American Journal of Plant Sciences, 2013, 4, 2199-2205
Published Online November 2013 (http://www.scirp.org/journal/ajps)
http://dx.doi.org/10.4236/ajps.2013.411273
Open Access AJPS
2199
Effect of Inoculation with Three Phytohormone Producers
Phytobacteria with ACC Deaminase Activity on Root
Length of Lens esculenta Seedlings
Natalia Elenes Zazueta1, Orlando Ortega Acosta2, Laura Martínez Herrera2, Raúl Alcalde Vázquez2,
Eugenia López López2, Angelica Guerrero Zúñiga3, Angelica Rodríguez Dorantes2*
1Advanced Technological Institute of Cajeme, Sonora, México; 2National School of Biological Sciences, I.P.N., México City,
México; 3Mexican Petroleum Institute, México City, México.
Received September 21st, 2013; revised October 20th, 2013; revised October 27th, 2013
Copyright © 2013 Natalia Elenes Zazueta et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Plant-associated bacteria that inhabit the rhizosphere may influence the plant growth by their contribution to the en-
dogenous pool of phytohormones and by the activity of ACC deaminase to decrease the ethylene concentration. The
aim of this study was to analyse the root length growth by the promoting effect of indole acetic acid producers phyto-
bacteria with ACC deaminase activity, on inoculated seeds of Lens esculenta as synergistic effect on root elongation. In
this study, although the roots of L. esculenta seedlings do not show a significant promotion, these phytobacteria could
be recommended to treat plants analyzing their added inoculum to increase plant biomass and retard the effect of ethyl-
ene on cultures supplied with Tryptophan and ACC.
Keywords: Plant Growth-Promoting Bacteria; Lens esculenta; Root Elongation Test; Indole Acetic Acid; ACC
Deaminase Activity
1. Introduction
The 1-aminocyclopropane-1-carboxylate (ACC) deami-
nase (ACC deaminase) hydrolyses the 1-aminocyclo-
propane-1-carboxylate (ACC) into ammonia and α-ke-
tobutyrate instead of its conversion into ethylene; the
uptake and cleavage of ACC by this enzyme decrease its
amount and as consequence the ethylene concentration in
plants [1-5]. Authors reported that the ACC deaminase
trait has been extensively studied in numerous soil mi-
croorganisms, but it is most common among plant
growth promoting rhizobacteria to protect plants from
both biotic and abiotic stresses and favor the increase of
plant biomass through the regulation of ethylene synthe-
sis in inoculated plants [5-14]. Bacteria that inhabit the
rhizosphere also may influence the plant growth by their
contribution to the endogenous pool of phytohormones,
such as auxins in plants; the production of the auxin Indole
Acetic Acid (IAA) is reported among plant-associated
bacteria [15]. The aim of this study was to analyze the
root length growth by the promoting effect of indole ace-
tic acid producers phytobacteria with ACC deaminase
activity on inoculated seeds of Lens esculenta as syner-
gistic effect on root elongation.
2. Materials and Methods
2.1. Evaluation of the IAA Production of the
Selected Phytobacteria
The phytobacteria employed: Lemna 2 strain, U-M1-4
strain and U-M1-5 strain, were isolated from the aquatic
plant Lemna gibba, collected from the Lake Xochimilco,
México. The selected phytobacteria were analyzed by
their Indole Acetic Acid (IAA) production [16,17] using
the Salkowski reagent according to the method of Bric et
al. [18] and Melo et al. [19], taking 4.9 mL of sterile
Luria-Bertani (LB) liquid media, added to culture tubes
(10 × 15 cm); the culture tubes were inoculated with 0.1
mL of each phytobacteria with an adjusted inoculum by
optical density of 5 × 107 cells/mL in sterile distilled wa-
ter and incubated at 28˚C for 120 h. After the incubation, *Corresponding author.

