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Effects of Plant Growth-promoting Rhizobacteria on the Seedling

Growth of Oat and Annual Ryegrass under Salt Stress

JI Yun-xiu1*

, HUANG Xiao-dong2

1 College of Environmental Science and Engineering, Dalian Maritime University, China, 1160262

Department of Biology, University of Waterloo, Canada, N2L 3G1fjxjyx@yahoo.com.cn

Abstract: Four strains of plant growth-promoting bacteria (PGPR) were isolated from the rhizosphereof salty plants, based on the ability to utilize the compound 1-aminocyclopropane-1-carboxylic acid(ACC) as a sole nitrogen source. These four bacterial isolates, especiallyPseudomonas sp. S1, alleviated

greatly salt stress and promoted significantly the seedling growth of oat and annual ryegrass under either5 or 10 g/kg NaCl stress in gnotobiotic growth pouch assay. Compared with control (no NaCl), moreplant promotion was observed under 10 g/kg NaCl stress. The greatly significant positive correlations

between ACC deaminase activity of four bacterial isolates and plant growth parameters (root length andshoot length) were observed (Pearson correlation co-efficients > 0.81). The results may imply thatinoculation with ACC deaminase-containing PGPR may serve as an environmentally-friendly and

economical alternative to the amelioration of increasingly saline soils.Key words: plant growth-promoting bacteria; 1 - aminocyclopropane - 1 carboxylic acid deaminase;salt stress

1 Introduction

Plant growth-promoting rhizobacteria (PGPR)can promote plant growth indirectly by the reductionor prevention of the action of plant pathogens, or directly via phosphorus solubilization, nitrogen

fixation, iron sequesterization by siderophores, phytohormone production (e.g. auxin, cytokinin, orgibberellin), and/or enzymatic lowering of plant ethylene levels

[1]. Particularly, PGPR containing

1-aminocyclopropane- l-carboxylic acid (ACC) deaminase were considered to effectively facilitate thegrowth of variety of plants, especially under stressful conditions such as flooding, heavy metals,phytopathogens, drought and high salt

[2-5]. Ethylene is an important phytohormone, but over-produced

ethylene under stressful conditions can result in the inhibition of plant growth or death, especially for

seedlings[6,7]

. PGPR containing ACC deaminase can hydrolyze ACC, the immediate precursor ofethylene, to -ketobutarate and ammonia, and in this way promote plant growth

[8].

Soils, especially cultivated soils, worldwide are becoming more saline from marginal irrigation

water, excessive fertilization, and desertification processes. Impacted soils are a major limiting production factor worldwide for every major crop

[9]. Inoculation of crops with ACC

deaminase-containing PGPR may assist plant growth by alleviating deleterious effects of salt stress

ethylene. Relative to the tolerance of PGPR to saline conditions, none of the most important cropstolerates high levels of salts, and NaCl is the most destructive salt affecting plant growth

[10]. Therefore,

here we report the isolation and initial characterization of four bacterial isolates with the ability to utilize

ACC from the rhizosphere of salty plants and examined their effects on the seedling growth of oat orannual ryegrass at various salt concentrations in gnotobiotic growth pouch assay.

2 Materials and Methods

2.1 Growth-promoting bacteriaPGPR were isolated from the rhizosphere of plants grown in the coastal wetland at Liaodong Bay,

*Corresonding author

e-mail address: fjxjyx@yahoo.com.cn

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China, based on the ability to utilize the compound 1-aminocyclopropane-1-carboxylic acid (ACC) as a

sole nitrogen source using the procedure of Penrose and Glick[11]

. Bacterial isolates were aerobicallycultured at room temperatures (211 C) in DF salts minimal medium with ACC as a sole nitrogensource (+ACC). Bacterial growth in liquid media was monitored by measuring the optical density (OD)

of the cultures at 600 nm.

2.2 ACC deaminase activity assayTo determine the amount of ACC deaminase activity of bacterial isolates, the amount of

-ketobutyric acid (-KA) generated from the cleavage of ACC was monitored using spectrophotometeras described by Penrose and Glick[11]previously. The amount of -KA produced during this reaction was

determined by comparing the absorbance at 540 nm of a sample to a standard curve of -ketobutarate.The ACC deaminase activity was expressed as the amount of -ketobutarate produced per 1 mg ofprotein per hour.

