounds into the soil by plants [1,2,37]. Researchers have identied numerous benecial free-living soil bacteria that associate closely with plants as plant growth-promoting bacteria [8,15,26]. Growth promotion can occur indirectly by the reduction or prevention of the action of plant pathogens, or directlyvia phosphorus solubilization, nitrogen xation, iron sequesterization by siderophores, phytohormone production (e.g. auxin, cytokinin, or gibberellin), and/or enzymatic lowering of plant ethylene levels [3,5,7,8,1316,18,30,31].

Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACC deaminase, 1-aminocyclopropane-1-carboxylic acid deaminase; AVG, L-a(aminoethoxyvinyl)-glycine; DW, dry weight; FW, fresh weight; IAA, phytohormone indole acetic acid. * Corresponding author. E-mail address: sghosh@dominican.edu (S. Ghosh). 2003 ditions scientiques et mdicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 9 8 1 - 9 4 2 8 ( 0 3 ) 0 0 0 1 9 - 6

Of particular interest, here is the lowering of ethylene levels in plants through bacterial enzymatic cleavage of 1-aminocyclopropane-1-carboxylate (ACC) to a-ketobutyrate and ammonia by ACC [10,11,22,24]. It is postulated that following binding of plant growth promotingbacteria to the plant root and/or seed coat [19], a sufcient population of bacteria are able to establish a sink for ACC and thereby lower endogenous ethylene levels; as a consequence, root elongation is enhanced [14,18,21,27]. This particular group of bacteria has also reduced the deleterious effects of ooding [17], phytopathogens [38], and heavy metals [3]. ACC deaminase genes have been isolated and characterized in a few bacterial and fungal strains [4,23,25,29,32,33]. In addition, the ACC deaminase gene from Enterobacter cloacae UW4 has been transferred to Escherichia coli, Pseudomonas putida, and Azospirillum brasilense and, in all cases, elongation of canola roots was promoted signicantly following inoculation with each bacterium [27,32]. Here we report the isolation and initial characterization of three bacterial strains with the ability to catabolize ACC and promote root elongation: Bacillus circulans DUC1, Bacillus rmus DUC2, and Bacillus globisporus DUC3. Bacillus can

278

S. Ghosh et al. / Plant Physiology and Biochemistry 41 (2003) 277281 Table 1 ACC deaminase activity of isolated bacterial strains and promotion of root elongation under gnotobiotic conditions. The root elongation data represents the growth of 5060 seedlings S.E. 6 d after seed germination. One hundred percent is equivalent to 46 mm, the length of a root from a germinated, non-treated seed Bacterial strain B.subtilis B.circulans DUC1 B.rmus DUC2 B.globisporus DUC3 nkat mg 1 0 2.24 10 9 4.64 10 11 2.30 10 9 5.57 10 11 2.28 10 9 5.10 10 11 Root elongation (% of control) 101 8.4 186 8.4 192 7.2 194 9.5

be grown vegetatively and then induced to sporulate, forming spores that remain viable during severe environmental conditions and long-term storage. It has been suggested that plant growth-promoting bacilli may yield more reproducible results in the eld and thus, be more attractive for commercialization than other less tolerant plant growth-promoting bacterial strains [15].

2. Results and discussion 2.1. Determination of ACC deaminase activity Three bacilli with the ability to grow on ACC selective minimal medium were isolated from soil, and identied as B. circulans DUC1, B. rmus DUC2, and B. globisporus DUC3. Representative growth curves are presented in Figs. 1 and 2. The above Bacilli species grew well in liquid DR salts minimal medium with either ACC or ammonium sulfate serving as the sole nitrogen source, whereas Bacillus subtilis grew well onlyin the presence of ammonium sulfate. The