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC
Deaminase Activity on Root Length of Lens esculenta Seedlings
2200
the cultures were centrifuged at 3,500 rpm, at 25˚C for
45 minutes to discard the bacteria pellets, the supernatant
was recovered and 2 mL of each supernatants were
mixed with 2 mL of Salkowski’s coloring reagent, the
development of a pink color indicates IAA production
and was quantified reading its absorbance at 535 nm. The
concentration of IAA was estimated by a standard curve
and the assays were performed by triplicate.
Evaluation of the ACC deaminase activity of the se-
lected phytobacteria The ACC deaminase activity of the
isolates was assayed according to the method of Penrose
and Glick [20] and Khandelwal and Sindhu [21], with
ACC and (NH4)2SO4 as sole nitrogen source. The assay
was done using agar plates with DF minimal medium [22]
supplemented with ACC (1mM) or (NH4)2SO4 (2 g/L),
equally divided into 24 sectors and spot inoculated with a
sterile toothpick each colony of the selected phytobacte-
ria. The plates were incubated at 28˚C for 48 to 72 h and
the presence of ACC deaminase activity, was recorded
by the measurement of the diameter of the colonies and
compared between the two nitrogen conditions. The as-
says were performed by duplicate.
2.2. Effect on Root Growth of Lens esculenta
Seedlings Inoculated with the Selected IAA
Producers with ACC Deaminase Activity
Phytobacteria
Bacterial inoculum were obtained by culturing the
phytobacteria strains on plates with LB agar medium for
48 h at 28˚C and re-suspending in sterile distilled water
to adjust by optical density an inoculum with cell density
of 5 × 107 cells/mL; 0.1 mL of the bacterial suspensions
were spread on Petri dishes containing mineral medium
with phytagel, added with Tryptophan (2 mg/L) and/or
ACC (1 mM). The plates were stand for 30 minutes for
the inoculum absorption. Twenty five commercially
seeds of Lens esculenta were surface-sterilized with 10%
sodium hypochlorite and then thoroughly rinsed with
sterile distilled water and placed in the Petri dishes, with
the respective conditions. Petri dishes with mineral
medium containing: 0.20 M NH4H2PO4, 0.50 M NH4NO3,
1.15 M Ca(NO3)2, 0.26 M CaCl2, 0.2 M MgCl2·6H2O,
0.20 M Mg(NO3)2·6H2O, 0.40 M MgSO4·7H2O, 0.20 M
KH2PO4, 1.2 M KNO3, 0.5 M K2SO4, 0.04 M
FeCl3·6H2O, 1.2 × 10−2 M H3BO3, 1.2 × 10−4 M
CuCl2·H2O, 2.3 × 10−3 M ZnCl2, 4.4 × 10−4 M
MnCl2·4H2O, 6 × 10−6 M Na2MoO 4·H2O, EDTA and
FeSO4·7H2O, pH = ±6.0, and 3 g of phytagel; un-
inoculated treatments were considered as blanks. All the
experiments were performed by duplicate and main-
tained at 30 °C in a growth chamber in dark for 4 days.
The plant root elongation promoting (PREP) activity
assay was employed to analyze the promoting and
synergistic effect of rhizobacteria strains on Lens esculenta
seeds, according to the modified root elongation assay of
Belimov et al. [23], the root length of the seedlings were
measure and the Growth Index (GI), expressed as the
ratio of the root lengths of plants grown in the presence
and absence of the phytobacteria, was obtained [14]; GI
= RLpb/RLc, where RLpb is the root length of plants
grown in the presence of the specific phytobacteria
inoculum and RLc is the root length of plants grown in
absence of inoculum (control).
2.3. Statistical Analysis
All data were analysed by One-way ANOVA analysis of
variance and the mean differences were compared ap-
plying a Tukey-Kramer post-test, using the statistics
program Graph Pad Instat Ver. 3.10.