2.3 Gnotobiotic growth pouch assayThe gnotobiotic growth pouch assay was to assess the effect of bacterial isolates on the seedling

growth of oat and annual ryegrass under gnotobiotic salt stress, according to a modification of themethod described by Penrose and Glick

[11]. Seed-pack growth pouches treated with different salt

solution ( 0, 5 and 10 g/kg NaCl) were autoclaved for gnotobiotic pouch assay. Sterilized oat or annual

ryegrass seeds were incubated for 1 h at room temperature(211 ) in 0.03 M MgSO4 (-PGPR) or in bacterial suspensions in 0.03 M MgSO4 (+PGPR, OD600=0.5). Following the incubation period, eightseeds were placed in each growth pouch, and 4 pouches were used for each treatment. The pouches were

incubated at room temperature. Measurements were made 5 d after seeding. The data were analyzed byone-way ANOVA with SPSS software.

3 Results

3.1 Determination of ACC deaminase activity

Four bacterial strains with the ability to grow on ACC selective minimal medium were isolatedfrom the rhizosphere soil of salty plants, and identified as Pseudomonas sp. S1, Citrobacter sp.S2, Enterobacter sp. S3 and Klebsiella sp.S4 by 16srDNA sequence analysis. The growth of these fourbacterial isolates in DF (+ACC) media with 15 g/kg NaCl (data not shown) did not show any changecompared with that in DF (+ACC) media with no NaCl (Fig.1). Among these four isolates, S1 grew best

in DF salts minimal medium with ACC substituting for ammonia, and S2 worst.S3 showed a similarlevel to S4.These results were in agreement with that of ACC deaminase activity assay (Fig.2). It wasobserved that bacterial isolates differed in their potential for ACC deaminase activity. S1 showed the

highest ACC deaminase activity of 4.9950.362 mol -KA (mgh)-1

, andS2 worst. S3 had a similarlevel of activity to S4.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 6 12 18 24 36 48 60

Time/h

OD600

S1

S2

S3

S4

0.0

1.0

2.0

3.0

4.0

5.0

6.0

S1 S2 S3 S4

Bacterial Isolates

ACCdeaminaseac

tivity/

mol-KA(mgh)-

1

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Figure 1 Growth curve of PGPR isolates in DF salts Figure 2 ACC deaminase activity

minimal medium with ACC

3.2 Effects of PGPR on the growth of oat seedlings under salt stress

The data of the effects of PGPR on the root length and shoot length of oat seedlings under saltstress in gnotobiotic growth pouch assay were summarized in Table 1.The results showed that withoutinoculation under 5 g/kg NaCl stress, the root length and shoot length decreased by 37.3% and 54.0%,respectively, compared with control(- NaCl, -PGPR), and the root length decreased by 59.2% under 10g/kg NaCl stress( no shoot). However, except that inoculation of oat seedlings with S2 had not

significant effect on root length under 5 g/kg NaCl, inoculation with these four isolates alleviated saltstress to oat seedlings and resulted in a significant increase of plant growth parameters under either 5 or10 g/kg NaCl. With increasing the concentration of NaCl, more plant growth promotion was observed.

Among these four bacterial isolates, S1 was the most effective, and S3 showed the similar effect to S4,and S2 showed the least plant growth promotion. Relative to uninoculation(-PGPR), inoculation with S1resulted in an increase of root length and shoot length by 24.2% and 33.2% under no NaCl, and 61.1%

and 192.7% under 5 g/kg NaCl, respectively. And much better under 10 g/kg NaCl, the average rootlength of oat seedlings inoculated with S1 increased over uninoculation by 137.2%.

