Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

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.)
B. Shaharoona, M. Arshad and Z.A. Zahir
Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan

Keywords 1-aminocyclopropane-1-carboxylic aciddeaminase, co-inoculation, ethylene, nodulation, plant growth promoting rhizobacteria, rhizobia. Correspondence Muhammad Arshad, Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan. E-mail: bio@fsd.comsats.net.pk

Abstract Aims: This study was conducted to test the hypothesis that the bacterial strains possessing 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity may also promote growth of inoculated plants and could increase nodulation in legumes upon co-inoculation with rhizobia. Methods and Results: Several rhizobacteria were isolated from maize rhizosphere through enrichment on ACC as a sole N source. Puried isolates were screened for growth promotion in maize under axenic conditions and for in vitro ACC-deaminase activity. A signicant positive correlation was observed between in vitro ACC-deaminase activity of bacterial cells and root elongation. None of the isolates produced auxins. Bradyrhizobium japonicum produced less amount of auxins but did not carry ACC-deaminase activity. Results of pot experiment revealed that co-inoculation with Bradyrhizobium and plant growth promoting rhizobacteria (PGPR) isolates enhanced the nodulation in mung bean compared with inoculation with Bradyrhizobium alone. Conclusions: It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolysed endogenous ACC into ammonia and a-ketobutyrate instead of ethylene. Consequently, root and shoot growth as well as nodulation were promoted. Signicance and Impact of the Study: The ACC-deaminase trait could be employed as an efcient tool to screen effective PGPR, which could be successfully used as biofertilizers to increase the growth of inoculated plants as well as nodulation in legumes.

2004/1202: received 19 October 2004, revised 16 July 2005 and accepted 18 July 2005
doi:10.1111/j.1472-765X.2005.01827.x

Introduction Ethylene is an important growth hormone, which is produced by almost all plants and mediates a wide range of different plant responses and developmental processes. The higher concentrations of ethylene are inhibitory to plant growth. Any factor/stimulus which causes a change in the endogenous levels of ethylene in a plant results in modied growth and development (Arshad and Frankenberger 2002). Recently, inoculation with specic bacteria has been shown to alter the endogenous levels of ethylene, which subsequently led to changes in the growth and development of inoculated plants (Glick et al. 1998).