3. Results
3.1. IAA Production and ACC Deaminase
Activity by the Tested Strains
Lemna 2 strain and U-M1-4 strain presented a higher
ACC deaminase activity (p < 0.001) than U-M1-5 strain,
according to their colonial diameter (Figure 1). The em-
ploy in the metabolic assay of ammonium sulfate as ni-
trogen source, was only taking it as control, compared to
the ACC assay, but in this study, the two phytobacteria
mentioned had a minor colony diameter, compared to the
ACC activity. The three phytobacteria presented an in-
crease in the IAA production as the tryptophan supply in
cultures increased, the basal production of this auxin
without the aminoacid presented this order: U-M1-5
strain (32.67 μg/mL) > U-M1-4 strain (30.7 μg/mL) >
Lemna 2 strain (19.32 μg/mL), all the bacterial isolates
were classified according to Khalid et al. [24] as higher
phytohormone producers (Figure 2).
3.2. Root Elongation Test of L. esculenta and the
Effect of Phytobacteria Inoculation
The response of L. esculenta roots showed that this spe-
cies was susceptible to the presence of Trp, ACC and Trp
+ ACC, with a significant reduction on the root devel-
opment compared to the roots grown on mineral medium:
U-M1-4 strain 55% > U-M1-5 strain 36.17% > Lemna 2
strain 27.95% (p < 0.001). In general, inoculation with
the three phytobacteria strains decrease the root length
compared to the control roots (U-M1-4 strain > U-M1-5
strain > Lemna 2 strain). Particularly, the effect of the
seeds inoculated with the strains and Trp showed that the
order of response was as follows: U-M1-5 strain >
U-M1-4 strain > Lemna 2 strain), the inoculated seeds
and ACC: Lemna 2 strain > UM1-5 strain > UM1-4 and
Open Access AJPS

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC
Deaminase Activity on Root Length of Lens esculenta Seedlings
Open Access AJPS
2201
Figure 1. ACC deaminase activity of the phytobacteria tested: (A) DF minimal medium with ACC and (B) DF minimal me-
dium with Ammonium Sulfate, where: “a and b” Lemna 2 strain, “c and d” U-M1-5 strain, “e and f” U-M1-6 strain. Mean
values ± S.D. from 48 replicates.
the inoculated seeds and Trp + ACC: UM1-5 strain >
Lemna 2 strain > U-M1-4 strain (Figures 3 and 4).
measured. It is known from application of exogenous
IAA [26] or application of diluted culture extracts or low
density inoculum of bacteria that produce high levels of
IAA [27,28] that low concentrations of IAA can stimu-
late primary root elongation; it is important to note that the
effect of bacterial IAA on plant growth depends on the
size of the inoculum of a single strain, but not always,
4. Discussion
Glick et al. [25] showed that the promotion of root
growth is one of the principal markers by which the
beneficial effect of plant growth-promoting bacteria is

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC
Deaminase Activity on Root Length of Lens esculenta Seedlings
2202
Figure 2. IAA in vitro production of the phytobacteria
tested. Mean values ± S.D. from three replicates.
the inoculum density employed means that more IAA is
available to the plant, and reports of bacterial mutants that
overproduce IAA show a root growth-inhibiting effect
[29-31]. Burd et al. [14] and Belimov et al. [32] reported
that there is a number of plant growth promoting bacteria
that contain the enzyme ACC deaminase and stimulate
the root growth of different plant species; ACC is exuded
from roots or seeds and cleaved by ACC deaminase to
NH3 and a-ketobutyrate, the bacteria utilize the NH3 as a
source of nitrogen and thereby decrease ACC within the
plant [9,33] with the concomitant reduction of plant eth-
ylene [4,14,34,35]. Patten and Glick [36] demonstrated
that the IAA secreted by a bacterium may promote root
growth directly by stimulating plant cell elongation or
cell division or indirectly by influencing bacterial ACC
deaminase activity. The authors mention that the IAA
and ACC deaminase work together to stimulate the root
elongation; this consideration is regarding to the exoge-
nous IAA that increase the transcription and activity of
ACC synthase; this enzyme catalyzes the production of
ACC in plants and therefore ACC stimulates the ACC
deaminase activity in bacteria [6,37], while the IAA
produced by the bacterial inoculum, stimulates the root
elongation or the formation of lateral and adventitious
roots [29-31,38]. In this study, the correlation between
the IAA production and the ACC deaminase activity by
the phytobacteria tested, showed a relationship between
the U-M1-4 and U-M1-5 strains that were joined together
as one group by this attribute, separated to the Lemna 2
strain; probably the density of the inoculum produced a
high IAA concentration that generated a inhibiting effect
on root growth, joined to the ACC metabolism activity
presented by the phytobacteria. This attribution is related
with the visible short and thick appearance of the roots
showed in the treatments with ACC and Trp + ACC. The
effect of the inoculation with these three phytobacteria
strains on Lens esculenta seeds, doesn’t show an evident
increase in the root length of seedlings; but particularly
was the response of two of the isolates: Lemna 2 strain and
Figure 3. Root length measurement of Lens esculenta seed-
lings: (a) Control experiments, (b) Trp, ACC and Trp +
ACC inoculated with Lemna 2 strain, (c) Trp, ACC and Trp
+ ACC inoculated with U-M1-4 strain and (d) Trp, ACC and
Trp + ACC inoculated with U-M1-5 strain. Mean values ±
S.D. from 50 replicates, the different bold letters show the
significant differences between experiments (p < 0.001).