ability to exploit ACC as a sole nitrogen source strongly suggests that these bacteria produce ACC deaminase [12]. This was supported when cell extracts of these bacterial strains were assayed for ACC deaminase activity with B. subtilis serving as a negative control (Table 1). In agreement with previous reports that indicated that all ACC deaminasecontaining bacteria act to facilitate plant growth, root elongation was signicantly promoted under gnotobiotic conditions by B. circulans DUC1, B. rmus DUC2, B. globisporus DUC3, whereas, B. subtilis had little effect (Table 1). Interestingly, the three newly isolated strains as well as B. subtilis strain produce similar levels of the phytohormone indole acetic acid (IAA) (data not shown). Thus, bacterial IAA does not appear to have any effect on root elongation of canola in the gnotobiotic, growth pouch assay. This implicates ACC deaminase as the primaryfactor for the observed enhancement of root growth (Table 1). Using B. globisporus DUC3 and B. subtilis along with a chemical generator of ethylene (ethophon) and a chemical inhibitor of ethylene L-a-(aminoethoxyvinyl)-glycine (AVG), we indirectly tested the role of lowered ethylene levels in the promotion of root elongation (Fig. 3). Root elongation was signicantly stimulated by treatment with B. globisporus DUC3, AVG, AVG + B. subtilis, and AVG +

Fig. 1. Growth of B. subtilis, B. circulans, B. rmus and B. globisporus on DF salts minimal medium plus ammonium sulfate. Data shown are from a typical experiment. The entire experiment was repeated ve times.

Fig. 2. Growth of B. subtilis, B. circulans, B. rmus and B. globisporus on DF salts minimal medium with ACC. Data shown are from a typical experiment. The entire experiment was repeated ve times.

Fig. 3. Root elongation of canola seedlings treated with B. globisporus DUC3 under gnotobiotic conditions. Seeds were treated with: A, MgSO4; B, B. globisporus DUC3; C, B. subtilis; D, AVG; E, B. globisporus DUC3 plus AVG; F, B. subtilis plus AVG; G, Ethophon; H, B. globisporus DUC3 plus Ethophon; I, B. subtilis plus Ethophon. Data represents 5060 seedlings S.E. after 10 d of growth.

S. Ghosh et al. / Plant Physiology and Biochemistry 41 (2003) 277281

279

Table 2 Effects of seed and soil inoculations with isolated bacterial strains on the growth of canola seedlings. The data represents 5060 seedlings S.E. Measurements were made 6 d after seed germination Agent MgSO4 (control) B.circulan DUC1 B.rmus DUC2 B.globisporus DUC2 Inoculations Seed Soil Seed Soil Seed Soil Seed Soil Root length (mm) 25 2.8 30 1.3 45 2.2 45 1.2 46 2.5 46 1.6 43 2.7 45 1.6 Root FW (mg per root) 2.3 0.15 2.5 0.14 3.7 0.15 3.9 0.16 3.7 0.12 3.8 0.15 3.6 0.16 3.6 0.14 Root DW (mg per root) 0.38 0.03 0.37 0.03 0.58 0.02 0.61 0.04 0.57 0.03 0.57 0.03 0.54 0.03 0.58 0.04 Shoot length (mm) 15 1.3 25 1.1 15 1.3 35 1.3 15 1.3 33 1.1 16 1.3 34 1.2

B. globisporus DUC3, whereas, root elongation was inhibited by ethophon alone or together with respective bacteria. These results are consistent with the proposed model on the mechanism of plant growth promotion by soil bacteria that lowers plant ethylene levels [14]. Relative to the MgSO4 control, seed or soil inoculation with the three isolated Bacillus strains resulted in an increase of root length by about 80% and 50%, respectively (Table 2). Both types of inoculations also resulted in an increase of root fresh and dry weights by about 50% and 57%, respectively (Table 2). However, the soil inoculation treatment also had a stimulatory effect on shoot elongation (Table 2). It was previously observed that ACC deaminase containing-bacteria do not need to colonize the root in order to stimulate root growth (i.e. mainly colonize seed coat) [19]. With soil inoculation, effective populations of plant growth promoting bacteria bind to both the seed and emerging root [13,37]. The difference in observations of shoot elongation may reect the efcacy of the soil inoculation in preventing the synthesis of ethylene in the entire root viz. an establishment of a greater sink for ACC. Using B. globisporus as a representative plant growthpromoting Bacillus strain, we analyzed the effects of a diurnal temperature regime [36], similar to temperatures occurring in spring in the Northern Hemisphere (i.e. 25 C days and 5 C nights) [9,34]. Using this regime, root and shoot length and fresh and dry weights were signicantly increased by soil application of B. globisporus DUC3 compared to the B. subtilis and MgSO4 controls (Table 3). 3. Conclusion In summary, we report here the isolation and preliminary characterization of three ACC deaminase-containing Bacil-