Table 1 Effects of PGPR on the growth of oat seedlings under salt stress

Treatments Control 5 g/kg NaCl 10 g/kg NaClRoot length Shoot length Root length Shoot length Root length Shoot length

(cm) (cm) (cm) (cm) (cm) (cm)

-PGPR 15.13 d 6.89 d 10.74 c 3.17 d 6.18 d

S1 18.79 a 9.18 a 17.31 a 9.28 a 14.66 a 5.82S2 16.68 c 7.55 c 11.33 c 4.27 c 8.59 c 2.02S3 17.86 b 7.96 bc 15.59 b 7.18 b 11.19 b 3.66

S4 17.60 b 8.40 b 15.80 b 7.68 b 10.90 b 3.05

Note: Sharing the same letters in a column do not differ significantly according to Duncans multiple-range test(P

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in an increase of root length and shoot length of annual ryegrass seedlings by 23.0% and 5.0% under no

NaCl, and 39.0% and 48.6% under 5 g/kg NaCl, respectively. And much better under 10 g/kg NaCl, theaverage root length of annual ryegrass seedlings inoculated with S1 increased over uninoculation by104.4%.

.3.4 Correlation studiesThe greatly significant positive correlations (>0.81) between ACC deaminase activity of the

bacterial isolates and root length or shoot length of either oat or annual ryegrass seedlings were observed

under either concentrations of NaCl in Table 3. The results also showed that the root length had asignificant positive correlation to shoot length. In addition, the results from the growth curve (Fig. 1)was in agreement with that in either ACC deaminase activity or seedling growth assay.

Table 3 Correlations between ACC deaminase activity and plant growth parameters

Treatment ACC deaminase activity Root length Shoot lengthPearson correlation

Control

Oat pouch 1 0.935* 0.963*Annual ryegrass 1 0.919* 0.812*5 g/kg NaCl

Oat pouch 1 0.932* 0.956*Annual ryegrass 1 0.976* 0.817*10 g/kg NaCl

Oat pouch 1 0.986* 0.974*Annual ryegrass 1 0.953* 0.929*

Note: *correlation is significant at the 0.01 level (2-tailed).

4 Discussion

Four bacterial isolates containing ACC-deaminase from different genera, Pseudomonas sp. S1,Citrobacter sp.S2,Enterobacter sp. S3 andKlebsiella sp.S4, differed in promoting the growth of oat or

annual ryegrass seedlings under salt stress in gnotobiotic growth pouch assay. Among these four strains,strain S1 was the most effective, and S3 showed the similar or a little better level to S4, and S2 showed

the least plant growth promotion. The variation in growth promotion by different isolates may be due tothe differences in their efficiency of colonizing the germinating roots and hydrolyzing the ACCsynthesized in roots. The significantly positive correlations between ACC deaminase activity of the

bacterial isolates and plant growth parameters supported the premise that the growth-promoting effect ofACC-utilising bacteria was due to a decrease in endogenous levels of ethylene through hydrolysis ofACC by bacterial ACC deaminase. This view is also strongly supported by the work reported by

others[8,12,13]

. The results also showed that IAA did not appear the positive correlation with the seedlinggrowth. This implicated that ACC deaminase, not IAA, may be the primary factor for seedling growth,which is in agreement with previous reports[7,14]. In addition, the results from the growth curve (Fig. 1)

was in agreement with that in either ACC deaminase activity or seedling growth assay. On the otherhand, strain S2 showed less plant growth promotion, although S2, S3 and S4 produced the similar level

of phytohormone IAA. This implicated that ACC deaminase, not IAA, may be the primary factor forseedling growth.

Gnotobiotic growth pouch assay, as a selection of effective PGPR, was used to study the effects ofPGPR on the seedling growth without the confounding effects of soil or environmental conditions. The

results showed that oat and annual ryegrass seedlings were sensitive to ethylene, and intolerant to saltstress. These four bacterial isolates promoted significantly the seedling growth especially under saltstress. With increasing the concentration of NaCl, more plant growth promotion was observed. This also

tested the mechanism that PGPR containing ACC deaminase promoted plant growth by degradingstress ethylene. PGPR containing ACC deaminase, when bound to the seed coat of a developingseedling, can regulate the endogenous ethylene level through hydrolysis of ACC to avoid the inhibition

of plant root elongation by over-produced ethylene, and also enhance the survival of some seedlings,