1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene derived from amino acid methionine in plants (Yang and Hoffman 1984). The synthesis of ethylene in plants is directly related with the concentration of ACC in the plant tissue (Machackova et al. 1997). It has been discovered that certain microorganisms contain an enzyme ACC-deaminase that hydrolyses ACC into ammonia and a-ketobutyrate (Mayak et al. 1999). Glick et al. (1998) have postulated that a signicant proportion of ACC produced by plants may be exuded from plant roots or seeds, which is hydrolysed by bacteria containing ACC-deaminase on the surface of root. Decreased concentration of ACC results in lower
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levels of endogenous ethylene, which eliminates the potential inhibitory effects of higher ethylene concentrations. Plant hormones have been assigned an important regulatory role in the development and establishment of nodulation (Hirsch and Fang 1994). Recent studies have strongly suggested that ethylene may function as an autoregulator to control nodule formation and development (Arshad and Frankenberger 2002). Different studies have revealed that exogenous ethylene applied directly as a gas, or indirectly as ACC or ethephon/ethrel acts as a strong inhibitor of nodulation (Yuhashi et al. 2000). Moreover, endogenous ethylene synthesis has been found to act as a potent negative regulator of nodulation as inhibitors of ethylene synthesis promoted nodule formation (Ligero et al. 1999). Restoration of nodulation in the presence of Ag+ (which inhibits ethylene action) and by aminoethoxyvinyl glycine (which is an inhibitor of endogenous ethylene biosynthesis) strongly supports this premise (Ligero et al. 1999; Guinel and Sloetjes 2000). Therefore, it is highly expected that presence of plant growth promoting rhizobacteria (PGPR) containing ACC-deaminase on the roots of legume could suppress accelerated endogenous synthesis of ethylene during the rhizobial infection and thus may facilitate nodulation. So, co-inoculation of legumes with competitive rhizobia and PGPR-containing ACC-deaminase could be an effective and novel approach to achieve successful and dense nodulation in legumes. Materials and methods Isolation Rhizobacteria were isolated from the maize rhizosphere by dilution plate technique using DF salt minimal medium (Dworkin and Foster 1958) containing ACC as sole nitrogen source (enrichment technique). Further streaking on fresh plates puried the collected rhizobacterial strains. Bradyrhizobium japonicum was obtained from National Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan (Hameed et al. 2004). Jar experiment Liquid medium was prepared by using DF salt minimal medium containing ACC as sole nitrogen source. Each strain was inoculated in 150 ml test-tube containing 60 ml of medium and was incubated at 28 1C for 3 days. An optical density of 05 recorded at a wavelength of 535 nm was achieved by dilution to maintain uniform cell density (108109 CFU ml)1). Two sterilized lter paper sheets were soaked and saturated with respective inoculum. Maize seeds were
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surface sterilized by dipping in 95% ethanol solution for 5 min, 02% HgCl2 solution for 3 min and washed thoroughly with sterilized water. Two surface disinfected seeds were sandwiched in between soaked lter papers and were rolled and placed in sterilized glass jars. In the case of the uninoculated control, sterilized inoculum was used. Sterilized Hoagland solution was added to bottom of jars for nutrients supply. Treatments in each jar were arranged using completely randomized design with ve replications of each treatment. Jars were placed in growth chamber at 25 1C adjusted to 16-h light and 8-h dark period. Data regarding root elongation, shoot length and fresh weight of seedlings (root + shoot) were recorded after 10 days and analysed statistically using a completely randomized design (Steel and Torrie 1980). Mean values were compared by Duncans multiple range test (Duncan 1955). The rhizobacteria exhibiting the highest growth promoting activity were identied by Biolog Identication System (Microlog System Release 4.2, Hayward, CA, USA). Rhizobacterial isolate Q7 was identied as Pseudomonas putida biotype A and Q14 as Pseudomonas uorescens. Determination of ACC-deaminase activity ACC-deaminase activity was determined by monitoring the amount of ammonia generated by the hydrolysis of ACC by the rhizobacterial isolates containing ACC-deaminase. In Erlenmeyer asks 500 ml broth of each strain was prepared in DF minimal salt medium containing 2 g l)1 ammonium sulfate as nitrogen source and incubated at 28 1C in orbital shaking incubator at 100 rev min)1 for 2 days. Then the broth was centrifuged at 8300 g for 10 min and cell pellets were collected in phosphate buffer (pH 7) and recentrifuged. Cell pellets were transferred to DF minimal salt medium containing 5 mmol l)1 of ACC substrate and incubated in orbital shaking incubator for 1 h at 28 1C. After 1 h the liquid broth was centrifuged at 8300 g for 10 min and ammonia was determined in supernatant solution by the protocol of Nagatsu and Yagi (1966). Determination of auxin production In vitro auxin production by selected isolates was investigated by colorimetry as IAA equivalents in the presence and absence of l-tryptophan by using the protocol described by Khalid et al. (2004). Mung bean nodulation pot trial The inoculum for the pot trial was prepared by growing the selected rhizobacterial isolates on DF minimal salt