lus strains, all of which are effective at promoting plant growth. This is the rst report of plant growth-promoting bacilli with ACC deaminase activity. Given the environmental persistence of bacilli compared to pseudomonads, these plant growth-promoting strains may be more attractive as microbial plant-inoculants than plant growth-promoting psuedomonad strains. 4. Method 4.1. Growth-promoting bacteria Plant growth-promoting bacteria were isolated from the rhizosphere of plants grown in southeastern Wisconsin, USA using the procedure of Glick et al. [12]. Isolated bacterial strains were characterized by biochemical tests performed at DEIBEL Laboratories, Inc. (Madison, WI, USA) in conjunction with API NE [(Non Fermentative) BioMerieux, Hazelwood, MO, USA]. Strain B. circulans DUC1 was isolated from the rhizosphere of alfalfa and soybean, B. rmus DUC2 from alfalfa and tomato, and strain B. globisporus DUC3 from corn and soybean. Bacteria were aerobically cultured at room temperature (22 1 C) in DF salts minimal medium [6] with ACC substituting for ammonia. Bacterial growth in liquid medium was monitored by measuring the optical density of the cultures at 600 nm. 4.2. ACC deaminase assay To determine the amount of ACC deaminase activityof isolated strains, the amount of a-ketobutyric acid generated from the cleavage of ACC was monitored using spectrophotometer as described by Honma and Shimomura [20] and modied by Shah et al. [32] previously. The amount of

Table 3 The effects of 25 C days and 5 C nights on the growth of canola seedlings inoculated with two Bacillus strains. Data represents 5060 seedlings S.E. the measurements were made 10 d after seed germination Treatment MgSO4 B.subtilis (control) B.globisporus DUC3 Root growth Length (mm) 45 1.3 46 1.6 67 1.4 FW (mg per root) 6.5 0.35 6.5 0.37 7.8 0.34 DW (mg per root) 0.52 0.022 0.51 0.025 0.86 0.024 Shoot growth Length (mm) 32.5 1.1 32.7 1.3 37.2 1.3 FW (mg per root) 12.5 0.2 12.6 0.3 14.2 0.3 DW (mg per root) 0.55 0.022 0.58 0.002 0.68 0.003

280

S. Ghosh et al. / Plant Physiology and Biochemistry 41 (2003) 277281

a-ketobutyric acid produced during this reaction was determined by comparing the absorbance at 540 nm of a sample to a standard curve of a-ketobutarate. B. subtilis ATCC 6051 was used as a negative control because it cannot utilize ACC as a sole nitrogen source. The bacterial cells for the ACC deaminase assay were prepared rst by growing to mid- up to late-log phase in a rich medium, tryptic soybean broth (Difco Laboratories, Detroit, MI, USA) [32]. 4.3. Preparation of seed and soil inoculations Plant growth-promoting bacterial stimulation of root elongation was evaluated under gnotobiotic conditions exactly as described by Hall et al. [18] plus using bacterial pellets resuspended in 100 mM MgSO4 and adjusted to 0.50 0.02 absorbance units at 600 nm. Canola seeds (Brassica campestris L. cv I-33) were kindly supplied by Agriculture Canada, Ottawa, Canada. Measurements were taken on the sixth dayafter seed germination. The effects of seed and soil inoculations on shoot and root growth were determined as follows. For the seed inoculation test, sterilized seeds were incubated for 1 h at 22 1 C in 100 mM MgSO4 or in bacterial suspensions in 100 mM MgSO4 (absorbance equals 1.2 0.02 at 600 nm). Bacterially coated seeds were planted in 15 cm plastic pots containing 100 g premium grade vermiculite (SUNGRO Horticulture, Seneca, IL, USA), which was soaked with 450 ml of water. For the soil inoculation test, 11 ml of each bacterial strains with an absorbance of 1.2 0.02 at 600 nm was mixed with 100 g premium grade vermiculite soaked with 450 ml of water and placed in 15 cm plastic pots. Seeds were placed under the soil and pots were maintained at 22 1 C under a 14-h photoperiod with an irradiance of 22 mol m2 s1 of mixed uorescent and incandescent light. Measurements were taken on the sixth dayafter seed germination. B. globisporus DUC3 was tested for its effects on root and shoot growth under diurnal conditions. Vermiculite (100 g in 450 ml plastic pots) was mixed with suspensions of B. subtilis or B. globisporus DUC3, respectively. Pots were maintained at 25 C during a 14-h light period and 5 C during a 10-h dark period. Measurements were taken the 10th day after seed germination. Biomass measurements were taken for each of the plants inoculated with the four Bacillus stains, with B. subtilis as the control. The plants were harvested, cleaned of all soil, if any, and then weighed. To obtain the dry weight, plants were frozen in liquid N2 and lyophilized for 48 h.