Open Access AJPS

Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC
Deaminase Activity on Root Length of Lens esculenta Seedlings
2203
Figure 4. Root length experiments of Lens esculenta seeds
inoculated with U-M1-5 strain: (a) Mineral Medium, (b)
Mineral Medium + ACC, (c) Mineral Medium + Trp and (d)
Mineral Medium + Trp + ACC.
U-M1-5 strain with a relationship according to their
measured attributes: Lemna 2 strain with a high ACC
activity (with a 47.32% of root growth compared to the
control roots) and U-M1-5 strain with the highest in vitro
IAA production (with a 94.98% of root growth compared
to the control roots) especially with the presence of Trp
(2 mg/L). Even the root length of the seedlings was lesser
than control seedlings for both strains; the seeds’ treat-
ment with Trp + ACC showed that the response of the
root length was higher in the experiments with TRP with
U-M1-5 strain (with a 78.26% of root growth compared
to the control roots) than Lemna 2 strain (with a 71.09%
of root growth compared to the control roots).
These results were according to the results obtained by
Zafar-Ul-Hye et al. [39]; these authors screened rhizo-
bacteria containing ACC deaminase to promote lentil
growth under axenic conditions and by Shaharoona et al.
[5], who analyzed different strains of rhizobacteria with
variations in their ACC deaminase activity, capability of
the rhizobacterial isolates of IAA production in the pres-
ence and absence of tryptophane and isolates also varied
in their ability to colonize etiolated pea roots; both ac-
cording to the reports by Shaharoona et al. [40] and
Zafar-Ul-Hye et al. [39] where the bacteria with more
ACC deaminase activity had more ability to decrease the
intensity of the called “ACC-induced classical triple re-
sponse”, the ACC deaminase activity of bacteria was
responsible for the decrease of endogenous and exoge-
nous ACC supply in inoculated plant with the inhibition
on the root length of L. esculenta seedlings (Lemna 2
strain to the control roots). Finally, although the roots of
L. esculenta seedlings do not show a significant promo-
tion with the presence of the strains, Trp and ACC in
medium; and even though these responses were adverse,
the results obtained in this work suggested a synergistic
effect between the two bacterial attributes probed and the
phytobacteria tested and could recommended that it is
important to consider the inoculum density to the in-
crease of plant biomass and retard the effect of ethylene
on cultures supplied with Tryptophan and ACC.
5. Acknowledgements
Authors are grateful to the Research Projects: SIP:
20131494 of the Secretaría de Investigación y Posgrado
del I.P.N. and ISITDF/325/11 AREAS PRIORITARIAS-
IPN and COFAA-IPN, EDI-IPN, SNI-CONACYT fel-
lowships.
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Effect of Inoculation with Three Phytohormone Producers Phytobacteria with ACC
Deaminase Activity on Root Length of Lens esculenta Seedlings
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