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medium, and Bradyrhizobium on yeast extract manitol. It was incubated at 28 1C for 3 days. The suspension (108109 CFU ml)1) of selected PGPR and Bradyrhizobium was injected into sterile peat (100 ml kg)1, seed to peat ratio 1 : 1 w/w) and incubated for 24 h at 28 1C before using it for seed coating. For inoculation, seed dressing was carried out with inoculated peat mixed with 10% sugar solution. In the case of the uninoculated control, the seeds were coated with the same but autoclaved inoculum suspension. Seeds were inoculated with P. putida biotype A and P. uorescens alone and in combination with B. japonicum. The coated seeds were sown in pots containing 12 kg per pot sandy clay loam soil [pH, 78; electrical conductivity of saturated soil extract (ECe), 35 dS m)1; organic matter, 078%; total nitrogen, 005%; Olsen phosphorus, 78 mg kg)1 and exchangeable potassium, 127 mg kg)1 soil]. There were ve replications and pots were arranged randomly according to a completely randomized design in a wire house under ambient light and temperature conditions. The fertilizers NPK were applied at 150372372 mg per pot at germination stage (as in this study ethylene has been suppressed by the inoculation, so effect of nitrogen on nodulation was eliminated). Pots were irrigated when needed. Data regarding root elongation, total biomass and nodulation were recorded 40 days after germinating. Data were analysed statistically (Steel and Torrie 1980) and mean values were compared by Duncans multiple range test (Duncan 1955). Results Screening of rhizobacteria containing ACC-deaminase Inoculation of maize seedlings with PGPR-containing ACC-deaminase had signicant effect on root elongation, shoot length and seedling (root + shoot) fresh weight (Table 1). Different rhizobacteria increased root elongation up to 18-fold over uninoculated control. Rhizobacterial isolate Q14 was the best that caused 18-fold increase in root elongation over uninoculated control and followed by Q7 and Q9. Shoot length of maize seedlings was also signicantly increased because of inoculation with rhizobacteria containing ACC-deaminase up to 19fold over uninoculated control. The most effective isolate was Q7. Isolates Q9 and Q14 were the next effective isolates, increasing shoot length 16-fold over the uninoculated control. Similarly signicant increases in fresh weight of seedlings (root + shoot) over uninoculated control were recorded in response to inoculation. Once again, rhizobacterial isolates Q7 and Q14 caused 20- and 16fold increase in fresh biomass of seedlings, respectively, over uninoculated control.

Table 1 Effect of rhizobacteria containing 1-aminocyclopropane-1carboxylic acid-deaminase on root elongation, shoot length and fresh weight of maize seedlings under axenic conditions (average of ve replicates)
Root elongation (cm) 76 d* 118 c 171 ab 830 cd 203 a 70 d 181 ab 210 a 169 b 161 b Shoot length (cm) 47 53 97 69 137 45 123 122 112 108 d cd abc bc a d a a ab ab Seedling fresh weight (g) 05 07 11 08 15 04 12 13 12 11 ef def abc cde a f abc ab abc abc

Treatment Control Q1 Q2 Q3 Q7 Q8 Q9 Q14 Q15 Q18

*Mean values sharing the same letter(s) in a column do not differ signicantly according to Duncans multiple range test (P 005).

ACC-deaminase activity All the rhizobacteria used for inoculation were assessed for their ACC-deaminase activity (Fig. 1). It was observed that isolates differed in their potential for ACC-deaminase activity. Highest total ACC-deaminase activity per hour was exhibited by rhizobacterial isolate Q7 (P. putida biotype A). However, ACC-deaminase activity calculated for unit biomass was almost same for all the isolates. A signicantly positive correlation (r 080; P 005) was observed between ACC-deaminase activity per unit time and root elongation caused by inoculation with
400 350 300 250 200 150 100 5 50 0 Q1 Q7 Q8 Q14 Q15 Rhizobacterial isolates Q18 0 10 15 Root elongation (cm) r = 080* 20 25

Figure 1 Relationship between 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity and root elongation in maize seedlings. h, ACC-deminase activity; r, Root elongation; *, P 005.