References
[1] D.A. Barber, K.B. Gunn, The effect of mechanical forces on the exudation of organic substances by the roots of cereal plants grown under sterile conditions, New Phytol. 73 (1974) 3945. G.D. Bowen, A.D. Tovira, The rhizosphere: the hidden half of the hidden half, in: Y. Waisel, A. Eshel, U. Kafka (Eds.), Plant Roots, The Hidden Half, Marcel Dekker, New York, 1991, pp. 641669. G.I. Burd, D.G. Dixon, B.R. Glick, A plant growth promoting bacterium that decreases nickel toxicity in plant seedlings, Appl. Environ. Microbiol. 64 (1998) 36633668. B.G.J.A. Campbell, Thomson, 1-Aminocyclopropane-1-carboxylate deaminase genes from pseudomonas strains, FEMS Microbid. Lott. 138 (1996) 207210. M. Caron, C. Patten, S. Ghosh, B.R. Glick, Effects of the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 on the physiology of canola roots, in: D.W. Green, C. David Fritz (Eds.), Proceedings of Twenty-second Annual Meeting of Plant Growth Regulation Society of America, Rhone-Poulenc Ag, 18-20 July 1995, pp. 279302. M. Dworkin, J. Foster, Experiments with some microorganisms, which utilize ethane and hydrogen, J. Bacteriol. 75 (1958) 592601. Y. Esashi, Ethylene and seed germination, in: A.K. Matoo, J.C. Suttle (Eds.), The Plant Hormone Ethylene, CRC Press, Boca Raton, FL, 1991, pp. 133137. B.R. Glick, The enhancement of plant growth by free-living bacteria, Can. J. Microbiol. 41 (1995) 109117. B.R. Glick, C.B. Jacobson, M.M.K. Schwarze, J.J. Pasternak, Does the enzyme 1-aminocyclopropane-1-carboxylate deaminase play a role in plant growth promotion by Pseudomonas putida GR12-2?, in: M.H. Ryder, P.M. Stephens, G.D. Bowen (Eds.), Improving Plant Productivity with Rhizosphere Bacteria, CSIRO, Adelaide, Australia, 1994, pp. 150152. B.R. Glick, C.B. Jacobson, M.M.K. Schwarze, J.J. Pasternak, 1-Aminocyclopropane-1 carboxylic acid deaminase mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 do not stimulate canola root elongation, Can. J. Microbiol. 1 (40) (1994) 911915. B.R. Glick, D.M. Karaturovc, P.C. Newell, A novel procedure for rapid isolation of plant growth-promoting pseudomonads, Can. J. Microbiol. 41 (1995) 533536. B.R. Glick, C. Liu, S. Ghosh, E.B. Dumbroff, Early development of canola seedlings in the presence of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, Soil Biol. Biochem. 29 (1997) 12331239. B.R. Glick, C.L. Patten, G.D.M. Holguin, D.M. Penrose, Biochemical and Genetic Mechanisms used by Plant Growth-Promoting Bacteria, Imperial College Press, London, 1999. B.R. Glick, D.M. Penrose, J. Li, A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria, J. Theor. Biol. 190 (1998) 6368. B.R. Glick, Y.C. Skof, Environmental implications of recombinant DNA technology, Biotechnol. Adv. 4 (1986) 261277. V.P. Grichko, B.R. Glick, Identication of DNA sequences that regulate the expression of the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylic acid deaminase gene, Can. J. Microbiol. 40 (2000) 11591165. V.P. Grichko, B.R. Glick, Amelioration of ooding stress by ACC deaminase-containing plant growth-promoting bacteria, Plant Physiol. Biochem. 39 (2001) 1117. J.A. Hall, D. Peirson, S. Ghosh, B.R. Glick, Root elongation in various crops by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2, Isr. J. Plant Sci. 44 (1996) 3742. Y. Hong, B.R. Glick, J.J. Pasternak, Plant-microbial interaction under gnotobiotic conditions: a scanning electron microscope study, Curr. Microbiol. 23 (1991) 111114.