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especially during the first few days after seeding [15]. Over-produced ethylene under salt stress can result

in the inhibition of plant growth or death, which is a major limiting crop production factor resulted fromsaline soils worldwide

[9]. Inoculation with PGPR containing ACC deaminase, as an effective way, can

increase the salt-resistance of plants. The results showed that these four bacterial isolates, especiallyPseudomonas sp. S1, alleviated plant salt stress to some extent and promoted significantly the growth ofoat and annual ryegrass seedlings under salt stress, therefore serving as an environmentally-friendly andeconomical alternative to the amelioration of increasingly saline soils.

AcknowledgementFinancial support for this work was provided by Dalian Maritime University Scholarship Fund

Program, Dalian, China and Dr. Bruce Greenbergs laboratory in the Department of Biology at theUniversity of Waterloo, Waterloo, Canada.

References

[1] Glick B R, Patten C L, Holguin G D M, et al. Biochemical and genetic mechanisms used by plantgrowth-promoting bacteria, London: Imperial College Press, 1999

[2] Grichko V P, Glick B R. Amelioration of flooding stress by ACC deaminase-containing plant

growth-promoting bacteria. Plant Physiol Biochem, 2001, 39:11-17

[3] Belimov A A, Hontzeas N, Safronova V I, et al. Cadmium-tolerant plant growth - promoting

bacteria associated with the roots of Indian mustard. Soil Biol Biochem, 2005, 37: 241-250

[4] Wang C, Knill E, Glick B R, et al. Effect of transferring 1-aminocyclopropane-1-carboxylic acid

(ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative

CHA96 on their growth-promoting and disease-suppressive capacities. Can J Microbiol, 2000, 46:

898-907

[5] Mayak, S, Tirosh T, Glick B R. Plant growth-promoting bacteria confer resistance in tomato plants

to salt stress. Plant Physiol Biochem, 2004, 42: 565-572

[6] Glick B R, Liu C, Ghosh S, et al. Early development of canola seedlings in the presence of theplant growth-promoting rhizobacteriumPseudomonas putida GR12-2, Soil Biol Biochem, 1997,

29:12331239.

[7] Ghosh S, Penterman J N, Little R D, et al. Three newly isolated plant growth-promoting bacilli

facilitate the seedling growth of canola, Brassica campestris. Plant Physiol Biochem, 2003,

41:277-281

[8] Glick B R, Penrose D M, Li J. A model for the lowering of plant ethylene concentrations by plant

growth-promoting bacteria, J Theor Biol, 1998, 190: 63-68

[9] Shannon M C, Grieve CM. Tolerance of vegetable crops to salinity. Sci Hortic, 1999, 78:5-38

[10] Bacilio M, Rodriguez H, Moreno M, et al. Mitigation of salt stress in wheat seedlings by

gfp-taggedAzospirillum lipoferum. Biol Fertil soils, 2004, 40:188-193

[11] Penrose D M, Glick B R. Methods for isolating and characterizing ACC deaminase-containing

plant growth-promoting rhizobacteria. Physiol Plant, 2003, 118:10-15

[12] Shah S, Li J, Moffatt B A, et al. Isolation and characterization of ACC deaminase genes from twodifferent plant grow - promoting rhizobacteria. Can J Microbiol, 1998, 44: 833-843

[13] Li J, Ovabin D H, Charles T C, et al. An ACC deaminase minus mutant ofEnterobacter cloacae

UW4 no longer promotes root elongation. Curr Microbiol, 2000, 41: 101-105

[14] Shaharoona B, Arshad M, Zahir Z A. Effect of plant growth promoting rhizobacteria containing

ACC-deaminase on maize ( Zea mays L.) growth under axenic conditions and on nodulation in

mung bean (Vigna radiata L.). Lett Appl Microbiol, 2006, 42: 155-159

[15] Glick B R, Jacobson C B, Schwarze M M K, et al. 1-A- minocyclopropane-1-carboxylic acid

deaminase mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR12 - 2

do not stimulate canola root elongation. Can J Microbiol, 1994, 40: 911-915