ACC-deaminase activity (nmol h1)

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Table 2 Effect of coinoculation of selected rhizobacteria containing 1-aminocyclopropane-1-carboxylic acid-deaminase on root elongation, total biomass and nodulation in mungbean in a pot trial (average of ve replicates)
Root elongation (cm) 155 c* 204 a 178 bc 207 a 195 ab 201 a 171bc 172 bc Total biomass (g) 172 203 198 204 226 247 214 229 c bc bc bc ab a a ab Number of nodules 3e 18 d 14 d 25 c 19 d 37 a 18 d 27 b Fresh weight of nodules (g) 006 047 042 034 033 075 043 033 d b b bc bc a bc bc Oven dry weight of nodules (g) 0001 0058 0021 0050 0020 0100 0020 0040 b ab b ab b a b abc

Treatment Control Q7 (Pseudonomas putida biotype A) Q14 (Pseudonomas uorescens) Bradyrhizobium japonicum Q7 + Q14 Q7 + Bradyrhizobium Q14 + Bradyrhizobium Q7 + Q14 + Bradyrhizobium

*Mean values sharing the same letter(s) in a column do not differ signicantly according to Duncans multiple range test (P 005).

rhizobacterial isolates. Rhizobacterial isolates Q14 and Q7 had maximum ACC-deaminase activity per unit time and they were also found to be most effective in promoting root elongation. Auxin production No auxin production by the rhizobacteria was observed in the absence of l-tryptophan; however, its addition resulted in auxin production (data not shown). B. japonicum synthesized less amount of auxin but lacked ACC-deaminase activity. Nodulation in mung bean Data revealed that inoculation/co-inoculation with Pseudomonas spp. and/or Bradyrhizobium had signicant effect on root elongation, total biomass and nodulation. Maximum root elongation was observed in the case of Bradyrhizobium and P. putida biotype A (Q7) applied alone or in combination. Total plant biomass was also maximum where same co-inoculation was employed. The most prominent effect of co-inoculation was observed in terms of number of nodules, and fresh and oven dry weight of nodules. Co-inoculation of Q7 with Bradyrhizobium resulted in 11-fold more number of nodules than uninoculated control and 48% than Bradyrhizobium alone (Table 2). Similarly, fresh weight of nodules was also increased signicantly in response to co-inoculation with the same isolates (12- and 12-fold higher than uninoculated control and Bradyrhizobium alone respectively). Similar effect was observed in the case of oven dry weight of nodules. Discussion Root elongation assay was used for selection of effective PGPR. Data revealed that rhizobacterial isolates signi158

cantly differed in their potential to promote root elongation. In vitro, ACC-deaminase activity of the rhizobacterial isolates used for inoculation also varied substantially. A signicant positive correlation between ACC hydrolysing activity of the rhizobacteria and root elongation in the inoculated plants supported the premise that root elongation might had increased due to hydrolysis of ACC resulting in decreased endogenous levels of ethylene. This view is also strongly supported by the work reported by other researchers (Hall et al. 1996; Glick et al. 1998; Mayak et al. 1999). None of the rhizobacteria produced auxins in vitro, which excludes the involvement of this hormone in the observed response to inoculation. The variation in growth promotion by different isolates may be due to the differences in their efciency of colonizing the germinating roots and hydrolysing the ACC synthesized in roots. Similar results have been reported by others (Li et al. 2000). Nodulation in mung bean was increased signicantly because of inoculation/co-inoculation of Pseudomonas spp. and/or Bradyrhizobium. As exogenously applied and endogenously produced ethylene has been reported to act as a potent negative regulator of nodulation (Ligero et al. 1999), it is highly expected that the presence of PGPR-containing ACC-deaminase on the roots of legume promoted nodulation by decreasing the synthesis of this hormone. Inoculation with PGPR-containing ACC-deaminase also promoted nodule formation by the indigenous competitive Bradyrhizobium. The results imply that co-inoculation of legumes with competitive rhizobia and PGPR-containing ACC-deaminase could be an effective approach for successful nodulation in legumes. Acknowledgement Financial support for this work was provided by Higher Education Commission (HEC), Islamabad, Pakistan.

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