[2]

[3]

[4]

[5]

[6] [7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15] [16]

[17]

Acknowledgements
[18]

The work described here was supported by grants from the National Science Foundation; #9512262 and #9552293, to S. Ghosh, and from the Natural Science and Engineering Research Council of Canada to B.R. Glick.

[19]

S. Ghosh et al. / Plant Physiology and Biochemistry 41 (2003) 277281 [20] M. Honma, T. Shimomura, Metabolism of 1-aminocyclopropane-1carboxylic acid, Agric. Biol. Chem. 42 (1978) 18251831. [21] M.B. Jackson, Ethylene in root growth and development, in: A.K. Matoo, J.C. Suttle (Eds.), The Plant Hormone Ethylene, CRC Press, Boca Raton, FL, USA, 1991, pp. 133137. [22] C.B. Jacobson, J.J. Pasternak, B.R. Glick, Partial purication and characterization of ACC deaminase from the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, Can. J. Microbiol. 40 (1994) 10191025. [23] Y.J. Jia, Y. Kakuta, M. Sugawara, T. Igarashi, N. Oki, M. Kisaki, T. Sholi, Y. Kanetuna, T. Horita, H. Matsui, M. Honma, Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum, Biosci. Biotech. Biochem. 63 (1999) 542549. [24] H. Kende, Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44 (1993) 283307. [25] H.J. Klee, M.B. Hayford, K.A. Kretzmer, G.F. Barry, G.M. Kishore, Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants, Plant Cell 3 (1991) 11871193. [26] J.W. Kloepper, R. Lifshitz, R.M. Zablotowicz, Free-living bacterial inocula for enhancing crop productivity, Trends Biotechnol. 7 (1989) 3943. [27] J. Li, D.H. Ovakim, T.C. Charles, B.R. Glick, An ACC deaminase minus mutant of Enterobacter cloacae UW4 no longer promotes root elongation, Curr. Microbiol. 41 (2000) 101105. [28] J.M. Lynch, The Rhizosphere, Wiley-Interscience, Chichester, England, 1990. [29] R. Minami, K. Uchiyama, T. Murakami, J. Kawai, K. Mikami, T. Yamada, D. Yokoi, H. Ito, H. Matsui, M. Honma, Properties, sequence, and synthesis in Escherichia coli of 1-aminocyclopropane1-carboxylate deaminase from Hansenula saturnus, J. Biochem. 123 (1998) 11121118.

281

[30] S. Nayani, S. Mayak, B.R. Glick, Effects of plant growth-promoting rhizobacteria on senescence of ower petals, Ind. J. Exp. Bio. 36 (1998) 836839. [31] D.M. Penrose, B.R. Glick, Levels of ACC and related compounds in exudate and extracts of canola seeds treated with ACC deaminasecontaining plant growth-promoting bacteria, Can. J. Microbiol. 47 (2001) 368372. [32] S. Shah, J. Li, B.A. Moffatt, B.R. Glick, Isolation and characterization of ACC deaminase genes from two different plant growth-promoting rhizobacteria, Can. J. Microbiol. 44 (1998) 833843. [33] R.F. Sheehy, M. Honma, M. Yamada, T. Sasak, B. Martineau, W.R. Hiatt, Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. Strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase, J. Bacteriol. 173 (1991) 52605265. [34] X. Sun, M. Grifth, J.J. Pasternak, B.R. Glick, Low temperature growth, freezing survival and production of antifreeze protein by the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, Can. J. Microbiol. 41 (1995) 776784. [35] V. Torsvik, J. Goksyr, F.L. Daae, High diversity in DNA of soil bacteria, Appl. Environ. Microbiol. 56 (1990) 782787. [36] C.Y. Wang, D.O. Adams, Chilling-induced ethylene production in cucumbers (Cucumis sativus L.), Plant Physiol. 69 (1982) 424427. [37] C. Wang, E. Knill, B.R. Glick, G. Defago, Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene into Pseudomonas uorescens strain CHA0 and its gacA derivatives CHA96 on their growth promoting and disease-suppressive capacities, Can. J. Microbiol. 46 (2000) 898907. [38] J.M. Whipps, Carbon utilization, in: J.M. Lynch (Ed.), The Rhizosphere, Wiley, Chichester, England, 1990, pp. 5997.